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Artif DNA PNA XNA. Jan 1, 2012; 3(1): 5–13.
PMCID: PMC3368815
(α,α-dimethyl)glycyl (dmg) PNAs
Achiral PNA analogs that form stronger hybrids with cDNA relative to isosequential RNA
Aland Gourishankar and Krishna N. Ganesh*
Indian Institute of Science Education and Research; Division of Organic Chemistry; National Chemical Laboratory; Pune, India
Current affiliation: Sinhgad Technical Education Society’s SKN College of Pharmacy; Kondhawa, Pune, India
*Correspondence to: Krishna N. Ganesh, Email: kn.ganesh/at/iiserpune.ac.in
The design and facile synthesis of sterically constrained new analogs of PNA having gem-dimethyl substitutions on glycine (dmg-PNA-T) is presented. The PNA oligomers [aminoethyl dimethylglycyl (aedmg) and aminopropyl dimethylglycyl (apdmg)] synthesized from the monomers 6 and 12) effected remarkable stabilization of homothyminePNA2:homoadenine DNA/RNA triplexes and mixed base sequence duplexes with target cDNA or RNA. They show a higher binding to DNA relative to that with isosequential RNA. This may be a structural consequence of the sterically rigid gem-dimethyl group, imposing a pre-organized conformation favorable for complex formation with cDNA. The results complement our previous work that had demonstrated that cyclohexanyl-PNAs favor binding with cRNA compared with cDNA and imply that the biophysical and structural properties of PNAs can be directed by introduction of the right rigidity in PNA backbone devoid of chirality. This approach of tweaking selectivity in binding of PNA constructs by installing gem-dimethyl substitution in PNA backbone can be extended to further fine-tuning by similar substitution in the aminoethyl segment as well either individually or in conjunction with present substitution.
Keywords: (α,α-dimethyl)glycyl PNA, gem-dimethylglycyl PNA, peptide nucleic acid, PNA-DNA binding, sterically constrained PNA analog, α-aminoisobutyric acid PNA
Peptide nucleic acid (PNA) is an interesting class of nucleic acid mimics first reported by Nielsen and Buchardt1 that is formally neither a peptide nor a nucleic acid, but embodies the hybrid structural features of both classes of bio-molecules. The structure of PNA consists of repeating units of 2-aminoethylglycine (aeg) to which the nucleobases (A, C, T and G) are attached via a tertiary amide linkage (Fig. 1). PNA hybridizes to cDNA and RNA sequences with almost equal avidity in a sequence specific manner.2,3 This feature, combined with its biological and chemical stability, promised potential applications for in vitro diagnostics and antisense therapeutics.4-7 PNA directed against genomic DNA around the transcription start site effectively knocks down the expression of the targeted gene and is an effective addition to the arsenal of gene silencing agents.8,9
Figure 1.
Figure 1.
General structures of PNA and DNA: B, nucleobase (T/A/C/G).
Despite several inherent advantages like sequence-specific binding and resistance to cellular enzymes, the major limitations of PNA for its effective applications are poor solubility in aqueous media, inefficient cellular uptake, almost equal affinity to cDNA and RNA (reducing selectivity of target by half) and ambiguity in parallel/antiparallel orientation selectivity of binding (non-specific targeting).10 To overcome these limitations, several efforts have been made to structurally alter the aeg-PNA backbone. Previous efforts from this laboratory11-14 and that of others15,16 have resulted in a number of acyclic, cyclic and chiral backbone modifications to generate a variety of PNA analogs. The aeg-PNA backbone composed of C-C and C-N bonds is highly flexible with a capacity to reorganize slowly into the preferred conformation for hybridization with cDNA or RNA. Pre-organizing the aeg-PNA backbone into “hybridization-competent conformation” is expected to impart entropic advantages for selective or preferential binding to DNA or RNA. Indeed, rational modifications of aeg-PNA backbone by introduction of cis-1,2-disubstituted cyclohexyl moiety to match the dihedral angle of the lone C-C bond in ethylenediamine segment to 60° found in PNA:RNA duplexes, resulted in achieving significant discrimination in binding of complementary isosequential DNA and RNA with preference for binding to RNA.17-19 Similarly, the introduction of chirality into aeg-PNA backbone has shown more selectivity for parallel or antiparallel binding depending on the nature of modifications,11,12 while introduction of cationic sidechains have improved solubility and enhanced cell uptake.20-22
