Synthesis and DNA/RNA complementation studies of peptide nucleic acids containing 5-halouracils

A series of peptide nucleic acids containing 5-halouracils have been synthesized using the Fmoc protection strategy, and their enhanced hybridizations have been studied.

Abstract

The monomers of peptide nucleic acids containing 5-halouracils (5-XU-PNA), incorporated into heptameric PNA in the middle position, have been synthesized. Thermodynamic analyses revealed that the heptameric PNA oligomer with DNA and RNA showed higher duplex stability compared to the unmodified PNA counterpart. NMR studies suggested that the electron withdrawing effect of the halogen atom increased the strength of the XU–A hydrogen bond.

Oligonucleotide analogs have received considerable attention during the past decade because of their present and potential therapeutic applications.1 Both modified and artificial nucleic acids have been investigated to increase their affinity and improve their nuclease resistance. In such context, a new type of artificial oligonucleotide with a unique backbone termed peptide nucleic acid (PNA) has been designed. PNAs, first described and synthesized by Nielsen in 1991,2 are completely artificial DNA analogues in which the sugar-phosphate backbone of natural nucleic acids is replaced by N-(2-aminoethyl)glycine units and nucleobases are attached via a methylene carbonyl linker. The spatial structure of PNAs is very similar to native oligonucleotides although PNAs are wholly synthetic nucleic acids. Their structure allows not only an effective recognition of complementary DNA or RNA obeying the Watson–Crick base-pairing rules, but also a remarkably high sequence specificity and affinity which are considered to be due to their uncharged backbone.3 For these reasons, PNAs are of great interest in biological medicine, with potential for development as antisense and/or antigene therapeutics. As research continues, chemical modifications of either the polyamide backbone or the nucleotide base have been performed for improved properties.4

Recently, introduction of halogen atoms into PNAs has attracted extensive interest because most of the halogenated modified PNAs have enhanced hybridization5 and cellular uptake.6 5-Halouracils (5-XU), which are formed when a halogen (fluorine, chlorine or bromine) substitutes the C5 hydrogen in uracil or the methyl group in thymine, may be one of the most important halogenated nucleobases.7 Substitution of the nucleobases of DNA with 5-XU has been reported, and the base pairing configuration and stability have been investigated.8 The synthesis of the 5-bromouracil (5-BrU) PNA monomer using the MMT protection strategy has been described.9 These lines of research led us to become interested in alternative 5-halouracils [5- fluorouracil (5-FU) and 5-chlorouracil (5-ClU)], particularly 5-FU, which is the most widely used chemotherapeutic agent in oncology, as a functionalized nucleobase for conjugation to the backbone of PNAs. Moreover, a number of prodrugs of 5-FU have been synthesized10 and the release of 5-FU from the prodrugs has been studied.11 Based on these studies, the combination of 5-FU and 5-FU-PNA may represent a promising new approach for cancer treatment.

In this paper, we report the synthesis of PNAs containing three kinds of 5-halouracils in order to meet the demands of urgent research. The Fmoc protection strategy has been adopted due to its mild reaction conditions and high coupling efficiency. The hybridization properties of 5-XU-PNA to DNA and RNA have been described, and the effect of the substituted 5-XU has been studied.

5-XU-PNA monomers were synthesized according to a similar previously reported procedure9 with minor modifications, as outlined in Scheme 1. The new protocol did not involve the MMT protection strategy, which caused either high expense or acid-lability. Therefore, we synthesized 5-XU-PNA monomers using the Fmoc protection strategy. tert-Butyl-N-(2-Fmoc-aminoethyl)glycinate, which is widely used as the backbone for PNA monomer synthesis, was prepared according to a literature procedure.12 Other key moieties are 5-halouracil acetic acids (2a–2c), which were prepared via direct alkylation of 5-XU with bromoacetic acid. Then using compound 2 coupled with tert-butyl-N-(2-Fmoc-aminoethyl)glycinate hydrochloride employing HATU as a condensing agent in the presence of DIEA, the Fmoc-protected PNA monomers (3a–3c) were produced. Finally, compound 3 was treated with TFA to remove the tert-butyl group to obtain the desired 5-XU-PNA monomers (4a–4c).

Scheme 1. Synthesis of 5-XU-PNA monomers.

