Synthesis and biochemical evaluation of Aminopropanolyl-Thymine tri-Phosphate (ap-TTP)

Article abstract of DOI:10.1080/15257770.2019.1688831

Deoxyribonucleoside triphosphates (dNTPs) are building blocks for the biosynthesis of DNA. Various modified dNTPs’ analogs have synthesized by structural changes of nucleoside’s susgar and nucleobases and employed for synthesis of modified DNA. A very few modified dNTPs have prepared from non-sugar nucleoside analogs. This report describes the synthesis of acyclic nucleoside triphosphate (NTP) analog from amino acid L-Serine as aminopropanolyl-thymine triphosphate (ap-TTP) and demonstrate its biochemical evaluation as enzymatic incorporation of ap-TTP into DNA with DNA polymerases with primer extension methods. Alanyl peptide nucleicacids (Ala-PNA) are the analogs of DNA which contains alanyl backbone. Aminopropanolyl–analogs are derivatives of alanyl back bone. Ap-TTP analog is nucleoside triphosphate analog derived from Ala-PNA. Importantly, this report also sheds light on the crystal packing arrangement of alaninyl thymine ester derivative in solid-state and reveals the formation of self-duplex assembly in anti-parallel fashion via reverse Watson-Crick hydrogen bonding and π–π interactions. Hence, ap-TTP is a useful analog which also generates the free amine functional group at the terminal of DNA oligonucleotide after incorporation.

Full text of DOI:10.1080/15257770.2019.1688831

Nucleosides, Nucleotides and Nucleic Acids
ISSN: 1525-7770 (Print) 1532-2335 (Online) Journal homepage: https://www.tandfonline.com/loi/lncn20
Synthesis and biochemical evaluation of
Aminopropanolyl-Thymine tri-Phosphate (ap-TTP)
Chandrasekhar Reddy Gade & Nagendra K. Sharma
To cite this article: Chandrasekhar Reddy Gade & Nagendra K. Sharma (2019): Synthesis
and biochemical evaluation of Aminopropanolyl-Thymine tri-Phosphate (ap-TTP), Nucleosides,
Nucleotides and Nucleic Acids, DOI: 10.1080/15257770.2019.1688831
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NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
https://doi.org/10.1080/15257770.2019.1688831
Synthesis and biochemical evaluation of
Aminopropanolyl-Thymine tri-Phosphate (ap-TTP)
a,b,c
a,b
Chandrasekhar Reddy Gade
and Nagendra K. Sharma
a
National Institute of Science Education and Research (NISER)-Bhubaneswar, Jatni, Khurda,
b
c
Odisha, India; HBNI-Mumbai, Mumbai, India Indian Institute of Science Education and Research,
Karakambadi Rd, Opp Sree Rama Engineering College, Rami Reddy Nagar, Mangalam, Tirupati,
Andhra Pradesh Andhra Pradesh, India
ABSTRACT
ARTICLE HISTORY
Deoxyribonucleoside triphosphates (dNTPs) are building blocks
for the biosynthesis of DNA. Various modified dNTPs’ analogs
have synthesized by structural changes of nucleoside’s susgar
and nucleobases and employed for synthesis of modified DNA.
A very few modified dNTPs have prepared from non-sugar
nucleoside analogs. This report describes the synthesis of acyc-
lic nucleoside triphosphate (NTP) analog from amino acid L-
Serine as aminopropanolyl-thymine triphosphate (ap-TTP) and
demonstrate its biochemical evaluation as enzymatic incorpor-
ation of ap-TTP into DNA with DNA polymerases with primer
extension methods. Alanyl peptide nucleicacids (Ala-PNA) are
the analogs of DNA which contains alanyl backbone.
Aminopropanolyl – analogs are derivatives of alanyl back bone.
Ap-TTP analog is nucleoside triphosphate analog derived from
Ala-PNA. Importantly, this report also sheds light on the crystal
packing arrangement of alaninyl thymine ester derivative in
solid-state and reveals the formation of self-duplex assembly in
anti-parallel fashion via reverse Watson-Crick hydrogen bond-
ing and p–p interactions. Hence, ap-TTP is a useful analog
which also generates the free amine functional group at the
terminal of DNA oligonucleotide after incorporation.
Received 21 June 2019
Accepted 31 October 2019
KEYWORDS
Aminopropanolyl-thymine
triphosphate; DNA
polymerase; primer
extension reaction; LC-MS;
gel electrophoresis
Introduction
Deoxyribonucleotide triphosphates (dNTPs) such as dTTP, dATP, dCTP &
dGTP are building blocks of deoxyribose nucleic acid (DNA) biosynthesis
1
(
Figure 1). Since 1953, after discovery of DNA duplex structure, structure
of dNTPs are modified to tune the functional and structural properties of
2–4
DNA. As resultant, various kind of modified dNTPs and their respective
DNA analogs are accomplished, and a few of them have shown astonishing
applications. There are two practical methods to modify the structure of
CONTACT Nagendra K. Sharma
nagendra@niser.ac.in
National Institute of Science Education and
Research (NISER)-Bhubaneswar, Jatni, Khurda, Odisha 75 2050, India.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/lncn.
Supplemental data for this article is available online at https://doi.org/10.1080/15257770.2019.1688831.
ß 2019 Taylor & Francis Group, LLC