A simpler way to impart steric constrain without incorporating rigid and chiral cyclic moieties is to introduce gem-dialkyl substitution into the flexible aminoethylglycine backbone of PNA (Fig. 2A and B). Such acyclic, achiral backbone having gem-dialkyl function would not only be rigid, but if the steric constraints imposed lead to favorable pre-organization, the inherent binding to cDNA or RNA will be enhanced due to entropic factors. The gem-dimethyl substitution on glycine segment of PNA can be achieved by replacing the glycine in backbone with α-aminoisobutyric (aib) moiety to obtain the aminoethyl-(α,α-dimethyl)glycyl (aedmg) PNAs (Fig. 2A). A number of aib-containing peptides occur naturally and the gem-dimethyl substitution on α-carbon is well known to promote helices in polypeptides.23 This feature, although not directly extendible to PNA (which is not a classical peptide), provided an initial rationale for us to synthesize the gem-dimethyl substituted aedmg-PNA. To examine the correlated effects of steric constrain developed due to gem-dimethyl glycine on the backbone in the adjoining aminoethyl segment in backbone, PNA analogs having aminopropyl moiety [Fig. 2B, R = Me, aminopropyl-(α,α-dimethyl)glycyl (apdmg)-PNA)] were also synthesized for comparative studies of the DNA/RNA complementation abilities. The unsubstituted aminopropylglycine–PNA (Fig. 2C, apg-PNA) was reported to induce destabilization of the derived PNA:DNA hybrids.24 Synthesis of PNA with spirocyclopentyl substitution (Fig. 2D) on glycine is known in literature,25,26 but no data on its complementation properties are reported. It is demonstrated here that the incorporation of α,α-dimethyl substitution on glycine in PNA stabilizes the derived PNA:DNA and PNA:RNA hybrids with significant preference for the PNA:DNA binding.
figure adna-3-5-g2
Figure 2. Structures of modified PNAs (A) aedmg, (B) apdmg, (C) apg and (D) spirocyclopentyl.
Synthesis of dmg-PNA-T monomers (6 and 12)
A straightforward route to obtain the desired dmg-PNA monomers 6 and 12 would be to directly N-alkylate the easily accessible ethyl α-aminoisobutyrate (H2N-CMe2-COOEt, aib). However, N-alkylation of aib with Boc-HNCH2CH2Cl or by reductive alkylation of Schiff base obtained by condensation with BocHN-CH2-CHO was not successful (Supplemental Material). Since this may be due to the steric problems imposed by the gem-dimethyl group, it was thought to reverse the N-alkylation strategy by using the gem-dimethyl component as the alkylating agent to react with N-protected dialkylamine (Fig. 3A). The synthesis of aedmg-PNA-T target monomer 6 was performed starting from 1,2-diaminoethane 1 which was treated with Boc-anhydride to give the N1-Boc-1,2-diaminoethane 2 that was alkylated at N2 with ethyl-2-bromoisobutyrate to obtain aminoethyl (α,α-dimethyl)glycine ethyl ester 3 in moderate yields. This was reacted with chloroacetyl chloride to yield the corresponding N-(chloroacetyl) derivative 4 which was used for alkylation to obtain N-(Boc-aminoethyl-α,α-dimethylglycyl)-thymine ethyl ester 5. It was hydrolyzed by methanolic sodium hydroxide to yield the desired aedmg-PNA monomer acid 6 for use in PNA synthesis (Fig. 3).
figure adna-3-5-g3
Figure 3. Synthesis of dmg-PNA monomers (A) aedmg-PNA-T 6 and (B) apdmg-PNA-T monomer 12. Values in brackets indicate yields. In Scheme B reagents a–e are same as in Scheme A.
The synthesis of the homologous aminopropyl (α,α-dimethyl)glycine (apdmg) acid monomer 7 (Fig. 3B) was performed starting from 1,2-diaminopropane by following the same route as in Figure 3A, through the intermediate steps yielding the products 810. The structural integrity of all new compounds of Figure 3 was confirmed by 1H, 13C NMR spectroscopic analysis and mass spectrometry (Supplemental Materials).
