Receptor-Specific Delivery of Peptide Nucleic Acids Conjugated to Three Sequentially Linked N-Acetyl Galactosamine Moieties into Hepatocytes

Article abstract of DOI:10.1021/acs.joc.0c00601

Peptide nucleic acids (PNAs) are DNA analogs that bind with high affinity to DNA and RNA in a sequence-specific manner but have poor cell permeability, limiting use as therapeutic agents. The work described here is motivated by recent reports of efficient gene silencing specifically in hepatocytes by small interfering RNAs conjugated to triantennary N-acetyl galactosamine (GalNAc), the ligand recognized by the asialoglycoprotein receptor (ASGPR). PNAs conjugated to either triantennary GalNAc at the N-terminus (the branched architecture) or monomeric GalNAc moieties anchored at Cγ of three consecutive PNA monomers of N-(2-aminoethyl)glycine (aeg) scaffolds (the sequential architecture) were synthesized on the solid phase. These formed duplexes with complementary DNA and RNA as shown by UV and circular dichroism spectroscopy. The fluorescently labeled analogs of GalNAc-conjugated PNAs were internalized by HepG2 cells that express the ASGPR but were not taken up by HEK-293 cells that lack this receptor. The sequential conjugate was internalized about 13-fold more efficiently than the branched conjugate into HepG2 cells, as demonstrated by confocal microscopy. The results presented here highlight the potential significance of the architecture of GalNAc conjugation for efficient uptake by target liver cells and indicate that GalNAc-conjugated PNAs have possible therapeutic applications.

Full text of DOI:10.1021/acs.joc.0c00601

Subscriber access provided by Uppsala universitetsbibliotek
Article
Receptor-Specific Delivery of Peptide Nucleic Acids Conjugated to Three
Sequentially Linked N-Acetyl Galactosamine Moieties into Hepatocytes
Krishna N. Ganesh, Pramod Bhingardeve, Bharath Raj Madhanagopal,
prashant jain, Hemanth Naick, and Muthiah Manoharan
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.0c00601 • Publication Date (Web): 12 Jun 2020
Downloaded from pubs.acs.org on June 13, 2020
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the
course of their duties.

Page 1 of 33
The Journal of Organic Chemistry
1
2
3
4
Receptor-Specific Delivery of Peptide Nucleic Acids Conjugated to Three
5
6
Sequentially Linked N-Acetyl Galactosamine Moieties into Hepatocytes
Pramod Bhingardeve,1 Bharath Raj Madhanagopal,2 Hemanth Naick,2 Prashant Jain,1
Muthiah Manoharan3 and Krishna Ganesh1,2,*
7
8
9
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
58
59
60
1. Indian Institute of Science Education and Research (IISER) Pune, Dr Homi Bhabha
Road, Pune 411008, Maharashtra, India
2. Indian Institute of Science Education and Research (IISER) Tirupati, Karkambadi
Road, Tirupati 517507, Andhra Pradesh, India
3. Alnylam Pharmaceuticals, Cambridge, Massachusetts 02142 USA.
*To whom correspondence should be addressed. kn.ganesh@iisertirupati.ac.in
1 PB. pramod.bhingardeve@iiserpune.ac.in; PJ. prashant.jain@students.iiserpune.ac.in
2 BM. madhang@iisertirupati.ac.in, HN. b.hemanth@iisertirupati.ac.in
3 MM. mmanoharan@alnylam.com
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 2 of 33
1
2
3
4
Table of contents
5
6
7
PNAs conjugated with N-acetyl galactosamine (GalNAc) in sequential vs. branched
architecture
8
9
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
58
59
60
ACS Paragon Plus Environment

Page 3 of 33
The Journal of Organic Chemistry
1
2
3
4
Keywords: N-acetyl galactosamine, GalNAc conjugate, peptide nucleic acid, hepatocyte,
5
6
cellular uptake
7
8
9
Abstract
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
58
59
60
Peptide nucleic acids (PNAs) are DNA analogs that bind with high affinity to DNA and
RNA in a sequence-specific manner but have poor cell permeability limiting use as therapeutic
agents. The work described here is motivated by recent reports of efficient gene silencing
specifically in hepatocytes by small interfering RNAs (siRNAs) conjugated to triantennary N-
acetylgalactosamine (GalNAc), the ligand recognized by the asialoglycoprotein receptor
(ASGPR). PNAs conjugated to either triantennary GalNAc at the N-terminus (the branched
architecture) or to monomeric GalNAc moieties anchored at C of three consecutive PNA
monomers of N-(2-aminoethyl)glycine (aeg) scaffolds (the sequential architecture) were
synthesised on solid phase. These formed duplexes with complementary DNA and RNA as
shown by UV and circular dichroism (CD) spectroscopy. The fluorescently labeled analogs of
GalNAc-conjugated PNAs were internalized by HepG2 cells that express the ASGPR but were
not taken up by HEK293 cells that lack this receptor. The sequential conjugatewas internalized
about 13-fold more efficiently than the branched conjugate into HepG2 cells as demonstrated
by confocal microscopy. The results present here highlight the potential significance of the
architecture of GalNAc conjugation for efficient uptake by target liver cells and indicate that
GalNAc-conjugated PNAs have possible therapeutic applications.
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 4 of 33
1
2
3
4
Introduction
5
6
7
8
Antisense oligonucleotide therapeutics originate from the specific molecular recognition
between the mRNA of the gene to be inhibited and synthetic single-stranded oligonucleotide
drugs.1-3 RNA interference (RNAi) is an endogenous pathway for post-transcriptional silencing
of gene expression triggered by double-stranded RNAs including endogenous microRNAs and
synthetic short interfering RNAs (siRNAs).4 As antisense oligonucleotide and siRNAs are too
large and hydrophilic to diffuse across cell membranes on their own, uptake into target cells
must be mediated by chemical modifications to the nucleic acid-based drug or adjuvants.5 The
conjugation of triantennary N-acetyl galactosamine (GalNAc3), which is the ligand for the
asialoglycoprotein receptor (ASGPR), to siRNA duplexes or single-stranded antisense
oligonucleotides enhances the potency of inhibition of gene expression in hepatocytes by 10 to
60 fold.6,7 Upon subcutaneous administration, a GalNAc3-siRNA conjugate (Figure 1) robustly
suppresses gene expression of the targeted mRNA in the liver of mice.6 It was also noted that
conjugation of optimized chemically modified siRNAs to GalNAc3 resulted in improved
systemic stability against nucleases and favorable pharmacokinetics relative to the
unconjugated siRNAs. The improved potency is a result of targeted delivery of the GalNAc3-
conjugated siRNA into hepatocytes via the ASGPR.8 This receptor is abundantly expressed
almost exclusively on hepatocytes and clears glycoproteins and desialylated platelets from
9
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
58
59
60
circulation.9,10 The first ever-approved oligonucleotide-GalNAc3 conjugate is an RNAi therapeutic,
Givosiran (GIVLAARI®), in which the sense strand of Givosiran siRNA is conjugated with GalNAc3
for the treatment of acute hepatic porphyrias.11 Numerous promising GalNAc-oligonucleotide
conjugates are in clinical development.
O
P
O
O
O
OH
OH
N
O
HO
HO
H
N
H
O
O
N
AcHN
O
O
O
OH
O
O
HO
HO
H
H
O
N
NH
N
O
O
AcHN
O
O
O
O
OH
O
HO
N
N
H
O
HO
H
AcHN
Figure 1. Structure of the GalNAc3-siRNA conjugate
ACS Paragon Plus Environment

