Cγ(S/ R)-Bimodal Peptide Nucleic Acids (Cγ- bm-PNA) Form Coupled Double Duplexes by Synchronous Binding to Two Complementary DNA Strands

Article abstract of DOI:10.1021/acs.joc.0c01853

Peptide nucleic acids (PNAs) are linear equivalents of DNA with a neutral acyclic polyamide backbone that has nucleobases attached via tert-amide link on repeating units of aminoethylglycine. They bind complementary DNA or RNA with sequence specificity to form hybrids that are more stable than the corresponding DNA/RNA self-duplexes. A new type of PNA termed bimodal PNA [Cγ(S/R)-bm-PNA] is designed to have a second nucleobase attached via amide spacer to a side chain at Cγon the repeating aeg units of PNA oligomer. Cγ-bimodal PNA oligomers that have two nucleobases per aeg unit are demonstrated to concurrently bind two different complementary DNAs, to form duplexes from both tert-amide side and Cγside. In such PNA:DNA ternary complexes, the two duplexes share a common PNA backbone. The ternary DNA 1:Cγ(S/R)-bm-PNA:DNA 2 complexes exhibit better thermal stability than the isolated duplexes, and the Cγ(S)-bm-PNA duplexes are more stable than Cγ(R)-bm-PNA duplexes. Bimodal PNAs are first examples of PNA analogues that can form DNA2:PNA:DNA1 double duplexes via recognition through natural bases. The conjoined duplexes of Cγ-bimodal PNAs can be used to generate novel higher-level assemblies.

Full text of DOI:10.1021/acs.joc.0c01853

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Article
C#(S/R)-Bimodal Peptide Nucleic Acids (C#-bm-PNA) form Coupled Double
Duplexes by Synchronous Binding to Two complementary DNA Strands
Pramod Bhingardeve, Krishna N. Ganesh, and Bharath Raj Madhanagopal
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.0c01853 • Publication Date (Web): 28 Sep 2020
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g
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C (S/R)-Bimodal Peptide Nucleic Acids (C -bm-PNA) form Coupled Double Duplexes by
Synchronous Binding to Two complementary DNA Strands
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Pramod Bhingardeve,a Bharath Raj Madhanagopal,b and Krishna N Ganesha,b,*
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a. Indian Institute of Science Education and Research (IISER) Pune, Dr Homi Bhabha Road,
Pune 411008, India
b. Indian Institute of Science Education and Research (IISER) Tirupati, Karkambadi Road,
Mangalam, Tirupati, 517507, India.
* To whom correspondence should be addressed. kn.ganesh@iisertirupati.ac.in
ABSTRACT
Peptide nucleic acids (PNA) are linear equivalents of DNA with a neutral acyclic
polyamide backbone that has nucleobases attached via t-amide link on repeating units of
aminoethylglycine. They bind complementary DNA or RNA with sequence specificity to form
hybrids that are more stable than the corresponding DNA/RNA self duplexes. A new type of
PNA termed bimodal PNA [Cg(4S/R)- bm-PNA] is designed to have a second nucleobase
attached via amide spacer to a sidechain at Cg on the repeating aeg units of PNA oligomer. Cg-
Bimodal PNA oligomers that have two nucleobases per aeg unit is demonstrated to
concurrently bind two different complementary DNAs, to form duplexes from both t-amide
side and Cg-side. In such PNA:DNA ternary complexes, the two duplexes share a common
PNA backbone. The ternary DNA 1:Cg(S/R)-bm-PNA:DNA 2 complexes exhibit better
thermal stability than the isolated duplexes, and the Cg(S)-bm-PNA duplexes are more stable
than Cg(R)-bm-PNAs duplexes. Bimodal PNAs are first examples of PNA analogues that can
form DNA2:PNA:DNA1 double duplexes via recognition through natural bases. The conjoined
duplexes of Cg-bimodal PNAs can be used to generate novel higher level assemblies.
INTRODUCTION
Peptide nucleic acids (PNA) are synthetic DNA analogues with an acyclic polyamide
backbone. PNA strands recognize the nucleobase sequences of complementary DNA and RNA
to yield highly stable derived PNA:DNA and PNA:RNA duplexes.1,2 The non-chiral PNA
backbone consists of recurring units of aminoethyl glycine (aeg) and in each aeg unit, one of
the nucleobases (A/T/C/G) is attached via tertiary amide function (Figure 1a). In PNA, the
distance between the neighbouring nucleobases is similar to that in DNA/RNA. This feature
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alongwith absence of charges, allows the PNA strands to form sequence-specific base pairing
with the complementary DNA/RNA strands.3 The resulting PNA duplexes with
complementary DNA/RNA have superior stability and sequence fidelity compared to that of
normal DNA duplexes.4 These attributes of PNA and its unique property of strand invasion5 of
DNA duplexes have been exploited in various applications including diagnostics6 and antisense
therapeutics.7 The simplicity of the PNA backbone provides ample opportunities for its
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structural variations and modulation of functional selectivity via conjugation with ligands for
8,9
cell recognition and transport across membrane.
The structural modifications involve
functionalization at Ca and Cg on the aeg backbone as they do not adversely impact the
complementation of PNA with DNA/RNA.8-10 In particular, PNAs that carry cationic
alkylamino, guanidino and polyethylene glycol substitutions at Ca or Cg show better DNA-
binding and cell-penetration.10,11 Another class of modifications involve constraining the aeg
unit through cyclization giving rise to substituted 5-membered proline/pyrrolidine rings or
fused cyclopentyl or cyclohexyl moieties on backbone, which may conformationally pre-
organize the backbone for preferential hybridization with DNA or RNA.8b,12
In this work, we have now embarked on a new strategy to enhance the versatility of
standard aeg-PNAs through installation of a second nucleobase on a sidechain hosted at Cg of
each aeg unit in PNA backbone (Figure 1b). Such PNA analogues (termed bimodal bm-PNA)
bear two nucleobases per aeg unit of PNA and can ideally form two duplexes (Figure 1c), one
from t-amide linked bases (as in normal PNA) and another from Cg-sidechain linked
nucleobases, both duplexes sharing the common aeg backbone. Further, the distance between
neighbouring nucleobases anchored on successive Cg-Cg sites (Figure 1c) is similar to that
between adjacent bases on t-amide chains of standard PNA. This geometrical compatibility
facilitates the concurrent binding of two DNA sequences one from each side. Employing such
an analogy with Ca sites on PNA backbone, we have recently demonstrated that Ca-bm-PNA
(Figure 1e) possessing nucleobases attached to sidechain at Ca through a triazole linker can
indeed form double duplexes (Figure 1f) providing the proof of concept.13 In this manuscript,
we show that Cg(S/R)-bm-PNAs with nucleobases anchored on Cg-sidechain in both S/R
configuration also recognize DNA from both sides, endorsing the generality of bimodal PNA
concept. Unlike many other DNA analogues, the simplicity of PNA backbone is ideally suited
to host two nucleobases per repeating unit. A previous approach to host two nucleobases on
DNA backbone via linkage at C4/C5 of sugar residue or at C5 of pyrimidine base (double
headed DNA) showed destabilization of derived DNA:DNA single duplexes and no attempt
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was made to hybridize the anchored base sequence with another complementary DNA to form
second duplex.14 The bimodal PNAs described here are conceptually quite different from DNA
/ PNAs made with synthetic Janus bases15 that recognize two strands of DNA from two faces
of nucleobase, which have recently found interesting applications.16 In our designs of bimodal
PNAs, natural bases are anchored on both t-amide and on Cg-sidechain (and at Ca-sidechain13)
with the backbone acquiring a Janus character for recognizing different complementary DNAs
from two sides. The control iso-PNAs (Figure 1d) wherein the nucleobases reside only on
Cg-sidechain (and on Ca-sidechain13), devoid of bases on t-amide link are new isomorphic
PNA analogues that can independently form duplexes with DNA.
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Ac
N
Ac
O
H
N
γ
(d)
α
(a)
B
B
N
N
H
O
O
O
O
O
γ
N
N
N
H
N
H
N
N
NH
B
O
α
N
B
Cγ(R)-bm-PNA
(b) Cγ(S)-bm-PNA
Cα(S)-bm-PNA
(e)
B
B
B
O
O
O
O
H
N
H
N
(S)
N
N
H
N
N
α
γ
γ
O
O
N
N
O
O
NH
NH
B
N
B
B
DNA
(f)
DNA
(c)
C
A
T
C
A
T
G
G
O
O
O
O
O
O
(S)
α
H
N
H
N
N
N
N
(S)
N
H
N
H
N
N
γ
O
O
N
N
N
O
NH
C
O
NH
N
N
A
T
C
G
A
G
T
DNA
DNA
Figure 1. (a) aeg-PNA (b) Cg(S/R)-bm-PNA (c) Cg-bm-PNA:DNA double duplex (d) Cg and
Ca-iso-PNA (e) Ca(S)-bm-PNA and (f) Ca-bm-PNA:DNA double duplex, B = T/A/G/C
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The target Cg(S/R)-bm-PNAs 1 and 2 (Figure 2) have 8 nucleobases (GT6C) linked via
t-amide sidechain as in standard PNA and each aeg unit carries a second nucleobase on
sidechain at Cg of ethylenediamine component. The nucleobase bearing ethylamido sidechain
at Cg in both S [Cg(S)-bm-PNA 1] and R [Cg(R)-bm-PNA 2] stereochemistry were synthesised
for comparison, although it is known that Cg(S) substituents are better for hybridization with
DNA.8e,11a The choice of spacer at Cg and the base sequences was due to ease of synthesis and
provide proof of the new concept rather than any structural or functional considerations.
Unsubstituted aeg-PNA-C/G unit was included at N and C- termini to avoid steric interference
during synthesis or in hybridization. The control Cg(S/R)-iso-PNAs (3 and 4) are new isomeric
PNA structures that bear nucleobases only on Cg-sidechain with absence of t-amide linked
nucleobases. They were used to test their independent abilities to base pair with DNA.
