Clicked polycyclic aromatic hydrocarbon as a hybridization-responsive fluorescent artificial nucleobase in pyrrolidinyl peptide nucleic acids

Article abstract of DOI:10.1016/j.tet.2012.03.062

Pyrene as well as other aromatic hydrocarbons could be successfully incorporated into pyrrolidinyl peptide nucleic acid bearing a d-prolyl-2-aminocyclopentane carboxylic acid backbone (acpcPNA) as a base surrogate via a triazole linker employing Cu-catalyzed alkyne-azide cycloaddition (click chemistry). The labeling can be performed via a pre-clicked pyrene monomer or by post-synthetic modification of azide-containing acpcPNA on solid support. Thermal denaturation experiments suggested that the pyrene-triazole unit can behave as a universal base in the acpcPNA system. The mode of base-pairing has been proposed based on molecular dynamics simulations. Importantly, the fluorescence spectra of the pyrene-labeled single stranded acpcPNA and its hybrid with DNA are quite different. The ratio of emissions at 380 and 460 nm changed significantly (up to a factor of 7) upon hybrid formation with complementary DNA.

Full text of DOI:10.1016/j.tet.2012.03.062

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Supporting information
Clicked polycyclic aromatic hydrocarbon as a hybridization-responsive fluorescent
artificial nucleobase in pyrrolidinyl peptide nucleic acids
Woraluk Mansawat,a Chalothorn Boonlua,a Khatcharin Siriwong,b and Tirayut Vilaivan a,*
aOrganic Synthesis Research Unit, Department of Chemistry, Faculty of Science, Chulalongkorn
University, Phayathai Road, Patumwan, Bangkok 10330, Thailand.
bDepartment of Chemistry and Center for Innovation in Chemistry, Faculty of Science, Khon
Kaen University, Khon Kaen 40002, Thailand.
*E-mail: vtirayut@chula.ac.th
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Fig. S1
Fig. S2
Fig. S3
Fig. S4
Fig. S5
Fig. S6
Fig. S7
Fig. S8
Fig. S9
1H (a) and 13C (b) NMR spectra of 4
Analytical HPLC chromatogram and MALDI-TOF mass spectrum of PNA1
Analytical HPLC chromatogram and MALDI-TOF mass spectrum of PNA2
Analytical HPLC chromatogram and MALDI-TOF mass spectrum of PNA3
Analytical HPLC chromatogram and MALDI-TOF mass spectrum of PNA1
Analytical HPLC chromatogram and MALDI-TOF mass spectrum of PNA5
Analytical HPLC chromatogram and MALDI-TOF mass spectrum of PNA6
Analytical HPLC chromatogram and MALDI-TOF mass spectrum of PNA7
HPLC chromatogram of crude PNA3 and after click reaction
Fig. S10 Thermal denaturation curves of hybrids with of PNA1 with DNA1–DNA4
Fig. S11 Photographs of PNA1 and its hybrids with DNA1–DNA4 under black light
Fig. S12 Selected snapshots of MD structures showing the hydrogen-bond formation
between TzPy and opposite base
Fig. S13 Fluorescence spectra of PNA5 (a) and PNA7 (b) in the absence (–) and
presence (···) of complementary DNA
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2
9.0
8.0
7.0
6.0
5.0
4.0
3.0
2.0
1.0
ppm (t1)
(a)
150
100
50
ppm (t1)
(b)
Fig. S1. 1H (400 MHz, DMSO-d6) (a) and 13C NMR (100 MHz, DMSO-d6) (b) spectra of 4.

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(a)
(b)
Fig. S2. Analytical HPLC chromatogram (a) and MALDI-TOF mass spectrum (b) of PNA1 (Bz-
GCATTAXAGATAC-LysNH2; X = TzPy) obtained by pre-formed TzPy monomer (CCA matrix)
(calcd. for [M+H]+: m/z = 4779.2).

