Electrostatics Favor PNA : DNA Stability over Stereochemistry in Pyrrolidine-Based Cationic Dual-Backbone PNA Analogues

Article abstract of DOI:10.1002/ejoc.202001581

Modifications to the peptide nucleic acid (PNA) backbone has been well known to alter the thermodynamical parameters of PNA : DNA complexes to broaden their utility for different applications. Electrostatic interactions between a modified PNA having a positively charged backbone and the negatively charged DNA has been shown to enhance thermal stabilities of PNA : DNA complexes at various instances. On the other hand, chiral introduction in PNA backbone leads to stereochemical preference that affects binding properties. However, the interplay between electrostatics and stereochemistry has not been systematically studied so far. Herein, we report the synthesis and biophysical characterization of cationic PNA named dapPNA, first of its kind, having a dual PNA backbone constituting of a pyrrolidine ring having a β-substitution. One of the aims of this study was to investigate the role of electrostatics over stereochemical preferences. The results show that electrostatic attraction between cationic dapPNA and negatively charged DNA overcomes the unfavorable stereochemical effects and enhances stability of PNA : DNA complexes. Moreover, two different PNA backbones derived from a single PNA monomer expands the repertoire of pyrrolidine based PNA analogues.

Full text of DOI:10.1002/ejoc.202001581

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Electrostatics Favor PNA:DNA Stability over
Stereochemistry in Pyrrolidine-Based Cationic Dual-
Backbone PNA Analogues
Ashwani Sharma,*[a, b] Shahaji H. More,[a] and Krishna N. Ganesh[a]
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Modifications to the peptide nucleic acid (PNA) backbone has
been well known to alter the thermodynamical parameters of
PNA:DNA complexes to broaden their utility for different
applications. Electrostatic interactions between a modified PNA
having a positively charged backbone and the negatively
charged DNA has been shown to enhance thermal stabilities of
PNA:DNA complexes at various instances. On the other hand,
chiral introduction in PNA backbone leads to stereochemical
preference that affects binding properties. However, the inter-
play between electrostatics and stereochemistry has not been
systematically studied so far. Herein, we report the synthesis
and biophysical characterization of cationic PNA named
dapPNA, first of its kind, having a dual PNA backbone
constituting of a pyrrolidine ring having a β-substitution. One
of the aims of this study was to investigate the role of
electrostatics over stereochemical preferences. The results show
that electrostatic attraction between cationic dapPNA and
negatively charged DNA overcomes the unfavorable stereo-
chemical effects and enhances stability of PNA:DNA complexes.
Moreover, two different PNA backbones derived from a single
PNA monomer expands the repertoire of pyrrolidine based PNA
analogues.
Introduction
and side chains imparting chirality, making them cyclic, adding
charges to enhance aqueous solubility and conjugating with
ligands to target them to specific cells.[7,10–15] Various five/six
membered carbocyclic and heterocyclic rings (Figure 1) preor-
ganize the PNA backbone with conformational constrain for
preferential DNA/RNA and parallel/antiparallel binding. The
resulting chiral and charged PNA backbones are good probes to
delineate the electrostatic and stereochemical effects on for-
mation of PNA:DNA complexes.[7,16]
Peptide nucleic acids (PNAs) are a class of designed synthetic
molecules, which have emerged as one of the potential and
effective analogues of DNA.[1,2] In PNA, the negatively charged
sugar-phosphate backbone of DNA is replaced with a neutral
and achiral polyamide backbone consisting of repeating N-(2-
aminoethyl)-glycine (aeg) units, wherein the nucleobases are
flanked through an acetyl linker.[3,4] PNA interacts with comple-
mentary DNA through sequence specific Watson-Crick base-
pairing and being neutral without the anionic charges as in DNA,
the PNA:DNA complexes are thermally more stable than the
corresponding DNA:DNA complexes.[4–6] PNA is not a natural
substrate for cellular enzymes and is thus resistant to both
nucleases and proteases. In spite of superior molecular recog-
nition properties towards DNA, the flexible and achiral backbone
of PNA renders ambiguity in its binding orientation with DNA
which can be both parallel (NÀ 5’) and antiparallel (NÀ 3’).[7]
Further, inefficient cell uptake and poor aqueous solubility due
to its non-ionic nature has limited its application as an antisense
drug.[8,9] In order to overcome these limitations and tune the
backbone conformation, several chemical modifications of PNA
have been employed by introducing substituents on backbone
Electrostatic plays an important role in stabilizing the
PNA:DNA complexes.[4] Extensive work on PNA modifications has
shown that cationic PNAs increase the stability of PNA:DNA
complexes due to electrostatic attraction of the positively
charged PNA with the anionic phosphate backbone of DNA.[23–25]
Creation of positively charged PNAs is achieved by anchoring
amines or guanidine groups on side chains and by conjugating
lysines at N/C-terminus that are protonated at physiological pH,
making PNA backbone cationic. The stereochemistry of substitu-
ents at the C2–C3 position on the backbone affects the stability
and imparts preferential binding of PNA to DNA (Figure 1).
Nielsen et al[17] reported trans cyclohexyl PNA (trans-chPNA) in
which the C2–C3 bond of ethylenediamine segment of PNA
backbone is fused with cyclohexane ring in trans configuration of
the substituents (Figure 1b). The (S,S) trans analogue showed
binding similar to unsubstituted aegPNA while the trans-(R,R)
showed weaker binding with DNA. On the other hand, both the
cis-chPNA analogues (R,S) and (S,R) showed preferential binding
with RNA compared to DNA with higher stability.[18] A similar
phenomenon was observed for cyclopentyl PNA (Figure 1c),
where the trans-(S,S)-cpPNA showed good thermal stability with
DNA while the PNA with opposite stereochemistry trans(R,R)
does not bind to DNA.[19] In comparison, both RNA duplexes from
the cis-cpPNA with (R,S) and (S,R) stereochemistry reported by
[a] Prof. Dr. A. Sharma, Dr. S. H. More, Dr. K. N. Ganesh
Department of Chemistry,
Indian Institute of Science Education and Research (IISER)
Tirupati 517507, India
E-mail: a.sharma@iisertirupati.ac.in
[b] Prof. Dr. A. Sharma
Department of Biology,
Indian Institute of Science Education and Research (IISER)
Tirupati 517507, India
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/ejoc.202001581
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Figure 1. Chemical structures of different PNAs indicating C2–C3 carbons of PNA backbone, marked in red (a) Aminoethyl glycyl, aegPNA[1] (b) Cyclohexyl,
chPNA[17,18] (c) Cyclopentyl, cpPNA[19,20] (d) Diaminopyrrolidine, dapPNA, (e) N-(Pyrrolidinyl-2-methyl)glycine, pmg-PNA[21], (f) pipecolyl-PNA[22] and (g)
pyrrolidinylPNA.[23]
Govindraju et al.[20] showed enhanced stability for both DNA and
RNA hybrids. As discussed above, both trans(R,R) cyclohexyl and
(R,R) cyclopentyl showed weaker or no binding to DNA which
was attributed to their unfavorable stereochemistry.