Several PNA oligomers were prepared on Wang resin using a manual device for solid-phase protocols. The sequences were: GGGTGTT-Lys-NH₂ (PNA1), GGG-FU-GTT-Lys-NH₂ (PNA2), GGG-ClU-GTT-Lys-NH₂ (PNA3) and GGG-BrU-GTT-Lys-NH₂ (PNA4), where FU, ClU, and BrU indicate the 5-FU-PNA unit, 5-ClU-PNA unit, and 5-FU-PNA unit, respectively (Fig. 1). The unmodified PNA1 was prepared for control studies, and the other embedded chimeric PNAs containing 5-XU-PNA units in the middle were designed to examine the effect of 5-XU on the stability of hybridization. The synthesis of PNA1–4 was carried out using HATU-DIEA as the coupling reagent. The deprotection of the side-chain amine was performed synchronously during the final cleavage of the oligomer from the polymeric support using TFA-TIS. The lysine residue was conjugated at the C-terminus of the PNA to promote solubility and prevent aggregation. All PNA oligomers were purified by reverse-phase HPLC, checked by analytical RP-HPLC, and characterized by mass spectrometry (Table 1).

Fig. 1. Structures of synthesized PNA oligomers. PNA1: X = CH₃, PNA2: X = F, PNA3: X = Cl, PNA4: X = Br.

Table 1. ESI-MS spectral and analytical RP-HPLC data of synthesized PNA oligomers.

Oligomer	Molecular formula	Mol wt calc.	Peaks observed	Purity (%)	Retention time (min)
PNA1	C83H109N43O25	2109.0	1055.4 [M + 2H]2+; 703.8 [M + 3H]3+; 528.2 [M + 4H]4+	97.24	9.212 a
PNA2	C82H106FN43O25	2113.0	1057.2 [M + 2H]2+; 705.0 [M + 3H]3+; 529.0 [M + 4H]4+	98.12	9.349 b
PNA3	C82H106ClN43O25	2129.5	1064.7 [M + 2H]2+; 710.3 [M + 3H]3+; 533.1 [M + 4H]4+	98.46	9.797 b
PNA4	C82H106BrN43O25	2173.9	1087.6 [M + 2H]2+; 725.5 [M + 3H]3+; 544.3 [M + 4H]4+	97.13	10.115 b

^aVenusil C-18 column (250 × 4.6 mm i.d., 5 μm); eluent A, 0.1% TFA in 100% water (v/v); eluent B, 0.1% TFA in 80% acetonitrile + 20% water (v/v); gradient, linear 12–32% B over 20 min; detection, 220 nm; flow rate, 1 mL min^–1.

^bAgilent Pursuit C-18 column (250 × 4.6 mm i.d., 5 μm); eluent A, 0.1% TFA in 100% water (v/v); eluent B, 0.1% TFA in 80% acetonitrile + 20% water (v/v); gradient, linear 10–30% B over 20 min; detection, 220 nm; flow rate, 1 mL min^–1.

The effects of halogen substitution in the base of PNA on the thermal stability T_m of the PNA duplexes with DNA and RNA (5′-AACYCCC-3′, Y = A, T/U, G or C) were investigated by UV-melting experiments (260 nm). The UV-T_m profiles of the perfectly matched duplexes are shown in Fig. 2, and the other UV-T_m profiles are given in the ESI‡ (S9–S11). The T_m values are determined by the maxima in the first derivative curves, and the results are shown in Table 2.

Fig. 2. UV-T_m profiles of PNA/DNA:DNA duplexes (top) and PNA/DNA:RNA duplexes (bottom). The T_m was measured in 10 mM phosphate buffer (pH 7.0); strand concentrations: 5 μM; temperature range: 10 to 80 °C; heating rate: 1 °C min^–1.

Table 2. T _m data for PNA/oligonucleotide complexes ^a .

Oligomers	T m (°C) b
	DNA2 c	DNA3 d	DNA4 d	DNA5 d	RNA1 c	RNA2 d	RNA3 d	RNA4 d
DNA1 e	20.9	<10	<10	<10	20.6	<10	<10	<10
PNA1	36.9	25.1	27.8	27.3	42.0	29.2	32.4	30.2
PNA2	40.9	25.4	28.9	27.5	45.7	29.4	33.8	30.0
PNA3	40.3	25.3	29.0	26.4	45.2	29.5	33.6	29.6
PNA4	38.7	25.6	27.3	26.2	44.5	29.7	33.2	29.4

^aSolutions of PNA/oligonucleotide were prepared in 10 mM phosphate buffer (pH 7.0); strand concentrations: 5 μM; temperature range: 10 to 80 °C; heating rate: 1 °C min^–1.

^b T _m was determined from the first derivative plot and the estimated error is ±0.5 °C.