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T
O
T
O
T
O
T
O
G
T
O
T
T
O
T
T
T
O
T
O
C
O
O
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O
O
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O
H
N
H
N
H
N
H
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H
N
H
N
H
N
(S)
NH2
H2N-Lys
HN
N
N
N
N
N
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N
N
O
O
O
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O
O
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O
CγS-bm-PNA 1
CγR-bm-PNA 2
O
NH
NH
NH
NH
NH
NH
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O
O
O
O
O
O
O
O
O
O
H
N
H
N
H
N
H
N
H
N
H
N
H
N
(R)
NH2
NH2
NH2
H2N-Lys
H2N-Lys
H2N-Lys
HN
HN
HN
N
N
N
N
N
N
N
N
O
O
O
O
O
O
O
O
O
O
O
NH
NH
NH
NH
NH
NH
C
T
G
A
C
T
H3C
O
H3C
H3C
O
O
H3C
O
O
O
H3C
O
H3C
O
H3C
O
H3C
O
O
O
O
H
N
H
N
H
N
H
N
H
N
H
N
H
N
N
N
N
N
N
N
N
N
O
O
O
O
O
O
O
O
NH
NH
NH
NH
NH
NH
CγS-iso-PNA 3
C
T
G
C
A
T
H3C
O
O
O
H3C
O
H3C
O
H3C
H3C
O
H3C
H3C
O
O
H3C
O
O
O
H
N
H
N
H
N
H
N
H
N
H
N
H
N
N
N
N
N
N
N
N
N
O
O
O
O
O
O
O
O
CγR-iso-PNA 4
O
O
NH
NH
NH
NH
NH
NH
C
G
A
T
C
T
g
g
Figure 2. Structures of C (S/R)-bm-PNAs and C (S/R)-iso-PNAs
RESULTS AND DISCUSSION
g(
)
g(
)
Synthesis of monomers C S/R -bm and C S/R -iso PNA monomers 1-4
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The Cg(S/R)-bm PNA monomers 1 and 2 that carry t-amide linked nucleobases on aeg
backbone with orthogonal amino protecting groups (NH-Boc on backbone and Fmoc on Cg-
ethylamino sidechain) and Cg(S/R)-iso PNA monomers 3 and 4 that have nucleobases only at
Cg sidechain (Figure 3) were used for solid phase synthesis of target bimodal PNAs (Figure 2).
The amino groups on nucleobases were suitably protected (iBu for 2-NH2 of G, Cbz for 6-NH2
of A and 4-NH2 of C), and can all be deprotected in a single step on the resin.
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O
O
NH
NH
N
O
N
O
O
CH3
O
CH3
O
O
(R) NHBoc
HO
(S) NHBoc
γ
HO
HO
NHBoc
HO
NHBoc
N
N
N
γ
N
O
O
O
O
NHFmoc
NHFmoc
NHFmoc
NHFmoc
2. γ-(R-eam) aeg-T
4. γ-(R-eam)-N(Ac) aeg
3. γ-(S-eam)-N(Ac) aeg
1. γ-(S-eam) aeg-T
Figure 3. Structures of protected monomers employed for the synthesis of iso-PNA and
bimodal PNA oligomers on solid phase. eam – ethylamino; aeg – aminoethylglycyl
The bimodal PNA monomers 1 and 2 with Cg(S/R) sidechain were synthesized starting
from L-glutamine and D-Glutamine as per synthetic route shown in Scheme 1. The Cg(S)-bm-
PNA monomer 1 was synthesised from compound 5 which was prepared from commercial L-
glutamine. The reaction of ester 5 with aq, LiOH in MeOH gave the acid 6.17 The NHCbz
protecting group in 6 was exchanged to NHFmoc to maintain orthogonality required for solid
phase synthesis. This was achieved by first hydrogenating 6 in presence of Pd/C in methanol
to give the free amine, which was in situ reprotected using Fmoc-Cl and 10% aq. Na2CO3 in
THF to yield the desired S-bm-PNA monomer 1.
The synthesis of Cg(R)-bm-PNA monomer 2 was done starting from D-glutamine 7
which was first protected at a-NH2 by reaction with Boc2O to yield a-NH-Boc-D-glutamine 8
(Scheme 1). This was followed by the reaction of 2(R)-NHBoc-D-glutamine 8 with
iodobenzene diacetate (PIDA) which led to the formation of 2(R)-NHBoc-4-aminobutanoic
acid 9. Reaction of compound 9 with CBzCl in toluene containing NaHCO3 produced the
orthogonally protected 2(R)-NHBoc-4-(NHCbz)-aminobutanoic acid 10 in quantitative yield.
It was converted using dimethyl sulfate to methyl ester 11, which was reduced using sodium
borohydride in absolute ethanol to give the alcohol 12. The primary hydroxyl group in 12 was
reacted with mesyl chloride in DCM in the presence of triethyl amine to obtain the O-mesylate
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derivative 13, which was immediately reacted with sodium azide in dry DMF to convert it to
the azide 14. The reduction of azide function using Raney Ni under hydrogenation conditions
yielded the free amine that was reacted with a-bromoethylacetate in-situ to obtain the alkylated
compound 15. The N-chloroacetylation of secondary amine in 15 was achieved by its reaction
with chloroacetyl chloride to get 16 that was condensed with thymine to afford the Cg(R)-
(NHFmoc-aminoethyl)-(NHBoc-aminoethyl) ethyl glycinate 17 in good yield. The appearance
of peaks at δ 7.05 ppm and 1.87 ppm in 1H NMR confirmed the presence of thymine in product
17. Subsequently, the ester was hydrolysed using 10% aq. LiOH/MeOH to obtain the acid 18,
which was hydrogenated in presence of Pd/C to yield the free amine. This was in-situ protected
using Fmoc-Cl and 10% aq. Na2CO3 in THF to generate the desired Fmoc derivative 2.
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Scheme 1. a.. 10% aq. LiOH, MeOH,3 h. b. Pd-C/H2, MeOH, 4 h; c. Fmoc-Cl, Na2CO3, THF-H2O; 4
h; d. (Boc)2O, NaOH, Dioxane, 1 h; e. PhI(OAc)2, EA:ACN:H2O (2:2:1); f. Cbz-Cl, NaHCO3,
Acetone; g. Me2SO4, K2CO3, Acetone, 4 h, 55 oC; h. NaBH4, EtOH, 4 h; i. MsCl, TEA, DCM, 0 oC; j.
NaN3, DMF, 80 oC, 8 h; k.H2, RaNi/EtOH, 80 h; Ethylbromoacetate, ACN, TFA, 12 h; l.
Chloroacetyl chloride, TEA, DCM, 8 h; m. Thymine, K2CO3, DMF, 8h.
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The monomer 3 necessary for synthesis of Cg(S)-iso-PNA 3 oligomer (Figure 2) was
prepared by route as shown in Scheme 2. Compound 19 an intermediate in the synthesis of 617
was N-acetylated to obtain 20 followed by hydrolysis of ester to yield the acid 21. The N-Cbz
function in 21 was deprotected by hydrogenation to the corresponding free amine which was
in-situ deprotected with the base labile Fmoc group to obtain the orthogonally protected
monomer 3. The Cg(R)-iso-PNA monomer 4 was synthesized by a similar set of reactions
13
starting from compound 15 as shown Scheme 2. NMR (1H and C) and mass spectral data
(ESI, S13-S31) were used to characterize the structures of all intermediates and monomers.
CH3
(S)
CH3
CH3
O
O
O
NHBoc
NHCbz
HO
NHBoc
C2H5O
NHBoc
NHCbz
C2H5O
NHBoc
NHCbz
HO
c, d
70%
N
N
N
N
b
a
H
O
O
O
O
85 %
80 %
NHFmoc
3
21
20
O
19
CH3
CH3
O
CH3
(R)
O
C2H5O
NHBoc
NHCbz
NHBoc
HO
NHBoc
C2H5O
a
b
N
N
N
c, d
66%
HO
NHBoc
H
O
O
O
N
90%
85 %
O
NHCbz
NHCbz
4
23
22
NHFmoc
15
Scheme 2. Synthesis of Cg(S/R)-iso-PNA monomers 3 and 4 needed for assembly of Cg(S)-
iso-PNA 3 oligomer and Cg(R)-iso-PNA 4 oligomers; a. DhbtOH, AcOH, DCM, 12 h; b.
10% aq. LiOH, MeOH,3 h.; c H2, Pd-C; d Fmoc-Cl, aq. NaHCO3, 2 h.
g
Solid phase synthesis of C (S/R)-bimodal and iso-PNA oligomers
The bimodal PNAs Cg(S)-bm-PNA 1 and Cg(R)-bm-PNA 2 having identical base
sequences but with different stereochemistry at Cg (Figure 2) were assembled on MBHA resin
using solid phase synthesis protocols,18 employing a combination of Boc (backbone) and Fmoc
(sidechain) strategy (Scheme 3). The PNAs were synthesized starting from C-terminus using
orthogonally protected monomers, Cg(R)-bm monomer 2 for Cg(R)-bm-PNA 2 oligomer and
Cg(S)-bm monomer 1 for Cg(S)-bm-PNA 1 oligomer. The sequence of steps employed for the
solid phase synthesis of Cg(S/R)-bm-PNAs is illustrated in Scheme 3. The synthesis was
initiated with first coupling on MBHA resin with the orthogonally protected L-lysine
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(wNHCBz,aNHBoc) as the C-terminal spacer amino acid. This was followed by deprotection
of aNHBoc to yield free NH2 group on resin, which was coupled with aeg-PNA-C monomer
and deprotected with TFA to regenerate free NH2 function on the resin. From this stage, each
backbone coupling was done by either Cg(S)-ethylamino-PNA monomer 1 for Cg(S)-bm-PNA
1 or Cg(R)-bm-PNA monomer 2 for Cg(R)-bm-PNA 2. The coupling of appropriate monomer
was followed by deprotection of Fmoc to free NH2 group on Cg-sidechain for subsequent
coupling with desired (ACbz/CCbz/GiBu/T)-acetic acid (shown in color) as per the sequence.
Successive cycles of couplings with monomer (1 or 2) and Fmoc deprotection (step a) and
nucleobase-acetic acid coupling and Boc deprotection (step b) were continued in stepwise
manner to build the target Cg(S/R)-bm-PNA oligomers. The final coupling was done with
unmodified PNA-G monomer. All coupling reactions were done under microwave conditions
to enhance the efficiency of coupling reactions. The protocol in Scheme 3 was also used for
synthesis of Cg(S)-iso-PNA 3 from monomer 3 and Cg(R)-iso-PNA 4 from monomer 4. Finally,
TFA-TFMSA (step c) was used to cleave the synthesized Cg(S/R)-bm-PNA and iso-PNA
oligomers from the resin. The PNA oligomers were purified using RP-HPLC and characterized
by mass spectral data Table 1 and ESI (S5-S9). The sequence of steps employed for solid phase
synthesis of Cg(S)-bm-PNA 1 oligomer is shown in Supporting Information (ESI, Scheme 2).