2
C. R. GADE AND N. K. SHARMA
Figure 1. Structure of dTTP/ddTTP, glycerol TTP and Aminopropanolyl-TTP
DNA: (a) enzymatic incorporation of modified dNTPs into DNA primer
with DNA ploymerase, and (b) chemical incorporation of modified nucleo-
side phosphoramidites at DNA synthesizer. The chemically modified
dNTPs are prepared by structural changes of sugar ring/nucleobases resi-
dues in target-specific nucleosides, and incorporated into sequence-specific
5–10
DNA primer for various motives.
For an example, glycerol nucleoside
triphosphate (gNTP), non-deoxyribose ring acyclic dNTP, is substrate of
DNA polymerase and successfully incorporated only at the terminal of
DNA primer, however, the further extension of gNTP incorporated DNA
1
1,12
primer are reportedly unsuccessful.
It has been explained that the fail-
ure of multiple incorporations of gNTP into DNA primer is possibly due
to following reasons: (i) The loss of contact between the single incorporated
DNA primer-template complex with polymerase enzyme; (2) The disturb-
ance of gNTP’s hydroxyl group alignment for nucleophilic attack at phos-
phate group of next gNTP (or dNTP) because of smaller size, greater
flexibility, or increased rotational freedom; (ii) destabilization duplex struc-
ture after incorporation of gNTP. Further, the conformationally constrained
synthetic cyclic analog of gNTP as threose nucleoside triphosphate (tNTP)
1
3
has been prepared which are also substrate of DNA polymerase. Another
0
modified nucleoside analog as 2 -amino threose nucleoside, bearing acti-
vated monophosphate ester, is capable for template guided extension on
1
4
0
0
primer without DNA polymerase. Similarly, 5 -amino and 3 -activated
monophosphate esters are also proficient for the template guided DNA
1
5,16
synthesis without DNA polymerase enzyme.
To explore the DNA
duplex/triplex structure stabilizing candidate, various the backbone-modi-
fied nucleic acid analogs are prepared and studied. For example, peptide
nucleic acid (PNA) is a potential DNA analog that has acyclic and achiral
backbone, aminoethyl glycinyl (aeg), in place of sugar-phosphate backbone
of native DNA. Nevertheless, PNA has considered as gene-based thera-
1
7
peutic drug candidates. The synthesis of aeg-PNA oligomers, now, are
customized using solid phase synthesis methods. Importantly, the