^cFully complementary oligonucleotide (antiparallel), DNA2: 5′-AACA[combining low line]CCC-3′, RNA1:5′-AACA[combining low line]CCC-3′.

^dSingle mismatch oligonucleotide (antiparallel), DNA3: 5′-AACT[combining low line]CCC-3′, RNA2:5′-AACU[combining low line]CCC-3′, DNA4: 5′-AACG[combining low line]CCC-3′, RNA3:5′-AACG[combining low line]CCC-3′, DNA5: 5′-AACC[combining low line]CCC-3′. RNA4:5′-AACC[combining low line]CCC-3′.

^eControl DNA: 5′-GGGT[combining low line]GTT-3′.

Firstly, DNA1 showed a T_m value of 20.9 °C (Table 2, entry 1) for the duplex with DNA2, whereas the T_m value of the PNA1 (no substitution) duplex with DNA2 was 36.9 °C (Table 2, entry 2) which is higher by 16.0 °C. When RNA1 was hybridized to DNA1 and PNA1, the T_m value of the duplex PNA1:RNA1 was 42.0 °C (Table 2, entry 2) which is higher by 21.4 °C than the duplex DNA1:RNA1 (T_m = 20.6 °C , Table 2, entry 1). The increased thermal stability of the PNA:DNA and PNA:RNA duplexes relative to their corresponding DNA:DNA or DNA:RNA duplexes is in agreement with previous studies.3b,9 Moreover, the thermal stability of the PNA:RNA duplex is slightly higher than the PNA:DNA duplex (42.0 versus 36.9 °C). This result is also consistent with a study reported previously.3b

Meanwhile, the thermal denaturation studies also showed the sequence specificity of 5-XU in PNA duplex hybridizations. The standard PNA1 gave T_m values of 25.1, 27.8 and 27.3 °C (Table 2, entry 2), respectively, when hybridized to DNA3–5 (single mismatch). The standard PNA1 formed a duplex with a single mismatch sequence with T_m values lower by 9.1–11.8 °C (25.1–27.8 versus 36.9 °C). In the case of 5-FU-PNA (PNA2), it gave T_m values of 25.4, 28.9 and 27.5 °C, respectively, when hybridized to DNA3–5, which were lower by 12.0–15.5 °C than the duplex PNA2:DNA2 (T_m = 40.9 °C, Table 2, entry 3). Similarly, the T_m values of the duplexes PNA2:RNA2–4 are lower by 11.9–16.3 °C than the duplex PNA2:RNA1 (29.4–33.8 versus 45.7 °C). For other 5-XU-PNAs (PNA3 and PNA4), destabilization was also observed when the PNAs were hybridized to DNA3–5 or RNA2–4 compared with DNA2 or RNA1. These results indicated that 5-XU preferred to bind to A, while T/U, G and C were unfavorable binding partners of 5-XU.

Furthermore, PNA2–4 containing one 5-XU unit showed 1.8–4.0 °C (for DNA) or 2.5–3.7 °C (for RNA) higher T_m values than control PNA1 (Fig. 3). The thermal stability of the XU:A base pairs varied as FU > ClU > BrU > T. Previous studies have suggested that the electron withdrawing effect of the halogen atom was a significant contributor to the higher stability of DNA duplexes.13 Therefore, we reason that the electron withdrawing effect of the halogen atom may be a favorable factor for the stability of PNA:DNA duplexes.

Fig. 3. Comparative T_m values for 5-XU-PNAs and unmodified PNA with complementary antiparallel DNA2 and RNA1. For each 5-XU-PNA, the ΔT_m value represents the amount of stabilization over that of the control (ΔT_m = T_{m XU–A} – T_{m T–A}).

To investigate the electronic effect of the halogen atom, NMR spectroscopy was employed to characterize the changes of chemical shift and the line width of the N₃ imino proton. In this work, a series of symmetrical structure PNA heptamers have been synthesized using the method described above, and the sequences were GGGXGGG (X = T, FU, ClU and BrU). The sequences have the advantage that the ¹H NMR spectra in the imino region should exhibit one imino proton resonance for T/XU and three imino proton resonances for G. Previous studies have shown that the chemical shift for the imino proton in T was more downfield than the chemical shift for the imino proton in G.14 Therefore, the peaks in the imino region can easily be assigned from the NMR spectra. The NMR measurements were obtained for the PNA-DNA duplexes containing intervening T/XU:A base pairs. The samples, 1 mM in single strand concentration, were dissolved in 100 mM NaCl, 10 mM Na₂HPO₄, 0.2 mM EDTA and 10% D₂O. After mixing the two strands, the oligonucleotides were heated to 80 °C and slowly cooled down to form the duplex. The NMR spectra were recorded using a previously reported method on a 500 MHz Bruker NMR system,13a and the spectra of the PNA–DNA duplexes are shown in Fig. 4. The peak of the T–A duplex at 13.44 ppm is lost upon substitution with XU; however, new peaks are observed at 14.20, 14.11 and 14.06 ppm, assigned to the XU imino proton. The chemical shift and the line width of the N₃ imino proton are listed in Table 3.

