store the information of possible interfering sequences for the
interpretation of biological data; we normally consider off
targets with almost complete complementarity (i.e., 16–17
bases/18) as severe interferent and redesign is needed if these
are biologically relevant; (d) when alternative sequences are
suitable, targeting of the “seed region” (an essential element
for miRNA function) [50] is preferred.
2. To verify the completion of the reaction, carry out a TLC
analysis (eluent: DCM/hexane 3/1, v/v) on a sample. The
spots of the products and intermediates are detected by irradiation of the TLC plate with a UV lamp. The desired compound
has an rf ¼ 0.8, while possible intermediate products due to
partial alkylation and containing free phenolic OH groups
show lower rf and can be revealed by treatment of the TLC
plate with a saturated aqueous solution of FeCl 3 followed by
gentle heating (brown coloration of the spot).
3. If the purification by recrystallization is not sufficient to remove
the persistent impurities and by-products (detected by TLC, see
Note 2), a flash chromatography column on silica gel using
hexane as eluent represents a convenient methods to obtain
pure p-tert-butyl-tetrahexyloxycalix[4]arene 2.
4. The reaction can conveniently be performed also in a mixture
DCM/TFA 5/7 (v/v) dissolving the calixarene in DCM, with
comparable results in terms of yields.
5. To verify the completion of the reaction, take a sample, and
check it by TLC (eluent: DCM). Due to the heterogenicity of
the reaction, be sure to sample also a portion of solid together
with an aliquot of solution. The reaction is complete when the
spot corresponding to the reagent 2 is disappeared and only a
spot with rf ¼ 0.30 corresponding to the product is present.
The spots on the TLC plate are visible under UV lamp.
6. The hydrated MgSO 4 should be filtered off on a pleated paper
filter and abundantly washed with DCM.
7. To verify the completion of the reaction, this is monitored by
TLC (eluent: DCM) to check the disappearance of the starting
calixarene (rf ¼ 0.30). Due to the reduction of the nitro groups
to amines, the product, as the partially reduced intermediates,
are retained at the TLC starting line. The TLC plate, after
elution, can be treated with an acidic solution of ninhydrin in
EtOH, followed by heating, to check the presence at the starting line of a spot that becomes purple/red corresponding to
amino containing calixarenes. Due to the difficulty in separating on TLC plate the partially reduced intermediates (calixarenes still bearing residual nitro groups) from the completely
reduced product, it could be convenient to monitor the
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Alessia Finotti et al.
interpretation of biological data; we normally consider off
targets with almost complete complementarity (i.e., 16–17
bases/18) as severe interferent and redesign is needed if these
are biologically relevant; (d) when alternative sequences are
suitable, targeting of the “seed region” (an essential element
for miRNA function) [50] is preferred.
2. To verify the completion of the reaction, carry out a TLC
analysis (eluent: DCM/hexane 3/1, v/v) on a sample. The
spots of the products and intermediates are detected by irradiation of the TLC plate with a UV lamp. The desired compound
has an rf ¼ 0.8, while possible intermediate products due to
partial alkylation and containing free phenolic OH groups
show lower rf and can be revealed by treatment of the TLC
plate with a saturated aqueous solution of FeCl 3 followed by
gentle heating (brown coloration of the spot).
3. If the purification by recrystallization is not sufficient to remove
the persistent impurities and by-products (detected by TLC, see
Note 2), a flash chromatography column on silica gel using
hexane as eluent represents a convenient methods to obtain
pure p-tert-butyl-tetrahexyloxycalix[4]arene 2.
4. The reaction can conveniently be performed also in a mixture
DCM/TFA 5/7 (v/v) dissolving the calixarene in DCM, with
comparable results in terms of yields.
5. To verify the completion of the reaction, take a sample, and
check it by TLC (eluent: DCM). Due to the heterogenicity of
the reaction, be sure to sample also a portion of solid together
with an aliquot of solution. The reaction is complete when the
spot corresponding to the reagent 2 is disappeared and only a
spot with rf ¼ 0.30 corresponding to the product is present.
The spots on the TLC plate are visible under UV lamp.
6. The hydrated MgSO 4 should be filtered off on a pleated paper
filter and abundantly washed with DCM.
7. To verify the completion of the reaction, this is monitored by
TLC (eluent: DCM) to check the disappearance of the starting
calixarene (rf ¼ 0.30). Due to the reduction of the nitro groups
to amines, the product, as the partially reduced intermediates,
are retained at the TLC starting line. The TLC plate, after
elution, can be treated with an acidic solution of ninhydrin in
EtOH, followed by heating, to check the presence at the starting line of a spot that becomes purple/red corresponding to
amino containing calixarenes. Due to the difficulty in separating on TLC plate the partially reduced intermediates (calixarenes still bearing residual nitro groups) from the completely
reduced product, it could be convenient to monitor the
134
Alessia Finotti et al.