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
3
Scheme 1. Synthesis of AminopropanolylThymine triphosphates (ap-TTP).
triphosphate derivatives of aeg-PNA are also synthesized and incorporated
1
8,19
into the DNA primer with the help of DNA polymerase.
Further the
achiral aeg-backbone of PNA is also modified by replacing with another
amino acid. For example, alanyl-PNA, bearing alanine amino acid residue,
is emerged as a potential PNA analog and studied the formation of stable
duplex, triplex, and tetraplex (i-motif) PNA:DNA (or PNA:PNA)
20–22
structures.
These reports inspired us to design the amino-functionalized dNTP ana-
logs from naturally ocurring chiral amino acids, and to demonstrate their
substrate activity with DNA polymerase. Recently, we have reported the
amino functionalized ddNTP analogs as pr-NTP from amino acid L-
Proline, and found that pr-NTPs are the substrate of Therminator DNA
2
3
polymerase. We, herein, describe the synthesis of rationally designed
amino-functionalized aminopropanolyl-Thymine triphosphate (ap-TTP)
from naturally occuring L-Serine amino acid and study their enzymatic
incorporations into DNA primer with DNA polymerase by primer exten-
sion reactions (PExR). The results of our studies would be significant for
the generation of amino-functionalized DNA primer.
Results and discussion
Synthesis of ap-TTP. We derivatized L-Serine into N-Boc-Serine methylate
(1), and converted into another reactive intermediate (2) as O-mesylate
compound (Scheme 1). The compound (2) was treated with nucleobase
Thymine (T) for N-alkylation under basic conditions which produced chiral
alanyl-PNA monomer as N-Boc-alanyl thymine methyl ester derivative (3).
The ester group of N-Boc-alanyl thymine ester (3) was reduced into
hydroxyl group as nucleoside derivative (4) with versatile reducing agents

4
C. R. GADE AND N. K. SHARMA
Figure 2. ORTEP diagram and Phase data of N-Boc-alanyl thymine methyl ester (3): Space-
group P 1 21/c 1 (14) – monoclinic; Cell a ¼ 5.2456(3) Å, b ¼ 26.0994(15) Å, c ¼ 12.1838(7) Å
ꢁ
3
b ¼ 99.467(4) ; V ¼ 1645.33(16) Å ; Z ¼ 4.
(
NaBH ). The hydroxyl group of nucleoside (4) was phosphorylated with
4
2
4
phosphorylating reagents by following the Ludwig-Eckstein method.
Finally, the desired product aminopropanolyl thymine triphosphate (5) was
isolated after purification by HPLC. The synthetic derivatives (1–4) and
1
13 31
desired compound ap-TTP (5) are characterized by NMR ( H/ C/ P), and
HRMS techniques, their spectral data are provided in Supplemental File.
Structural studies of N-Boc-alanyl thymine Ester (3). We crystalized N-Boc-
alanyl thymine ester (3) and obtained its single crystal. Our X-ray diffractom-
eter studies have confirmed the chemical structure of the compound (3), and
X-ray data are deposited to Cambridge crystallographic data center (CCDC)
with reference number CCDC 1568315. The ORTEP diagram of compound 3
is depicted in Figure 2.
Since DNA/RNA nucleobases have strong abilities to exhibit non-cova-
lent interactions such as hydrogen bonding and p–p interactions. Thus we
extracted the crystal packing arrangement of compound (3) using software
(
(
Diamond 3.2) to explore the supramolecular self-assembly structure
Figure 3). The packing arrangement of thymine ester (3) (along c-axis)
exhibits the formation of Thymine-Thymine dimer via hydrogen bonding
3
and p–p interactions. The N -H (Thymine residue) of compound 3 is
2
hydrogen bonded with O¼C (another thymine residue) of the same com-
3
2
pound as two N -H—O¼C at the distance ꢀ2.0 Å. Importantly, these
hydrogen bond interactions are reversely different from natural DNA
3
4
Watson-Crick hydrogen bonding (N -H—O¼C ). In literature, such type

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
5
Figure 3. Structural studies of single crystal of N-Boc-alanyl thymine methyl ester (3) by X-ana-
lyse: (a) Packing diagram (top left); (b) diagram of selected region (top right); (c) Hydrogen
bonding pattern (bottom left) and (d) Detail of distance for hydrogen bonding and p–p inter-
action (Bottom right).
of hydrogen bonding, also called reverse Watson-Crick hydrogen bonding,
are reported in the supramolecular assemblies structure of ferrocene linked
2
5,26
thymine/uracil conjugated compounds.
Generally, Watson-Crick and
Hoogsteen types of hydrogen bonding are most common in nucleobase
2
7–31
supramolecular self-assembly structure by modified nucleosides.
Recently we reported the formation of helical supramolecular self-assembly
3
2
by prolamide thymidine/uridine analogs. Besides, the hydrogen bonding,
the side view of compound 3’s packing diagram shows the formation of the