Page 5 of 33
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
Peptide nucleic acids (ex. Figure 2, PNA 1) are DNA analogs that bind sequence
specifically and with high affinity to both DNA and RNA.12,13 In PNAs, the nucleobases are
linked via tertiary amide to an N2-aminoethylglycyl (aeg) backbone. Several PNA chemical
modifications have been designed to improve their selectivity for DNA or RNA and to enhance
their uptake into cells.14-19 Biessen et al. have reported the delivery of PNA conjugated to two
GalNAc moieties into hepatocytes in a mouse model and have demonstrated reduction of
expression of microsomal triglyceride transfer protein encoded by the targeted MTP
mRNA.20,21 Motivated by the potency of GalNAc3-conjugated siRNAs and antisense
oligonucleotides in hepatocytes and subsequent approval of an RNAi therapeutic, we have
synthesized two classes of PNA conjugates for evaluating their efficiency of their cell uptake.
These are PNAs conjugated to triantennary GalNAc3 through a spacer chain (“branched”
conjugates, Figure 2, PNA 2) and a PNA in which GalNAc monomers are anchored to C on
the aeg backbone at three sequential PNA units at N-terminus, (“sequential” conjugates Figure
2, PNA 3) The sequential GalNAc PNA conjugates were designed to examine to what extent
the linear substitution can mimic the geometry of branched GalNAc3 in binding to the ASGPR
on hepatocytes. In addition to synthesis of branched and sequential GalNAc-PNA conjugates
and their fluorescent derivatives, this manuscript reports on efficiencies of their hybridization
with DNA and RNA and relative uptake by cells that do and do not express the ASGPR. The
sequential PNA analogue was more efficiently taken into cells than was the branched PNAs
(Figure 2).
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
58
59
60
Figure 2. Structures of control, unconjugated PNA 1, branched GalNAc3 conjugate PNA 2, and
sequential GalNAc conjugate PNA 3
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 6 of 33
1
2
3
4
Results and Discussion
5
6
7
8
The synthesis of triantennary GalNAc3 (1c) fragment (Figure 3) required for the
synthesis of PNA 2 was done starting from the commercial D(+)-galactosamine to first obtain
1a as per the literature procedure6,7,22 and then coupling with N,N’-protected lysine benzyl ester
followed by debenzylation of ester to acid. The synthesis of [C(S)-GalNAc-T]3 PNA monomer
(2) required for the synthesis of linear GalNAc conjugated PNA 3 was done starting from (-
NHBoc)-(-NHFmoc)-L-Lys-OH (1) (Scheme 1).
9
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
58
59
60
Figure 3. Chemical structures of target GalNac monomers 1c and 2 and synthesis of 1c
The activation of acid function 3 by N,N-methoxymethyl amine gave the activated ester
4, which was oxidized in the presence of lithium aluminum hydride in THF to obtain the
aldehyde 5. This was coupled with glycine benzyl ester under reductive amination conditions
to obtain NHFmoc-[C(S)--NHBoc]-aminoethyl benzyl glycinate 7. The coupling of thymine
acetic acid 8 with amine 7 in the presence HBTU, HOBt and DIEA yielded C(NHFmoc) (CS-
butyl-4-NHBoc)-2-aminoethyl (N-acetamido-thyminyl) glycyl benzyl ester 9. The
deprotection of NHBoc function gave the free amine 10, which was coupled with 5-[3,4,6-tri-
O-acetyl-2-(acetylamino)-2-deoxy-β-D-galactopyranosyl]oxy]-pentanoic acid7 (11) to yield
the C(S)-GalNAc substituted PNA-T ester 12. Finally, catalytic hydrogenation of 12 over 10%
Pd-C in the presence of acetic acid-methanol afforded the target CS-butyl-4-amido (GalNAc)
aeg-PNA -T monomer 2. All intermediate compounds were characterized by 1H, 13C NMR and
mass spectral data.
Synthesis of GalNAc-PNA oligomers. The synthesis of GalNAc conjugated PNA oligomers
were done using standard solid phase synthesis protocol using Fmoc strategy (SI) on Rink
amide resin. The assembly of PNA oligomer PNA 1 was done from C-terminus to the N-
ACS Paragon Plus Environment

Page 7 of 33
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
terminus using appropriate standard protected PNA (A/G/C/T) monomers.23 The amino group
on the resin was first coupled with orthogonally protected (CNHFmoc/CS-Boc)-L-
lysine-OH24-27 at the C-terminus with in situ activation of acid with HBTU/HOBt, followed by
removal of Fmoc by 20% piperidine in DMF treatment. The chain assembly was continued by
sequential coupling with appropriate PNA monomers and deprotection using piperidine before
each coupling step. At the end of PNA 1 oligomer synthesis, additional coupling with the
triantennary GalNAc3 acid 1 using Pybop, HOBt and DIEA gave resin bound 15-mer GalNAc3-
PNA oligomer PNA 2 (Scheme 2).
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
58
59
60
Scheme 1. Synthesis of Cγ(S)-butyl-4-amido(GalNAc)-aeg-PNA-T monomer (2)
O
NHBoc
NHBoc
NHBoc
NH
NHBoc
OBn
H2N
EDC-Cl, HOBt,
DIEA
LAH, THF
-150C
N
O
O
6
O
8
O
H
OMe
HN
AcOH, NaCNBH4
37%
H
N
N
OH
OH
FmocHN
FmocHN
OMe
95%
FmocHN
OBn
FmocHN
O
80%
O
O
HBTU, HOBt, DIEA
47%
3
7
5
4
O
O
O
N
NH
OAc
NH
O
AcO
AcO
O
O
NHAc
H2N
NH
N
O
O
N
O
NH
BocHN
OH
H2, Pd/C
20 0C, 50%
O
O
b
a
50% TFA, DCM
11
O
N
O
FmocHNgS
OH
FmocHN
OBn
O
N
N
O
O
O
O
HBTU, HOBt, DIEA
O
N
N
FmocHN
OBn
63%
AcO
OAc
FmocHN
OBn
OAc
AcO
AcO
O
O
O
NH
O
NH
AcO
NHAc
NHAc
O
O
12
10
2
9
In a separate synthesis, after the completion of 12-mer PNA, three successive coupling
cycles (coupling-deprotection) with GalNAc-PNA monomer 2 units, gave resin bound 15-mer
[C(S)-GalNAc-T]3-PNA 3 in which one GalNAc fragment is attached to each of the 3 terminal
PNA-T units at C through a butylamido spacer chain (Scheme 3). The corresponding
fluorescent PNA oligomers Cf-PNA 4, Cf-GalNAc3-PNA 5 and Cf-[C(S)-GalNAc-T]3-PNA
6, required for cell permeation studies were synthesized after the final coupling of PNA
oligomers at N-terminus with GalNAc derivative and deprotection of terminal NHFmoc on
resin by DIEA and coupling with 5/6-carboxy fluorescein using Pybop and HOBt. The
unmodified PNA 1 without any N-terminal GalNAc unit, and its fluorescent derivative PNA 4
were synthesized in a similar way as control PNAs for biochemical and biophysical studies.
Scheme 2. Solid phase synthesis of (GalNAc)3-PNA 2
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 8 of 33
1
2
3
4
5
6
7
8
9
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
58
59
60
ACS Paragon Plus Environment

Page 9 of 33
The Journal of Organic Chemistry
1
2
3
4
Scheme 3. Solid phase synthesis of [C(S)-GalNAc-T]3-PNA 3
NH2
5
6
7
8
9
O
O
H
N
FmocHN
R
e
s
in
NH
N
H
C - A -T - A - A - T - G - C -T - G - G - C
O
T
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
58
59
60
O
1) 20% Piperidine
in DMF
b
a
OH
FmocHN
H2N
N
g
Step 1
O
2) Coupling in
Pybop,HOBt,DIEA
OAc
O
AcO
AcO
NH
O
NH2
NHAc
O
T
O
O
H
N
O
H
g
FmocHN
N
N
R
e
s
i
n
NH
N
H
C - A -T - A - A - T - G - C -T - G - G - C
o
O
OAc
O
NH
O
H2N
O
AcO
AcO
Repeat step 1
NHAc
NH2
T
T
O
O
g
H
g
O
O
H
NH
N
FmocHN
N
N
N
R
e
s
in
C - A -T - A - A - T - G - C -T - G - G - C
Repeat step 1
NH
N
H
O
O
O
O
O
HN
HN
H2N
O
O
O
O
AcO
AcO
NHAc
AcO
AcO
NHAc
O
AcO
NH2
AcO
T
T
T
O
O
H
H
g
g
O
g
O
H
NH
FmocH
N
N
N
N
N
N
R
e
s
i
n
NH
C - A -T - A - A - T - G - C -T - G - G - C
N
H
N
O
O
O
O
HN
HN
HN
H2N
O
Repeat step 1
O
O
O
O
O
O
O
AcO
AcO
O
AcO
AcO
NHAc
NHAc
NHAc
AcO
AcO
AcO
AcO
AcO
1.Cleavage from resin
90%TFA:DCM / TIPS
2.Deprotection of OAc
30% aq.NH3
NH2
T
T
g
T
O
O
H
O
H
N
O
O
H
H
g
N
g
NH2
C - A -T - A - A - T - G - C -T - G - G - C
N
H
H2N
N
N
N
N
N
O
O
O
O
H2N
HN
HN
HN
O
O
O
g
[C (S)-GalNAc-T]3-PNA 3
O
O
O
O
O
HO
HO
O
HO
HO
HO
HO
NHAc
NHAc
NHAc
HO
HO
HO
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 10 of 33
1
2
3
4
5
6
7
8
9
After the assembly, all PNA oligomers were cleaved from the solid support (L-lysine
derivatized Rink amide resin), using TFA in DCM in the presence of TIPS to yield completely
deprotected PNA oligomers having L-lysine amides at their C-termini. The PNA oligomers in
solution were precipitated by addition of cold diethyl ether and purified by semi-preparative
C18 reverse phase high performance liquid chromatography (RP-HPLC). The purity of PNA
oligomers was re-checked by analytical C18 RP column and characterized by MALDI-TOF
mass spectral data (Table 1).
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
58
59
60
The fluorescent oligonucleotides Cf-PNA 4, Cf-GalNAc3-PNA 5 and Cf[C(S)-
GalNAc-T)3]-PNA 6 need for cell uptake studies were synthesised on solid support as shown
in Scheme 4 and characterised by mass spectrometry data (Table 1). The UV absorption
spectra of fluorescent PNAs showed absorbance maxima at 260 nm due to nucleobases and a
lower intensity broad absorbance with maxima at 458 nm and 484 nm which are indicative of
the presence of 5(6)-carboxyfluorescein in the PNA oligomers (Supporting Information, S33).
Scheme 4. Synthesis of fluorescent PNA oligomers
Table 1. MALDI-TOF spectral analysis of the synthesized PNA oligomers
Sr.
PNA sequence
Molecular
Formula
Calc
Mass
Obs
Mass
Rt
(min)
No. Code
174 228 86 50
C H N O
1
2
3
4
5
PNA 1
4321.77
5907.08
5448.57
4680.80
6392.58
4321.26
5907.44
5487.18[M +K]+ 11.5
4681.60[M +H]+ 14.9
14.2
10.9
245 351 95 80
C H N O
GalNAc3-PNA 2
[C(S)-GalNAc-T]3 PNA 3
Cf-PNA 4
225 317 91 72
C H N O
195 238 86 56
C H N O
272 374 98 86
C H N O
Cf-GalNAc3-PNA 5
6392.86
11.2
ACS Paragon Plus Environment