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T
T
T
NHX
NHY
NHX
NHY
O
C
a
C
T
LysNHC
T
C
T
C
T
LysNHC
C
NHX
OH
LysNH2
NHX
NH2
NH
NH2
LysNHC
O
NHX
NH2
LysNHC
a
b
O
a
HN NH2
O
O
O
O
T
T
T
T
C
G
NHX
O
NHX
O
C
T
C
T
T
T
T
C
T T
C
T T T
LysNHC
T
T
OH
NHY
NHY
OH
NH2
NH NH
LysNHC
NH2
NHX
NH2
LysNHC
O
NHX
LysNHC
O
a
b
O
a
O
b
NH
NH
NH2
NH NH
NH
NH
NH
NH
O
O
O
O
O
O
O
O
O
O
T
G
G
T
G
T
C
G C
T
T
T
A
C
NHX
O
O
NHX
NHY
C
C
T
T T
T T
T T T
C
T
T
T T
T
T
NHY
OH
OH
NHX
NH2
NH
NH2
LysNHC
O
LysNHC
O
LysNHC
a
b
b
O
a
NH NH NH
NH NH NH NH NH2
NH NH NH
NH NH
O
O
O
O
O
O
O
O
O
O
O
O
O
C
A
T G C
A
A
T G C
T
G
C
T
O
G
C
T
C
LysNHC
T
T T
T T T
T
T T T
T
C
T
T
T T T G
T
NHX
OH
NH2
NHX
NH2
LysNHC
LysNHC
O
NHX
a
O
O
b
NH NHHN HN
O
NH
NH NH NH NH NH
NH
NH NH NH NH NH
NH
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
C
C
A
A
C
T
T G C
T G C
T
A
T
G C
B
C
T
T
T T T G
T
B
c
O
H2N
C
O
O
NH2
O
B
HO
NHX
NH NH NH NH NH
NH
O
B
NHX
N
N
O
=
=
NHX BocHN
OH
O
O
O
O
O
NHY
C
B = CCbz/GiBu/ACbz/T
A
C
T
G
T
X = Boc; Y = Fmoc NHY
1 - 4
B = ACbz / GiBu /CCbz /T
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Scheme 3. Common protocol for synthesis of Cg(S/R)-bm-PNA oligomers from monomers 1 and 2
and Cg(S/R)-iso-PNA oligomers from monomers 3 and 4. Wavy line represents Cg sidechain with S or
R stereochemistry. Typical coupling reaction: Microwave 25 W, 5 min, rt 6 h. The resin 10 mg (~0.2
mmol/g), monomers (12 mg, 3 eq) a (i) HOBt (6 mg, 3 eq), HBTt (6 mg, 3 eq), DIPEA (10 µL ),
DMF (ii) 20% piperidine in DMF; b (i) HOBt, HBtU, DIPEA, DMF (ii) 50% TFA in DCM; c TFA-
TFMSA. Coupling of monomers on t-amide side shown by bold italics (in black) and coupling on Cg-
side shown by bold letters (in colour) for bases A,T, C and G.
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Table 1. MALDI-TOF spectral data of Cg-iso- and Cg-bm-PNA oligomers
Entry
1
PNA Oligomers Mol. formulae
HPLC Rt
Calcd. Mass
Obs. Mass
3542.50
3543.07
2580.69
2580.66
Cγ-S-bm-PNA 1
Cγ-R-bm-PNA 2
Cγ-S-iso-PNA 3
Cγ-R-iso-PNA 4
C145H187N65O44
C145H187N65O44
C112H136N70O19
C112H136N70O19
13.3
3543.43[M + H]+
3543.43 [M + H]+
2580.79 [M + K]+
2580.79 [M + K]+
2
3
4
13.5
13.1
13.5
g
Thermal stability of C (S/R)-bm and iso(S/R)-PNA:DNA duplexes
The Cg(S/R)-bm-PNA and Cg(S/R)-iso-PNA oligomers were independently hybridised
with DNA 1 or DNA 2 that are complementary to Cg-side and t-amide side base sequences
respectively to generate different Cg-amide duplexes with DNA 1 (Figure 4, A-B, D-E) and t-
amide duplexes with DNA 2 (Figure 4, C-F). When both DNA 1 and DNA 2 are present, the
bimodal Cg(S/R)-bm-PNAs can form double duplexes (Figure 4, G and H). After mixing the
PNA and DNA components in appropriate stoichiometry, the samples were annealed by
o
heating the mixtures to 80 C in buffer at pH 7.2 and cooled slowly to room temperature to
enable efficient hybridization. The DNA sequences (DNA 1 and DNA 2) were chosen to yield
PNA:DNA antiparallel duplexes in which the N-terminus of PNA is oriented towards the 3’
end of DNA. The thermal stabilities of various duplexes were obtained by recording UV
absorbance at 260 nm by continuous variation of temperature. The melting temperatures (Tms)
of the PNA:DNA hybrids were extracted from the corresponding UV-T plots (Figure 5).
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Figure 4. iso-PNA and bm-PNA duplexes. A. Cg(S)-iso-PNA 3:DNA 1; B. Cg(S)-bm-PNA 1:DNA 1;
C. DNA 2:Cg(S)-bm-PNA 1; D. Cg(R)-iso-PNA 3:DNA 1; E. Cg(R)-bm-PNA 2:DNA 1; F. DNA
2:Cg(R)-bm-PNA 2; G. DNA 2:Cg(S)-bm-PNA 1:DNA 1 (double duplex); H. DNA 2:Cg(R)-bm-PNA
2:DNA 1 (double duplex)
iso-PNA and bm-PNA single duplexes with DNA. The establishment of PNA:DNA duplexes
is confirmed by the observed sigmoidal transitions in UV-T plots,18,19 in which the melting
point Tm of duplexes correspond to the midpoint of transitions. This was determined from the
the first derivative plot in which the maximum provides a more accurate estimation of the Tm
value (Figure 5). The single sigmoidal transitions seen for Cg-iso-PNA duplexes A and D
clearly indicate that the new isomeric PNA structure carrying nucleobases only on Cg can form
o
perfect duplex with DNA 1. The (S)-iso-PNA 3 duplex (A) has higher stability (Tm 49.2 C)
than the (R)-iso-PNA 4 duplex (D, Tm 42.2 oC) with DTm (S-R) of +7 oC. The designed (S/R)
bimodal PNAs also formed corresponding Cg-amide Cg(S)-bm:PNA:DNA 1 (B) and Cg(R)-
bm:PNA:DNA 1 (E) duplexes with Tms of 51.7 oC and 38.1 oC respectively. These data suggest
that the presence of unpaired nucleobases on t-amide side can influnece the stability of Cg-
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amide duplexes. The Cg(S)-bm:DNA 1 duplex (B) had a higher melting (+2.5 C) than the
corresponding (S)-iso-PNA:DNA1 duplex (A), while Cg(R)-bm:DNA1 duplex (E) had a lower
o
melting (-4.1 C) than (R)-iso-PNA:DNA 1 duplex (D). The Cg(S/R)-bm-PNAs also formed
duplex on t-amide side with DNA 2 similar to that of standard PNA:DNA duplexes, with Tms
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of 45.5 oC for Cg(S)-bm duplex (C) and 33.4 oC for Cg(R)-bm duplex (F) respectively; again
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the S-duplex was significantly more stable than R-duplex by +12.1 C. Thus stereocenter at
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Cg dictates the stability of individual duplexes derived from iso-PNAs and Cg-bm-PNAs. The
results are consistent with the literature reports that Cg(S) substitution stabilises PNA:DNA
duplexes better than Cg(R)-substitution.11 Significantly, the present results demonstrate that
bases attached on sidechain at Cg on backbone instead of t-amide link on aeg backbone (as in
standard PNA) can successfully form duplexes, both in the new isomeric iso-PNA and in the
designed Cg-bimodal PNA.
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Figure 5. UV-melting and first derivative plots of duplexes. Normalised melting curve (red line) and
first derivative plots (blue dashed line). Numbers in plots correspond to Tm of duplexes.
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Double duplex from C (S/R)-bm-PNA:DNA complexes. The Cg(S/R)-bm-PNAs were
hybridized with an equimolar mixture of DNA 1 complementary to Cg-side and DNA 2
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complementary to t-amide side. Figure 5 (G and H) shows the melting curves of the derived
hybrids. In contrast to single sigmoidal melting noticed for single duplexes, both complexes
exhibited double sigmoidal melting, indicating the presence of two distinct transitions. The Tms
of 52.7 oC and 73.9 oC (Figure 5G) for the ternary complex DNA 2:Cg(S)-bm-PNA:DNA 1 are
higher than the Tms of the corresponding isolated duplexes. Similarly, the ternary complex
PNA:DNA DNA 2:Cg(R)-bm-PNA:DNA 1 (Figure 5H) from R-bimodal PNA exhibited two
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o
o
Tms at 38.7 C and 69.8 C. Though the transitions are broad, they are well resolved, with
distinct and discernible transitions having difference in Tms of 21.2 oC for S-bm-PNA complex
and 31.1 oC for R-bm-PNA complex. The ternary complex in both Cg(S) and Cg(R) bm-PNAs
are more stable than the individual binary complexes by >20 oC. The double sigmoidal UV-Tm
pattern indicated the melting process to be biphasic (three state) and can originate from either
a sequential melting of two duplexes as in DNA triplexes19 or simultaneous disassociation of
both DNA strands from PNA as in PNA2:DNA melting.20 A sequential melting process for two
duplexes should not have any influence on the second transition since after the dissociation of
first DNA strand from the ternary complex, the melting of second duplex should be similar to
that of the corresponding isolated duplex. A single step dissociation of ternary complex as in
PNA2:DNA triplexes20 should lead to only a single transition representing true melting of
ternary complex. Since both Tms in each bimodal (S/R) PNA:DNA2 complex (G/H) are higher
than individual duplexes (B,C/D,E) the melting process of two duplexes are coupled to each
other, mutually enhancing their stability. The difference in the Tms of higher transitions of the
ternary complexes of Cg(S)-bm-PNA 1 and Cg(R)-bm-PNA 2 is +4 °C, which is 3-times less
than the difference in Tms of their isolated duplexes [DTm(B-E) Cg-amide: +13.6 °C; (C-F) t-
amide: +12.1°C). Thus the formation of ternary complexes compensates the larger difference
in thermal stabilitites of individual Cg(R) and Cg(S) duplexes.