6
C. R. GADE AND N. K. SHARMA
anti-parallel self-duplex type of structure. The strand of that duplex struc-
ture is formed by hydrogen bonding between C ¼ O and N–H of two adja-
cent Boc residue of compound 3 as C ¼ O—H–N at a distance 2.4 Å. Also,
unique p–p interactions are noticed at a distance 3.7Å, between two thymine
residues such as thymine of one stand with the thymine of another. We also
extracted the diameter of the self-assembled duplex as 10 Å, and pitch value
5.2 Å (Figure 3). Overall, non-covalent interactions firmly stabilized the self-
duplex structure of aminopropanol thymine ester (3) in the solid-state crystal
(Figure 3, bottom right).
Biochemical Evaluation of ap-TTP (5). After structural studies of pro-
tected ap-nucleoside ester, its triphosphate was attempted to incorporate
into DNA primer using a primer extension reaction method. For compara-
tive studies, we used known substrates of DNA polymerase (dTTP &
ddTTP) as control studies with the various commercially available DNA
-
-
polymerases: Taq, Vent exo , Deepvent exo , Bst, and Therminator. The
DNA primers (P1/FAM-P1) and DNA Templates (T1/T2) were used for
incorporation of ap-TTP/control templates are following
0
0
Primers: (P1) 5 -TGTAAAACGACGGCCAGT-3 ;
0 0
Fam-P1)Fam-5 TGTAAAACGACGGCCAGT-3
(
Template (the underlined sequence is complementary to the primers):
0
ꢂ
(
T1) 3 ACATTTTGCTGCCGGTCAA GTCGAGGCAT-5’
0
(
T2) 3 ACATTTTGCTGCCGGTCAAAAAAAAAAAA-5
0
For the gel-electrophoretic studies, the FAM-labeled primer (5 Fam-P1:
0
0
FAM-5 TGTAAAACGACGGCCAGT-3 ) was treated with ap-TTP in the
ꢁ
presence of DNA polymerase and DNA template (T1) at temperature 55 C
for primer extension reactions. Then the reaction mixture was separated
and visualized with the desaturating PAGE gel electrophoretic technique
for compression with labeled primer. The similar experiments were per-
formed with control substrates (dTTP/ddTTP). Their gel images are
depicted in Figure 2. In Figure 2a, the gel-band of lane L1 belongs to the
0
labeled DNA primer (5 -FAM-P1), while the gel-bands of L2/L3/L4 belong
to the primer extension reaction with respective dTTP/ddTTP/ap-TTP with
therminator DNA polymerase (Figure 2a). The gel shifts show that ap-TTP
0
has incorporated into primer (5 -FAM-P1) as like control substrate ddTTP.
It has known that ddTTP is a terminator of DNA synthesis after single
33
incorporation into DNA primer. These results strongly support the exten-
sion of labeled primer with ap-TTP after single incorporation as like ddTTP.
However, the gel band shifts with both ap-TTP and ddTTP are lower than
another control natural substrate dTTP under similar conditions. It shows