Solid phase synthesis of aedmg-PNA and apdmg-PNA oligomers
PNA oligomers containing gem-dimethylglycyl unit (dmg PNA-T) were assembled by solid-phase peptide synthesis on MBHA resin derivatized with lysine and the sequences are shown in Table 1. The unmodified PNA (aeg-PNAs 1 and 12) and the modified dmg PNA-T oligomers (Table 1, aedmg-PNA 2–6 and 13 and apdmg-PNA 711 and 14) were designed to test the effect of dmg modifications on the stability of the derived PNA:DNA and PNA:RNA duplexes and triplexes. The aedmg-PNA-T unit (t) was incorporated into the aeg-PNA-T8 sequence at pre-defined positions by solid phase synthesis to yield aedmg-PNAs (PNA 25) and the all-modified aedmg-PNA 6. Similarly, the apdmg PNA-T unit (t) was incorporated to obtain the apdmg-PNA-T oligomers (PNA 711). The mixed base PNA sequences having three modified units of aedmg-PNA-T (t) (PNA 13) and apdmg-PNA-T (t) (PNA 14) were synthesized along with the unmodified aeg-PNA 12 to examine the effect of modifications on the stability of PNA:DNA and PNA:RNA duplexes. All PNA oligomers were cleaved from the solid support by treatment with TFA-TFMSA to yield PNAs carrying lysine at the carboxyl terminus (to enhance their aqueous solubility). The PNA oligomers were purified by HPLC on a PepRPC column and finally characterized by MALDI-TOF mass spectrometry (Table 1). The complementary homoadeninyl DNA 15 having a CG/GC lock at the ends to prevent slippage in triplexes and the mixed base sequences (Table 1, footnote) DNA 1618 complementary to PNAs 13 and 14 were synthesized on a DNA synthesizer using standard phosphoramidite chemistry. Poly rA (RNA 19) was used for triplex studies with PNA oligomers 111. The mixed base RNA oligonucleotides RNA 20 and 21 (Table 1, footnote) complementary to PNA oligomers (13 and 14) were obtained from commercial sources along with their HPLC purity and mass spectral data.
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Table 1. PNA sequences with their HPLC and mass spectral data and cDNA and RNA sequences*
UV–Tm studies on aedmg-PNA2:DNA and aedmg-PNA2:RNA triplexes
The homothymine aeg-PNA sequences with cDNA-dA8 are known to form PNA2:DNA triplexes.1,2 The binding stoichiometry of aedmg-PNA-T8:DNA 15 complexes was established as 2:1 expected for a PNA2:DNA triplex from UV Job’s plot (see Supplemental Material).27 The thermal stabilities of triplexes from unmodified PNA 1 and dmg-PNA-T8 (PNA 211) with cDNA 15 and RNA 19 (polyrA40) were determined from temperature-dependent UV absorbance at 260 nm. The UV-Tm plots show a single sigmoidal transition, characteristic of PNA2:DNA triplex melting, wherein both the PNA strands dissociate simultaneously from DNA in a single step. The derivative plot of selected UV-melting data are shown in Figure 4 and the Tm values corresponding to peaks from such plots for various PNA2:DNA and PNA2:RNA triplexes of aedmg and apdmg-PNAs with cDNA 5 or RNA 19 are shown in Table 2.
figure adna-3-5-g4
Figure 4. First derivative UV absorbance-temp plots of (A) aedmg-PNA2:DNA and (B) aedmg-PNA2:RNA triplexes. See Supplemental Material for corresponding data on apdmg-PNA2:DNA/RNA triplexes. The numbers associated with each curve corresponds to PNAs in (more ...)
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Table 2. UV-Tm (°C) of dmg-PNA2:DNA and dmg-PNA2:RNA triplexes*
It is seen from the UV-melting data (column 1) that introduction of single aedmg-PNA-T modification at N-terminus enhanced the Tm of derived PNA2:DNA 15 triplex by 8.1°C (entry 2) over unmodified triplex (entry 1) and a successive increase in the number of modifications (entries 3–6) effected stepwise enhancement in stability. Since each triplex has two PNA strands, the total number of modifications per triplex is twice that number in a single PNA strand and this corresponds to an average stabilization of around 3.5–4.5°C per modification in triplex. Significantly, the fully modified oligomer (aedmg-PNA-T8 6) formed triplex with DNA 15 (entry 6) so strongly that no melting was seen even up to 85°C. A progressive increase in Tm per unit of aedmg-PNA-T modification in homothymine sequences suggests that the nature of the structural factors causing stabilization is additive and cumulative.