Towards this goal, we designed trans(R,R)-3,4-diaminopyrroli-
dine based PNA monomer (dapPNA) in which the ethylenedi-
amine unit of aegPNA is structural part of the pyrrolidine ring
(Figure 1d). The orthogonally protected (Boc/Fmoc) dapPNA
monomer (Figure 2A) can be employed in standard solid phase
peptide synthesis (SPPS) through either Boc or Fmoc chemistry.
Notably depending upon the direction of synthesis and choice of
amino functionality at side chain, dapPNA monomer can give rise
to two different PNA backbones from a single monomer unit.
One PNA has backbone starting from a primary amine (Fig-
ure 2B), while the other PNA backbone initiated from a secondary
cyclic amine (Figure 2C). Earlier attempts of using cyclic
pyrrolidinyl amine in PNA backbone involved N-(pyrrolidinyl-2-
methyl) glycine[21] (pmgPNA, Figure 1e) that binds to DNA weakly
compared to the standard aegPNA and the weaker binding was
attributed to lack of positive charge in the PNA backbone.[21] In
the secdapPNA, backbone (Figure 2C) is cationic due to proto-
nated amine (NH3+) in the pyrrolidine ring. Additionally, dapPNA
To examine whether the introduction of electrostatic forces
between the cationic PNA and negatively charged DNA can
overcome unfavorable stereochemical effects in PNA/DNA bind-
ing, we hypothesized that insertion of a N atom in the
cyclopentyl ring of trans(R,R)-cpPNA would make it cationic and
thereby improve its binding to anionic DNA in addition to
improving aqueous solubility of PNA. Such an introduction of N
atom in cyclopentyl ring will also affect the 5-membered
pyrrolidine ring puckering leading to better pre-organization of
PNA backbone for favorable binding. Reports on Pyrrolidinyl
based PNA having a modular dipeptide backbone by Vilaivan et
al (Figure 1g) also suggests the importance of structural
constraints over electrostatic interactions.[23]
Figure 2. Chemical structures of (A) dapPNA monomer 13, (B) dapPNA and (C) secdapPNA.
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constitutes a new class of PNA analogues having backbones
Hydroxyl group in compound 7 was converted to O-mesyl
derivative 8 followed by SN2 inversion to give azide 9 in the
presence of sodium azide under heating conditions. Compound
8 and 9 were obtained as crystalline solids and were charac-
terized by single crystal X-ray diffraction. Azide 9 was further
hydrogenated using Raney-Ni to obtain the corresponding
amine, which was N-alkylated using ethyl bromoacetate to
obtain 10. Acylation of 10 with chloroacetyl chloride provided
compound 11 in good yield that was employed further for N1-
alkylation of thymine using mild base K2CO3 to yield ester 12.
This ester was hydrolyzed with aq. sodium hydroxide gave the
corresponding acid, which was subjected to insitu hydrogenation
for N1-benzyl deprotection using Pd/C to obtain corresponding
amine. This was protected with Fmoc chloride to obtain the
desired dapPNA monomer 13 in overall good yield. The structural
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derived from pyrrolidine ring having β-substitution instead of
well-known α-substituted pyrrolidines derived from proline.[21,23]
Results and Discussion
Synthesis of (3R,4R)-Diaminopyrrolidinyl (dapPNA) Monomer
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The synthesis of target monomer 13 (Scheme 1) was accom-
plished starting from the commercially available L-tartaric acid
that was condensed with benzyl amine to obtain imide 1.
Complete reduction of imide 1 was achieved using reducing
agent LAH to get the diol 2 by slightly modifying the reported
procedure.[26] The mono O-protection of diol 2 was attempted
using t-butyldimethylsilyl chloride (TBDMSCl) in THF and using
sodium hydride as base to yield compound 3 that was subjected
to mesyl chloride to obtain O-mesylate 4. Compound 5 was
1
integrity of all the compounds was confirmed by H, 13C NMR &
DEPT spectroscopic analysis and mass spectrometry.
À
obtained by SN2 attack of azide (N3 ) to O-mesylate 4 with
Solid Phase Peptide Synthesis of PNA Oligomers
inversion of stereochemistry using sodium azide in dry DMF
under heating conditions. The reaction also yielded azidoalcohol
6 as the minor product that was required for the next step. To
obtain azidoalcohol 6 in good yield, compound 5 was O-
desilylated to 6 using TBAF. The presence of azide group in both
5 and 6 was confirmed by IR. The azido alcohol 6 was reduced to
amine selectively without N-benzyl removal using Raney-Ni and
further insitu protected with Boc to obtain the compound 7.
Peptide nucleic acids that have a pseudo peptide backbone can
be synthesized by standard solid phase peptide synthesis (SPPS)
using Boc/Fmoc strategy. dapPNA monomer 13 contains orthog-
onal protection with Boc and Fmoc groups and thus can be
subjected to separate SPPS using either Boc or Fmoc chemistry
giving rise to two different isomeric PNA backbones as shown in
Figure 2. Assembly by Boc chemistry will build the dapPNA
oligomer utilzing the primary amino function in the PNA
Scheme 1. a) Benzyl amine, Xylene, reflux, 6–7 h, (90%); b) LiAlH4, THF, reflux, 12 h, (73%); c) NaH, TBSÀ Cl, dry THF (80%); d) MsCl, Et3N, dry DCM, (75%); e)
°
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NaN3, DMF, 100 C, 12 h, (48%); f) 1 M TBAF, dry THF, 2 h, (86%); g) H2, Raney-Ni, (Boc)2O, EtOAc, (74%); h) MsCl, Et3N, dry DCM, (83%); i) NaN3, dry DMF, 70 C,
18 h, (86%); j) H2, Raney-Ni, EtOH; k) Ethyl bromoacetate, K2CO3, dry ACN, (71%); l) Chloroacetyl chloride, Et3N, dry DCM, (85%); m) Thymine, K2CO3, dry DMF,
18 h, (85%); n) 1 N aq. NaOH, EtOH; o) H2, Pd/C, EtOH:H2O (8:2), 18 h; p) FmocÀ Cl, NaHCO3, 12 h, (57%).
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backbone while Fmoc chemistry provides secdapPNA oligomers
that involves secondary amine of pyrrolidine ring in the PNA
backbone.