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
7
Figure 4. Gel-shift images of DNA primer (Fam-P1) after dTTP/ddTTP/ap-TTP incorporation
with different DNA polymerase Enzymes and DNA template (T1): (a) the only primer no dNTPs
or its analogs (L1); with enzyme Therminator (L2–L4)/Vent-Exo (L5 and L6)/Taq (L7 and L8).
(
b) Only primer no dNTPs or its analogs (L1); with enzyme Bst (L2 and L3)/Deepvent (L4 and
L5). (c) Full-length extension of primer Fam-P1 other three dNTPs and respective TTP with
Therminator DNA polymerase and DNA template (T2)
that more than one dTTP (control) have incorporated into the DNA primer
0
(
5 -FAM-P1). In literature, the multiple incorporations of dNTP into primer
34
are reported with high fidelity DNA polymerase enzyme. We tested ap-
TTP with other DNA polymerases such as Vent-exo, Taq, Bst, or Deepvent
and then compared with control ddTTP by the visualization of respective gel
shifts under similar primer extension reaction conditions (Figure 4a,b). In
Figure 4a, the gel shifts of ap-TTP and ddTTP are depicted in respective lane
L4 & L5 for Vent-exo polymerase, L5 & L6 for Taq polymerase. However,
the gel shift of ap-TTP and ddTTP, with Bst/Deepvent polymerase, are pro-
vided in lane L2-L4 in Figure 4b, and then compared with DNA primer L1
of Figure 4b. The gel shifts of ap-TTP consistently match with the gel-shift
of ddTTP (control) in the presence of other given DNA polymerases. These
gel studies strongly support the single incorporation of ap-TTP into DNA
primer as like control ddTTP with various DNA polymerases. The incorpor-
ation of dTTP/ddTTP/ap-TTP into labeled primer with DNA polymerase has
also summarized in Table 1. Hence ap-TTP is also substrate of DNA poly-
merase as like as like dTTP and ddTTP.
Further, to investigate the multiple incorporations of ap-TTP into the
primer, we have designed a template (T2) that could incorporate multiple

8
C. R. GADE AND N. K. SHARMA
Table 1. DNA polymerase vs. aminopropanolyl-TPP & control (dNTP &ddNTP) reaction results.
Sr. No
DNA Polymerase
dTTP
ddTTP
Ap-TTP
1
2
3
4
5
Bst
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Deepventexo-
Taq
Vent exo-
Therminator
dTTP. Herein, we have performed primer extension reactions with
designed primer/template, ap-TTP and control dTTP by gel-electrophoretic
methods, and their gel-shifts are depicted in Figure 4c. In case of dTTP, we
noticed the multiple incorporations of dTTP into the DNA primer up to
almost full-length of DNA sequence (Figure 4c, L2). However, with ap-
TTP, the gel-shift band (Figure 4c, L3) is almost equal to that of terminator
ddTTP (Figure 3, L4). Herein, ap-TTP could not get incorporated into
DNA after single incorporation, unlike dTTP. The similar results have
reported with gNTP. Hence ap-TTP is another terminator of DNA biosyn-
thesis after single incorporation.
Liquid Chromatography-Mass Spectrometry (LC-MS) Studies. To ensure the
incorporation of ap-TTP into DNA primer, we analyzed the reaction PCR
mixture by LC-ESI-Mass methods. We recorded the mass spectra of DNA pri-
mer (P1) and Template (T1) before and after treatment with ap-TTP in the
presence of DNA polymerase (Vent-exo). A similar experiment was repeated
with the ddTTP (control) for comparative studies. The ESI-Mass spectra of
primer/extended primer with ap-TTP and control ddTTP are provided in the
supplemental file. The selected region of mass spectra for the primer extension
reaction of ap-TTP is depicted in Figure 5. The mass spectrum of DNA pri-
mer P1 exhibits a prominent peak at 1381.99 (m/z) which belongs to molecu-
4ꢃ
lar mass ion (M ) of primer as [M -4H] , while the mass of template (T1)
p
p
has appeared at 1454.16. In the presence of Ventexo- DNA polymerase and
ap-TTP, the mass of DNA primer (P1) appeared at 1447.28 (m/z), higher than
lone primer, which belong to the molecular mass ion of single ap-TTP
4
ꢃ
ꢂ
ꢂ
extended DNA primer (M ) as [M -4H] . Similarly, the mass of DNA pri-
p
p
4ꢃ
ꢂꢂ
mer P1 was appeared at 1447.28 (m/z) which belongs to [Mp -4H] , the
mass of single ddTTP extended DNA primer (Mp ). This mass is also higher
than primer under similar primer extension reaction conditions as like ap-
TTP. Hence, these mass results confirm only single incorporation of ap-TTP
into DNA primer in the presence of DNA Template and DNA polymerase.
ꢂꢂ
Conclusion
In summary, we have described the synthesis, characterization, and bio-
chemical evaluation of ap-TTP with various DNA polymerases. We have
also described the supramolecular self-assembly duplex structure in