Fig. 4. NMR spectra in the imino region of the PNA–DNA duplexes: (A) T:A, (B) FU:A, (C) ClU:A, (D) BrU:A. The spectra were recorded at 5 °C with 512 scans using a water suppression WATEGATE program with d19 of 40 μs. The oligonucleotide was dissolved in a solution of 100 mM NaCl, 10 mM Na₂HPO₄, and 0.2 mM EDTA, pH 7.0 in 10% D₂O. Each spectrum was calibrated using TSP as an internal standard reference.

Table 3. Chemical shifts and line width of imino protons.

Nucleobase	Chemical shifts of N3 (ppm)			Line width (half height width, Hz)
	Monomer a	PNA1–4:DNA2 duplexes b	Δδ c
T	11.30	13.44	2.14	20
FU	11.88	14.20	2.32	36
ClU	11.90	14.11	2.21	25
BrU	11.87	14.06	2.19	22

^aChemical shifts of the N₃ imino proton of PNA monomers in DMSO-d₆.

^bChemical shifts of the N₃ imino proton of the duplexes PNA-DNA2. ¹H NMR spectra were recorded at 5 °C with 512 scans using a water suppression WATEGATE program with d19 of 40 μs. The oligonucleotide was dissolved in a solution of 100 mM NaCl, 10 mM Na₂HPO₄, and 0.2 mM EDTA, pH 7.0 in 10% D₂O.

^cΔδ = δ_oligo – δ_monomer.

In the PNA monomers, the chemical shifts of the N₃ imino proton in DMSO-d₆ were observed at 11.27, 11.88, 11.90 and 11.87 ppm, respectively (ESI‡). The imino protons of 5-XUs are more acidic than the corresponding imino proton of T. Similarly, significant downfield shifts in the N₃ resonance of 0.62–0.76 ppm in the XU:A relative to the T:A were observed in the NMR studies of PNA-DNA duplexes (Table 3, column 3). The difference in the chemical shift of N₃ imino protons between 5-XU and T is due to the different electronic effect of the halogen atom versus methyl. The 5-halo substituent of XU is electron withdrawing whereas the T methyl group is electron donating. The electron withdrawing properties of all three halogens result in the low electron density of the pyrimidine ring, and increase the acidity of N₃ protons. The increased acidity of the N₃ protons of 5-XU would be expected to strengthen the hydrogen bond between N₃–H and adenine. Moreover, upon substitution of T with 5-XUs, the N₃ chemical shifts in the duplexes move further downfield (Table 3, column 4). The NMR measurements indicate that the strength order of the hydrogen bond is FU:A > ClU:A > BrU:A > T:A. This is consistent with the results of UV melting studies.

On the other hand, the N₃ imino proton line widths for duplexes containing 5-XU:A were slightly broader than those of corresponding T:A duplex (Table 3, column 5). Previous studies have suggested that the broadening of the ClU:A imino proton peak in the DNA duplex might result from the proton exchange from within the intact base pair.13a Therefore, the observed broadening of the XU:A imino proton peak is not directly related to the duplex stability.

Conclusions

In conclusion, we have synthesized the monomers of peptide nucleic acids containing 5-XU using the Fmoc protection strategy for the first time. PNA carrying 5-XU forms a duplex with complementary DNA or RNA with higher affinity than unmodified PNA. The XU:A base pair is more stable than the T:A base pair in the PNA:DNA or PNA:RNA duplex. Based on the significant roles of 5-XU in pharmaceutical applications, we can envision that 5-XU-PNA might be useful as a potent anti-tumor agent. Further work that includes the cellular delivery of 5-XU-PNA and the antisense effect of the functionalized nucleobase of 5-XU-PNA oligomers, in combination with the release of 5-XU from 5-XU-PNA, is in progress.

This work was supported by the Foundation and Advanced Research Project of CQ CSTC (2013jjB0011), Fund Project for Transformation of Scientific and Technological Achievements from the Ministry of Science and Technology (2014KXWQT071242) and Animal Disease Prevention and Food Safety Key Laboratory of Sichuan Province.