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(a)
(b)
Fig. S3. Analytical HPLC chromatogram (a) and MALDI-TOF mass spectrum (b) of PNA2 (Bz-
GCATTAXAGATAC-LysNH2; X = C) (CCA matrix) (calcd. for [M+H]+: m/z = 4621.0).

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(a)
(b)
Fig. S4. Analytical HPLC chromatogram (a) and MALDI-TOF mass spectrum (b) of PNA3 (Bz-
GCATTAXAGATAC-LysNH2; X = N3) (CCA matrix) (calcd. for [M+H]+: m/z = 4552.9).

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(a)
(b)
Fig. S5. Analytical HPLC chromatogram (a) and MALDI-TOF mass spectrum (b) of PNA1 (Bz-
GCATTAXAGATAC-LysNH2; X = TzPy) obtained by post-synthetic click reaction (CCA
matrix) (calcd. for [M+H]+: m/z = 4779.2).

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(a)
(b)
Fig. S6. Analytical HPLC chromatogram (a) and MALDI-TOF mass spectrum (b) of PNA5 (Bz-
GCATTTXTGATAC-LysNH2; X = TzPy) (CCA matrix) (calcd. for [M+H]+: m/z = 4761.2).

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(a)
(b)
Fig. S7. Analytical HPLC chromatogram (a) and MALDI-TOF mass spectrum (b) of PNA6 (Bz-
GCATTTXTGATAC-LysNH2; X = TzPh) (CCA matrix) (calcd. for [M+H]+: m/z = 4637.0).

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(a)
(b)
Fig. S8. Analytical HPLC chromatogram (a) and MALDI-TOF mass spectrum (b) of PNA7 (Bz-
GCATTTXTGATAC-LysNH2; X = TzAn) (CCA matrix) (calcd. for [M+H]+: m/z = 4737.1).

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Fig. S9. HPLC chromatogram of crude PNA3 (Bz-GCATTAN3AGATAC-LysNH2) before (top)
and after (bottom) click reaction with 1-ethynylpyrene.

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1.30
1.25
1.20
1.15
1.10
1.05
1.00
0.95
PNA1 + DNA1 (G)
PNA1 + DNA2 (A)
PNA1 + DNA3 (C)
PNA1 + DNA4 (T)
PNA3 + DNA4 (T)
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30
40
50
60
70
80
90
T (oC)
Fig. S10. Thermal denaturation curves of hybrids between PNA1 (TzPy) with DNA1–DNA4 and
PNA3 (azide) with DNA4. Conditions: 2.5 µM PNA, 3.0 µM DNA in 10 mM sodium phosphate
buffer pH 7.0.

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PNA1
+DNA1
+DNA2
+DNA3
+DNA4
ss
Y = G
Y = A
Y = C
Y = T
Fig. S11. Photographs of PNA1 and its hybrids with DNA1–DNA4 viewed under black light.
Conditions: 2.5 µM PNA, 3.0 µM DNA in 10 mM sodium phosphate buffer pH 7.0.

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TzPy-Guanine (PNA1·DNA1)
TzPy-Adenine (PNA1·DNA2)
TzPy-Cytosine (PNA1·DNA3)
TzPy-Thymine (PNA1·DNA4)
Fig. S12. Selected snapshots of MD structures showing the hydrogen-bond formation between
TzPy and opposite base for four simulated systems. The hydrogen bond acceptor-donor distances
are shown.

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350
300
250
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150
100
50
350
300
250
200
150
100
50
PNA
PNA+DNA
PNA
PNA+DNA
0
0
350
400
450
500
550
600
350
400
450
500
550
600
Wavelength (nm)
Wavelength (nm)
Fig. S13. Fluorescence spectra of PNA5 (a) and PNA7 (b) in the absence (···) and presence (–) of
complementary DNA (5'-GTATCAGAAATGC-3') (conditions: 2.5 µM PNA, 3.0 µM DNA in
10 mM sodium phosphate buffer pH=7.0, λexcit = 350 nm).