Since PNA2:RNA triplexes have slightly different conformational features,10 the stabilizing effect of the aedmg-PNA units on the derived triplexes (PNA2:RNA 19) was examined (Table 1, column 2). Single N-terminal modification gave only marginal positive effect (ΔTm = 1.4°C, entry 2) over control triplex (entry 1), while further increase in the number of aedmg-PNA units (entry 3–6), gave a Tm enhancement of about 2–2.5°C per modification. Again, PNA 62:RNA 19 triplex from the fully modified aedmg-PNA (entry 6) failed to show complete melting even at 85°C. It was noticed that the melting of aedmg-PNA2:DNA triplexes with PNAs 4 and 5 having three or more modifications had a slightly larger change in hyperchromicity compared with unmodified PNA2:DNA triplex suggesting that aedmg-modified PNAs have better stacking of the bases. It should be pointed out that the Tms for DNA and RNA triplexes are not directly comparable since RNA used is poly rA. Nevertheless, the relative trends are similar and the enhancement per modification in RNA triplexes is less than that for DNA triplexes.
To examine whether the observed enhancement in stability is at the cost of selectivity, the triplex Tm of completely modified aedmg-PNA-T8 (PNA 6) with DNA 16 having single mismatch in the middle (C instead of A) was measured. A larger destabilization (ΔTm = -24.4°C, entry 8) was observed for mismatched triplex from fully modified aedmgPNA 6 in comparison with analogous triplex from unmodified control PNA 7 (ΔTm = -12.8°C, entry 7). This not only indicated the formation of triplex by fully modified PNA 6 (although no melting was seen till 85°C) but also confirmed that the stability of aedmg-PNA:DNA triplex was not achieved at the expense of loss of sequence selectivity.
The UV-Tm data in Table 2, columns 3 and 4 are for the corresponding triplexes derived from apdmg–PNA T8 oligomers, having propylene chain instead of ethylene in the dmg-PNA oligomers (PNA 711). While single N-terminal modification resulted in a slight destabilization, further modifications enhanced the Tm by 4.1°C per modification, which is less than 7–9°C seen for triplexes from aedmg-PNA (PNA 26). PNA2:RNA triplexes derived from apdmg-PNA showed destabilization up to two modifications, but increasing them stabilized the triplexes over control. However, the degree of stabilization with apdmg-PNAs was lower compared with that seen with aedmg-PNA:RNA triplexes. The stabilization of apdmg-PNA:DNA/RNA triplexes is interesting, since it was earlier reported that aminopropylglycyl PNA24 destabilized the derived hybrids compared with aminoethylglycyl-PNA. Thus, the gem-dimethyl substitution on glycine in both aminoethyl and aminopropyl PNAs stabilize the constituted DNA and RNA triplexes over that of triplexes of unmodified PNA and the stabilization is significantly better for DNA triplexes in comparison to RNA triplexes.
UV–Tm studies on aedmg-PNA:DNA/RNA and apdmg-PNA:DNA/RNA duplexes
The PNA:DNA and PNA:RNA duplexes differ in conformation and structural features compared with PNA2:DNA and PNA2:RNA triplexes,10 and hence the mixed base sequence aeg (12), aedmg (13) and apdmg (14) PNAs were examined for their duplex forming abilities with antiparallel (ap) cDNA 17 and RNA 20 sequences (Fig. 5). PNAs with mixed base purine-pyrimidine sequences can form duplexes also in parallel orientations (parallel: N-terminus of PNA aligned with 5′ of DNA/RNA; antiparallel: N-terminus of PNA aligned with 3′-end of DNA/RNA) and hence this possibility was examined with parallel (p) cDNA 18 and RNA 21 oligonucleotides. This is not possible in triplexes wherein the identity of parallel-antiparallel binding orientation of the two PNA strands involved in complex formation cannot be distinguished.
figure adna-3-5-g5
Figure 5. First derivative UV absorbance-temp plots of PNA:DNA duplexes. p and ap refer to parallel and antiparallel duplexes. For PNA numbers see Table 1.
The duplexes were constituted by mixing equimolar amounts of aeg-PNA 12, aedmg-PNA 13 and apdmg-PNA 14, individually with the cDNA 17, RNA 20 for antiparallel duplexes and DNA 18, RNA 21 for parallel PNA:DNA duplexes. The Job’s plot completely supported formation of 1:1 duplexes in both aedmg-PNA 13 and apdmg-PNA 14 complexes with DNA 17 (see Supplemental Material). The Tm data (Table 3) was obtained from the plot of UV absorbance vs temperature and show that the antiparallel duplex (aedmg-PNA 13:DNA 17) with 3 modifications exhibits very high stabilization (entry 1, ΔTm = +31.1°C, 10°C/modification) compared with the corresponding unmodified duplex (PNA 12:DNA 17, entry 1). The parallel duplex of modified aedmg-PNA (PNA 13:DNA 18) is also significantly stabilized (entry 2, ΔTm = +19°C, 6°C/modification) compared with the unmodified parallel duplex (PNA 12:DNA 18, entry 2). apdmg-PNA 14 showed enhanced stabilization of its antiparallel duplex with cDNA 17 (entry 1, ΔTm = +23°C, 7°C/modification) over that of unmodified PNA 12, but about the same stability for parallel duplex (entry 2).