Page 11 of 33
The Journal of Organic Chemistry
1
2
3
4
Cf[C(S)-GalNAc-T]3 PNA 6
246 327 91 78
C H N O
6
5806.88
5806.19
12.4
5
6
7
Incorporation of three GalNAc moieties in PNA was intended to aid the selective
uptake of PNAs into hepatocytes that exhibit ASGPR on their surface. Since the utility of
GalNAc3 modified PNAs depends on whether or not they form stable PNA:RNA or PNA:DNA
hybrids under physiological conditions, the thermal stability of the hybrids formed by the
modified PNAs with cDNA (DNA 1) and cRNA (RNA 1) were determined using temperature
dependent UV spectroscopy.
8
9
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
58
59
60
Thermal stability of PNA:DNA and PNA:RNA duplexes
The control PNA 1 and triantennary GalNAc3-PNA 2 and [C(S)-GalNAc-T]3-PNA 3
were hybridized with DNA 1 and RNA 1 to generate corresponding antiparallel
PNA:DNA/RNA duplexes (Figure 4). The effect of conjugation of GalNAc on the thermal
stability of derived PNA:DNA hybrids was investigated by temperature dependent UV
absorbance experiments (Figure 4). All PNA:DNA and PNA:RNA hybrids showed well
defined sigmoidal transition suggesting successful formation of duplexes. The DNA duplex of
unmodified aeg-PNA (PNA 1:DNA 1) showed a melting (Tm) of 48.9 °C, while the Tm of
GalNAc conjugated PNA 2:DNA 1 duplexes were slightly higher: triantennary GalNAc3-PNA
o
o
2:DNA 1 duplex Tm of 52.5 C and [C(S)-GalNAc-T]3-PNA 3:DNA 1 duplex Tm ~51.3 C.
Thus, conjugation of GalNAc to PNA showed slight stabilization of corresponding DNA
duplexes by 3.6 °C and 2.4 °C, respectively (Table 2), with triantennary duplex being slightly
more stable than mono-GalNAc PNA duplexes.
In comparison, the Tm of duplex PNA 1:RNA 1 from unmodified aeg-PNA was 59.5
°C, the Tm of triantennary GalNAc3-PNA 2:RNA 1 duplex was 55.8 oC and the Tm of [C(S)-
GalNAc-T]3-PNA 3:RNA 1 duplex was 53.8 oC (Figure 4). Thus, GalNAc conjugated PNAs
showed a slight destabilization (Tm ~ 4-6 oC) of their duplexes with RNA over that of control
o
PNA:RNA duplex. The PNA:RNA duplexes were slightly more stable (Tm ~ +2-3 C) than
the corresponding PNA:DNA duplexes. The destabilization of PNA:RNA hybrids induced by
o
GalNAc3 modification was only marginal (Tm ~ -1 to -2 C/modification) and the melting
temperatures were still higher than the corresponding PNA:DNA hybrids.
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 12 of 33
1
2
3
4
5
6
7
8
9
PNA:DNA
(B)
PNA:RNA
(A)
1.2
1.0
0.8
0.6
0.4
0.2
0.0
1.2
1.0
0.8
0.6
0.4
0.2
0.0
GalNAc -PNA:DNA
GalNAc -PNA:RNA
3
3
[C(S)-GalNAc-T] PNA:DNA
[C(S)-GalNAc-T] PNA:RNA
3
3
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
58
59
60
20 30 40 50 60 70 80 90
20 30 40 50 60 70 80 90
o
o
Temperature ( C)
Temperature ( C)
(C)
Figure 4. Thermal melting curves for unmodified PNA 1, branched GalNAc3-conjugated PNA 2, and
sequential GalNAc-conjugated PNA 3 hybridized to (A) DNA or (B) RNA and (C) Structures of
PNA:DNA duplexes. Buffer: 10 mM sodium cacodylate, pH 7.2, NaCl 10 mM. Concentration of PNA
and DNA 3 M of each strand
Table 2. UV-Tm values of complementary PNA:DNA duplexes with triantennary-GalNAc3 PNA
andtrivalent [C(S)GalNAc-T]3-PNA units
DNA 1
RNA 1
Tm ∆Tm
Entry
PNA Sequence
Tm
∆Tm
PNA 1 H-TTTCATAATGCTGGC-Lys-Lys-NH2
PNA 2 GalNAc3-TTTCATAATGCTGGC-Lys-Lys-NH2
PNA 3 [C(S)GalNAc-T]3-CATAATGCTGGC-Lys-Lys-NH2
1
2
3
48.9
-
59.5
52.5 + 3.4 55.8 -3.7
51.3 + 2.4 53.8 -5.7
Buffer: 10 mM sodium cacodylate, pH =7.2, NaCl 10 mM. Tms are accurate to within  0.5 oC
CD studies of PNA:DNA and PNA:RNA duplexes
The effect of conjugation of triantennary GalNAc3 to N-terminus of PNA or monomeric
GalNAc substituted at C in the side chain of PNA backbone on the conformation of derived
PNA:DNA/RNA duplexes was studied by CD spectroscopy. The CD-spectra for ss PNA 1,
and GalNAc conjugated PNA 2 and PNA 3 did not show any significant CD bands (SI). The
chiral induction from GalNAc3 substituents on PNA stacking or conformation is negligible.
The CD spectra of single stranded DNA and RNA show low intensity positive bands in the
region of 260 to 280 nm.
ACS Paragon Plus Environment

Page 13 of 33
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
The CD-spectra of duplexes PNA 1:DNA 1, GalNAc3-PNA 2:DNA 1 and [C(S)-
GalNAc-T]3-PNA 3:DNA 1 showed almost similar characteristics with weak positive bands at
222 nm, strong positive band at 275 nm and a weak negative band at 250 nm (Figure 5A). In
case of corresponding RNA duplexes, positive band at 220 nm was relatively strong,
accompanied by strong positive band at 265 nm and weak negative band at 245 nm (Figure
5B). The almost identical CD profiles of the control and GalNAc conjugated PNA:DNA
duplexes and similar profile of control and GalNAc conjugated PNA:RNA duplexes suggested
that GalNAc modifications do not alter the PNA:DNA/RNA duplex conformation and retain
the same as unsubstituted PNA:DNA/RNA duplexes.
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
58
59
60
(B)
20
15
10
5
(A)
9
6
PNA:DNA
PNA:RNA
GalNAc -PNA:DNA
GalNAc PNA:RNA
3
3
[C(S)-GalNAc-T] PNA:DNA
[C(S)-GalNAc-T] PNA:RNA
3
3
3
0
0
-5
-3
-6
-9
-10
-15
-20
200 220 240 260 280 300
Wavelength (nm)
200 220 240 260 280 300
Wavelength (nm)
Figure 5. Circular dichroism spectra of (A) PNA:DNA duplexes and (B) PNA:RNA duplexes
formed by unmodified PNA 1, branched GalNAc-conjugated PNA 2, and sequential GalNAc-
conjugated PNA 3. Buffer: 10 mM sodium cacodylate, pH 7.2, NaCl 10 mM. Concentration of
PNA and DNA 5 M of each strand.
Cell uptake studies
The cell uptake studies were conducted with fluorescent PNA oligomers Cf-PNA 4, Cf-
GalNAc3-PNA 5 and Cf-[C(S)-GalNAc-T]3-PNA 6. The fluorescent PNAs upon excitation at
484 nm showed the emission spectra with maximum at 520 nm. The fluorescent intensities of
duplexes were slightly lower than that of single-stranded fluorescently labeled PNAs when
excited at either at 458 nm or 484 nm. The GalNAc conjugated PNAs (PNA 2 and PNA 3)
were designed for selective uptake by hepatocytes (HepG2 cells) that have ASGPR on the cell
surfaces, which bind to triantennary GalNAc3. HepG2 cells were treated with 5(6)-
carboxyfluorescein tagged Cf-GalNAc3-PNA 5 and Cf-[C(S)-GalNAc-T]3-PNA 6 and their
uptake was studied by confocal microscopy. In these experiments, 5(6)-carboxyfluorescein
tagged isosequential aeg-PNA was used as control. To confirm the selectivity of uptake, the
experiments were conducted with HEK-293 cells, which lack ASGPR, as negative control.8
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 14 of 33
1
2
3
4
5
6
7
8
9
Cell uptake studies with HepG2 cells. HepG2 cells were grown in MEM medium at 37°C
under 5% CO2 to reach a confluency of 50%. They were then treated with 5(6)-
carboxyfluorescein functionalized PNAs Cf-GalNAc3-PNA 5, Cf-[C(S)-GalNAc-T)3]-PNA 6
or unmodified control Cf-PNA 4 each at 4 M concentrations. The internalized PNAs were
visualized by imaging the cells with confocal laser scanning microscopy (Figure 6). To stain
the nucleus, 4' 6-diamidino-2-phenylindole (DAPI) was used. Figures 6A-C show the overlay
of the images from the DAPI and carboxyfluorescein channels. The fluorescence images were
merged with the corresponding differential interference contrast (DIC) images to confirm that
the peptides were within the cell boundaries (Figures 6D-F).
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
58
59
60
In case of cells treated with Cf-GalNAc3-PNA 5 or Cf-[C(S)-GalNAc-T)3]-PNA 6, the
presence of green fluorescence indicated that the PNAs were taken up by HepG2 cells. This is
evident from Figures 6B and 6E, which show the nuclei and the cell boundary along with green
puncta that appear within the cells. A comparison of the fluorescence intensity and the number
of cells that show PNA uptake indicated that the extent of internalization of Cf-[C(S)-GalNAc-
T)3]-PNA 6 with three monovalent GalNAc substitutions was higher than that of triantennary Cf-
GalNAc3-PNA 5. The localization of Cf-[C(S)-GalNAc-T)3]-PNA 6 within the cell was clearly
observed as punctate of green fluorescence in Figure 6D. The appearance of discrete punctate
in the vicinity of the nucleus and distributed throughout the cell suggested that the
internalization could be a result of endocytosis, as expected for ASGPR mediated cellular
uptake. In contrast to the GalNAc modified PNAs, the unmodified Cf-PNA 4 did not show any
green fluorescence (Figure 6E and 6F) in the cells, indicating no internalization. These results
showed that the cell penetrating ability of the GalNAc3 modified PNAs (PNA 5 and PNA 6)
was indeed conferred by the GalNAc3 functionalization.
ACS Paragon Plus Environment