The monomer 13 was incorporated at the desired sites within
homo-oligomeric and the mixed aegPNA sequences as dapPNA
(represented by t*) using boc chemistry and as secdapPNA
(represented by t*) using Fmoc chemistry during solid phase
synthesis (Table 1). All PNA oligomers were purified by reverse
phase HPLC and characterized by MALDI-TOF.
with cDNA were recorded. All single-stranded PNA oligomers
exhibited very low induced CD signals (Figure S1, ESI). However
upon binding with cDNA, all the PNA:DNA complexes showed
strong CD signals corresponding to PNA2:DNA triplex for
homopyrimidine PNAs (PNA1–PNA4) and PNA:DNA duplex for
mixed sequences (PNA5–PNA8). A low intensity positive band at
258 and 285 nm (generally covering whole wavelength range
255–290 nm) along with moderate negative band at 250 nm
characteristic of PNA:DNA:PNA triplex for homopyrimidine PNA
(PNA1–PNA4) were observed (Figure 3A). However, mixed
sequences (PNA5–PNA8), generally form PNA:DNA duplex and
shows appearance of moderate positive band between 260–
270 nm and a weaker negative band around 240 nm (Figure 3B).
The modifications in control aegPNA might alter base stacking
patterns in their complexes with DNA leading to slight shift in
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Circular Dichroism (CD) Studies of PNA:DNA Complexes
To confirm the binding specificity and stoichiometry of PNA:DNA
complexes, CD spectra for PNA oligomers, and their complexes
Table 1. HPLC Retention time, and MALDI-TOF characterization of PNA1–PNA14.
Figure 3. CD spectras (A) Triplexes of PNA1–PNA4 with complementary DNA1 (CGA8GC) (B) Duplexes of PNA5–PNA8 with antiparallel DNA 3 (AGTGATCTAC).
Conditions: PNA concentration 2 μM in buffer, 10 mM sodium phosphate pH 7.4, 10 mM NaCl.
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CD bands. PNA2:DNA triplex formation in case of homopyrimi-
Thermal Stability of PNA:DNA Duplex
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dine PNA3 was also confirmed by UV-Job’s plot that confirms
that homopyrimidine PNA3 bind to DNA1 in 2:1 stoichiometry
(Figure S2, ESI).
To check the thermal stability of PNA:DNA duplexes, a model
sequence GTAGATCACT was chosen, which has been used in a
number of publications to evaluate the thermal melting of
modified PNA:DNA duplexes.[17–20] dapPNA monomer was in-
corporated at desired position in the synthesized mixed purine-
pyrimidine aegPNA control sequence PNA5 to obtain PNA6–8.
The thermal stabilities of duplexes of PNA5–PNA8 with comple-
mentary DNA3 (AGTGATCTAC, antiparallel) and DNA4 (CATC-
TAGTGA, parallel) were studied by temperature dependent UV
absorbance experiments. The melting temperature (Tms) for
PNA:DNA duplexes extracted from the UV-melting curves are
shown in Table 3.
Thermal Stability of PNA2 :DNA Triplex
The homo-oligomers of modified dapPNA1–4 were hybridized
with perfect matched DNA1 (CGA8GC) in PNA2 :DNA (2:1) ratio
for triplex formation. The stability of the triplexes containing the
modification were examined by variable temperature UV
absorbance. The formation of PNA:DNA complexes is indicated
by sigmoidal transition with a maxima in derivative curve.[2,27]
which represents the Tm for the complex. The results of Tm for
various PNA2 :DNA triplexes incorporating modified t* at C and N
termini and in the center are shown in Table 2. The modified t*
at N and C- termini stabilize the derived triplexes, while t* at the
center destabilizes the triplex, compared to the control unmodi-
fied aegPNA (PNA1). The stabilization of PNA2 :DNA triplex
having N-terminal modification (PNA2) and C-terminal modifica-
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Depending on the site of incorporation, all dapPNA (t*)
sequences PNA6–PNA8 show substantial enhancement of
derived antiparallel PNA:DNA duplex stability (ΔTm3 = +5.1 to
°
+13.4 C) compared to the unmodified control PNA5. Single
incorporation at N-terminus (PNA 6) resulted in enhancement of
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Tm by 5.1 C while in the middle (PNA 7), it stabilized the duplex
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by 12.9 C. The incorporation of two units of dapPNA (PNA8)
enhanced the stability even better (+13.4 C) in comparison to
duplex of control unmodified aegPNA (PNA5).
°
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tion (PNA4) was 2.7 C and 7.7 C respectively over Tm of
unmodified PNA1:DNA1 triplex (Table 2). PNA3:DNA1 triplex
°
with t* in the center was destabilized by 13.1 C. The sequence
To scrutinize the role of electrostatic interactions, the effect
of salt concentration on thermal stability of dapPNA:DNA
complexes was studied. The results show marginal decrease of
specificity of PNA binding was investigated by measuring
thermal stabilities of triplexes with DNA2 having a single
mismatch base C in the middle shown in red (CGA4CA3GC). The
Tms of mismatch triplex PNA1 (control) and PNA2 were lowered
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À 0.7 C in Tm for unmodified aegPNA sequence when the salt
concentration was increased from 10 mM to 100 mM. However, a
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°
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by 10.6 C and 10.8 C respectively, while triplexes PNA3 and
PNA4 with modifications in center and C-terminus did not bind
to mismatch DNA2 (Table 2). These results suggest incorporation
of dapPNA monomer in aegPNA sequence can discriminate
between triplexes with perfect complementary and single-
mismatch complementary DNA. These results with triplexes
encouraged us to synthesize mixed purine-pyrimidine PNA
sequences incorporating t* and study duplex formation with
complementary DNA.
sharp decrease of upto À 11.1 C in melting temperature was
observed for modified dapPNAs (Figure S7, ESI) clearly suggest-
ing the electrostatic role-play in the stability of PNA:DNA
complexes.
Since dapPNA is chiral, comparative selectivity in binding of
DNA in parallel/antiparallel orientation was studied with dapPNA
duplexes with DNA4 that can form parallel duplexes with PNA5–
PNA8. It is seen from Table 3 that dapPNAs can form parallel
duplexes with DNA4 but stability is lower than that of antiparallel
°
Table 2. UVÀ Tm ( C) Studies of PNA2 :DNA triplexes with matched DNA1 and mismatched DNA2.
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Table 3. UVÀ Tm ( C) Studies of PNA:DNA duplexes with antiparallel DNA3 and parallel DNA4.
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[a] cpPNA sequence from literature with trans(R,R)-stereochemistry.[19b]
°
°
duplexes by À 8.3 C to À 12.8 C. This shows distinctly better
orientation selectivity of all the modified PNAs (PNA6–PNA8)
binding to antiparallel DNA3 in comparison to parallel DNA4.