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
9
Figure 5. Mass spectrum of the extended mass of primer with Ap-TTP.
protected solid-state monomer crystal as a unique anti-parallel self-duplex
via unique reverse Watson-Crick hydrogen bonding and p–p interactions.
Importantly, the enzymatic studies support the single incorporation of ap-
TTP into DNA primer as like control therminator ddTTP. Hence ap-TTP
is a different substrate of DNA polymerase to introduce an amine function-
0
ality at 3 -position of DNA primer.
Experimental section
Materials. All reagents and solvents were purchased from commercial sour-
ces and used as received unless otherwise specified. DNA primers and
Template oligonucleotides were purchased from IDT. DNA polymerase
enzymes purchased from NEB. DMF and DCM were distilled over CaH2
and stored on molecular sieves. THF and dioxane were dried over sodium
benzophenone and stored over sodium. Pyridine was distilled over potas-
sium hydroxide and stored on CaH . Triethyl ammonium bicarbonate
2
(
TEAB) prepared by passing CO gas into 2 M aqueous triethylamine solu-
2
ꢁ
tion and stored at 4 C. HPLC was performed on Waters semipreparative
HPLC using Sunfire column(C18; 4.6ꢄ 150 mm) using 20 mM TEAB and
Acetonitrile (0–10% 30 min).
ESI-Mass experiments: LC-MS experiments were performed on Bruker
microTOF-Q II coupled with a Waters Acquity UPLC autosampler. Primer
extension reaction products (25 ul) were diluted with 25 ll Milli-Q water
and injected about 10 ll vis autosampler by the following program as flow
rate 0.2 ml/minute linear gradient.0–10 min80% A, 10–12 min 30% A,
12–13 min 30% A isocratic, 13 and 14min 100% A linear gradient and 14
and 15 min 100% A isocratic. UPLC column (C-18 RP, 2.1 ꢄ 5.0 mm,
1
.7 lm, Acquity Waters) used for LC-MS experiments. The column tem-
ꢁ
perature maintained at 30 C during the experiments (buffer A (5 mM
Ammonium acetate with pH 7.0) and buffer B (Methanol).
Primer extension reaction Method for mass analyses: Primer extension
reactions for LC-MS analysis were carried out with 5 mM of primer and