Page 15 of 33
The Journal of Organic Chemistry
1
2
3
4
B
A
5
6
7
8
9
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
58
59
60
D
C
E
F
Figure 6. Confocal microscopy images of HepG2 cells treated with Cf-GalNAc3-PNA 5 (A-B), and Cf-
[C(S)-GalNAc-T]3-PNA 6 (C-D) and Cf-PNA 4 (E-F). A, C, and E are merged images of DAPI and
carboxyfluorescein channels. B, D, and F show merged images from the DAPI and carboxyfluorescein
channels along with differential interference contrast image showing the intact cells (gray), nuclei
(blue), and internalized PNAs (green). Scale bars represent 25 m.
Cellular uptake studies with HEK-293 cells. The HEK-293 cells in which ASGPR are not
expressed were used as negative controls to confirm that observed internalization of GalNAc3
modified PNAs was indeed mediated by specific interactions with the cell surface receptor
ASGPR. The cells were then treated with Cf-GalNAc3-PNA 5, Cf[C(S)-GalNAc-T]3-PNA 6
or unmodified Cf-PNA 4 at same concentrations (4 M) and experimental conditions as in the
experiment with HepG2 cells, followed by imaging under confocal laser scanning microscopy
(Figure 7). The panels, A, D, and G correspond to the DAPI signals indicating the nuclei of the
cells. The panels, B, E, and H show the images obtained from the detector capturing
carboxyfluorescein emission signals. The panels C, F and I show the merged images of the
DAPI and carboxyfluorescein channels along with the respective DIC image.
In case of Cf-GalNAc3-PNA 5 treated cells, no green fluorescence emission was
observed. This is evident from Figure 7C, which shows the nuclei and the cell boundary but no
PNA within the cells. Similar results were obtained with Cf[C(S)-GalNAc-T]3-PNA 6 and
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 16 of 33
1
2
3
4
unmodified Cf-PNA 4 as well. These results clearly indicate that PNAs, with or without GalNAc
5
6
modifications, are incapable of cell penetration into HEK 293 cells.
7
8
9
B
A
C
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
58
59
60
E
F
D
H
I
G
Figure 7. Confocal microscopy images of HEK-293 cells treated with Cf-GalNAc3-PNA 5 (A-C),
Cf[C(S)-GalNAc-T]3-PNA 6 (D-F) and Cf-PNA 4 (G-I). A, D, and G are images of DAPI. B, E, and
H show signals from carboxyfluorescein channel. C, F, and I show the merged image of DAPI channels
and carboxyfluorescein channels along with differential interference contrast image showing the intact
cells (grey), nuclei (blue), and internalized PNAs (green). Scale bars represent 25 m.
Flow cytometry studies. For quantitative comparison of the uptake of the GalNAc modified
PNAs by HepG2 cells flow cytometry studies were carried out. HepG2 cells were cultured as
mentioned above. The cells were treated with 5(6)-carboxyfluorescein functionalized PNAs
Cf-GalNAc3-PNA 5, Cf[C(S)-GalNAc-T)3]-PNA 6 or unmodified control Cf-PNA 4 each at 4
M concentrations for 12 hours. The results show cells treated with GalNAc modified PNAs
showed significant uptake as compared to untreated cells and cells incubated with unmodified
control Cf-PNA 4 (Figure 8). Similar to the results of the confocal microscopy studies, the cells
were found to internalize Cf[C(S)-GalNAc-T)3]-PNA 6 more than Cf-GalNAc3-PNA 5. While
ACS Paragon Plus Environment

Page 17 of 33
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
the measured uptake was only 3.1% for triantennary Cf-GalNAc3-PNA 5, it was 39.5% for the
Cf[C(S)-GalNAc-T)3]-PNA 6. Thus, conjugation of three GalNAc moieties to the last three
PNA monomeric residues at the N terminus was found to effect a 12.7 fold increase in the
uptake of the PNA by HepG2 cells compared to the triantennary GalNAc modification.
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
58
59
60
Figure 8. Uptake of unmodified control Cf-PNA 4, Cf-GalNAc3-PNA 5, and Cf[C(S)-
GalNAc-T)3]-PNA 6 by HepG2 cells as measured by flow cytometry. The results shown are
average of two independent measurements. The error bars represent standard deviations. The
percentage refers to the fraction of total number of cells that are fluorescent among the total
number of cells analysed by FACS in each case.
Cytotoxicity studies. For ultimate therapeutic application of the GalNAc modified PNAs, it is
essential to confirm whether the modified PNAs elicit any adverse effects in the target cells.
We assessed the cytotoxicity of the GalNAc modified PNAs GalNAc3-PNA 2 and [C(S)-
GalNAc-T)3]-PNA 3 towards HepG2 cells and compared them with the unmodified control
PNA 1 using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
(Figure 9). The cells were incubated with PNAs at the concentration of 4 μM for 12 hours, as
in the cellular uptake studies, prior to treatment with MTT. The results show that the viability
of the cells were not significantly altered by the treatment of the cells with the PNAs. The
viability of the cells were marginally reduced when they were treated with GalNAc3-PNA 2,
while treatment with [C(S)-GalNAc-T)3]-PNA 3 did not lead to any reduction in the viability
compared to the control PNA. On the contrary, treatment of the cells with [C(S)-GalNAc-T)3]-
PNA 3 slightly enhanced the cell viability compared to the control and untreated PNAs;
however, this effect was not significant.
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 18 of 33
1
2
3
4
5
6
7
8
9
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
58
59
60
Figure 9. Cell viability measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay for HepG2 cells after treatment with 4 μM of PNA 1, GalNAc3-PNA 2
or [C(S)-GalNAc-T)3]-PNA 3 for 12 hours. The data shown are average of three
measurements. The error bars represent standard deviations.
Discussion
From detailed structure-activity relationship studies on oligonucleotide-GalNAc
conjugates28 it was found that the branched triantennary (GalNAc)3 moiety linked to siRNA
through spacer chain as depicted in Figure 1 was the best in terms of its binding to the ASGPR.
This is perhaps due to the fact that this cluster displays the appropriate inter-GalNAc distance
and cluster geometry to match the receptor binding site.29-33 The siRNA conjugates in which
the GalNAc moieties are linearly displayed with conjugation on successive bases, ribosugars,
acyclic and cyclic linkers or to phosphate groups on backbone in a sequence rather than in
cluster have also been examined.33,34 It was found that conjugates having sequential GalNAc
moiety linked to ribosugars or bases were as good in potency as the branched triantennary
GalNAc3 linked siRNAs.35 In the only report so far on PNA-GalNAc conjugate, wherein the
two GalNAc units are linked via lysine at N-terminus to PNA, it was found that the bioactivity
and distribution of the PNA conjugate was better than the unconjugated PNA.20,21 In this
context, the present comparative results show that the linear PNA conjugate with three
monovalent GalNAc ligands linked to successive C site of aeg PNA backbone (PNA 3) shows
40 times times more internalization in HepG2 cells than the unconjugated PNA and 13 times
more uptake than the N-terminus branched GalNAc3 conjugate (PNA 2). These PNAs are not
internalized in HEK293 cells that lack the ASGP receptor and hence the GalNAc induced
enhancements in cell uptake of linear conjugates are significant and interesting. It may be
ACS Paragon Plus Environment