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Table 3. UV-Tm of dmg-PNA:DNA and dmg-PNA:RNA duplexes*
The mixed unmodified PNA 12 and the modified aedmg-/apdmg-PNA 13 also forms antiparallel and parallel duplexes with the complementary RNAs 20 and 21 respectively. The Tm data of these duplexes are shown in Table 3 (entry 3 and 4). While both aedmg and apdmg PNAs stabilize the corresponding antiparallel PNA:RNA duplexes (entry 3, aedmg ΔTm = +22.2°C and apdmg ΔTm = +14.6°C) they actually destabilized the corresponding parallel PNA:RNA duplexes (entry 4) by 8.9°C and 7.6°C respectively compared with the unmodified PNA. Thus the Tm results clearly suggest that both aedmg and apdmg-PNAs enormously stibilize (1) duplexes with DNA and RNA over that of unmodified PNA, (2) antiparallel duplexes with DNA and RNA are more stable than the corresponding parallel duplexes and (3) show stronger preference for duplexing with cDNA relative to isosequential RNA.
It was generally observed that the width of the derivative UV-Tm curves of the complexes increased with the degree of substitution, which suggests slow association-dissociation equilibrium due to dmg substitution. However, the change in the intensity of the different derivative curves could not be correlated with the number of modifications in triplexes and perhaps a consequence of differential changes in base stacking for each modification. It is possible that all modified (PNA 6 and 12) and alternative (PNA 5 and 11) site modified triplexes induce base stacking patterns in phase toward a global uniformity, while changes from single or random modifications may result in only local effects. However, in cases of duplexes, all modified aedmg-PNA 13 and apdmg-PNA 14 showed much higher amplitudes of first derivative curves indicating much better stacking in the dmg-PNA:DNA duplexes.
CD studies on apdmg-PNA:DNA and apdmg-PNA:RNA complexes
In order to examine if aedmg-PNAs have any significant differences in their overall conformational features, the CD spectra of PNA complexes with DNA were examined (Fig. 6). The PNA2:DNA triplexes exhibit two maxima at 260 nm and 280 nm, a minimum at 246 nm and crossover points around 250 nm. The double hump profile in the region 260–280 nm is characteristic of polyT-polyA:polyT (PNA2:DNA) triplexes.28 The CD profiles of dmg-PNA:DNA duplexes were similar to those of the control achiral aeg-PNA:DNA duplexes indicating that the overall conformational effects induced by dmg-PNA-T units in the derived PNA complexes with cDNA seem to be only marginal.
figure adna-3-5-g6
Figure 6. CD spectra of (A) aedmg-PNA2:DNA triplexes and (B) apdmg-PNA:DNA triplexes. The number shown above each curve refers to the PNA sequence shown in Table 1.
The aib residues in peptides severely restrict the possible rotations about N-Cα and Cα-C′ bonds toward promoting α-helix or 310 helix and the non-chiral nature of substitution may induce right or left handed structures equally well, depending on the nature of other residues in sequence.23 aedmg-PNA is not a classical peptide and hence the exact molecular origin and details of structural consequences of gem-dimethyl substitution in PNA backbone remains to be seen. The comparative studies on aedmg and apdmg-PNA backbones clearly suggest that applying conformational constrain on glycine segment by introduction of gem-dimethyl substitution leads to both better hybridization by pre-organizing the backbone for better stacking interaction of the base pairs. This gem-dilkyl effect on glycine is transmitted to adjacent ethylenediamine/propylenediamine chain since unlike ap-PNA (with only adjacent glycine in backbone), which destabilize the DNA complexes, apdmg-PNA stabilize both DNA and RNA hybrids.
For general experimental conditions see Supplemental Material.