To investigate if the higher thermal stability seen for dapPNA
complexes is sequence independent, another mixed purine-
pyrimidine sequence PNA9 (control) and PNA10 that incorpo-
rates t* at N-terminus and in the center were synthesized. Their
duplex stability in antiparallel and parallel orientations with
complementary DNA5 (ACTGAGGTAA) and DNA6 (AATGGAGT-
CA) were studied (Table 4). The UV-melting studies on modified
PNA sequence (PNA10) incorporating two dapPNA units showed
direction involving secondary amine group to yield isomeric
secdapPNA. Such isomeric PNA was synthesized (Figure 2C) using
Fmoc chemistry in SPPS. Incorporation of secdapPNA monomer
at desired positions of control aegPNAT8 sequence (PNA1) gave
PNA11–PNA13, which were evaluated for stability of secdapP-
NA:DNA complexes. The octameric secdapPNA poly T sequences
(PNA11–PNA13) upon hybridization with DNA1, showed triplex
formation, as evidenced by the UV Job’s plot and CD spectra
(Figure S9, S10 ESI). UV-melting studies on secdapPNA2 :DNA
triplexes showed that secdapPNA modification at N-terminal
°
stabilizes triplex formation by 3.5 C, whereas middle and C-
°
high thermal stability (ΔTm5 = +6.3 C) for antiparallel DNA5 and
terminal modifications showed destabilizing effect (Table 5). The
results were slightly better when compared to the analogous
pmgPNA (Figure 1e) reported in literature where all modified
triplexes were destabilizing. pmgPNA derived from proline is not
cationic (at pH 7.0) in its backbone unlike secdapPNA which adds
a cationic amino group to pmgPNA making secdapPNA triplex
(PNA 11) thermally more stable. In case of mixed purine-
better orientation selectivity against parallel DNA6 compared to
unmodified aegPNA control sequence PNA9 (Table 4). This
confirmed the generality of stabilization of DNA duplexes by
dapPNAs.
As discussed in the beginning, dapPNA structure can also
lead to another backbone in which the chain runs in a different
°
Table 4. UVÀ Tm ( C) Studies of PNA:DNA duplexes with antiparallel DNA5 and parallel DNA6.
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Table 5. UVÀ Tm ( C) Studies of PNA2 :DNA triplexes with matched DNA1 and mismatched DNA2.
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pyrimidine sequence, secdapPNA:DNA duplexes showed no
detectable melting transition.
lowering of stability of duplexes with mismatch, suggested
retention of sequence selectivity in binding despite of the
additional stability offered by electrostatic interactions.
Another structural/conformational effect influencing stability
is the ring puckering of pyrrolidine ring in dapPNA, that is
different from that of cyclopentyl ring in cpPNA. The five-
membered rings interconvert between envelope and twist
conformation and the distortion made by the out-of-plane atoms
generates ring pseudorotation. In saturated alicyclic rings such as
cyclopentane, pseudorotaional barrier is negligible whereas in
pyrrolidine it is 1.2 Kcal/mol.[28] This suggests imposition of
rigidity in pyrrolidine ring of dapPNA, locking the conformation
for DNA binding. Solution state NMR study of unmodified
Discussion
Electrostatic interactions and stereochemistry play an important
role in thermal stability of PNA:DNA complexes.[4,23–25] Introduc-
tion of a cationic group like amine group in the neutral PNA,
which undergoes protonation under physiological conditions,
increases backbone electrostatic interaction of the cationic PNA
with anionic DNA. This electrostatic interactions increase the
thermal stability of PNA:DNA complexes. On the other hand, the
presence of chirality in PNA provides orientation selectivity for
antiparallel or parallel binding of PNA with chiral DNA. The
stereochemistry at the chiral center/s is a contributing factor in
PNA backbone pre-organization and one PNA stereomer may
preferentially bind to DNA than the other stereomer.[7,17–20] We
surmised that the thermal stability of weakly binding stereomeric
PNA can be improved by electrostatic interactions by addition of
a cationic group in the PNA backbone. Introduction of a cationic
amino group in weakly binding trans-(R,R) cyclopentyl ring of
cpPNA lead to (R,R)-dapPNA monomer (Figure 1). PNAs incorpo-
rating dapPNA (PNA7) bound to DNA with higher thermal
stability (Table 3) compared to the same sequence with cpPNA at
same site (GTAGATCACT), which did not bind to DNA as reported
in literature.[19b] All dapPNA incorporated sequences PNA6–PNA8
showed high thermal stability for PNA:DNA duplexes with
PNA:DNA complexes suggested the β-dihedral angle between
[29]
°
C2–C3 to be 141 . One of the intermediate 9 for the synthesis
of (1R,2R) dapPNA monomer 13 showed endo ring-pucker for the
pyrrolidine ring with β dihedral angle between C2–C3 to be 103
that is close to the required β-dihedral angle for PNA:DNA
complex formation, The β-dihedral angle in (1R,2R) cpPNA as
°
°
calculated by molecular dynamics is in the range of 70 to
[19a]
°
90 .
The puckering of pyrrolidine ring thus reorganizes the
PNA backbone, to a favorable β-dihedral angle between C2–C3
and thereby enhance the thermal stability with DNA.
A special characteristic of monomer 13 is that it can give rise
to another backbone secdapPNA that has a free primary amino
group and can become cationic in physiological conditions. The
role of electrostatic interactions on cationic secdapPNA was
evaluated by UV-thermal melting of corresponding PNA com-
plexes with DNA and compared with reported pmgPNA which
lacks the amino group. The Tm data showed that cationic
secdapPNA forms stronger PNA2:DNA triplexes compared to
neutral pmgPNA, further reinstating the role of electrostatic
interactions in PNA:DNA stability. However, the stability of
secdapPNA duplex with DNA was generally lower than unmodi-
fied aegPNA. This might be due to an unfavorable ring pucker
due to the amide nitrogen in secdapPNA, which becomes part of
the ring.
°
maximum ΔTm of 12.9 C (compared to unmodified), and better
°
selectivity for antiparallel duplexes with +9.2 C for a single
dapPNA middle modification (PNA7). The electrostatic interac-
tions can be disrupted by addition of salt and in presence of
increasing amounts of NaCl (10 mM–100 mM), PNA7 duplex with
°
DNA, showed a sharp decrease of Tm up to À 11.1 C. This pointed
to the role of electrostatic interaction in dapPNA duplexation.