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Mismatch C( )-bm-PNA:DNA duplexes. The sequence fidelity in the complementation of
base-pairing from Cg-side in bm-PNA:DNA duplexes was examined by determining the Tms
of mismatched duplexes. Cg(S/R)-bm-PNAs (1 and 2) were hybridized individually with DNA
1m (5’-AGCGCA-3’) and DNA2m (5’-GAAACAAC-3’) that carry single C:Abase mismatch
on Cg-side and t-amide side respectively (ESI, S11-S12). All mismatched duplexes showed
lower Tms compared to perfect duplexes. (Figure 6, green bars). The mismatch duplex Cg(S)-
o
bm-PNA 1:DNA 1m was destabilized by (−)13 C and Cg(R)-bm-PNA 1:DNA 1m duplex
o
destabiized by (−)3.9 C. The corresponding t-amide mismatch duplexes with DNA 2m also
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showed lower stability with DTms of (−)11.3 oC and (−)3.6 oC for S-bm PNA 1 for R-bm-PNA
2 respectively. In the corresponding mismatched double duplexes from Cg(S)-bm-PNA 1 and
Cg(R)-bm-PNA 2, the two transitions collapsed into single transitions with Tms of 61.5 oC and
52.3 oC, with detsbilization DTms of (−)12.4 oC and (-)17.5 oC respectively (Figure 6). Thus, in
mismatched bimodal complexes, both DNA strands clearly dissociate in single step, similar to
that in PNA2:DNA triplex.20 Notably, even in the set of mismatched complexes, the Tm of
ternary complex [DNA 2m:Cg(S/R)-bm-PNA:DNA 1m] was higher than the Tms of isolated
duplexes, substantiating the generality of enhanced stability in double duplexes. Compared to
perfect duplexes, the destabilization seen in mismatched duplexes of S-bm-PNA were almost
similar to destabilization per base mismatch seen in unmodified PNA:DNA duplexes.19 This
substantiated that co-existence of two duplexes sharing a common PNA backnone as in
bimodal PNA:DNA2 ternary complexes mutually confer higher stabilty on each other.
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Figure 6. Comparative Tms of duplexes and ternary complexes. Labels on X-axis correspond
to duplexes shown in in Figure 5
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CD spectra of C -bm-PNA:DNA duplexes
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The CD spectral profiles of Cg(S/R)-bm-PNA duplexes and double duplexes (Figure 7)
resemble the characteristic CD profiles of PNA:DNA duplexes,21 with minor changes in
relative intensities among the duplexes and double duplexes. The double duplexes (green
curve) indicated prominent positive bands at 265 nm and 218-220 nm. The Cg(S)-bm-PNA 1
exhibited relatively higher band at 260 nm, suggesting a better stacking of bases compared to
that in Cg(R)-bm-PNA 2. The handedness of two stereomeric Cg(S/R)-bm-PNA:DNA duplexes
remain same as both of them have L-lysine at C-terminus and the CD spectra are dominated
by DNA contribution with well-established right handedness of DNA:PNA duplexes. The
chiral effects Cg (S) and Cg(R) on PNAbackbone seems to be weak to influence the handedness
of base stacks and the stability of derived duplexes are affected through minor conformational
effects. The CD spectra also ruled out the formation of any bm-PNA2:DNA 2 triplexes arising
from t-amide side of bimodal PNAs.
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Figure 7. CD spectra of Cg-bm-PNA:DNA duplexes. Labels for duplexes as in Figure 4.
Plausible dissociation model for bimodal PNA:DNA ternary complexes
The double sigmoidal transition seen in bimodal PNA:DNA2 duplexes with both Tms
higher than that of isolated duplexes is interesting and results in enhanced stability of both
duplex components of the ternary complex. As mentioned earlier, this observation does fit not
into either a sequential melting model (like in DNA triplexes) or simulatneous dissociation
model (as in PNA2:DNA triplexes). It may be mentioned that in PNAs that carry Janus bases
and bind two DNA strands, the original stability of individual duplexes was retained (Figure 4
in ref 16b). The origin of double melting process in bimodal PNAs, which do not retain the
original duplex stability, may be ascribed to a process of premelting in the ternary complex
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during the first transition, followed by complete melting during the second transition. Such
premelting processes are well documented in DNA duplexes and involves co-operative
perturbations in base stacking accompanied by non-cooperative changes in sugar-phosphate
backbone conformation.22 In the intermediate conformations adopted during premelting
transitions termed premeltons22d the bases retain H-bonding with slight distortion. In DNA-
drug interactions, this may involve additional reorganization in the spine of hydration.
Premelting transitions are quite broad and ultimately lead to sharp transition with complete
dissociation of both strands through breaking of H-bonds.
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The kinetics of association – dissociation transition of PNA:DNA hybrids is slow and
leads to broad melting transitions. In Cg-bm-PNA:DNA2 complex where two duplexes share a
common PNA backbone, the premelting of the tight ternary complex would involve partial
unwinding of both duplexes with co-operative unstacking of bases accompanied by changes in
PNA backbone conformation. The unwinding of the complex with destacking of bases may
lead to the first transition. (Figure 8, path A). Compared to DNA:DNA duplexes, the pre-
melting transition in bimodal PNA:DNA2 double duplexes, seems to be more prominent. This
is due to a tighter winding of two duplexes that co-operatively uncoil with a greater degree of
unstacking of bases. The two DNA strands are still held in place by H-bonding between
nucleobases of bimodal PNA. The common PNA backbone thus couples the premelting of the
two duplexes which is followed by complete dissociation in a subsequent step to result in
second transition. The mismatched ternary complex yields only a single transition, with the
mismatches on both sides presumably relieving the strain compared to the tight perfect double
duplex. A similar pattern of double transitions with Tms higher than the isolated duplexes were
also seen with Ca-bm-PNA,13 suggesting the generality of the phenomenon. The difference in
the Tms of the S and R ternary complexes [Cg(S)-bm-PNA 1 and Cg(R)-bm-PNA 2] is only 4
°C, about 3 times lower than the Tm differences between the respective isolated duplexes [DTm
Cg-amide (B-E): 13.6 °C and t-amide (C-F), 12.1 °C)]. This points to the additional stabilizing
effects arising when the two duplexes are coupled through a shared backbone, which also
overrides the lower stabilizing effects in Cg(R) stereomeric duplex compared to Cg(S) duplex.
Understanding the structure and biophysical origin of two transitions in bimodal PNA ternary
complexes and their higher stability compared to isolated duplexes needs further
experimentation such as identifying the intermediates by careful temperature dependent CD
experiments and computational studies.
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Figure 8. Cartoon representation of (A) pre-melting-dissociation and (B) sequential
dissociation models for melting of bimodal PNAs
CONCLUSIONS
A new type of PNA derivatives termed “bimodal PNA”s intended to concurrently
hybridise to two complementary DNA strands have been designed by installing a set of
nucleobases on Cg-sidechain of each aeg repeating unit of the standard PNA oligomer. The
orthogonally protected (Fmoc/Boc) stereomeric Cg(S/R)-bimodal PNA monomers were
employed for solid phase synthesis of bimodal Cg(S/R)-bm-PNAoligomers having mixed bases
on both sides. Cg-iso-PNA monomers with bases only on Cg-sidechain on aeg backbone were
used for synthesis of isomeric Cg(S/R)-iso-PNA oligomers. The UV-melting data on (S/R)-iso-
PNA:DNA hybrids suggested formation of stable duplexes from the new isomeric PNA that is
devoid of nucleobases linked to the t-amide as in aeg-PNA.
The designed bimodal Cg(S/R)-bm-PNAs individually bind DNAs complementary on
each side to yield the corresponding binary Cg-amide and t-amide duplexes. Together, they
hybridize on both sides of bimodal PNA backbone to generate ternary complexes, which show
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biphasic double transition with higher stability compared to independent duplexes. Cg(S)-
dupexes show significantly higher stability than Cg(R) duplexes. The enhanced Tms in two step
melting of ternary complex suggests neither a sequential melting mechanism as in DNA
triplexes or by a single step simultaneous dissociation as in PNA2:DNAtriplexes. The complete
dissociation of ternary complex is perhaps preceded by a pre-melting transition involving a
partial unstacking of bases and backbone conformational change (Figure 8). The UV-melting
profiles of Cg(S/R)-bm-PNA double duplexes observed in this work are consistent with that
we recently reported for Ca-bm-PNA,13 validating the generality of the design principles of
bimodal PNAs to create two duplexes that are structurally coupled with a shared PNA
backbone. The proposed mechanism of dissociation of such structurally coupled double
duplexes needs further study.
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The Cg-bm-PNA duplexes studied here along with recently published Ca-bm-triazole
PNA oligomer13 are first examples of PNA derivatives in which single PNA backbone hosts
two PNA:DNAduplexes through base pairing of natural bases. The base pairing of nucleobases
on Ca and Cg-sidechains are probably Watson−Crick type, because of the high duplex stability
and sequence specificity. This was further substantiated by lower Tms in mismatched duplex.
The control iso-PNAs (Cg and Ca-iso-PNAs11) are new isomorphs of PNAs that successfully
form strong duplexes and open avenues for studying entirely new types of PNA:DNA
complexes.
Based on the choice of the sequences on either side
(polypurines/polypyrimidines/Gn/Cn), the Cg and Ca-bimodal PNAs can be used to generate
welded PNA:PNA binary duplexes, ternary double triplexes, and tetraplexes. The enhanced
stability and augmented molecular recognition properties of the bimodal PNAs can be
exploited to design complex supramolecular nanoassemblies with defined functions tailored
for various applications in biotechnology as well as material science.25 Among the probable
biological applications, bimodal PNAs can be used to specifically and concurrently target two
genes, inhibit micro RNA structures, to probe biological processes and modulate them for
therapeutics.26 Further, bimodal PNAs can used like stapler strands in DNA origami to expand
the limits of programmable folding in nucleic acid nanotechnology.27
EXPERIMENTAL SECTION
The chemicals used were of laboratory or analytical grade. All the solvents used were
distilled or dried to carry out different reactions. Thin layer chromatography (TLC) were used
to monitor reactions. Typically, workup involved washing of the organic layer sequentially
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with water and brine, drying the organic extract with anhydrous sodium sulphate and
subsequent removal of the solvent in roatary evaporator under vacuum. Pre-coated silica gel
GF254 sheets (Merck 5554) were used for TLC analysis and spots detected under UV
illumination, exposure to iodine vapour or by ninhydrin solution spray. Silica gel (60-120 or
100-200 mesh) was used for column chromatographic separations. The PNA oligomers were
purified using reverse phase HPLC system equipped with a semipreparative BEH130 C18 (10
X 250 mm) column. The DNA oligonucleotides were obtained commercially from Integrated
DNA Technologies (IDT). Salts and reagents used in buffer preparation such as NaCl, sodium
cacodylate etc. were obtained from Sigma-Aldrich. The pH of the buffer solutions was adjusted
using HCl (Sigma Aldrich). Compounds 6 and 19 were prepared from L-Glutamine by
procedure described in reference 17.