Page 19 of 33
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
pointed out that the relative enhancement in internalization of linear vs branched GalNAc-PNA
conjugates is much higher than that between the corresponding GalNAc-siRNA conjugates.
This suggests that PNA conformation provides a favourable display geometry for the linearly
distributed GalNAc units on PNA backbone compared to the N-terminus cluster for recognition
of the ASGPR. The binding of linear PNA conjugate PNA 3 may also be assisted by some non-
specific interaction of hydrophobic PNA strand with the receptor facilitating a favourable
orientation for binding with the receptor, a feature that is perhaps not possible in PNA 2 where
the N-terminus branched (GalNac)3 cluster is far removed from PNA chain. Further
understanding requires a study evaluating the binding affinities of PNA conjugates with the
receptor.
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
58
59
60
Conclusions
Motivated by GalNAc3-conjugated oligonucleotide drugs that are efficiently taken up by
liver cells and by the approval of givosiran (GIVLAARI®), PNA was conjugated with GalNAc
in two different ways: (i) a triantennary branched structure linked at the N-terminus of the PNA
via a spacer chain and (ii) a sequential architecture in which three monovalent GalNAc units
were conjugated at the C of the backbone on three successive N-terminal PNA residues. Both
type of GalNAc-conjugated PNAs formed stable duplexes with complementary DNA with
thermal stability enhanced compared to unmodified PNA, and both had slightly lower stability
with complementary RNA than the unmodified PNA. The CD spectral profiles showed that
conjugation of the bulky branched GalNAc3 or of the three successive GalNAc-linked residues
did not alter the conformations of duplexes formed with DNA or RNA relative to the duplexes
formed with the unconjugated PNA. The GalNAc-conjugated PNAs were fluorescently labeled
with carboxyfluorescein for cell permeability studies. The PNA with three sequential
monovalent GalNAc units was more efficiently taken up by HepG2 cells, which express the
ASGPR, than either the GalNAc3-PNA conjugate or the unmodified PNA. None of the PNAs
were efficiently internalized by HEK293 cells, which do not express the receptor. These results
indicate that in the case of PNAs the architecture of the GalNAc conjugation tunes the
efficiency of uptake. This is rather significant, as it is not the case for antisense oligonucleotides
and siRNAs.34-36 On the other hand, the hydrophobic nature of the anionic backbone of
antisense oligonucleotide conjugates (phosphodiester vs phosphorothioate) has been shown to
affect the receptor binding.36 As PNAs are neutral and in the present work they are slightly net
positive due to terminal lysines, the ligand-receptor recognition may get modified which
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 20 of 33
1
2
3
4
5
6
deserves further evaluation. Nevertheless, our data indicate that appropriately designed
GalNAc-conjugate PNAs have potential in therapeutic applications.
7
8
9
Experimental Methods
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
58
59
60
The chemicals used were of laboratory or analytical grade. All the solvents used were
distilled or dried to carry out different reactions. Reactions were monitored by thin layer
chromatography (TLC). Usual workup involved sequential washing of the organic extract with
water and brine followed by drying the organic layer over anhydrous sodium sulfate and
evaporation of solvent under vacuum. TLCs were carried out on pre-coated silica gel GF254
sheets (Merck 5554). TLCs were analyzed under UV lamp, by iodine spray and by spraying
with ninhydrin solution, followed by heating of the plate. Column chromatographic separations
were performed using silica gel (60-120 or 100-200 mesh). The scheme for synthesis of
Compound 1 is shown in Supporting Information (Scheme 4, S4) following reported
procedures.6,7,22 Compounds 34 and 1 were characterized using 1H, 13C NMR and mass spectral
data (Supporting Information).
1H and 13C NMR spectra were recorded using JEOL 400 MHz NMR spectrometer (400
MHz for 1H and 100 MHz for 13C). The delta (δ) values for chemical shifts are reported in ppm
and are referred to internal standard TMS or deuterated NMR solvents. 13C NMR were
recorded using broad band 1H decoupling conditions 13C{1H}. The optical rotation values were
obtained on Rudolph Research Analytical Autopol V polarimeter. Mass spectra for reaction
intermediates were obtained by Applied Biosystems 4800 Plus MALDI-TOF/TOF mass
spectrometry using TiO2 or 2,5-dihydroxybenzoic acid (DHB) and the integrity of PNA
oligomer was checked on the same instrument using SA, DHB or CHCA as matrix. High
resolution mass spectra for final PNA GalNAc3 and [C(S)-GalNAc-T]3 were recorded on
Synapt G2 High Definition Mass Spectrometry. PNA oligomers were purified on Agilent
technologies 260 infinity HPLC system using semi-preparative BEH130 C18 (10 x 250 mm)
column. The unmodified PNA monomers were obtained from ASM technologies Ltd.
Germany. The complementary DNA and RNA 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 NaOH or HCl, from Sigma Aldrich.
ACS Paragon Plus Environment

Page 21 of 33
The Journal of Organic Chemistry
1
2
3
4
N-(Decanoic)-tris-[(carboxyethoxymethyl)aminopropyl )-tri(O-acetyl) N(acetyl
5
6
galactosamine]aminomethane [(Fmoc)methoxy)carbonyl]-benzyl lysinate 1b
7
8
9
ω
α
To a solution of [N H(Boc),N H(Fmoc)]-lysine benzyl ester (0.5 g, 0.25 mmol) in
DMF (2 mL) were added HBTU (0.3 g, 0.76 mmol) and DIEA (0.14 mL, 0.92 mmol) and the
resulting mixture was stirred for few minutes. A solution of compound 1a (0.42 g, 0.76
mmol) in DMF (2 mL) was added, and stirring was continued at room temperature overnight.
Solvents and volatiles were removed under reduced pressure, and the residue was dissolved
in DCM (20 mL), washed with saturated NaHCO3 (5 mL) and water (5 mL). After drying
over anhydrous Na2SO4 the solvent was evaporated under reduced pressure. The residue was
purified by silica gel chromatography (eluent: 3-15% MeOH in DCM) to obtain compound
1b (0.6 g, 50%). 1H NMR (400 MHz, DMSO-d6) δ 7.91 – 7.69 (m, 14H), 7.44 – 7.28 (m,
8H), 6.99 (s, 1H), 5.75 (s, 1H), 5.21 (d, J = 3.4 Hz, 2H), 5.12 (s, 1H), 4.97 (dd, J = 11.2, 3.4
Hz, 2H), 4.49 (d, J = 8.5 Hz, 2H), 4.32 – 4.18 (m, 2H), 4.07 – 3.96 (m, 9H), 3.88 (dd, J =
18.9, 10.0 Hz, 3H), 3.70 (dt, J = 10.8, 5.6 Hz, 3H), 3.55 (m, J = 12.0, 5.7 Hz, 12H), 3.44 –
3.36 (m, 4H), 3.07 – 2.96 (m, 13H), 2.28 (t, J = 6.3 Hz, 6H), 2.10 (s, 8H), 2.04 (dd, J = 13.1,
6.2 Hz, 9H), 1.99 (d, J = 2.5 Hz, 9H), 1.89 (s, 8H), 1.77 (s, 8H), 1.55 – 1.40 (m, 22H), 1.33
(s, 1H), 1.24 – 1.16 (m, 9H), 1.10 (s, 2H). 13C{1H} NMR (101 MHz DMSO-d6) δ 172.5, 172.3,
172.0, 170.0, 169.9, 169.6, 169.4, 156.2, 143.8, 140.7, 136.0, 128.4, 128.00, 127.6, 127.0,
125.2, 120.1, 115.6, 101.0, 70.5, 69.8, 68.6, 68.3, 67.3, 66.7, 65.8, 64.9, 61.4, 59.5, 54.9,
54.0, 49.4, 46.6, 39.5, 36.4, 36.3, 36.0, 35.1, 29.4, 28.7, 22.8, 21.8, 20.5 ppm. MS (MALDI-
TOF) m/z calcd. for C117H172N12O42 [M+H]+, 2418.70 ; found: 2441.00 [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
58
59
60
N-(decanoic) -tris-[(carboxyethoxymethyl)aminopropyl)-tri-(O-acetyl) N-acetyl
galactosamine]aminomethane [(Fmoc)methoxy)carbonyl]-Lysine (1c):
Compound 1b (0.5 g, 0.22 mmol) was dissolved in methanol, evaporated under reduced
pressure to remove traces of chlorinated solvent from previous step, re-dissolved in MeOH, 10
wt% Pd-C (1.0 g, wet Degussa type) was added at 0 oC. The reaction was hydrogenated under
o
normal pressure by maintaining temperature 20-25 C for 1h. After completion of reaction
(monitored by TLC), the reaction content was filtered through celite and washed with MeOH.
The combined filtrates were evaporated under reduced pressure to afford 1c (0.16g, 40%) as a
white solid. 1H NMR (400 MHz, DMSO-d6) δ 7.90 (t, J = 7.9 Hz, 8H), 7.81 (t, J = 5.5 Hz, 3H),
7.77 – 7.67 (m, 3H), 7.41 (t, J = 7.4 Hz, 2H), 7.32 (t, J = 7.4 Hz, 2H), 7.00 (d, J = 6.0 Hz, 1H),
5.21 (d, J = 3.4 Hz, 3H), 5.00 – 4.94 (m, 3H), 4.50 (d, J = 8.5 Hz, 3H), 4.28 – 4.19 (m, 3H),
4.05 – 3.96 (m, 9H), 3.87 (dt, J = 11.0, 8.9 Hz, 3H), 3.70 (dt, J = 10.8, 5.6 Hz, 3H), 3.53 (dd,
J = 13.6, 7.1 Hz, 14H), 3.43 – 3.37 (m, 5H), 3.07 – 2.96 (m, 14H), 2.28 (t, J = 6.3 Hz, 6H),
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 22 of 33
1
2
3
4
5
6
7
8
9
2.10 (s, 9H), 2.03 (dd, J = 13.1, 6.2 Hz, 10H), 1.99 (s, 10H), 1.88 (s, 9H), 1.77 (s, 9H), 1.49
(dq, J = 19.4, 6.5 Hz, 24H), 1.33 (s, 2H), 1.25 – 1.18 (m, 18H).13C{1H} NMR (101 MHz DMSO-
d6) δ 174.0, 172.5, 172.0, 171.9, 170.0, 169.9, 169.4, 167.0, 155.6, 153.4, 144.0, 143.9, 142.6,
140.7, 140.2, 139.4, 138.9, 137.4, 134.1, 131.7, 128.9, 127.6, 127.1, 125.2, 123.2, 122.7, 121.4,
120.0, 115.6, 114.6, 109.8, 101.0, 70.5, 69.8, 68.6, 68.2, 67.3, 66.7, 65.4, 61.4, 59.5, 39.5, 36.3,
35.8, 35.0, 31.5, 31.3, 29.4, 28.7, 25.30, 22.8, 22.0, 21.8, 20.5, 13.9, 10.8 ppm. MS (MALDI-
TOF) m/z calcd. for C110H166N12O42 [M+H]+, 2328.58 ; found: 2351.57 [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
58
59
60
NH[(Fmoc)-NH(Boc)-N,N methoxymethyl lysine amide (4)
To a stirring solution of Fmoc-protected Boc Lysine 3 in (5 g, 10.7 mmol) CH2Cl2 (50
ml) was added EDC (1.8 g, 11.8 mmol) and HOBt (2 g, 13 mmol). After stirring for 10 min at
o
23 C, N,O-dimethylhydroxylamine hydrochloride (1.2 g, 12 mmol) in EtiPr2N (3 ml, 24
mmol) was added and the reaction mixture was stirred 12 h. It was diluted with EtOAc and
washed aq. HCl (0.1 N), 10% aq. K2CO3, dried over anhydrous MgSO4 and concentrated. The
residue obtained was purified on silica gel (100−200 mesh) using petroleum ether and EtOAc
to give compound 4 as a white powder (4.5 g, 80%). 1H NMR (400 MHz, CDCl3) δ 7.75 (d, J
= 8.1 Hz, 2H), 7.60 (t, J = 6.6 Hz, 2H), 7.39 (t, J = 7.5 Hz, 2H), 7.33 – 7.28 (m, 2H), 5.61 (d,
J = 8.9 Hz, 1H), 4.68 (d, J = 49.4 Hz, 2H), 4.36 (d, J = 7.2 Hz, 2H), 4.21 (t, J = 7.0 Hz, 2H),
3.76 (s, 3H), 3.21 (s, 3H), 3.11 (d, J = 4.5 Hz, 2H), 1.92 (s, 1H), 1.76 (td, J = 13.4, 7.6 Hz, 1H),
1.61 (dd, J = 13.9, 5.5 Hz, 2H), 1.51 (dd, J = 14.7, 7.5 Hz, 2H), 1.42 (s, 9H), 1.28 – 1.18 (m,
2H). 13C{1H} NMR (101 MHz, CDCl3) δ 172.5, 156.0, 155.8, 143.7, 143.5, 141.0, 127.4,
126.8, 124.9, 119.5, 78.9, 77.1, 67.0, 61.4, 50.5, 46.9, 40.0, 32.2, 31.8, 29.3, 28.2, 22.2, 20.8
ppm. HRMS (ESI-TOF) m/z calcd. For C28H37N3O6: 511.2682 and found 534.2571[M+ Na]+.
NH(Boc),NH[(Fmoc) pentanaldehyde (5)
Compound 4 (3 g, 6 mmol) was dissolved in THF (15ml), cooled to -20°C, and LiAlH4
(0.2 gm, 5.2 mmol) was added slowly. After 1h, the reaction was quenched with EtOAc and
the organic layer washed with aq. HCl (0.1N), dried over MgSO4 and concentrated. The residue
obtained was purified on silica gel (100−200 mesh) using petroleum ether and EtOAc to give
compound 5 as a pale yellow oil (2.6 g, 95 %). 1H NMR (400 MHz, DMSO-d6) δ 7.39 – 7.27
(m, 5H), 7.24 (m, J = 5.2 Hz, 1H), 7.19 (m, J = 5.9 Hz, 1H), 6.70 (m, J = 8.7 Hz, 1H), 5.00 (d,
J = 5.2 Hz, 2H), 4.75 (d, J = 16.8 Hz, 2H), 4.4 (d, J = 16.7 Hz, 2H), 4.20 (dd, J = 33.7, 18.8
Hz, 1H), 3.95 (dd, J = 44.8, 17.4 Hz, 1H), 3.81 – 3.70 (m, 1H), 3.49 – 3.18 (m, 4H), 3.05 (dd,
J = 19.4, 7.5 Hz, 2H), 1.75 (d, J = 1.8 Hz, 2H), 1.64–1.47 (m, 2H), 1.36 (s, 9H). 13C{1H} NMR
(101 MHz, CDCl3) δ 199.5, 156.3, 143.9, 141.5, 127.9, 127.2, 125.2, 120.1, 79.4, 77.1, 67.1,
ACS Paragon Plus Environment