However, electrostatic interactions are sequence independent
and may lower sequence fidelity in binding. The considerable
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dissolved in water and extracted with ethyl acetate. The solvent was
Conclusion
1
2
3
4
5
6
7
8
9
evaporated under reduced pressure and product was purified on
column chromatographic as light reddish oil. Yield: (4.2 g, 80%);
[α]D25 = +26 (c 1.0, MeOH); 1H NMR (200 MHz, CDCl3): δ 7.34–7.27 (m,
5H, Ph), 4.18–4.08 (m, 1H, <C-OCH), 4.00–3.92 (m, 1H, À CH<C-OCH),
3.74–3.56 (m, PhCH2 diastereoscopic, 2J=2.3 Hz, 2H), 3.21 (dd, J=
6.2 Hz, 10.1 Hz, 1H, À CH-OTBS), 2.80–2.60 (m, 3H), 2.20 (dd, J=4.6 Hz,
10.0 Hz, 1H), 0.87 (s, 9H, TBS), 0.05 (d, J=3.9 Hz, 6H, TBS); 13C NMR
{H}(50 MHz, CDCl3): δ 137.5, 128.9, 128.2, 127.2, 79.1, 78.4, 61.0, 60.4,
60.1, 25.7, 17.9, - 5.0; MS (EI) m/z: Calculated for C17H30NO2Si [M+H]+
is 308.20, Found 308.19 [M+H]+.
In this study we designed and synthesized for the first time a
dual dapPNA monomer that give rise to two different backbones
named dapPNA and secdapPNA. The design was based on our
hypothesis that introduction of a cationic group in stereochemi-
cally unfavorable PNA backbone for duplex formation should
enhance the thermal stability of its complexes with DNA. The
results showed that both dapPNA and secdapPNA with unfavor-
able stereochemistry for binding significantly stabilize its com-
plexes with DNA mainly due to electrostatic interactions. The
enhanced stabilization of PNA:DNA complexes is also accom-
panied by greater discrimination of mismatched sequences and
better orientation selectivity between parallel and antiparallel
DNA sequences. This highlights the role of interplay between
electrostatic interactions and stereochemistry and might be
useful in designing future PNA analogues.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
(3S,4S)-1-benzyl-3-O-mesyl-4-(tert-butyldimethylsilyloxy)
pyrrolidine (4). To a stirred solution of 3 (4.5 g, 14.6 mmol) and
°
triethylamine (8.3 ml, 58.6 mmol) in dry DCM (40 ml) at 0 C under
nitrogen, was added methanesulfonyl chloride (2.3 ml, 29.2 mmol) in
dry DCM (10 ml) over a period of 15 min. The reaction mixture was
stirred for another 20 min. at room temperature, diluted with DCM
(20 ml), washed successively with water, brine and dried over sodium
sulphate. The organic layer was concentrated and purified to get
mesyl 4 as a colourless oil. Yield: (4.2 g, 75%); [α]D25 = +38 (c 1.0,
1
MeOH); H NMR (200 MHz, CDCl3): δ 7.37–7.25 (m, 5H, Ph), 4.89–4.80
(m, 1H, À CHÀ OMs), 4.41 (dt, J=6.2 Hz, 1H, À CHÀ OTBS), 3.75–3.51 (m,
PhCH2-diastereoscopic, 2J=14.0 Hz, 2H) 3.10 (dd, J=6.6 Hz, 9.6 Hz,
1H), 3.00 (s, 3H, mesyl CH3), 2.95–2.80 (m, 2H), 2.32 (dd, J=5.9 Hz,
9.7 Hz, 1H), 0.88 (s, 9H, TBS), 0.07 (d, J=5.7 Hz, 6H, TBS); 13C NMR
{H}(50 MHz, CDCl3): δ 137.7, 128.6, 128.2, 127.1, 86.7, 76.6, 60.1, 59.8,
57.6, 38.1, 25.6, 17.8, À 5.0. ; MS (EI) m/z: Calculated for C18H32NO2SSi
[M+H]+ is 386.18, Found 385.73 [M+H]+, 407.69 [M+Na]+.
Experimental Section
All starting materials and reagents available commercially were used
without further purification. Other details are provided in the
Supporting Information.
(3R,4R)-1-benzyl-3,4-dihydroxypyrrolidine-2,5-dione (1). L-tartaric
acid (45.0 g, 0.3 mol) in xylene (200 ml) stirred at room temperature,
benzyl amine (32.7 ml, 0.3 mol) was added and refluxed using a dean
stark apparatus till the appropriate amount of water (10.8 ml,
0.6 mol) was collected during 7–8 hrs. After cooling, the resulting
solid was filtered and washed with ethanol followed by acetone to
obtain product 1 as a light yellow solid which was recrystallized from
ethanol to get a white solid. This was used for next step without
(3R,4S)-3-azido-1-benzyl-4-(tert-butyldimethylsilyloxy)pyrrolidine
(5). A mixture of mesyl 4 (17.0 g, 44.2 mmol) and sodium azide
(23.0 g, 353.8 mmol) in dry DMF (200 mL) under nitrogen was heated
°
at 100–110 C for 12 hrs. After completion of reaction the solvent
was removed under reduced pressure. The residue dissolved in ethyl
acetate and washed with water, brine, dried over sodium sulphate
and purified by column chromatography to yield 5 as light yellow
oil. Yield: (7.0 g, 48%); [α]D25 = +50 (c 1.0, MeOH); 1H NMR (200 MHz,
CDCl3): δ 7.30–7.15 (m, 5H, Ph), 4.33 (q, J=6.2 Hz, 1H, À CHÀ OTBS),
3.68–3.45 (m, 3H, CHÀ N3 and À CH2À Ph), 3.05–2.88 (m, 2H, À CH2),
2.54–2.36 (m, 2H, À CH2), 0.86 (s, 9H, TBS), 0.35 (d, J=7.2 Hz, 6H, TBS);
13C NMR {H}(50 MHz, CDCl3): δ 138.3, 128.4, 128.2, 127.0, 72.8, 61.2,
60.3, 60.1, 56.3, 25.6, 18.0, À 5.2.; MS (EI) m/z: Calculated for
C17H29N4OSi [M+H]+ is 333.21, Found 333.85 [M+H]+.
further purification. Yield (59.8 g, 90%); [α]D25 = +138 (c 1.0, MeOH);
1
°
M.P.=196–198 C; H NMR (200 MHz, DMSO-d6) δ 7.26–7.11 (m, 5H,
Ph), 6.25–6.17 (m, 2H, 2×OH), 4.53–4.37 (m, 2H, benzyl CH2), 4.32–
4.24 (m, 2H, 2×CH); 13C NMR {H}(50 MHz, DMSO-d6): δ 174.8, 136.2,
128.8, 127.8, 74.8, 41.4.; MS (EI) m/z: Calculated for C11H12NO4 [M+
H]+ is 222.07, Found 222.04 [M+H]+, 244.15 [M+Na]+.