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1H and C NMR spectra were recorded on Bruker AC-400 (400 MHz) or JEOL 400
MHz NMR spectrometers using CDCl3 or DMOSO-d6 as solvents. The chemical shift values
are reported in parts per million (ppm, d scale) with reference to the internal TMS as standard.
The optical rotation were measured on Rudolph Research Analytical Autopol V polarimeter.
All UV-Visible spectral measurements were done on Perkin Elmer Lambda 45 double beam
spectrometer. The mass spectra of all compounds were obtained by HRMS and the mass of
PNA oligomers was confirmed by MALDI-TOF/TOF data using DHB (2,5-Dihydroxybenzoic
acid) or CHCA (α-Cyno-4-hydroxycinnamic acid) as matrix.
N-ethylamino(tertbutoxycarbonyl)-N(acetamido-N1-thyminyl)-2(S)-[(Fmoc)
aminoethyl] glycine (1)
To compound 6 (1 g, 2 mmol) dissolved in ethanol, 10% Pd/C on charcoal was added
under H2 atmosphere and stirred at room temperature for 6 h. After the reaction was complete
as monitored by TLC, the reaction mixture was filtered on celite-545 pad and the filtrate
evaporated to get solid residue (crude weight 0.8 g). The free amine (1 g, 2 mmol) was
dissolved in 10 mL THF:H2O (1:1) taken in a clean flask and stirred at 0 ºC. Na2CO3 (0.5 g, 5
mmol) was added to the reaction mixture followed by slow dropwise addition of Fmoc-Cl (0.6
g, 2 mmol). The reaction mixture was stirred at 0 ºC for 1 h and then at 25 ºC for 4 h. The
solvent was evaporated on a rotary evaporator. The residue was dissolved in water (10 mL)
and washed with of diethyl ether (20 mL). It was neutralized with 10% aq. HCl and extracted
with EtOAc (25 mL × 3). The resisdue obtained after concentration of the combined organic
extract was purified by column chromatography to yield compound 1 (1.0 g, 86%). Rf = 0.50
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Methanol/DCM (20:80); [α]D25= -3.21 (c= 0.5%, MeOH); 1H NMR (400 MHz, DMSO-d6) δ
11.28 (s, 1H), 7.89-7.87 (d, 2H, J = 8 Hz), 7.68- 7.67 (d, 2H, J = 4 Hz), 7.40 (t, 2H, J = 16 Hz),
7.32 (t, 2H, J = 16 Hz), 7.21 (s, 1H), 7.17 (s, 1H), 4.80-4.46 (m, 2H), 4.31-4.20 (m, 4H), 4.03-
3.60 (m, 3H), 3.23-2.97 (m, 4H), 4.03-360 (m, 3H), 3.23-2.97 (m, 4H), 1.74 (maj) 1.72 (min)
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(S, 2H), 1.37 (maj) 1.35 (min) (s, 9H) ppm. C{1H} NMR (100 MHz, DMSO-d6) δ 172.0,
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170.6, 170.4, 167.9, 167.3, 164.3, 156.1, 155.1, 155.6, 150.1, 143.1, 143.9, 141.9, 141.7, 140.7,
127.6, 127.1, 125.1, 120.1, 108.2, 78.0, 77.8, 66.4, 65.3, 59.8, 47.6, 46.8, 46.3, 39.5, 37.5, 31.9,
28.3, 21.1, 20.8, 14.1, 12 ppm. HRMS (ESI-TOF) m/z calcd for C33H39N5O9 [M+Na]+
672.2648, found 672.0918.
5-Amino-2(R)-[(tert-butoxycarbonyl)amino]-5-oxopentanoic acid (8)
A solution of di-tert-butyl dicarbonate [(Boc)2O] (16.5 g, 17 mL, 75 mmol) in dioxane
(100 mL) was added in portion to an ice-cold solution of D-glutamine 7 (10 g, 68 mmol) in aq.
o
NaOH (1 N, 100 mL). The reaction mixture was stirred at 0 C for 1 h, after which dioxane
was completely removed under vacuum. The aqueous layer was washed with diethyl ether to
remove excess [(Boc)2O], acidified by addition of saturated aq. KHSO4 to pH 2-3 under cooling
and extracted with EtOAc (3 × 150 mL). The combined organic extracts dried over an. Na2SO4,
was evaporated to obtain compound 8 as a white powder which was used without further
purification (14.5 g, 86% yield). HRMS (ES-TOF) m/z calcd for C10H18N2O5 [M+ H]+
246.1216, found 246.1494.
4-amino-2-(R)-[(tert-butoxycarbonyl)amino] butanoic acid (9)
A slurry of compound 8 (5 g, 20 mmol), EtOAc (25 mL), ACN (25 mL), water (12 mL)
and iodobenzene diacetate (8 g, 24 mmol) was stirred at 16 °C for 30 min and stirring continued
at 20 oC for 4 h. It was filtered under vacuum and the filter cake was washed with EtOAc and
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dried in vacuum to obtain compound 7 (2.6 g). Rf = 0.2 EtOAc/MeOH (50:50); H NMR (400
MHz, DMSO-d6) 6.18 (1 H, s), 3.61 (2 H, s), 3.02 (2H, d, J 7.9), 2.82 – 2.64 (1 H, m), 1.57 –
1.06 (10 H, m). 13C {1H} (101 MHz, DMSO-d6) 171.10, 155.13, 78.17, 50.98, 40.57, 39.52,
31.31, 28.15. HRMS (ESI-TOF) m/z calcd for C9H18N2O4 [M + Na]+ 218.1267, found
218.1756.
4-amino (benzyloxy)carbonyl)-2(R)-[tert-butoxycarbonyl)amino]butanoic acid (10)
To an ice-cold solution of compound 9 (1 g, 4.6 mmol) in acetone (25 mL), aq. NaHCO3
(1 g, 15 mL, 14 mmol) was added and stirred at 0 oC for 10 min. To this, benzylchloroformate
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(2 g, 2 mL, 5.5 mmol) in toluene was added and stirring of the reaction mixture was continued
overnight at room temperature. The organic solvent was removed under vacuum, the aqueous
layer was washed with diethyl ether (2 x 30 mL), acidified with saturated aq. KHSO4 to pH 2-
3 and extracted with EtOAc (3 x 60 mL). The combined organic layer was dried and evaporated
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to give compound 10 as sticky oil (1.45 g, 90% yield). Rf = 0.67 EtOAc/MeOH (50:50). H
NMR (400 MHz, CDCl3) δ 7.37 – 7.27 (m, 5H), 5.58 (d, J = 49.7 Hz, 2H), 5.35 (m, J = 40.1
Hz, 1H), 5.14 – 5.02 (m, 2H), 4.40 – 4.28 (m, 1H), 3.71 (s, 2H), 3.54 – 3.41 (m, 1H), 3.14 –
3.00 (m, 1H), 2.07 (d, J = 3.9 Hz, 1H), 1.69 (dd, J = 9.0, 4.8 Hz, 1H), 1.46 – 1.38 (m, 9H).
13C{1H} NMR (101 MHz, CDCl3) δ 175.0, 173.2, 156.9, 156.6, 136.6, 128.6, 128.2, 80.4, 77.2,
66.8, 52.6, 51.0, 37.2, 33.6, 30.1, 28.4. HRMS (ESI-TOF) m/z calcd for C17H24N2O6 [M + H]+
353.1713, found 353.1454.
4-amino(benzyloxy)carbonyl)-2(R)-[(tert-butoxycarbonyl)amino]methylbutanoate (11)
To a stirred solution of compound 10 (5 g, 15 mmol) and K2CO3 (5.0 g, 36 mmol) in
acetone (70 mL), dimethyl sulfate (2 mL, 17.5 mmol) (caution: dimethylsulphate is hazardous
and must refer to material safety sheet befor handling) was added. The reaction mixture was
heated to 55 °C for 5 h under reflux, the solvent was evaporated and to the concentrate, water
(90 mL) was added. It was then extracted with EtOAc (3 × 50 mL) and the combined organic
layer was washed with brine, dried and concentrated. The residue obtained was purified on
column chromatography to give compound 11 as white solid (2.4 g, 93% yield). mp = 65-68
°C; Rf = 0.5 petroleum ether/EtOAc (70:30); H NMR (400 MHz, CDCl3) δ 7.38 – 7.28 (m,
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5H), 5.50 (s, 1H), 5.26 (s, 1H), 5.10 (q, J = 12.3 Hz, 2H), 4.37 (t, J = 8.5 Hz, 1H), 3.72 (s, 3H),
3.54 – 3.43 (m, 1H), 3.11 – 3.00 (m, 1H), 2.07 (dd, J = 8.4, 4.4 Hz, 1H), 1.89 (s, 1H), 1.73 –
1.62 (m, 1H), 1.43 (s, 9H). 13C{1H} NMR (101 MHz, CDCl3) δ 173.15, 156.5, 156.0, 136.7,
128.6, 128.2, 80.4, 77.2, 66.8, 52.6, 51.0, 37.2, 33.7, 28.4. HRMS (ESI-TOF) m/z calcd for
C18H26N2O5 [M]+ 366.4088, found 366.9160.
4-Amino(benzyloxycarbonyl)-2(R)-[(tert-butoxycarbonyl)amino]-butanol (12)
To a solution of compound 11 (2.5 g, 13.0 mmol) in absolute EtOH (30 mL) was added
NaBH4 (0.6 gm, 20.5 mmol) and stirred for 6 h under N2 atmosphere at room temperature.