Page 23 of 33
The Journal of Organic Chemistry
1
2
3
4
5
6
60.2, 47.3, 39.9, 29.9, 28.6, 28.5, 22.2 ppm. HRMS (ESI-TOF) m/z calcd. For C28H37N3O6
452.2311 and found 452.5510[M+H]+.
7
8
N-[ethylamino(NHFmoc)-C(S)(NHBoc)aminobutyl] benzyl glycinate (7)
9
To a solution of the aldehyde 5 in ethanol, benzyl glycine ester hydrochloride (3 g, 66
mmol) was added DIPEA and neutralized with (0.5 mL, 40 mmol) at ice cold condition, stirred
for 5-10 min, followed by addition of sodium cyanoborohydride (1.7 g, 26 mmol) with
continued stirring for 4 h. After completion of reaction (monitored by TLC), solvent was
evaporated and the product was extracted with EtOAc. The organic layer was concentrated,
subjected to column chromatography purification, and the product was eluted with 35%
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
58
59
60
1
EtOAc: Pet-ether to obtain compound 7. Yield (1.5 g, 37%). H NMR (400 MHz, CDCl3) δ
7.75 (d, J = 7.5 Hz, 2H), 7.61 (d, J = 7.1 Hz, 6H), 7.41 – 7.27 (m, 2H), 5.15 (t, J = 6.8 Hz, 2H),
4.60 (s, 1H), 4.41 (d, J = 6.4 Hz, 1H), 4.21 (t, J = 6.7 Hz, 2H), 3.69 (s, 1H), 3.45 (q, J = 17.5
Hz, 2H), 3.10 (d, J = 4.9 Hz, 1H), 2.67 (dd, J = 12.5, 4.5 Hz, 1H), 1.54 – 1.29 (m, 13H), 1.28
– 1.18 (m, 2H). 13C{1H} NMR (101 MHz, CDCl3) δ 173.6, 156.5, 144.2, 141.5, 128.9, 127.7,
127.1, 125.2, 121.14, 120.1, 108.1, 77.2, 66.8, 66.6, 53.5, 53.0, 52.0, 50.9, 49.7, 47.9, 47.5,
34.2, 28.6, 23.0, 22.5, 22.4 ppm. HRMS (ESI-TOF) m/z calcd. For C35H43N3O6 601.3152 and
found 601.7440 [M+H]+.
N-[ethylamino(NH(Fmoc)-C(S)butylamino(NHBoc)]-N[acetamido(N1-thyminyl)]
benzyl glycinate (9)
To thymine acetic acid 8 (1 g, 55 mmol) in anhydrous DMF (4 mL), the amine 7 (5 g,
o
80 mmol) was added and cooled to 0 C. After 5 min stirring, HBTU (3 g, 81.5 mmol) and
DIPEA (1.5 mL, 12 mmol) were added to the reaction mixture and stirred for 12 h at RT. After
completion of reaction, it was poured into water (25 ml), neutralized with sat.aq. KHSO4 (10
mL), and extracted with ethyl acetate (3 x 20 mL). The organic layer was dried over anhydrous
Na2SO4. The solvent was removed under reduced pressure and product was purified by column
chromatography, eluting with petroleum ether/EtOAc (1:1) to obtain the compound 9. (Yield
2.8 g, 47%). 1H NMR (400 MHz, CDCl3) δ 9.06 (d, J = 51.6 Hz, 1H), 7.73 – 7.25 (m, 13H),
6.78 (t, J = 54.4 Hz, 1H), 5.52 (d, J = 8.0 Hz, 1H), 5.21 – 4.97 (m, 2H), 4.74 – 4.45 (m, 2H),
4.41 – 3.96 (m, 4H), 3.86 – 3.66 (m, 2H), 3.43 (dd, J = 25.6, 5.1 Hz, 2H), 3.14 – 2.75 (m, 3H),
1.80 (t, J = 7.4 Hz, 3H), 1.42 (d, J = 18.8 Hz, 12H), 1.24 (s, 2H). 13C{1H} NMR (101 MHz,
CDCl3) δ 169.3, 169.1, 168.4, 168.0, 164.4, 156.7, 156.5, 156.3, 152.1, 151.54 151.3, 144.1,
144.0, 143.9, 143.8, 141.5, 141.3, 141.1, 135.2, 134.9, 129.0, 128.9, 128.8, 128.6, 128.4, 127.8,
127.8, 127.2, 125.3, 120.1, 110.9, 79.3, 77.2, 68.0, 67.4, 66.8, 66.5, 66.3, 50.5, 50.0, 49.4, 48.2,
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 24 of 33
1
2
3
4
5
6
47.4, 47.2, 40.1, 39.9, 31.9, 31.5, 29.7, 28.5, 23.0, 22.9, 12.3. HRMS (ESI-TOF) m/z calcd.
For C42H49N5O9 767.3530 and found 768.3619 [M+H]+.
7
8
9
N-[ethylamino(NH(Fmoc)-C(S)butylamine]-N[acetamido(N1-thyminyl)] benzyl
glycinate (10)
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
58
59
60
Compound 9 (2 g, 2.8 mmol) was dissolved in DCM (75 mL) along with trifluoroacetic
acid (15 mL) and the reaction was stirred at room temperature for 30 minutes. The reaction
mixture was diluted with toluene (100 mL) and concentrated under reduced pressure. The
residue was co-evaporated with toluene (2 x 100 mL) and dried under reduced pressure using
a high vacuum pump to yield 10 as the TFA salt (1.7 g) which was used for the next reaction
without any further purification. HRMS [ESI-TOF] m/z calculated for C37H41N5O7. 667.3006;
Found 690.1689 [M+Na]+.
N-ethylamino(NHFmoc)-C(S)[(2-N-acetyl galactosamino-3,4,6-tri-O-acetyl-1-O-
butylcarboxy)amidobutyl]-N-[acetamido-N1-thyminyl)]benzyl glycinate (12)
To a solution of tri-O-acetyl-N(acetyl)-galactosamine pentanoic acid 11 (1.6 g, 3.5
mmol) in DMF (2 mL) were added HBTU (2 g, 5 mmol) and DIEA (1 mL, 7.3 mmol). The
resulting mixture was stirred for few minutes. A solution of compound 10 (3.5 g, 5.1 mmol) in
DMF (2 mL) was added, and stirring was continued at room temperature overnight. Solvents
and volatiles were removed under reduced pressure, and the residue was dissolved in DCM (20
mL), washed with saturated NaHCO3 (5 mL) and water (5 mL). After drying over anhydrous
Na2SO4 and concentration gave a residue was purified by silica gel chromatography (eluent: 3-
15% MeOH in DCM) to obtain compound 12 (1.6 g, 63 %).1H NMR (400 MHz, DMSO-d6) δ
11.29 (s, 1H), 7.89 – 7.84 (m, 2H), 7.81 (d, J = 9.2 Hz, 1H), 7.73 – 7.64 (m, 3H), 7.42 – 7.24
(m, 10H), 7.18 (s, 1H), 7.11 (d, J = 18.3 Hz, 1H), 5.19 (d, J = 9.2 Hz, 2H), 5.10 (s, 1H), 4.96
(d, J = 14.6 Hz, 1H), 4.69 (dd, J = 44.0, 16.7 Hz, 1H), 4.47 (d, J = 8.5 Hz, 2H), 4.39 – 4.17 (m,
4H), 4.15 – 3.97 (m, 4H), 3.87 (dd, J = 19.7, 9.1 Hz, 1H), 3.69 (dd, J = 8.2, 6.9 Hz, 2H), 3.45
– 3.35 (m, 2H), 2.99 (dd, J = 13.4, 6.9 Hz, 2H), 2.11 – 1.85 (m, 12H), 1.73 (dd, J = 26.6, 6.5
Hz, 6H), 1.51 – 1.21 (m, 10H). 13C{1H} NMR (101 MHz, DMSO-d6) δ 171.8, 170.0, 169.9,
169.6, 169.3, 168.7, 167.8, 164.3, 156.1, 150.9, 143.8, 141.8, 140.7, 135.8, 135.5, 128.5,
128.2, 128.0, 127.8, 127.6, 127.0, 125.1, 120.0, 108.2, 101.0, 70.5, 69.8, 68.6, 66.7, 66.6,
65.9, 65.3, 61.4, 54.9, 49.8, 49.3, 48.3, 47.7, 46.8, 39.5, 38.4, 35.0, 31.2, 29.5, 28.6, 23.0, 22.7,
21.8, 20.5,13.6, 11.90. HRMS (ESI-TOF) m/z calcd. For C56H68N6O17; 1097.1814 and found
1097.4727 [M+H]+.
ACS Paragon Plus Environment