(3S,4S)-1-benzylpyrrolidine-3,4-diol (2). To an ice-cooled solution of
1 (11.0 g, 49.7 mmol) in THF (300 ml) under nitrogen, Lithium
Aluminium Hydride (5.7 g, 149.1 mmol) was added and refluxed for
12 hrs after which reaction was quenched with aqueous sodium
sulphate solution. The resulting mixture was filtered, and the salt
obtained was taken in diethyl ether and stirred for 2–3 hrs and
filtered. Extraction in diethyl ether was repeated four times. The
combined filtrate was concentrated and the residue was purified by
(3S,4R)-4-azido-1-benzylpyrrolidin-3-ol (6). A 1 M solution of tetra-
butyl ammonium fluoride in THF (23 mL) was added to a solution of
°
5 (5.0 g, 15.1 mmol) in THF (50 mL) under nitrogen at 0 C. The
reaction mixture was stirred at room temperature for 2 hrs, solvent
was removed and extracted with ethyl acetate. The combined
organic layers washed with brine, dried over sodium sulphate and
purified by column chromatography to afford 6 as a colourless oil.
Yield: (2.8 g, 86%); [α]D25 = +66 (c 1.0, MeOH); 1H NMR (200 MHz,
CDCl3): δ 7.38–7.20 (m, 5H, Ph), 4.38–4.28 (m, 1H, À CHÀ OH), 3.94 (q,
J=6.1 Hz, 1H, À CHÀ N3), 3.65 (s, 2H, À CH2À Ph), 2.96–2.84 (m,
2H,À CH2), 2.75–2.55 (m, 2H, À CH2), 2.39 (br s, 1H, À OH); 13C NMR
{H}(50 MHz, CDCl3): δ 137.7, 128.7, 128.2, 127.2, 71.2, 62.0, 60.1, 59.9,
56.2; MS (EI) m/z: Calculated for C11H15N4O [M+H]+ is 219.12, Found
219.11 [M+H]+.
column chromatography to obtain compound 2 as a white solid.
Yield: (7.0 g, 73%); [α]D25 = +34 (c 1.0, MeOH); M.P.=101–103 C; H
1
°
NMR (200 MHz, CDCl3): δ 7.32–7.22 (m, 5H, Ph), 4.15–3.95 (m, 4H, 2×
OH (2H) and 2×CH (2H)), 3.67–3.47 (m, PhCH2-diastereotopic, J=
12.6 Hz, 2H), 2.89 (dd, J=5.8 Hz, 10.1 Hz, 2H, À CH2), 2.41 (dd, J=
3.9 Hz, 10.3 Hz, 2H, À CH2); 13C NMR {H}(50 MHz, CDCl3): δ 137.0, 129.3,
128.3, 127.4, 78.1, 60.3, 60.0 ; MS (EI) m/z: Calculated for C11H16NO2
[M+H]+ is 194.11, Found 194.13 [M+H]+.
2
(3R,4S)-1-benzyl-3-Boc-amino-4-hydroxypyrrolidine (7). Azido-alco-
hol 6 (2.3 g, 10.6 mmol) in ethyl acetate (15 ml) taken in a
hydrogenation flask in which Boc-anhydride (2.8 g, 12.7 mmol) and
Raney-Ni (1.0 g) was added. The reaction mixture was hydrogenated
in a parr apparatus maintaining the pressure 40–45 psi for 3 hrs and
monitored by TLC. When reaction mixture showed disappearance of
starting azide on TLC, catalyst was filtered off and the solvent was
evaporated under reduced pressure. The product was purified by
(3S,4S)-1-benzyl-4-(tert-butyldimethylsilyloxy)pyrrolidin-3-ol
(3).
Into the cold solution of diol 2 (3.3 g, 17.1 mmol) in THF (50 ml)
under nitrogen, was added NaH (0.49 g, 20.5 mmol) and the reaction
mixture was stirred in an ice bath for 15 min followed by the
addition of tert-butyldimethylsilyl chloride (2.58 g, 17.1 mmol). The
reaction mixture was stirred at room temperature for 12 hrs. Reaction
monitored on TLC, after the completion of reaction as observed by
TLC, solvent was evaporated to dryness and the residue was
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column chromatography to yield 7 as a white solid. Yield: (2.3 g,
N-[(3R,4R)-1-benzyl-4-Boc- aminopyrrolidin-3-yl]-N-(chloroacetyl)-
1
2
3
4
5
6
7
8
9
74%); [α]D25 = +9 (c 1.0, MeOH); M. P.=79–81 C; H NMR (200 MHz,
CDCl3): δ 7.36–7.20 (m, 5H, Ph), 5.31 (br s, 1H, À NH), 4.30–4.17 (m, 1H,
À CHÀ OH), 4.14–3.97 (m, 1H, À CHÀ NH), 3.59 (s, 2H, À CH2À Ph), 3.05 (br
s, 1H, À OH), 2.86–2.67 (m, 2H, À CH2), 2.65–2.45 (m, 2H, À CH2), 1.42 (s,
9H, t-Boc); 13C NMR {H}(50 MHz, CDCl3): δ 155.8, 137.7, 128.8, 128.1,
127.0, 79.2, 69.8, 60.7, 60.0, 58.0, 52.1, 28.2.; MS (EI) m/z: Calculated
for C16H25N2O3 [M+H]+ is 293.18, found 293.75 [M+H]+, 316.19 [M+
Na]+.
