Major Project (Draft 2)

Major Project (Draft 2)

Reductive Alkylation and Sequential Reductive Alkylation-Click Chemistry for On-Solid-Support Modification of Pyrrolidinyl Peptide Nucleic Acid

Ditmangklo et al. (2013) conducted a study to develop the methodology for the site-specific attachment of fluorophores to the backbone of pyrrolidinyl peptide nucleic acids (PNAs) with an α/β-backbone derived from D-prolyl-(1S,2S)-2-aminocyclopentanecarboxylic acid (acpcPNA). The reductive N-alkylation of the acpcPNA, previously modified with a (3R,4S)-3-amino pyrrolidine-4-carboxylic acid (azaACPC) spacer, was carried out on solid support by first reacting the azaACPC-modified acpcPNA with the aldehyde-containing labels in the presence of NaBH₃CN under mildly acidic conditions. The reductive alkylation reaction is remarkably efficient and compatible with a range of reactive functional groups including azide and alkynes. The azide/alkyne-modified acpcPNA was further labeled with pyrene (Py)/thiazole (TO), a representative azide/alkyne-functionalized fluorophore, using Cu(I)-catalyzed Huisgen azide-alkyne cycloaddition (click chemistry). The two-step reaction sequence proceeded in quantitative yield without side reactions as verified by Matrix Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) mass spectrometry after cleavage of the acpcPNA from the solid support. The acpcPNA probe in this way does not negatively affect the affinity and specificity of the pairing to its DNA target. This methodology can be applied in creating self-reporting pyrene- and thiazole orange-labeled acpcPNA probes that can yield a change in fluorescence in response to the presence of the correct DNA target have also been explored. The excellent fluorescence was observed with thiazole orange-labeled acpcPNA in the presence of DNA. The specificity could be further improved by enzymatic digestion with S1 nuclease, providing a 9- to 60-fold fluorescence enhancement with fully complementary DNA and a less than 3.5-fold enhancement with mismatched DNA targets. The researchers suggested that this strategy offers a convenient and effective way for the development of internally-labeled fluorescent acpcPNA probes.

This study provides a new strategy for site-specific modification of pyrrolidinyl peptide nucleic acid consisting of an alternating sequence of nucleobase-modified
D-proline/(1S,2S)-2-aminocyclopentanecarboxylic acid (acpcPNA). However, the strategy for fluorophores-labeled to acpcPNA are some limitations.

1) The synthesis of internally-labeled fluorescent acpcPNA probes in this study were synthesized only click with pyrene and thiazole orange as fluorescence dyes in the presence of tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine37 (TBTA), tetrakis(acetonitrile) copper(I) hexafluorophosphate in 3:1 (v/v) DMSO:^tBuOH and (+)-sodium-L-ascorbate  in H₂O at room temperature overnight. Although this condition is an effective way for pyrene and thiazole orange, but some fluorophore is not stable in the presence (+)-sodium-L-ascorbate. According to Wen-hai et al. (2005), the alkyne-fluorophores (styryl dye, fluorescein) were conjugated to biomolecules in the presence of CuSO₄,  tris(carboxyethyl)phosphine (TCEP) as the reducing agent, and tris(triazolyamine) as the ligand.

2) An internally-labeled fluorescent acpcPNA probes in this study were synthesized in two steps (Reductive Alkylation and Click Chemistry) while the DNA probes (Moritz et al., 2012) were produced only one step (Click Chemistry). Therefore, the acpcPNA probes may be used large-consumer for synthesis more than the DNA probes.

The strength of this study is that the confirmation of this strategy by using the MALDI-TOF mass spectroscopy and HPLC techniques. This method is widely acceptable for research in the synthesis of fluorescent probes field and this is convenient and efficient way to site-specific attachment of fluorophores to acpcPNA. Furthermore, these probes can apply in the area of molecular diagnostics.

References

Wen-hai, Z., Hannah, N. B., Krishnamoorthy, S., He, T., Qian, W. (2005). Synthesis of hemicyanine dyes for ‘click’ bioconjugation. Tetrahedron Lett., 46, 1691-1695.

Moritz, M. R., Carolin. H., Peggy, R. B., Hans-Achim, W. (2012).  A “Clickable” Styryl Dye for Fluorescent DNA Labeling by Excitonic and Energy Transfer Interactions. Chem. Eur. J., 18, 1299-1302.

Boonsong, D., Chalothorn, B., Chaturong, S., Tirayut, V. (2013). Reductive Alkylation and Sequential Reductive Alkylation-Click Chemistry for On-Solid-Support Modification of Pyrrolidinyl Peptide Nucleic Acid. Bioconjugate Chem., 24, 614-625.