Page 25 of 33
The Journal of Organic Chemistry
1
2
3
4
5
6
N-ethylamino(NHFmoc)-C(S)[(2-N-acetyl galactosamino-3,4,6-tri-O-acetyl-1-O-
butylcarboxy)amidobutyl]-N-[acetamido-N1-thyminyl)] glycine (2)
7
8
9
Compound 12 (0.6 g, 0.6 mmol) was dissolved in MeOH containing Pd-C (1.0 g, 10
wt% wet, Degussa type) at 0o C and the reaction was hydrogenated under normal pressure by
maintaining temp 20-25 oC for 1 h. After completion of reaction (monitored by TLC), reaction
contents was filtered through celite, washed with MeOH and the combined filtrate was
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
58
59
60
1
concentrated to afford 2 as a white solid (0.3 g, 50 %); H NMR (400 MHz, DMSO-d6) δ 11.26
(s, 1H), 8.71 (s, 2H), 7.85 (d, J = 7.6 Hz, 2H), 7.74 (t, J = 5.2 Hz, 1H), 7.58 (d, J = 7.2 Hz, 1H),
7.40 – 7.28 (m, 3H), 5.21 (d, 1H), 4.97 (d, J = 11.2 Hz, 1H), 4.49 (d, J = 8.4 Hz, 1H), 4.43 (d,
J = 8.6 Hz, 2H), 4.02 (s, 3H), 3.97 – 3.82 (m, 25H), 3.72 (dd, J = 21.6, 11.6 Hz, 2H), 3.54 (d,
J = 13.2 Hz, 1H), 3.38 (dd, J = 16.4, 10.1 Hz, 5H), 3.26 (s, 2H), 3.20 – 3.12 (m, 1H), 3.01 (d,
J = 5.5 Hz, 2H), 2.10 (s, 3H), 2.01 (m, J = 15.7 Hz, 6H), 1.89 (s, 4H), 1.75 (d, J = 15.1 Hz,
6H), 1.46 (m, J = 7.4 Hz, 8H), 1.23 (m, 2H). 13C{1H} NMR (101 MHz, DMSO-d6) δ 171.9,
171.5, 170.0, 169.7, 169.4, 168.5, 168.0, 167.3, 164.5, 151.0, 148.6, 142.4, 142.2, 139.9,
127.0, 124.2, 120.0, 108.0, 101.0, 70.5, 69.8, 68.7, 66.7, 61.4, 57.5, 51.3, 50.8, 50.0, 49.4,
48.5, 47.89, 46.6, 41.9, 39.5, 38.2, 35.0, 31.5, 30.4, 28.8, 28.6, 23.1, 22.8, 22.5, 22.1, 21.9,
20.6, 19.2, 18.1, 13.5, 11.9. HRMS (ESI-TOF) m/z calcd. For C49H62N6O17; 1006. 4171 and
found 1029.4069 [M+Na]+.
Solid phase synthesis of GalNAc3 conjugated PNA
The PNA oligomers were assembled on rink amide resin using Fmoc synthesis protocol
from C-terminus to N-terminus direction. The PNA oligomers were synthesized on solid phase,
by using Fmoc strategy.26,27 The resin-bound amine PNA oligomers (25 mg, 0.22 mmol/g) in
DMF were reacted with corresponding PNA monomer A/T/G/C (3 eq) in the presence of
HBTU (6 mg, 17 μmol, 3 eq), HOBt (2 mg, 17 μmol, 3 eq), and DIPEA (3 μL, 17 μmol, 3 eq).
o
The reaction was done for 5 min in microwave at 65 C and 25 W and then 6h at room
temperature (Discover SPS Manual Microwave Peptide Synthesizer, CEM Corporation, NC).
Excess reagents were removed by filtration and the resin was washed with DMF, DCM and
MeOH.
After all the PNA monomers were coupled, the resin-bound 15 mer PNA oligomer (25
mg, 0.22 mmol/g) in DMF (1:1) was coupled with corresponding (GalNAc)3 derivative acid 1
(40 mg, 17 μmol 3 eq) in the presence of Pybop (9 mg, 17 μmol 3 eq), HOBt (2 mg, 17 μmol,
3 eq) and DIPEA (3 μL, 17 μmol 3 eq). The reaction was done for 10 min in microwave at 75
oC and 40 W and then 24 h at room temperature. Excess reagents were removed by filtration
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 26 of 33
1
2
3
4
5
6
7
8
9
and the resin was washed with DMF, DCM and MeOH. For synthesis of trivalent-
[C(S)−GalNac-T]3 PNA oligomer 3, the resin bound 12-mer was coupled with the the
monomer 2 in three consecutive cycles of addition-deprotection. The resin was treated with
90% TFA in DCM (200 μL) and triisopropylsilane (10 μL) in cold for 10 min followed by
stirring for 1.5 to 2 h at room temperature. The resin was filtered out, and the filtrate was
concentrated. The residue was dissolved in methanol and the product was precipitated with
cold dry ether. It was dissolved in MilliQ water and purified by RP-HPLC using semi-
preparative C18 (10×250 mm) column using water-acetonitrile solvent system gradient from
0.1% TFA in CH3CN:H2O (5:95) to 0.1% TFA in CH3CN:H2O (95:5) in 20 min, flow rate of
2 mL/min, monitoring at 260 nm wavelength.
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
58
59
60
Temperature dependent UV absorbance for Tm measurement. UV-melting experiments
were carried out on Varian Cary 300 UV spectrophotometer equipped with Peltier heating
system. The samples for Tm measurements were prepared by mixing the calculated amounts of
respective oligonucleotides in the stoichiometric ratio (1:1, duplex) in sodium cacodylate
buffer (10 mM) and NaCl (10 mM) at pH 7.2 to achieve a final strand concentration of 3 μM
for each strand. The samples were annealed by heating at 90 °C for 10 min. followed by slow
cooling to room temperature for 8-10 h and then refrigerated for 12 to 24 h. The samples (500
μL) were transferred to quartz cell and equilibrated at the starting temperature for 5 min. The
OD at 260 nm was recorded from 20-92 °C with temperature ramping of 1 °C/min. Each
melting experiment was repeated at least twice. From the plot of normalized absorbance at 260
nm as a function of the temperature, the Tm was determined from maximum in its first
derivative using Origin 8.5. The concentrations of all oligonucleotides were calculated from
absorbance and the molar extinction coefficients of the corresponding nucleobases i.e. T = 8.8
cm2/μmol; C = 6.6 cm2/μmol; G = 11.7 cm2/μmol and A = 13.7 cm2/μmol.37,38
Circular dichroism. CD spectra were recorded on JASCO J-815 spectropolarimeter. The
duplexes were constituted as described above. The CD spectra were recorded as an
accumulation of 3 scans from 300 to 190 nm using 1 mm cell, band-width of 1 nm, response
of 1 sec with resolution of 0.1 nm and a scan speed of 100 nm/min.
Fluorescence spectroscopy. UV-Visible spectra for all PNA oligomers and complementary
oligonucleotides were recorded on Perkin Elmer Lambda-45 UV-Visible Spectrophotometer
and fluorescence spectra were recorded on Horiba Jobin Yvon Fluorolog 3 spectrophotometer.
The samples for fluorescence spectra were prepared by mixing calculated amounts of PNA and
DNA in stoichiometric ratio (1:1 for duplex) in sodium cacodylate buffer (10 mM) and NaCl
ACS Paragon Plus Environment