glycine ethyl ester (11). To an ice-cooled stirred solution of 10
(2.55 g, 6.8 mmol) in dry DCM (30 ml) was added triethylamine
(3.8 mL, 27.3 mmol). The reaction mixture was stirred for 15 min
followed by the addition of chloroacetyl chloride (1.1 mL,
13.8 mmol) and continued to stir for another 10 hrs. The reaction
mixture was diluted with 100 mL of DCM and washed with water,
brine and dried over sodium sulphate and concentrated to obtain
chloro compound 11 as a colourless liquid which was used without
1
°
1
further purification. Yield: (2.6 g, 85%). H NMR (200 MHz, CDCl3): δ
(3S,4R)-1-benzyl-4-Boc-amino-3-O-mesylpyrrolidine (8). To a stirred
solution of 7 (2.3 g, 7.9 mmol) and triethylamine (4.4 mL, 31.6 mmol)
7.38–7.18 (m, 5H, Ph), 5.23 (br s, 1H, À NHBoc), 4.51–4.05 (m, 6H,
À CO2CH2, À CH2À CO2Et, 2×CH ring (2H)), 4.03–3.90 (m, 2H, À CH2Cl),
3.71–3.38 (m, 2H, À CH2-Ph), 2.91–2.57 (m, 2H, À CH2), 2.52–2.17(m,
2H, À CH2), 1.43 (s, 9H, t-Boc), 1.24 (t, J=7.1 Hz, 3H, À CH3). MS (EI) m/
z: Calculated for C22H33ClN3O5 [M+H]+ is 454.21, Found 454.41 [M+
H]+, 476.39 [M+Na]+.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
°
in dry DCM (20 mL) at 0 C under nitrogen, was added methanesul-
fonyl chloride (1.2 ml, 15.8 mmol) in dry DCM (5 ml) over a period of
°
15 min and stirred for half an hour at 0 C. The reaction mixture was
diluted by adding 20 mL of DCM and washed successively with
water, brine and dried over sodium sulphate. The organic layer was
concentrated and purified to get mesylate 8 as a white crystalline
N-[(3R,4R)-1-benzyl-4-Boc-aminopyrrolidin-3-yl]-N-(thymin-1-
acetyl)-glycine ethyl ester (12). To a solution of 11 (0.7 g, 1.5 mmol)
in 10 mL dry DMF was added potassium carbonate (0.258 g,
1.9 mmol) followed by thymine (0.236 g, 1.9 mmol) at room temper-
ature. The reaction mixture was stirred for 18 hrs at room temper-
ature after which it was diluted with 100 ml of ethyl acetate and
washed with water, brine and dried over sodium sulphate. The crude
product purified by column chromatography to afford pure com-
pound 12 as white solid. Yield: (550 mg, 85%); [α]D25 =À 12 (c 1.0,
solid. Yield: (2.4 g, 83%); [α]D25 = +37 (c 1.0, CHCl3); M. P.=130–
1
°
132 C; H NMR (200 MHz, CDCl3): δ 7.40–7.20 (m, 5H, Ph), 5.10–4.87
(m, 2H, À NH and À CHÀ OMs), 4.50–4.27 (m, 1H, À CHÀ NH), 3.65 (s, 2H,
À CH2À Ph), 3.01 (s, 3H, mesyl CH3), 2.97–2.80 (m, 2H, À CH2), 2.49 (dd,
J=7.5 Hz, J=9.2 Hz, 2H, À CH2), 1.44 (s, 9H, t-Boc).; 13C NMR {H}(MHz,
CDCl3): δ 155.3, 137.5, 128.6, 128.3, 127.2, 79.8, 79.0, 59.6, 58.8, 56.5,
51.2, 37.7, 28.2.; MS (EI) m/z: Calculated for C17H27N2O5S [M+H]+ is
371.16, Found 372.09 [M+H]+, 394.13 [M+Na]+.
1
DMSO); H NMR (200 MHz, CDCl3): δ 7.38–7.15 (m, 5H, Ph), 7.10–6.95
(3S,4R)-4-azido-1-benzyl-3-Boc-aminopyrrolidine (9). To a stirred
solution of mesyl 8 (2.5 g, 6.7 mmol) in dry DMF (30 mL) was added
sodium azide (3.5 g, 53.8 mmol) at room temperature under nitro-
(m, 1H, Thymine À NH), 4.90–3.93 (m, 8H, 2×CH (2H), ester À CH2,
À CH2À CO2Et, À CH2-Thymine), 3.70–3.40 (m, 2H, À CH2-Ph), 3.35–3.10
(m, 1H), 2.90–2.55 (m, 2H, 2×CH), 2.45–2.15 (m, 2H, 2×CH), 1.90 (d,
J=8.5 Hz, 3H, Thymine CH3), 1.41 (d, J=9.5 Hz, 9H, t-Boc), 1.30–1.15
(m, 3H, À CO2CH2CH3); 13C NMR {H}(50 MHz, CDCl3): δ 169.7, 169.3,
167.7, 166.8, 164.4, 155.6, 151.3, 141.1, 137.9, 128.5, 128.3, 127.3,
127.2, 110.6, 62.1, 62.0, 61.2, 60.9, 60.3, 59.5, 56.6, 55.0, 47.5, 45.0.; MS
(EI) m/z: Calculated for C27H38N5O7 [M+H]+ is 544.27, Found 544.59
[M+H]+, 566.57 [M+Na]+.
°
gen. The reaction mixture was heated at 60–70 C for 18 hrs. The
solvent was removed under reduced pressure and the residue was
dissolved in water and extracted with ethyl acetate three times. The
combined organic layer was washed with water, brine and dried
over sodium sulphate and purified by column chromatography to
afford azide 9 as a white solid. Yield (1.85 g, 86%); [α]D25 =À 12 (c 1.0,
1
°
MeOH); M. P.=70–72 C; H NMR (200 MHz, CDCl3): δ 7.40–7.20 (m,
5H, Ph), 5.10–4.75 (m, 1H, À NH), 4.15–3.90 (m, 1H, À CHÀ NH), 3.85–
3.70 (m, 1H, À CHÀ N3), 3.60 (s, 2H, À CH2À Ph), 3.04 (dd, J=6.8 Hz, J=
10.2 Hz, 1H), 2.84 (dd, J=6.6 Hz, J=9.8 Hz, 1H), 2.54–2.32 (m,2H,
À CH2), 1.45 (s, 9H, t-Boc).; 13C NMR {H}(50 MHz, CDCl3): δ 154.9, 137.7,
128.6, 128.2, 127.2, 79.7, 66.6, 59.4, 58.0, 57.8, 56.5, 28.2.; MS (EI) m/z:
Calculated for C16H24N5O2 [M+H]+ is 318.19, Found 318.90 [M+H]+,
340.93 [M+Na]+.
N-[(3R,4R)-1-Fmoc-4-Boc-aminopyrrolidin-3-yl]-N-(thymin-1-acetyl)-
glycine (13). To an ice cooled solution of compound 12 (150 mg,
0.28 mmol) in 5 mL of ethanol was added 1.5 mL of 1 N sodium
hydroxide solution in water and the reaction mixture was stirred in
cold condition for 30 min. Water (10 mL) was added to the reaction
mixture and solvent was removed on rotary evaporator to half of its
volume. The remaining mixture was neutralized with Dowex H+ resin
to pH 6 and concentrated to get 130 mg of corresponding acid as
white solid. This was taken in 10 mL of ethanol: water (8:2), added
10% PdÀ C (65 mg, 50% by wt.) and was hydrogenated at 60 psi for
18 hrs. The reaction mixture thus obtained was filtered on celite pad
and was concentrated to get 100 mg of crude amine. The crude
amine was dissolved in 10 mL of acetone: water (2:1), cooled in an
ice bath and sodium bicarbonate (24 mg, 0.28 mmol) was added
followed by the addition of Fmoc-chloride (94 mg, 0.36 mmol). The
reaction mixture was stirred at room temperature for 12 hrs after
which the acetone was removed and the water layer was neutralized
with saturated solution of potassium hydrogen sulphate and
extracted with ethyl acetate three times. The combined ethyl acetate
layers washed with water, brine and dried over sodium sulphate and
purified by column chromatography to obtain monomer 13 as white
solid. Yield: (100 mg, 57%); [α]D25 =À 9 (c 1.0, DMSO); 1H NMR
(200 MHz, CDCl3): δ 10.26 (br s, 1H, À COOH), 7.80–7.15 (m, 8H, fmoc),
7.10–6.85 (m, 1H, Thymine À NH), 4.60–3.98 (m, 6H, À CH2 & À CH fmoc,
À CH ring, À CH2À CO2H), 3.95–3.55 (m, 3H, À CH2-Thymine, À CH ring),
3.45–3.10 (m, 2H, 2×CH), 2.40 (t, J=8.6 Hz, 1H, À CH), 2.10–1.90 (m,
1H, À CH), 1.71 (s, 3H, Thymine À CH3), 1.39 (s, 9H, t-Boc); 13C NMR
{H}(50 MHz, CDCl3): δ 175.6, 143.7, 141.1, 127.8, 127.7, 127.1, 125.1,
125.0, 119.9, 49.5, 47.0, 30.6, 29.6, 28.2, 17.5.; MS (EI) m/z: Calculated
for C33H37N5O9 [M+Na]+ is 670.24, Found 671.92 [M+Na]+.