1
0
C. R. GADE AND N. K. SHARMA
template, 100 mM ap-TTP, dTTP or ddTTP in a total reaction volume of
25 ll with 2.0 units of Vent exo DNA polymerase.
Primer extension reaction Method for PAGE gel studies. All primer exten-
sion reactions were performed with 1 mM Fam-labeled primer, 1 mM tem-
plate 100 mM NTP (dTTP, dTTP, and Ap-TTP) in 10.0 ll reaction buffers
(
5
provided by the supplier with enzymes). All reactions were performed at
5 C for 60 min with two units of Therminator; Vent exo or Deep Vent exo
ꢁ
DNA polymerases in 10.0 ll reactions. Though for Bst and Taq DNA poly-
merases, we used 8.0 and 10.0 units, respectively. Denaturating Gel experi-
ment performed with 20% PAGE (29: 1 Acrylamide, bis-acrylamide) and
7.0 M urea. Gels were prepared and run with 1xTBE buffer and visualized
under trans-UV using the Bio-Rad gel doc system.
Experimental procedures for the synthesis of compounds (2–5)
O-Mesyl-N-Boc Serine methyl ester (2): N-Boc serine methyl ester (2.0 g,
ꢁ
9
.13 mmol) was dissolved in anhydrous pyridine (10 ml) and stirred at 0 C
under nitrogen atmosphere. Mesyl chloride (1.06 ml, 13.7 mmol) was added
ꢁ
dropwise with a syringe and stirred at 0 C for one hour. The reaction
mixture was concentrated under vacuum using the rota vapor and then
dissolved into water and extracted with organic solvent DCM. The DCM
layer was washed with sodium bicarbonate solution followed by brine
solution and kept over anhydrous sodium sulfate. The DCM solution was
concentrated and purified by silica gel column with Ethyl acetate:
Hexanes (1:9) that produced 1.89 g of the oily compound in 70% yield. Rf
(
0.62) was determined on TLC 40% EtOAc/Hexane.1H-NMR (400 MHz,
CDCl ) d ppm 1.36 (9 H, s), 2.95 (3 H, s), 3.71 (3 H, s), 4.39–4.42(1H, m),
3
4
2
.47–4.53(2H, m), 5.53-5.55(1H, m) 13C-NMR (100 MHz, CDCl ) d ppm
8.11, 37.17, 52.89, 53.01, 68.99, 80.43, 155.07, 169.15.HRMS (ESI-TOF)
3
Calculated (C H O N S þ Na) 320.0774, found 320.0775
1
0
17 7
N-Bocalanyl Thymine ester (3): Thymine (0.2 g, 1.58 mmol), K CO
2
3
(
0.328g, 2.38 mmol) and 18-Crown-6 (0.083 g, 0.31 mmol) was dissolved
into anhydrous DMF (5 ml) under a nitrogen atmosphere and stirred for
0 min. The mesyl derivative 2 (0.471 g, 1.58 mmol) was added to DMF
1
reaction mixture and stirred at room temperature for overnight under
nitrogen atmosphere. The reaction mixture concentrated under vacuum
and purified over silica gel column using solvent system (MeOH and
DCM) which gave 0.181 g white solid in 35% yield. R (0.46) was
f
1
determined on TLC using 5% MeOH/DCM. H NMR (400 MHz, DMSO-d
6
þCD OD) d ppm 1.27 (9 H, s), 1.72 (3 H, s), 3.54–3.60(1H, m), 3.63
3
(
(
3 H, s), 4.18–4.23(1H, m), 4.44–4.46(1H, m), 7.18(1 H, m). 13C-NMR
100 MHz, DMSO-d þ CD OD) d ppm 11.23, 27.49, 49.19, 51.78, 51.88,
6
3

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
11
79.40, 109.05, 142.20, 151.43, 156.01, 165.26, 17.42 HRMS (ESI-TOF)
Calculated (C H O N þNa) 328.1503, found 328.1509.
1
4
21 6 3
N-Boc-aminopropanolyl Thymine (4): Thymine methyl ester (3) (0.15 g,
0.45 mmol) was dissolved in anhydrous ethanol under nitrogen atmosphere
by stirring for 10 minutes followed by addition of Sodium borohydride
0.052 g, 1.37 mmol) and stirred overnight under nitrogen atmosphere at
(
room temperature. The concentrated reaction mixture had extracted with
ammonium chloride and ethyl acetate. The ethyl acetate layer was washed
with brine solution and kept over anhydrous sodium sulfate. The organic
layers were concentrated with rota-vapor under vacuum and purified by
silica gel column chromatography with solvent system MeOH and DCM,
which gave 0.109 g of white solid in 80% yield. R (0.24) was determined
f
1
on TLC using 5% MeOH/DCM. H-NMR (400 MHz, DMSO-D ) d ppm
6
1.29 (9 H, s), 1.72 (3 H, s), 3.23-3.29 (1H, m), 3.34–3.41 (2H, m), 3.74–3.80
(
1H, m), 3.95–4.00 (1H, m), 4.80–4.83 (1H, m), 6.54-6.57 (1H, m),
7.31(1H, s), 11.12 (1H, s) 13C-NMR (100 MHz, DMSO-D d ppm 12.51,
6)
28.52, 49.98, 51.18, 61.90, 78.25, 107.90, 142.58, 151.43, 155.72, 164.81.
ESI-HRMS (ESI-TOF) Calculated (C H O N ) 300.1554, found 300.1556.
1
3
21 5 3
Aminopropanolyl thymine triphosphate (5). Triphosphorylationof synthe-
sized nucleoside alcohol was performed by following the Ludwig-Eckstein
18
method. N-Boc-aminopropanolyl Thymine alcohol 4 (0.04 g, 0.133 mmol)
was dissolved in 3 ml of anhydrous pyridine. Pyridine was evaporated
in the Rota vapor under vacuum followed further drying in a vacuum
desiccator at ambient temperature for one hour. Desiccator was opened
under an argon atmosphere and the round bottom flask was closed with
a septum. All the next steps were performed under argon pressure.
Salicylchlorophosphidite (0.032 g, 0.160 mmol) dissolved in dry dioxane
(
1.0 ml) was added and stirred at room temperature for 10 min.
Immediately the cocktail of tri- butylammonium pyrophosphate (0.146 g,
.267 mmol) and tri-butylamine (0.267 mL, 1.07 mmol) in anhydrous
0
dimethylformamide (DMF) (1 ml) was injected into the flask and stirred
for 10 min followed by the addition of iodine (1.0%) in pyridine:water
(
98:2) solution. After 10 min, 10 ml of 100 mM TEAB buffer (pH 8.0) was
added and stirred for one hour, followed by lyophilization. The reaction
mixture was dissolved in 5 ml-distilled water and extracted with ethyl
acetate. The aqueous layer was lyophilized, and Purified on DEAE
Sephadex A-25 anion exchange column with elution buffer (0.1 M-1.0M
TEAB). The Fractions containing compounds were concentrated and
treated with 1.0 N HCl for removal of Boc. The reaction was quenched
with aqueous ammonia and lyophilized before HPLC purification. The
1
purified sample was characterized as ap-TTP as a white powder. H NMR
(
400 MHz, D O) d 1.94 (s, 3 H), 3.94 (s, 1 H), 4.04 – 4.20 (m, 2 H), 4.26
2