Mono-N-Boc-diaminoethane (2)
1,2-diaminoethane (1) (20 g, 0.33 mol) was taken in dioxane:water (1:1, 500 ml) and cooled in an ice bath. Boc-azide (5 g, 35 mmol) in dioxane (50 ml) was slowly added with stirring and the pH was maintained at 10.0 by continuous addition of 4M NaOH. The mixture was stirred for 8 h and the resulting solution was concentrated to 100 ml. The N1, N2-di-Boc derivative not being soluble in water, precipitated, and it was removed by filtration. The corresponding N1-mono-Boc derivative was obtained by repeated extraction from the filtrate in ethyl acetate. Removal of solvents yielded the mono-N-Boc-diaminoethane 2 (3.45 g, 63% yield). 1H NMR (CDCl3) δ: 5.21 (br s, 1H, NH), 3.32 (t, 2H, J = 8 Hz), 2.54 (t, 2H, J = 8 Hz), 1.42 [s, 9H, C(CH3)3]. Mcalc (C7H16N2O2) = 160.21 and Mobs = 161.18 (M+1).
N1-Boc-1,3-diaminopropane (14)
1,3-diaminopropane 13 (20 g, 0.27 mol) on reaction with Boc-anhydride (5.9 g, 27 mmol) under identical reaction conditions as above gave a mixture of N1,N3-di-Boc and the N1-mono-Boc derivative 14 from which the former compound was precipitated and removed by filtration. The product obtained by repeated extraction from ethyl acetate and removal of solvents yielded the mono-N-Boc-diaminopropane 14 (4 g, 60% yield). 1H NMR (CDCl3) δ: 4.78 (br s, 1H, NH), 3.32 (q, 2H), 2.74 (t, 2H), 1.59 (t, 2H), 1.42 [s, 9H, C(CH3)3]. Mcalc (C7H16N2O2) = 160.21 and Mobs = 174.18 (M+1).
Ethyl N–Boc-(aminoethyl)-α,α-dimethylglycinate (3)
The N1-(Boc)-1,2-diaminoethane 2 (3.2 g, 20 mmol) was treated with 2-bromoethylisobutyrate (3 ml, 20 mmol) in dry acetonitrile (50 ml) in the presence of anhydrous K2CO3 (5.5 g, 40 mmol) and the mixture was stirred at 70°C for 15 h. The reaction mixture was concentrated to remove the acetonitrile and work-up with ethylacetate-water and purification by silica gel column chromatography gave ethyl-N-Boc-(aminoethyl)-α,α-dimethylglycinate 3 as a yellowish oil (4.2 g, 76.6% yield). 1H NMR (CDCl3) δ: 5.29 (br s, 1H, NH), 4.22–4.11 (q, 2H), 3.26–3.21 (t, 2H), 2.7–2.65 (t, 2H), 1.43 [s, 9H, C (CH3)3], 1.34 [s, 6H, C(CH3)2], 1.30–1.23 (t, 3H); 13C NMR (CDCl3) δ: 176.8, 171.2, 156.6, 79.7, 62.5, 61.6, 43.9, 37.7, 28.1, 22.1, 21.1 and 13.8. Mcalc(C13H26N2O4) = 274.36 and Mobs = 275.21 (M+H+).
Ethyl N-Boc-(aminopropyl)-α,α-dimethylglycinate (9)
The N1-Boc-1,3-diaminopropane 14 (3.12 g, 18 mmol) was reacted with 2-bromoethylisobutyrate (2.65 ml, 18 mmol) under similar conditions, followed by usual work-up and chromatography gave ethyl N-Boc-(aminopropyl)-α,α-dimethylglycinate 15 as yellowish oil (4.5 g, 87% yield). 1H NMR (CDCl3) δ: 5.02 (br s, 1H, NH), 4.18–4.07 (q, 2H), 3.17–3.11 (t, 2H), 2.51–2.45 (t, 2H), 1.63–1.56 (t, 2H), 1.40 [s, 9H, C(CH3)3], 1.26 [s, 6H, C(CH3)2], 1.23–1.20 (t, 3H). 13C NMR (CDCl3) δ: 176.8, 155.9, 60.7, 58.8, 41.9, 38.9, 30.5, 28.3, 25.2, and 14.2. Mcalc (C14H28N2O4) = 286.38 and Mobs = 289.17 (M+3H+).