N-[(3R,4R)-1-benzyl-4-Boc-aminopyrrolidin-3-yl]-glycine ethyl ester
(10). To solution of azide 9 (3.3 g, 10.4 mmol), in ethanol (20 mL),
was added Raney-Ni (1.4 g) and the mixture was hydrogenated in a
parr apparatus at 45 psi for about 3 hrs. After completion of reaction,
the solvent was filtered over a celite pad and concentrated over rota
evaporator to get colourless oil. The oil was taken in dry acetonitrile
(50 mL), to which K2CO3 (2.5 g, 18.1 mmol) was added and the
°
mixture was stirred under nitrogen at 0 C for 15 min followed by
the addition of ethyl bromoacetate (1.4 mL, 12.5 mmol). The mixture
was further stirred at room temperature for about 12 hrs. The solvent
was evaporated, the residue was taken in ethyl acetate and washed
with water, brine, dried over sodium sulphate and purified to get the
compound 10 as a light yellow oil. Yield: (2.8 g, 71%); [α]D25 = +8.3
(c 1.2, CHCl3); 1H NMR (200 MHz, CDCl3): δ 7.38–7.15 (m, 5H, Ph),
5.13–4.93 (m, 1H, À NHBoc), 4.17 (q, J=7.1 Hz, 2H, À CO2CH2), 3.93–
3.72 (m, 1H, À NH), 3.59 (s, 2H, À CH2À Ph), 3.49 (s, 2H, À CH2À NH), 3.22–
3.00 (m, 2H, 2×CH), 2.83–2.68 (m, 1H), 2.64–2.48 (m, 1H), 2.20–2.05
(m, 2H, À CH2), 1.43 (s, 9H, t-Boc), 1.26 (t, J=7.1 Hz, 3H, À CO2CH2CH3).;
13C NMR {H}(50 MHz, CDCl3): δ 172.3, 155.2, 137.9, 128.7, 128.2, 127.1,
79.2, 65.2, 60.6, 59.9, 59.7, 58.7, 56.1, 48.9, 28.2, 14.1.; MS (EI) m/z:
Calculated for C20H32N3O4 [M+H]+ is 378.23, Found 377.13 [M+H]+,
399.02 [M+Na]+.
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Full Papers
doi.org/10.1002/ejoc.202001581
Circular dichorism: CD spectra were recorded on a Jasco J-715
Keywords: aegPNA
nucleic acid · Stereochemistry
· Antisense oligonucleotides · Peptide
1
2
3
spectropolarimeter. The CD spectra of the PNA:DNA complexes and
the relevant single strands were recorded in 10 mM sodium
phosphate buffer (pH 7.4) with appropriate NaCl concentration at
°
10 C. The CD spectra of the oligothymine T8 single strands and
4
5
6
7
8
9
mixed sequence PNAs and their complexes with DNA were recorded
with accumulation of 5 scans from 300 to 195 nm using a 1 cm cell,
a resolution of 0.1 nm, band-width of 1.0 nm, sensitivity of 2 mde-
grees, response 2 sec and a scan speed of 50 nm/min. The PNA:DNA
complexes were constituted by mixing appropriate strands in a 2:1
stoichiometry for triplexes and 1:1 stoichiometry for duplexes in
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°
buffer followed by heating to 90 C for 5 mins and annealed by slow
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°
cooling to 4 C.
UV Job’s Plot: To a solution of DNA in 10 mM sodium phosphate at
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make 10 different fractions with mole ratio from 0–100% with fixed
concentration. Then UV of each sample was scanned at temperature
of 10 C and the absorbance at 260 nm was recorded. This was
plotted as a function of the mole fraction of PNA.
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UV-Tm measurements: UV melting experiments were performed on
Cary 300 Bio UV-Visible Spectrophotometer equipped with a thermal
melt system. The sample for Tm measurement was prepared by
mixing calculated amount of stock oligonucleotide and PNA
solutions together in 1 mL of 10 mM sodium phosphate buffer
(pH 7.4). The samples 1 mL were transferred to quartz cell, sealed
with Teflon stopper after degassing with nitrogen gas for 15 min and
equilibrated at the starting temperature for at least 30 min. The
°
optical density at 260 nm was recorded in steps from 10–85 C with
°
temperature increment of 0.5 C/min. Nitrogen gas was purged
°
through the cuvette chamber below 20 C to prevent the condensa-
tion of moisture on the cuvette walls. Each melting experiment was
repeated at least thrice. The results were normalized and analysis of
data was performed using Microcal Origin (Microsoft Corp.). The
absorbance or the percent hyperchromicity at 260 nm was plotted as
a function of the temperature. The Tm was determined from the
°
peaks in the first derivative plots and is accurate to �1 C.
Supporting Information (see footnote on the first page of this
article): Electronic Supporting Information (ESI) includes additional
UV-Tm curves, CD spectras and spectroscopic investigation such as
LC–MS, NMR, IR, HPLC and MALDI-TOF for the synthesized com-
pounds and PNA oligos.
Acknowledgements
This work was supported by the Indian Institute of Science
Education and Research (IISER) Tirupati and Department of
Biotechnology (DBT grant BT/PR23487/NNT/28/1295/2017), Govt of
India. The work was initiated at National Chemical Laboratory
(NCL), Pune, India.
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Conflicts of interest
Manuscript received: December 4, 2020
Revised manuscript received: January 11, 2021
Accepted manuscript online: January 11, 2021
There are no conflicts to declare.
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www.eurjoc.org
1155
© 2021 Wiley-VCH GmbH