After complete removal of ethanol under vaccum, water (60 mL) was added to the concentrate
and extracted with EtOAc (3 x 30 mL). The pooled organic extract was washed with brine,
dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by column
chromatography to give compound 12 as white solid (2.0 g, 89 % yield). M.p = 80-82 oC; Rf =
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0.4 petroleum ether/EtOAc (50:50); H NMR (400 MHz, DMSO-d6) δ 7.82 – 7.72 (m, 5H),
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6.27 (s, 1H), 5.65 (d, J = 8.4 Hz, 1H), 5.53 (q, J = 12.3 Hz, 2H), 4.16 – 3.94 (m, 4H), 3.87 (dd,
J = 12.8, 6.1 Hz, 1H), 3.47 (d, J = 5.3 Hz, 1H), 3.04 (s, 8H), 2.22 – 2.10 (m, 1H), 2.08 – 1.96
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(m, 1H), 1.93 – 1.83 (m, 9H). C{1H} NMR (101 MHz, CDCl3) δ 156.7, 136.8, 128.6, 128.2,
80.0, 77.1, 66.7, 65.6, 49.9, 37.8, 32.3, 28.5. HRMS (ES-TOF) m/z calcd for C17H26N2O5 [M]+,
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338.1842, found 338.3427
4-Amino(benzyloxycarbonyl)-2(R)-[(tert-butoxycarbonyl)amino]butylmethane sulfonate
(13)
To an ice-cold solution of compound 12 (2 g, 6 mmol) in dry DCM (30 mL), triethyl
amine (2.0 mL, 15 mmol) and mesyl chloride (0.6 mL, 8 mmol) were added and the mixture
was kept stirring for 30 min at 0 oC under N2 atmosphere. DCM (30 mL) was added and washed
with water (30 mL) and brine (20 mL). The organic layer was dried and concentrated to give
compound 13 (2.3 g). Rf = 0.53 petroleum ether/EtOAc (50:50). This compound was used for
next step without further purification.
4-Amino(benzyloxy)carbonyl)-2(R)-[(tert-butoxycarbonyl)amino]-butylazide (14)
A solution of compound 13 (2.0 g, 5.5 mmol) and sodium azide (5.5 g, 82.5 mmol) in
dry DMF (55 mL) was heated for 6 h at 80 °C. Water (60 mL) was added, extracted with ethyl
acetate (3 x 30 mL) and the combined extract was washed with water (25 mL) followed by
brine (25 mL). The dried organic layer was concentrated to obtain a residue that was purified
by column chromatography to give compound 14 as sticky yellowish oil (1.4 g, 80% yield). Rf
= 0.73 petroleum ether/EtOAc (50:50); 1H NMR (400 MHz, CDCl3) δ 7.44 – 7.30 (m, 31H),
5.47 (s, 1H), 5.11 (q, J = 12.3 Hz, 2H), 4.73 (s, 1H), 3.84 (s, 1H), 3.45 (d, J = 16.0 Hz, 3H),
3.08 – 2.96 (m, 1H), 1.74 (dd, J = 8.3, 4.3 Hz, 1H), 1.59 (dt, J = 13.7, 7.2 Hz, 1H), 1.46 (s,
9H). 13C{1H}NMR (101 MHz, CDCl3) δ 156.5, 155.9, 136.6, 128.5, 128.1, 80.1, 66.7, 55.2,
47.7, 37.5, 33.1, 28.3. HRMS (ESI-TOF) m/z calcd. for C17H25N5O4 [M]+, 363.1907, found
363.2510
N-ethylamino-(tert-butoxycarbonyl)amino)-2(R)-[(benzyloxycarbonyl)amino]ethyl
glycinate (15)
A solution of compound 14 (0.7 mg, 2.0 mmol) in absolute EtOH (15 mL) taken in
hydrogenation flask (Parr reactor) was mixed with Raney Nickel (2 mL). It was hydrogenated
with H2 pressure of 50-55 psi for 6 h at room temperature . The catalyst was filtered off from
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reaction mixture, and the filtrate was concentrated to yield a residual yellowish oil of amine
(630 mg, 1.8 mmol). This was treated with ethylbromo acetate (0.2 mL, 1.7 mmol) and triethyl
amine (0.8 mL) in ACN (20 mL) and the reaction mixture was stirred at room temperature for
12 h. The solvent was evaporated under vacuum, water (50 mL) was added to the concentrate
and The aqueous layer was extracted with EtOAc (3 × 40 mL). The combined organic extract
was washed with sat. NaHCO3 followed by brine, dried and concentrated on rotary evaporator.
The residue obtained was purified on silica gel (100-200 mesh) using petroleum ether and ethyl
acetate to give compound 15 as yellowish oil (0.65 mg, 80%). Rf = 0.48 petroleum ether/EtOAc
(20:80); 1H NMR (400 MHz, CDCl3) δ 7.40 – 7.31 (m, 5H), 5.61 (s, 1H), 5.05 (ddd, J = 26.6,
19.7, 9.8 Hz, 4H), 4.24 – 4.14 (m, 3H), 4.03 (s, 1H), 3.98 – 3.67 (m, 2H), 3.50 (ddd, J = 21.0,
18.4, 6.3 Hz, 2H), 3.17 (d, J = 10.4 Hz, 1H), 3.10 – 2.99 (m, 1H), 1.90 – 1.65 (m, 2H), 1.42 (s,
9H), 1.32 – 1.27 (m, 3H). 13C{1H} NMR (101 MHz, CDCl3) δ 168.9, 168.6, 156.7, 136.8,
136.6, 128.6, 128.3, 79.8, 77.2, 66.7, 61.7, 53.1, 51.0, 50.0, 49.0, 47.5, 47.2, 41.0, 40.8, 37.8,
33.6, 32.8, 28.5, 14.2. HRMS (ESI-TOF) m/z calcd for C21H33N3O6 [M]+ 423.2369, found
423.0408.
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N-ethylamino(tertbutoxycarbonyl)-N(chloroacetamido)-2(R)-[(benzyloxycarbonyl)
aminoethyl] ethyl glycinate (16)
To an ice-cold solution of compound 15 (3.0 g, 7.3 mmol) and triethyl amine (3 g; 4
mL, 30 mmol) in dry DCM (50 mL) was treated with chloroacetyl chloride (0.6 mL, 7.3 mmol)
and the reaction mixture was stirred for 8 h. DCM (20 mL) was added to the reaction mixture
and washed with water (50 mL) and brine (50 mL). The dried organic extract was concentrated
and the residue was purified on silica gel (100-200 mesh) using petroleum ether and EtOAc to
give compound 16 as colourless sticky oil (2.5 g, 72 %). Rf = 0.59 petroleum ether/EtOAc
1
(40:60); H NMR (400 MHz, CDCl3) δ 7.33 (dt, J = 8.3, 4.0 Hz, 5H), 5.08 (q, J = 12.8 Hz,
2H), 4.41 (dd, J = 39.3, 16.3 Hz, 2H), 4.30 – 4.08 (m, 4H), 3.86 – 3.61 (m, 2H), 3.37 (dd, J =
46.5, 9.9 Hz, 2H), 3.20 – 2.85 (m, 4H), 2.05 (d, J = 9.5 Hz, 2H), 1.91 – 1.88 (m, 2H), 1.40 (d,
J = 8.1 Hz, 9H), 1.25 (t, J = 7.1 Hz, 3H). 13C{1H} NMR (101 MHz, CDCl3) δ 169.2, 168.6,
167.6, 164.4, 156.9, 151.5, 141.0, 136.8, 128.6, 128.2, 111.0, 79.9, 77.2, 62.5, 61.7, 60.5, 52.4,
51.7, 50.0, 47.9, 37.9, 33.2, 32.5, 28.5, 14.3, 12.4. HRMS (ESI-TOF) m/z calcd for
C23H34ClN3O6 [M]+ 499.2085, found 499.1733.
N-ethylamino(tertbutoxycarbonyl)-N(acetamido-N1-thyminyl)-2(R)-[(benzyloxy
carbonyl) aminoethyl] ethyl glycinate (17)
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A solution of compound 16 (1 g, 2 mmol), K2CO3 (0.3 g, 2.5 mmol) and thymine (0.3
g, 2.5 mmol) in dry DMF (20 mL) was stirred at room temperature for 12 h. Water (50 mL)
was added to the reaction mixture and product was extracted into EtOAc (3 x 40 mL). The
organic layer after work up was dried and concentrated to yield a residue which was purified
on silica gel (100-200 mesh) using petroleum ether and ethyl acetate to give compound 17 as
white solid (0.97 g, 88%); Rf = 0.47 EtOAc (100); 1H NMR (400 MHz, CDCl3) δ 9.70 (d, J =
120.7 Hz, 1H), 7.35 – 7.27 (m, 5H), 7.00 (d, J = 26.8 Hz, 1H), 5.70 – 5.51 (m, 1H), 5.06 (q, J
= 12.5 Hz, 2H), 4.79 (d, J = 16.2 Hz, 1H), 4.47 – 4.32 (m, 1H), 4.30 – 4.08 (m, 4H), 3.91 (d, J
= 17.2 Hz, 1H), 3.76 (d, J = 41.0 Hz, 1H), 3.65 – 3.48 (m, 2H), 3.47 – 3.26 (m, 1H), 3.06 (ddd,
J = 19.7, 14.2, 6.1 Hz, 3H), 2.15 (s, 1H), 1.87 (d, J = 7.2 Hz, 3H), 1.73 – 1.58 (m, 1H), 1.39
(d, J = 9.8 Hz, 9H), 1.26 (dt, J = 21.1, 7.1 Hz, 3H). 13C{1H} NMR (101 MHz, CDCl3) δ 169.3,
168.6, 167.6, 164.5, 156.8, 156.2, 151.6, 151.3, 141.3, 136.7, 128.5, 128.1, 111.0, 80.0, 79.8,
66.7, 62.4, 61.6, 52.2, 51.6, 49.9, 48.7, 47.9, 47.3, 37.9, 37.6, 33.0, 32.3, 28.4, 14.2, 12.4 ppm.
HRMS (ESI-TOF) m/z calcd for C28H39N5O9 [M + Na]+ 612.2748,found 612.2722
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N-ethylamino(tertbutoxycarbonyl)-N(acetamido-N1-thyminyl)-2(S)-[(benzyloxy
carbonyl) aminoethyl] glycine (18)
To a stirred solution of compound 17 (0.5g, 1 mmol) in MeOH was added 10% aq.
LiOH and reaction mixture was stirred at room temperature for 3-4 h. The solvent was removed
under vacuum and the aqueous layer was neutralized with activated Dowex H+ resin till pH of
the solution reached 5.0. The resin was filtered out and the filtrate was concentrated to obtain
compound 18 as white solid (0.4 g, 85%). M.p = 241-245 °C; Rf = 0.5 EtOAc/MeOH (50:50);
1H NMR (400 MHz, CDCl3) δ 7.78 (d, J = 8.4 Hz, 2H), 7.66 – 7.50 (m, 2H), 7.35 (dd, J = 28.0,
8.6 Hz, 4H), 5.55 – 5.10 (m, 1H), 4.65 – 4.32 (m, 2H), 4.22 (s, 1H), 3.96 (d, J = 14.0 Hz, 1H),
3.64 (d, J = 72.1 Hz, 1H), 3.11 (s, 1H), 2.78 (d, J = 46.7 Hz, 1H), 2.08 – 1.86 (m, 1H), 1.84 –
1.61 (m, 2H), 1.46 (dd, J = 16.2, 4.8 Hz, 9H). 13C{1H} NMR (101 MHz, DMSO-d6) δ 172.3,
170.8, 168.1, 167.5, 164.5, 156.2, 155.8, 151.0, 142.0, 137.4, 128.6, 128.0, 108.5, 78.3, 65.4,
51.3, 48.8, 47.9, 46.6, 39.5, 37.9, 32.0, 28.4, 21.3, 12.2 ppm. HRMS (ESI-TOF) m/z calcd for
C26H35N5O9 [M]+ 561.2435, found 561.2143.