Page 27 of 33
The Journal of Organic Chemistry
1
2
3
4
5
6
(10 mM) at pH 7.2 to achieve a final strand concentration of 3 μM for each strand. The annealed
samples were used to record fluorescence spectra at ambient temperature with λex 458 and 484
nm and λem 520 nm with excitation slit width of 1 nm and emission slit width of 6 nm.
7
8
9
Cell Permeation study. HEK293 cells without any ASGPR as negative control were obtained
from American Type Culture Collection (ATCC, CRL-1573). HepG2 cells (ATCC HB-8065)
having ASGPR on cell surface were procured from National Centre for Cell Science (NCCS),
Cell Repository, Pune. The cells were cultured using Eagle’s minimum essential medium
(MEM), which was supplemented with 10% fetal bovine serum (FBS, Sigma Aldrich). The
cells were incubated at 37°C with 5% CO2 and maintained at the exponential growth phase.
About 1-2 million cells were transferred to the new flask and the cells were split. The medium
was discarded, and the cells were gently washed with 2 mL of sterile phosphate buffered saline
(PBS). Following PBS wash, the adhered cells were treated with 3 mL of 0.25% trypsin and
0.2% EDTA. After trypsin treatment for about 3 minutes at 37°C, 8 mL cell medium was added
to stop trypsinization. The concentration of live cells was estimated using a cell counter. The
cells suspension was centrifuged to and the pellet was resuspended in MEM. Finally, 1-2
million cells were passaged to a new flask.
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
58
59
60
Confocal laser scanning microscopy analysis
The cells were seeded in 35 x 15 mm cell imaging dishes containing 500 L MEM with
10% FBS and allowed to grow at 37°C in a humidified atmosphere containing 5% CO2 for at
least 24 h. After the cells have grown to reach 50% confluency they were treated with PNAs.
The media from all the imaging dishes were discarded carefully and 500 L fresh media
containing respective 5(6)-carboxyfluorescein tagged PNAs (4 M) to the corresponding
dishes. The adhered cells were incubated at 37°C under 5% CO2 for another 24 h. After
treatment with PNAs, the medium was carefully aspirated out and the adhered cells were
washed with PBS three times. The cells were then replenished with 500 L. Prior to imaging,
20 L of DAPI was added to stain the nuclei of the cells. The confocal microscopy imaging
has been repeated at least twice for each PNA.
Imaging of cells treated with caboxyfluorescein tagged PNAs were done on Leica TCS
SP8 microscope. The pin hole was set to 1 airy unit. For visualization of internalized PNAs,
the carboxyfluorescein dye tagged to the PNAs were excited with 514 nm laser and the
fluorescence emission signals from the molecules were detected from 550 nm to 660 nm using
the HyD detector. For visualization of nuclei, DAPI was excited at 405 nm with UV laser and
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 28 of 33
1
2
3
4
5
6
the signals over the range of 420 nm to 500 nm were detected. Bright field images were
captured using UV laser illumination and PMT detector.
7
8
Flow cytometry
9
HepG2 cells were seeded in a 12-well plate (1 x 105 cells/well) and cultured for 24
hours in DMEM, which was supplemented with 10% fetal bovine serum (FBS, Sigma Aldrich)
at 37°C with 5% CO2. Then, media were removed and replaced with fresh media containing
either no PNA or 4 μM PNA. After an incubation period of 12 hours, the media from all the
wells were discarded and the cells were washed with cold PBS containing 2 mM EDTA and
harvested by trypsinization. The detached cells were diluted and suspended in PBS. The cells
were then analyzed using BD FACS celesta (BD Biosciences, San Jose, CA, USA).
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
58
59
60
MTT assay
HepG2 cells were seeded in a 96-well plate with a density of 1 x 104 cells/well, each
containing 100 μL of DMEM. After culturing them overnight at 37 °C in a 5% CO2 incubator,
they were treated the PNAs (4 μM). Untreated cells were used as controls. After an incubation
of 12 hours, the media was discarded. Fresh media (96 μL) was added to all the wells. Then, 4
μL of MTT solution (1 mg/mL) was added to each well and the cells were incubated for 4 h at
37 °C in a 5% CO2 incubator. Finally, the media was discarded and 100 μL of the DMSO was
added to each well to solubilize the formazan crystals. After 1 hour, absorbance at 570 nm was
measured using EnSight multimode plate reader (Perkin Elmer, USA).
Acknowledgment. P.B thanks UGC for the award of research fellowship. The authors thank
Dr Raghavendra Kikkeri, IISER Pune for permission to use lab facilities for biological studies.
P. J. thanks IISER Pune for research fellowship. B. M. and H.N. acknowledges IISER Tirupati
for post doctoral fellowship.
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
ACS Paragon Plus Environment

Page 29 of 33
The Journal of Organic Chemistry
1
2
3
4
5
6
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
7
8
9
Authors contributions
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
58
59
60
P. B., Chemical synthesis, Biophysical studies, Indian Institute of Science Education and
Research (IISER) Pune, Dr Homi Bhabha Road, Pune 411008
B. M. and H. N. B., Cell uptake experiments, Indian Institute of Science Education and
Research (IISER) Tirupati, Karkambadi Road, Tirupati 517507, Andhra Pradesh, India
B. M. and P. J., Fluorescence cytometry and MTT assay experiments
M. M. Alnylam Pharmaceuticals, Boston, USA and K. N. G. IISER, Pune and IISER,
Tirupati, Design of experiments and Project Investigators
References
1. van der Ree, M. H.; de Vree, J. M; Stelma, F.; Willemse, S.; van der Valk, M.; Rietdijk,
S.; Molenkamp, R.; Schinkel, J.; van Nuenen, A. C.; Beuers, U.; Hadi, S.; Harbers, M.; van der
Veer, E.; Liu, K.; Grundy, J.; Patick, A. K.; Pavlicek, A.; Blem, J.; Huang, M.; Grint, P.; Neben,
S.; Gibson, N. W.; Kootstra, N. A.; Reesink, H. W. Safety, tolerability, and antiviral effect of
RG-101 in patients with chronic hepatitis C: a phase 1B, double-blind, randomised controlled
trial. Lancet 2017, 389, 709–717.
2. Rottiers, V.; Obad, S.; Petri, A.; McGarrah, R.; Lindholm, M. W.; Black, J. C.; Sinha,
S.; Goody, R. J.; Lawrence, M. S.; deLemos, A. S.; Hansen, H. F.; Whittaker, S.; Henry, S.;
Brookes, R.; Najafi-Shoushtari, S. H.; Chung, R. T.; Whetstine, J. R.; Gerszten, R. E.;
Kauppinen, S.; Näär, A. M. Pharmacological inhibition of a microRNA family in nonhuman
primates by a seed-targeting 8-mer antimir. Sci. Transl. Med. 2013, 5, 212ra162.
3. Janssen, H. L.; Reesink, H. W.; Lawitz, E. J.; Zeuzem, S.; Rodriguez-Torres, M.;
Patel, K.; van der Meer, A. J.; Patick, A. K.; Chen, A.; Zhou, Y.; Persson, R.; King, B. D.;
Kauppinen, S.; Levin, A. A.; Hodges, M. R. Treatment of HCV infection by targeting
microRNA. N. Engl. J. Med. 2013, 368, 1685–1695.
4. Wittrup, A.; Lieberman, J. Knocking down disease: a progress report on siRNA
therapeutics. Nat. Rev. Genet. 2015, 16, 543–552.
5. Whitehead, K. A.; Langer, R.; Anderson, D. G. Knocking down barriers: advances in
siRNA delivery. Nat. Rev. Drug Discov. 2009, 8, 129–138.
ACS Paragon Plus Environment