Ethyl N-(Boc-aminoethyl)-N-(chloroacetyl)-α,α-dimethylglycinate (4)
The ethyl N-Boc-(aminoethyl)-α,α-dimethylglycinate 3 (4.0 g, 14.6 mmol) was taken in 10% aqueous Na2CO3 (75 ml) and dioxane (60 ml). Chloroacetyl chloride (3.5 ml, 43.8 mmol) was added in two portions with vigorous stirring. The reaction was complete within an hour and was brought to pH 8.0 by addition of 10% aqueous Na2CO3 and concentrated to remove the dioxane. The product after work-up was purified by column chromatography to obtain the ethyl N-Boc-(aminoethyl)-N-(chloroacetyl)-α,α-dimethylglycinate 4 as a yellowish oil. (4.2 g, 82% yield). 1H NMR (CDCl3) δ: 5.13 (br s, 1H, NH), 4.27–4.17 (q, 2H), 4.13 (s, 2H), 3.57–3.5 (t, 2H), 3.36–3.30 (t, 2H), 1.49 [s, 6H, C(CH3)2], 1.43 [s, 9H, C(CH3)3], 1.27–1.20 (t, 3H). 13C NMR (CDCl3) δ: 173.9, 167.3, 156.0, 79.8, 72.6, 61.6, 61.2, 43.7, 41.8, 41.2, 28.2, 23.8 and 13.9. Mcalc (C15H27N2O5Cl) = 350.16 and Mobs = 352.01 (M+2).
Ethyl N-Boc-(aminopropyl)-N-(chloroacetyl)-α,α-dimethylglycinate (10)
The ethyl N-Boc-(aminopropyl)-α,α-dimethylglycinate 15 (3.6 g, 12.5 mmol) reacted with chloroacetyl chloride (5 ml, 62.5 mmol) under similar conditions, work up and chromatographic purification gave ethyl N-Boc-(aminopropyl)-N-(chloroacetyl)-α,α-dimethylglycinate 16 as a yellowish oil (3.8 g, 83.5% yield). 1H NMR (CDCl3) δ: 4.77 (br s, 1H, NH), 4.17–4.06 (q, 2H), 4.04 (s, 2H), 3.48–3.40 (t, 2H), 3.17–3.10 (t, 2H), 1.91–1.82 (t, 2H), 1.46 [s, 6H, C(CH3)2], 1.43 [s, 9H, C(CH3)3], 1.23–1.17 (t, 3H). 13C NMR (CDCl3) δ: 173.64, 166.22, 156.09, 61.66, 61.05, 42.19, 41.70, 32.33, 28.30, 23.75, and 13.97.Mcalc(C16H29N2O5Cl) = 364.16 and Mobs = 365.13 (M+1).
N-(Boc-aminoethyl-α,α-dimethylglycyl)-thymine ethyl ester (5)
Ethyl N-Boc-(aminoethyl)-N-(chloroacetyl)-α,α-dimethyl-glycinate 4 (4.0g, 11.4 mmol) was stirred with anhydrous K2CO3 (1.68 g, 12.2 mmol) and thymine (1.58 g, 12.5 mmol) in DMF. After completion of reaction (TLC), solvent DMF was removed under reduced pressure followed by aqueous work-up and column chromatographic purificationgave N-Boc-(aminoethyl-α,α-dimethyl glycyl)-thymine ethyl ester 5 (4.8 g, 96.5%). 1H NMR (CDCl3) δ: 6.94 (s, 1H), 5.43 (br s, 1H, NH), 4.57 (s, 2H), 4.17–4.06 (q, 2H), 3.57–3.50 (t, 2H), 3.41–3.34 (t, 2H), 1.87 (s, 3H, CH3), 1.48 [s, 6H, C(CH3)2], 1.42 [s, 9H, C(CH3)3], 1.23–1.16 (t, 3H). Mcalc (C20H32N4O7) = 440.49 and Mobs = 463 (M+Na+).
N-Boc-(aminopropyl-α,α-dimethylglycyl)-thymine ethyl ester (11)
Ethyl N-Boc-(aminopropyl)-N-(chloroacetyl)-α,α-imethyl-glycinate 16 (2.0 g, 5.5 mmol) was reacted with thymine (0.76 g, 6.1 mmol) under similar conditions in presence of K2CO3 in DMF followed by work-up and column chromatographic purification to give N-Boc-(aminopropyl)-α,α-dimethyl-glycyl-thymine ethyl ester 17 as white solid (2.2 g, 88% yield). 1H NMR (CDCl3) δ: 6.98 (s, 1H), 4.95 (br s, 1H, NH), 4.49 (s, 2H), 4.16–4.06 (q, 2H), 3.49–3.41 (t, 2H), 3.24–3.18 (t, 2H), 1.89 (s, 3H), 1.47 [s, 6H, C(CH3)2], 1.42 [s, 9H, C(CH3)3], 1.23–1.16 (t, 3H). 13C NMR (CDCl3) δ: 173.7, 166.4, 164.2, 156.4, 151.2, 110.6, 61.9, 61.19, 41.49, 31.92, 28.39, 23.98, 14.03, 12.32. Mcalc(C21H34N4O7) = 454.61 and Mobs = 477.18 (M+Na+).