N-ethylamino(tertbutoxycarbonyl)-N(acetamido-N1-thyminyl)-2S-[(Fmoc) aminoethyl]
glycine (2)
Compound 18 (1 g, 2 mmol) was dissolved in ethanol containing 10% Pd/C and
subjected to H2 atmosphere in a ballon with stirring at room temperature for 6 h. After the
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reaction was complete as monitored by TLC, it was filtered on celite-545 pad and the filtrate
was evaporated to get solid product (crude weight 0.8 g). The free amine was dissolved in
THF:H2O (1:1, 10 mL) in a flask, stirred with solid Na2CO3 (0.5 g, 5 mmol) at 0 ºC, followed
by dropwise addition of Fmoc-Cl (0.6 g, 2 mmol). After 1 h at 0oC, temp was raised to 25 ºC
and kept for 3 h. The reaction mixture was concentrated on a rotary evaporator and the residue
was dissolved in water (10 mL) and washed with of diethyl ether (20 mL). After neutralization
with 10% HCl, it was extracted with EtOAc (25 mL × 3) and the dried organic layer on
concentration gave a residue that was purified by column chromatography to yield compound
2 (1.0 g, 86%). Rf = 0.50 Methanol/DCM (20:80); [α]D25= + 3.19 (c= 0.5% , MeOH); 1H NMR
(400 MHz, DMSO-d6) δ 12.58 (s, 1H), 11.28 (d, J = 3.0 Hz, 1H), 7.88 (d, J = 7.5 Hz, 2H), 7.68
(d, J = 6.5 Hz, 2H), 7.41 (t, J = 7.4 Hz, 2H), 7.36 – 7.27 (m, 2H), 7.23 – 7.14 (m, 1H), 6.79
(dd, J = 66.5, 9.0 Hz, 1H), 4.58 (ddd, J = 46.1, 28.4, 12.1 Hz, 2H), 4.37 – 4.12 (m, 3H), 3.98
(dt, J = 39.4, 12.3 Hz, 1H), 3.51 – 3.15 (m, 4H), 3.05 (ddd, J = 18.4, 13.5, 8.0 Hz, 2H), 1.95
(d, J = 30.2 Hz, 1H), 1.77 – 1.69 (m, 2H), 1.37 (d, J = 10.7 Hz, 9H), 1.17 (t, J = 7.1 Hz, 1H).
13C{1H} NMR (101 MHz, DMSO-d6) δ 172.0, 170.6, 167.9, 167.3, 164.3, 156.1, 155.1, 155.6,
150.1, 143.1, 143.9, 141.7, 140.7, 127.6, 127.1, 125.1, 120.1, 108.2, 78.0, 77.8, 66.4, 65.3,
59.8, 47.6, 46.8, 39.5, 37.5, 31.9, 28.3, 21.1, 20.8, 14.1, 12 ppm. HRMS (ESI-TOF) m/z calcd.
for C33H39N5O9 [M] + 649.2748, found 649.2596
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N(Acetyl)-N-ethylamino(tertbutoxycarbonyl)-2(S)-[(benzyloxycarbonyl)aminoethyl]
ethyl glycinate (20)
To a stirred solution of 19 (1 g, 2.5 mmol) in dry DCM (10 mL) was added DCC (0.7
g, 4 mmol) and DhbtOH (0.6 g, 4 mmol) and the reaction mixture was stirred at room
temperature for 10 min. Glacial acetic acid (0.2 mL, 4 mmol) was added and stirring continued
overnight. Removal of solvent under reduced pressure gave a residue which was dissolved in
EtOAc (300 mL), washed with saturated aq. NaHCO3 (300 mL) followed by aq. KHSO4 (10%,
300 mL). After usual work up, the organic layer was concentrated under reduced pressure to
obtain crude product that was purified by column chromatography to afford compound 20 (1.5
gm, 80%) as a sticky oil. 1H NMR (400 MHz, CDCl3) δ 7.37 – 7.27 (m, 5H), 5.63 (s, 1H), 5.13
– 5.03 (m, 2H), 4.27 – 4.10 (m, 3H), 4.08 – 3.96 (m, 2H), 3.88 (d, J = 17.1 Hz, 3H), 3.82 –
3.62 (m, H), 3.51 – 3.35 (m, 2H), 3.17 – 2.83 (m, 2H), 2.07 (d, J = 41.0 Hz, 3H), 1.91 – 1.55
(m, 3H), 1.40 (d, J = 4.9 Hz, 9H), 1.26 (dt, J = 11.2, 7.1 Hz, 3H). 13C{1H} NMR (101 MHz,
CDCl3) δ 172.6, 171.5, 170.1, 169.2, 156.8, 136.8, 128.6, 128.1, 80.1, 79.6, 77.2, 66.6, 61.9,
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61.5, 51.0, 50.5, 47.4, 37.9, 37.6, 33.5, 32.6, 28.5, 21.5, 14.2 ppm. HRMS (ESI-TOF) m/z
calcd. for C23H35N3O7 [M]+ 465.2475, found 465.1733.
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carbonyl) aminoethyl] ethyl glycine (21)17
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A solution of compound 20 (0.5g, 1 mmol) in MeOH was treated with 10% aq. LiOH
and reaction mixture was stirred at room temperature for 3-4 h. The solvent was removed under
vacuum and the aqueous layer was washed with diethyl ether. It was neutralized with activated
Dowex H+ resin till pH of the solution reached 5.5, The resin was removed by filtration and
the filtrate concentrated to obtain compound 21 as white solid (0.4 g, 85%); Rf = 0.5
EtOAc/MeOH (50:50); 1H NMR (400 MHz, CDCl3) δ 7.38 – 7.28 (m, 5H), 5.50 (d, J = 89.1
Hz, 1H), 5.13 – 5.03 (m, 2H), 4.18 – 3.92 (m, 3H), 3.75 (t, J = 23.4 Hz, 2H), 3.46 (ddd, J =
34.8, 10.5, 7.1 Hz, 2H), 3.09 (dd, J = 28.3, 9.8 Hz, 1H), 2.17 – 2.02 (m, 3H), 1.83 – 1.60 (m,
2H), 1.38 (dd, J = 17.1, 6.5 Hz, 9H). 13C NMR (101 MHz, CDCl3) δ 173.0, 171.3, 157.0, 136.3,
128.3, 77.2, 66.7, 50.3, 47.7, 37.5, 29.7, 28.2, 21.4,20.8 ppm. HRMS (ESI-TOF) m/z calcd for
C21H31N3O7 [M + K]+. 476.1799, found 476.0979.
N(Acetyl)-N-ethylamino(tertbutoxycarbonyl)-2(S)-[(Fmoc)aminoethyl] glycine (3)
To compound 21 (1 g, 2 mmol) dissolved in EtOH, 10% Pd/C on charcoal was added
under H2 atmosphere and stirred at room temperature for 6 h, when the reaction got completed
as monitored by TLC. The charcoal was filtered on celite-545 pad and filtrate was evaporated
to get solid product (0.8 g) which was dissolved in 10 mL THF:H2O (1:1). It was stirred at 0
ºC with Na2CO3 (0.5 g, 5 mmol) followed by dropwise addition of Fmoc-Cl (0.6 g, 2 mmol).
The stirring was continued at 0 ºC for 1 h and then at 25 ºC for 3 h to complete the reaction.
Evaporation of solvent gave a residue that was dissolved in water (10 mL) and washed with
diethyl ether (20 mL). It was then neutralised with 10% aq. HCl and extracted with EtOAc (25
mL×3). The organic layer upon concentration gave a residue that was purified by column
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chromatography to yield compound 3 (1.0 g, 70 %). H NMR (400 MHz, CDCl3) δ 7.74 (d, J
= 7.5 Hz, 2H), 7.64 – 7.50 (m, 2H), 7.38 (t, J = 7.4 Hz, 2H), 4.81 – 4.28 (m, 7H), 4.24 – 3.89
(m, 1H), 3.84 – 3.63 (m, 2H), 3.55 – 3.28 (m, 5H), 3.27 – 2.87 (m, 6H), 2.19 – 1.98 (m, 3H),
1.82 – 1.47 (m, 2H), 1.36 (t, J = 32.1 Hz, 9H). 13C{1H} NMR (101 MHz, CDCl3) δ 175.4,
141.1, 127.5, 126.9, 119.7, 77.2, 47.1, 28.1, 20.4 ppm. HRMS (ESI-TOF) m/z calcd. for
C28H35N3O7 [M +K]+ 564.2112, found 564.0740.
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ethyl glycinate (22)
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A stirred solution of 15 (1 g, 2.5 mmol) in dry DCM (10 mL) was treated with DCC
(0.7 g, 4 mmol) and DhbtOH (0.6 g, 4 mmol). The resulting mixture was stirred at room
temperature for 10 min after which glacial acetic acid (0.2 mL, 4 mmol) was added and the
stirring continued for overnight. The organic solvent was removed under reduced pressure, to
yield a residue that was dissolved in EtOAc (300 mL), followed by aqueous work up and
concentration of organic extract gave a residue which was purified by column chromatography
to afford 22 (1.5 gm, 85%) as a sticky oil. 1H NMR (400 MHz, CDCl3) δ 7.37 – 7.27 (m, 5H),
5.63 (s, 1H), 5.13 – 5.03 (m, 2H), 4.27 – 4.10 (m, 3H), 4.08 – 3.96 (m, 2H), 3.88 (d, J = 17.1
Hz, 3H), 3.82 – 3.62 (m, H), 3.51 – 3.35 (m, 2H), 3.17 – 2.83 (m, 2H), 2.07 (d, J = 41.0 Hz,
3H), 1.91 – 1.55 (m, 3H), 1.40 (d, J = 4.9 Hz, 9H), 1.26 (dt, J = 11.2, 7.1 Hz, 3H). 13C{1H}
NMR (101 MHz, CDCl3) δ 172.6, 171.5, 170.1, 169.2, 156.8, 136.8, 128.6, 128.1, 80.1, 79.6,
77.2, 66.6, 61.9, 61.5, 51.0, 50.5, 47.4, 37.9, 37.6, 33.5, 32.6, 28.5, 21.5, 14.2 ppm. HRMS
(ESI-TOF) m/z calcd. for C23H35N3O7 [M +H]+ 466.2553, found 466.3684.
