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.

Major Project (Draft 1)

Major Project (Draft 1)

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 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 limitation.

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. Forthemore, these probes can apply in DNA detection as a diagnosis of some disease.

MINOR PROJECT

Pyrrolidinyl peptide nucleic acid internally-labeled with cyanine-styryl dye as a fluorescence DNA probe

My research question is to find excellent the environment-sensitive fluorescence labels, alkyne-modified cyanine-styryl derivatives (STR), which were labeled onto pyrrolidinyl peptide nucleic acid with D-proline/(1S,2S)-2-aminocyclopentanecarboxylic acid backbone (acpcPNA) via sequential reductive alkylation-Click strategy with the aim to create self-reporting PNA probes that exhibit fluorescence change in response to the correct DNA target. The styryl dye family has received increasing attention as a photostable DNA label that revealed the excellent colorimetric response in difference microenvironments.

Researchers who have looked at this subject are Wen-hai Zhan and Rubner. Both of studies focused on the synthesis and evaluation of styryl dyes capable of exhibiting turn-on fluorescence through sequence-specific interaction with nucleic acid in fluorescence spectroscopy and diagnostic application.

Zhan et al. (2005) argue that the 2-propynyl substituted hemicyanine dyes were synthesized in a facile route and showed good optical properties that have a strong absorbance as well as a large strokes shift of the fluorescence emission maxima. The terminal alkynyl group of dyes was successfully employed for bioconjugation through a Click strategy with azide-modified cowpea mosaic virus (CPMV).

Rubner et al. (2012) argue that the alkyne-modified indole-quinoline styryl dye (CyIQ) was synthesized and incorporated it into DNA via click chemistry. The CyIQ dye exhibit good brightness and give excellent photostability.

Debate centers on this basic issue of the fluorescent properties of cyanine-styryl dyes which exhibit good brightness. Thus, we are interested in synthesizing new clickable styryl dyes for attachment onto acpcPNAs with the aim to use them as DNA sensing probes.

My work will be closer to Rubner’s in the focus on the synthesis of the alkyne-modified styryl family for fluorescent labeling and incorporated into nucleic acid as fluorescent probes.

Hopefully my contribution will be to synthesize and incorporate into acpcPNA which discriminate between the complementary DNA and single mismatch DNA and apply in SNP detection for diagnosis.

Reference list

Zhan, W-h., Barnhill, H. N., Sivakumar, K., Tian, H., Wang, Q. (2005). Synthesis of hemicyanine dyes for ‘click’ bioconjugation. Tetrahedron Letters, 46, 1691-1695.

Rubner, M. M., Holzhauser, C., Bohlander, P. R., Wagenknecht, H-A. (2012). A “Clickable” Styryl Dye for Fluorescent DNA labeling by Excitron and Energy Transfer Interactions. Chemistry - A European Journal, 18, 1299-1302. 

Assignment 2: Writing Introduction

Stage 1: DNA sequence analysis is widely used in several applications ranging from clinical diagnosis, food and agricultural sciences and forensic sciences. The development of rapid, sensitive and accurate method for DNA sequence determination is therefore a very important research area. There are two main approaches to investigate a DNA sequence, by direct sequencing and by the use of a hybridization probe. The latter method is more attractive for routine diagnosis because there is no requirement for expensive instruments.

Stage 2: The fluorescent hybridization probe is a short oligomer of DNA or its analogue that has a base sequence complementary to the region of interest in the DNA target. The conventional molecular beacon (MB) probe was designed and improved by Kramer et al.   (1996). There found that the MB probes are suited method for SNP detection. However, the MB probes was cannot performed well at room temperature. The limitation was avoided by using binary-probes for SNP analysis at room temperature. Therefore, the base-discriminating fluorescent (BDF) probes containing pyrenecaboxamide-labeled uracil (^PyU) and cytosine (^PyC) nucleobases were developed by Okamoto et al. (2004). Moreover, Seio et al. (2008) had synthesized and studied properties of N⁶-[N-(pyren-1-ylmethyl)carbamoyl]-deoxyadenosine (dA^pymcm) which was incorporated into internal oligonucleotides and showed fluorescence increasing in the presence of DNA target. Peptide nucleic acid (PNA) is one of DNA analogue in which the deoxyribose phosphate backbone is replaced by N-(2-aminoethylglycine) unit.  PNA has been first reported by Nielsene et al.  in 1991 (also known as aegPNA or Nielsen's PNA). Appella et al. (2005) synthesized fluorescent aegPNA probes as a sequence-free molecular beacon for DNA analysis. This PNA probe was increased fluorescent signal in the presence of the perfectly complementary DNA. Moreover, the thaizole orange modified FIT probes was described by Socher et al. (2008). The duplex of FIT probes with complementary DNA is exhibited increasing fluorescent signal.

Stage 3: Vilaivan et al. (2005) had reported a new version of pyrrolidinyl PNA called acpcPNA. This new PNA system contains a rigid backbone consisting of D-prolyl-2-aminocyclopentanecarboxylic acid (ACPC) subunits. Previously research in this area, the DNA and aegPNA was only developed for fluorescent probes. Nevertheless, the pyrrolidinyl PNA (acpcPNA) are no reported on the optical properties. Therefore, the fluorescent acpcPNA probes were developed.

Stage 4: The objective of this research is to develop fluorescent acpcPNA probes with styryl fluorescent dyes labeling via a sequential reductive alkylation/click chemistry. In addition, the optical properties of dyes-labeled acpcPNA in the absence and presence of target DNA are investigated.

Stage 5: The fluorescent acpcPNA probes can be discriminated the absence and presence of target DNA on ‘naked-eye’ detection and applied in SNP detection for diagnosis. 

A NEW FAMILY OF BIOORTHOGONALLY APPLICABLE FLUOROGENIC LABELS

Abstract: Synthetic procedures for the construction of fluorogenic azido-labels were developed. Photophysical properties were elaborated by experimental and theoretical investigations. Of the newly synthesized fluorogenic and bioorthogonally applicable dyes two were selected on the basis of their fluorogenic performance and further subjected to in vitro and in vivo studies. Both tags exhibited excellent fluorogenic properties as in aqueous medium, the azide form of the selected dyes is virtually non-fluorescent, while their “clicked” triazole congeners showed intense fluorescence. One of these labels showed a very large Stokes shift. To the best of our knowledge this is the first reported case of mega-Stokes type of fluorogenic labels. These studies have justified that these two fluorogenic tags are remarkably suitable for bioorthogonal tagging schemes. The developed synthetic approach together with the theoretical screen of possible fluorogenic tags will enable the generation of libraries of such tags in the future.

Reference

Herner, A.; Nikić, I.; Kállay, M.; Lemke, E. A.; Kele, P. Org. Biomol. Chem. 2013, 11, 3297–3306.

Results/Finding

The azide-alkyne groups in click chemistry are suitable methodology for bioorthogonal tagging schemes.

Citation

1) Herner et al. (2013) reported that the bioorthogonally applicable dyes were labeled via click chemistry in aqueous medium condition (p. 3297).

2) Herner et al. (2013) found that the azide form exhibited non-fluorescent properties while the triazole-linkage form revealed excellent fluorescent (p. 3297).