Hydrolysis of the PNA ethyl esters 5 and 11
The ethyl ester 5 was hydrolyzed using aqueous NaOH (2N, 5 ml) in methanol (5 ml) and the resulting acid was neutralized with activated Dowex-H+ till the pH of the solution was 7.0. The resin was removed by filtration and the filtrate was concentrated to obtain the resulting Boc-protected acid 6 in excellent yield (85%), 1H NMR (CDCl3) δ: 10–11 (2H, thymine NH and –COOH), 7.18 (s, 1H), 5.43 (br s, 1H.NH), 4.2–4.16 (q, 2H), 3.5–3.56 (t, 2H), 3.39–3.32 (t, 2H), 1.87 (s, 3H, CH3), 1.48 [s, 6H, C(CH3)2], 1.42 [s, 9H, C(CH3)3]. Mcalc (C18H28N4O7) = 412.44 and Mobs = 435.57 (M+Na+).
Solid phase synthesis
The solid phase synthesis was performed on MBHA resin (Sigma, 100–200 mesh) having initial amine loading value of 0.85 mmol/g, which was lowered to 0.25 mmol/g by capping with acetic anhydride in dry DMF/DCM and pyridine as a base. The MBHA resin with free amine was functionalized by coupling with N (α)-Boc-N(ε)-ClCbz- l-lysine using DCCI as coupling reagent. The lysine loaded resin was swollen in dry CH2Cl2 (30 min) and treated with 50% TFA in CH2Cl2 (1 ml × 2, 15 min each). After washing with CH2Cl2, neutralization of TFA salt with 5% DIPEA in CH2Cl2 (1 ml × 3, 2 min each) and washed further with CH2Cl2. The unmodified/modified PNA monomers were coupled to resin in presence of HBTU/HOBt/DIPEA in DMF/NMP and the synthesis continued using the same coupling agent to make PNA oligomers (Table 1). The efficiency of each coupling was monitored by Kaiser’s test. At the end of the assembly the PNA oligomers were cleaved from the resin with TFA/TFMSA in presence of scavenging agent thioanisole.
A typical cleavage reaction consisted of treating of resin-bound PNA oligomer (10 mg) in an ice bath with thioanisole (20 μl) and 1,2-ethanedithiol (8 μl) for 10 min and TFA (120 μl) followed by TFMSA (16 μl) and stirring continued for 2 h. After filtering the resin, the filtrate was evaporated under vacuum. The residue was dissolved in methanol (~0.1 ml) and precipitated by adding ether. The PNA oligomers were purified by RP-HPLC (C18 column) and characterized by MALDI-TOF mass spectrometry. The overall yields of the crude PNA oligomers were in the range 70–80%.
UV-Tm Measurements
The concentration of PNA and DNA was calculated on the basis of absorbance using the molar extinction coefficients of nucleobases for A = 15.4, t = 8.8, C = 7.3 and G = 11.7 M−1cm−1. The complexes were prepared in sodium phosphate buffer (10 mM, pH 7.4) by annealing the samples at 85°C for 5 min followed by slow cooling to room temperature. Absorbance vs. temperature profiles were obtained by monitoring at 260 nm with a UV-VIS spectrophotometer scanning from 5 to 85°C at a ramp rate of 0.2°C per minute. Experiments were repeated at least thrice and the Tm values were obtained from the peaks in the first derivative plots. The reported Tm values are accurate to (±) 0.5°C.
In summary, the incorporation of gem-dimethyl substituted aedmg-PNA monomer into the aeg-PNA backbone preferentially increased the Tm of the derived complexes with DNA as compared with RNA. This perhaps arises from a structural pre-organization of the backbone due to steric constrains imposed by the gem-dimethyl unit leading to a better base stacking of base pairs in DNA/RNA complexes. This is interesting since the earlier study from our group17-19 has shown that the cyclic cyclohexanyl PNAs show strong preference for RNA binding and the present results allow tuning of PNA backbone to DNA binding by introduction of rigidity without any associated chirality. Further work is in progress in our group to extend these features toward the study of PNA derivatives with the gem-dimethyl substitution shifted to the adjacent aminoethyl segment, combining subsitution in both fragments, thermodynamic analysis and molecular dynamic simulations of the derived PNA:DNA and PNA:RNA duplexes.
Supplementary Material
Additional material
Acknowledgments
A.G. thanks CSIR, New Delhi for a research fellowship. K.N.G. acknowledges DST, New Delhi for award of JC Bose Fellowship.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
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