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N(Acetyl)-N-ethylamino(tertbutoxycarbonyl)-2(R)-[(benzyloxycarbonyl) aminoethyl]
glycine (23)
To a stirred solution of compound 22 (0.70 g, 1.5 mmol) in THF (15 mL), aq. NaOH
(2 M, 15 mL) was added at 0 °C and kept stirred for 1 hr at 0 °C. Water (50 mL) was added,
and the resulting mixture was extracted with EtOAc (2 × 35 mL). The aqueous layer containing
the product was neutralized with saturated aq. KHSO4 at 0 °C to pH 3−4 and extracted into
EtOAc (4 x 35 mL). The combined organic extracts after drying and concentration gave crude
product that was purified by column chromatography to afford 23 as a white solid (0.63 g,
90%). 1H NMR (400 MHz, CDCl3) δ 7.31 (dt, J = 12.0, 4.1 Hz, 5H), 5.09 (t, J = 9.6 Hz, 2H),
4.21 – 3.86 (m, 2H), 3.74 (t, J = 18.5 Hz, 5H), 3.44 (d, J = 32.9 Hz, 1H), 3.17 – 3.00 (m, 8H),
2.15 – 2.02 (m, 3H), 1.84 – 1.54 (m, 2H), 1.48 – 1.34 (m, 9H). 13C{1H} NMR (101 MHz,
CDCl3) δ 173.0, 171.3, 157.0, 136.3, 128.3, 77.2, 66.7, 50.3, 47.7, 37.5, 29.8, 28.2, 21.4, 20.8
ppm. HRMS (ESI-TOF) m/z calcd. for C20H29N3O7 [M +Na]+ 460.2060, found 460.2320.
N(Acetyl)-N-ethylamino(tertbutoxycarbonyl)-2(R)-[(Fmoc)aminoethyl] glycine (4)
To the compound 23 (1 g, 2 mmol) dissolved in ethanol, 10% Pd/C on charcoal was
added under H2 atmosphere and stirred at room temperature for 6 h. after which it was filtered
on celite-545 pad. The filtrate was evaporated to get crude amine product (crude weight 0.8 g)
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which was dissolved in 10 mL THF:H2O (1:1), stirred at 0 ºC with Na2CO3 (0.5 g, 5 mmol).
followed by dropwise addition of Fmoc-Cl (0.6 g, 2 mmol). The reaction mixture was kept
stirred at 0 ºC for 1 h and then at 25 ºC for 3 h. after which solvent was evaporated and the
residue was dissolved in of water (10 mL) and washed with diethyl ether (20 ml). After
neutralization with with 10% aq. HCl, product was extracted with ethyl acetate (25 mL × 3),
the dried organic layer was concentrated to obtain a residue obtained that was purified by
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column chromatography to yield compound 4 (1.0 g, 66 %). H NMR (400 MHz, CDCl3) δ
7.74 (d, J = 7.5 Hz, 2H), 7.64 – 7.50 (m, 2H), 7.38 (t, J = 7.4 Hz, 2H), 4.81 – 4.28 (m, 7H),
4.24 – 3.89 (m, 1H), 3.84 – 3.63 (m, 2H), 3.55 – 3.28 (m, 5H), 3.27 – 2.87 (m, 6H), 2.19 – 1.98
(m, 3H), 1.82 – 1.47 (m, 2H), 1.36 (t, J = 32.1 Hz, 9H). 13C{1H} NMR (101 MHz, CDCl3) δ
175.4, 141.1, 127.5, 126.9, 119.7, 77.2, 47.1, 28.1, 20.4 ppm. HRMS (ESI-TOF) m/z calcd. for
C28H35N3O7 [M +K]+ 564.2112, found 564.2699.
Solid-Phase Synthesis
The PNA oligomers were synthesized on MBHA (4-methyl–benzhydryl amine) resin
using combination of Boc and Fmoc protocols.27 The solid-phase synthesis was carried out in
a reactor with a sintered glass bottom, using MBHA resin with loading value of 0.20 mmol/g.
The deprotected NH2 group on resin was treated with N,N’-protected lysine acid, activated
with the coupling reagent mixture of HOBt (6 mg, 3 eq), HBTt (6 mg, 3 eq), DIPEA (10 µL ) in
DMF. The terminal N-t-Boc group of lysine on resin was first deprotected with 50% TFA in
DCM (3 × 15 min) followed by washing with DCM and DMF (3 × 10 mL) to give the TFA
salt of amine. This was neutralized using 10% N,N-diisopropylethylamine (DIPEA) in DCM
(3 × 10 min) to liberate free amine on resin. The resin was then coupled with activated
carboxylic acid group of C-aeg-PNA monomer using same coupling reagents. The deprotection
of NHBoc of aeg-C monomer on the resin with TFE was followed by reaction with the
incoming Cg-bimodal monomer 1 using (i) HOBt (3 eq), HBTU ( 3 eq), DIPEA (3eq ) in DMF
(Scheme 3) The deprotection of sidechain NHFmoc group after coupling was done using 20%
piperidine in DMF followed by side chain coupling with appropriate nucleobase acetic acid
(A/T/C; 3 eq) done using HOBt (3 eq), HBTU (3 eq), and DIPEA (3 eq) in DMF (500 μL).
After ecery coupling and deprotection step, the resin was successively washed with DCM and
DMF (3 x 1 mL) before the next reaction. These cycles were repeated as in Scheme 3 to reach
the desired PNA oligomer sequence.
Cleavage of the bm-PNA oligomers from solid support
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The MBHA resin (10 mg) after complete assembly of Cg-bm-PNA oligomers was
stirred with thioanisole (20 μL) and 1,2-ethanedithiol (8 μL) in an ice bath for 10 min. Cold
TFA (200 μL) and TFMSA (16 μL) were then added and the reaction mixture with resin was
kept stirred for further 1.5 to 2 h at room temperature. The resin was filtered out and washed
twice with TFA and the filtrate was concentrated in a microfuge tube and treated with cold
diethyl ether to precipitate the PNA prodcut. Upon centrifugation, the sediment was dissolved
in 40% MeNH2 solution for deprotection of the isobutyryl group on 2-NH2 of G nucleobase at
rt for 8 h. It was then concentrated on Speedvac for purification by HPLC.
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The purification of PNAs was carried out on Agilent HPLC system with semi-
preparative BEH130 C18 (10×250 mm) Phenomenex column using solvents water and
acetonitrile with composition A: 0.1% TFA in CH3CN:H2O (5:95) and B= 0.1% TFA in
CH3CN:H2O (1:1). The gradient for elution was 100% A to 100% B in 20 min, with flow rate
of 2 mL/min. The HPLC elutions were monitored at 220 and 254 nm wavelength.
Temperature-UV absorbance measurements
The temperature dependent UV-absorbance experiments were carried out on Varian
Cary 300 UV spectrophotometer equipped with a Peltier heating system. Sodium cacodylate
buffer (10 mM, pH 7.2) containing NaCl (10 mM) was used for preparing the samples. The
respective PNA and DNA complementary sequences in stoichiometric ratio (1:1, duplex) were
mixed to achieve a final concentration of 2 μM for each strand. The samples were heated to 90
°C for and maintained at 10 min. followed by slow cooling over 8-10 h to room temperature.
The samples were refrigerated for 24 h and for the experiments 500 μL of sample was
transferred to quartz cell and equilibrated at the starting temperature for 5 min. The absorbance
at 260 nm was recorded from 20-90 °C with temperature gradient of 0.5 oC/min. The melting
experiments were repeated thrice to get consistent values. The normalized absorbance at 260
nm was plotted as a function of the temperature. The data were fitted by sigmoidal curve,
function - Boltzmann for one face and biphasic dose response for two face binding, where R
square value in range of 0.96 to 0.99. The Tm values were obtained from the peak in first
derivative plot of normalized absorbance with respect to temperature and are accurate to ± 1.0
°C. The data were processed using OriginPro 8.5. The concentration of all oligonucleotides
were calculated on the basis of UV absorbance at 260 nm from the molar extinction coefficients
of the corresponding nucleobases: T = 8.8 cm2/μmol; C = 6.6 cm2/μmol; G = 11.7 cm2/μmol
and A = 13.7 cm2/μmol as per literature.28
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Circular dichroism spectra
CD spectra were recorded at a temperature 10 oC on a JASCO J-815 spectropolarimeter,
with accumulation of 3 scans from 300 to 200 nm using 2 mm cell, a resolution of 0.1 nm,
band-width of 1 nm, sensitivity of 2 mdeg, response of 2 sec and a scan speed of 50 nm/min.
The calculated amounts of bm-PNA oligomers and the complementary DNA were mixed
together in stoichiometric ratio (1:1 for duplex) in sodium cacodylate buffer (10 mM)
containing and NaCl (10 mM), pH 7.2 to achieve a final strand concentration of 10 μM for
each strand. The samples were annealed as in UV-T experiments.
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ASSOCIATED CONTENT
Supporting Information: The Supporting Information is available free of charge. Schemes
for synthesis of known compounds, 1H and 13C NMR spectra of all new synthetic compounds
and key intermediates, HRMS of new compounds, HPLC and MALDI-TOF spectral data of
all PNA oligomers
AUTHOR INFORMATION
Corresponding Author
Krishna N Ganesh, Indian Institute of Science Education and Research, Karkambadi Road,
Mangalam, Tirupati 517507, India. orcid.org/0000-0003-2292-643X, Email:
kn.ganesh@iisertirupati.ac.in
Authors contributions
P. B., Chemical synthesis and Biophysical studies, Indian Institute of Science Education and
Research (IISER) Pune, Dr Homi Bhabha Road, Pune 411008.
pramod.bhingardeve@students.iiserpune.ac.in
B.M. Contributed to interpretation of UV-melting results, Department of Chemistry, Indian
Institute of Science Education and Research (IISER) Tirupati, Tirupati 517507, India;
orcid.org/0000-0003-1043-8754 250
Acknowledgements
PB thanks IISER Pune for a research fellowship, BM is a recipient of Post-doctoral
fellowshiptp from IISER Tirupati and KNG acknowledges DST, New Delhi, Govt of India
for a research grant (EMR/2016/007601).
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