evolution of the reaction by electrospray mass spectrometry.
This technique can easily evidence the presence of possible
intermediates still bearing nitro groups.
8. The Pd/C catalyst is a very thin solid. At least two superimposed paper filters are necessary to efficiently filter it off from
the reaction mixture. However, the catalyst could not be
completely retained and the filtrate could still present a pale
black/gray color. In this latter case, the filtrate must be filtered
again using the same procedure.
9. Alternatively, the reaction can be conveniently carried out in a
microwave reactor performing two cycles (1 h each) at 200 W
and 25
C.
10. The completion of the reaction is monitored by TLC (eluent:
DCM/MeOH 96/4, v/v) and spots revealed under the UV
lamp. The product has an rf ¼ 0.32.
11. The progression of the reaction is monitored by TLC (eluent:
DCM/MeOH 96/4, v/v) to verify the disappearance of the
starting fully protected calixarene (rf ¼ 0.32) or, even better,
by ESI–Mass Spectrometry. In fact, the removal of even a single
protecting group is sufficient to retain the corresponding product at the starting line in the TLC plate. An ESI-MS analysis in
positive ionization mode is then necessary to verify the possible
presence of intermediates due to partial deprotection in addition to the final fully deprotected compound.
12. The repetition of this operation helps the complete removal of
residual TFA.
13. With this procedure, the trifluoroacetate anions of the resulting
TFA octa-salt are replaced by chloride anions.
14. In order to determine the concentrations of argininocalix[4]
arene 1 to be employed for in vitro studies on target cells, the
IC 50 after treatment for different days should be always determined. In order to avoid to use antiproliferative (and possibly
cytotoxic) concentrations, the argininocalix[4]arene (1)
should be used at concentration lower than the IC 50 values.
In any case, experiments similar to those depicted in Fig. 3
might be considered [62, 63].
15. Unlike commercially available anti-miRNAs, which needs continuous administrations, a single administration of antimiRNA PNAs complexed to argininocalix[4]arene 1 is sufficient to obtain the biological effects on microRNA activity
[47]. In order to have a PNA to be used as positive control,
PNAs carrying an octaarginine (R8) peptide conjugated at
N-terminus of the PNA chain should be considered. We have
reported that the delivery of these R8-carrying PNAs
approaches 100% (i.e., uptake in 100% of the target cell
PNA Delivery with Argininocalix[4]arene
135
This technique can easily evidence the presence of possible
intermediates still bearing nitro groups.
8. The Pd/C catalyst is a very thin solid. At least two superimposed paper filters are necessary to efficiently filter it off from
the reaction mixture. However, the catalyst could not be
completely retained and the filtrate could still present a pale
black/gray color. In this latter case, the filtrate must be filtered
again using the same procedure.
9. Alternatively, the reaction can be conveniently carried out in a
microwave reactor performing two cycles (1 h each) at 200 W
and 25
C.
10. The completion of the reaction is monitored by TLC (eluent:
DCM/MeOH 96/4, v/v) and spots revealed under the UV
lamp. The product has an rf ¼ 0.32.
11. The progression of the reaction is monitored by TLC (eluent:
DCM/MeOH 96/4, v/v) to verify the disappearance of the
starting fully protected calixarene (rf ¼ 0.32) or, even better,
by ESI–Mass Spectrometry. In fact, the removal of even a single
protecting group is sufficient to retain the corresponding product at the starting line in the TLC plate. An ESI-MS analysis in
positive ionization mode is then necessary to verify the possible
presence of intermediates due to partial deprotection in addition to the final fully deprotected compound.
12. The repetition of this operation helps the complete removal of
residual TFA.
13. With this procedure, the trifluoroacetate anions of the resulting
TFA octa-salt are replaced by chloride anions.
14. In order to determine the concentrations of argininocalix[4]
arene 1 to be employed for in vitro studies on target cells, the
IC 50 after treatment for different days should be always determined. In order to avoid to use antiproliferative (and possibly
cytotoxic) concentrations, the argininocalix[4]arene (1)
should be used at concentration lower than the IC 50 values.
In any case, experiments similar to those depicted in Fig. 3
might be considered [62, 63].
15. Unlike commercially available anti-miRNAs, which needs continuous administrations, a single administration of antimiRNA PNAs complexed to argininocalix[4]arene 1 is sufficient to obtain the biological effects on microRNA activity
[47]. In order to have a PNA to be used as positive control,
PNAs carrying an octaarginine (R8) peptide conjugated at
N-terminus of the PNA chain should be considered. We have
reported that the delivery of these R8-carrying PNAs
approaches 100% (i.e., uptake in 100% of the target cell
PNA Delivery with Argininocalix[4]arene
135