1
2
C. R. GADE AND N. K. SHARMA
(
3
dd, J ¼ 14.7, 7.3 Hz, 1 H), 4.41 (dd, J ¼ 6.7, 3.1 Hz, 1 H), 7.57 (s, 1 H),
1PNMR (162MHz, D O):(ꢃ22.13, t, 1P, P ), (ꢃ11.16, ꢃ11.04,d, 1 P, P ),
2
b
c
(
ꢃ10.4, ꢃ10.16, d, 1P, P ) (J ¼19.44Hz, J ¼19.44Hz) HRMS (ESI-TOF)
a
ab
bc
Calculated for [C H NO P (M-H)] m/z437.9863, found m/z 437.9969.
8
15
12 3
Funding
CRG thanks DAE for fellowship, and also thanks to Ranjay Tiwari for solving crystal struc-
ture. This project financially supported by DST-SERB.
References
[
1] Dadov ꢀa , J.; Cahov ꢀa , H.; Hocek, M. Polymerase Synthesis of Base-Modified DNA. In
Modified Nucleic Acids; Springer, 2016; p 123–144.
[
2] Watson, J. D.; Crick, F. H. Molecular Structure of Nucleic Acids: A Structure
for Deoxyribose Nucleic Acid. Nature 1953, 171, 737–738. DOI: 10.1038/
1
71737a0.
[
3] Hocek, M. Enzymatic Synthesis of Base-Functionalized Nucleic Acids for Sensing,
Cross-Linking, and Modulation of Protein-DNA Binding and Transcription. Acc.
Chem. Res. 2019, 52, 1730–1737. DOI: 10.1021/acs.accounts.9b00195.
[4] Flamme, M.; McKenzie, L. K.; Sarac, I.; Hollenstein, M. Chemical methods for the
modification of RNA. Methods 2019, 161, 64–82.
[
5] Diafa, S.; Ev ꢀe quoz, D.; Leumann, C. J.; Hollenstein, M. Enzymatic Synthesis
0
0
of 7 ,5 -Bicyclo-DNA Oligonucleotides. Chem Asian J. 2017, 12, 1347–1352. DOI:
0.1002/asia.201700374.
1
[
[
6] Liu, C.; Cozens, C.; Jaziri, F.; Rozenski, J.; Mar ꢀe chal, A.; Dumbre, S.; Pezo, V.;
Marli ꢁe re, P.; Pinheiro, V. B.; Groaz, E.; Herdewijn, P. Phosphonomethyl
Oligonucleotides as Backbone-Modified Artificial Genetic Polymers. J. Am. Chem.
Soc. 2018, 140, 6690–6699. DOI: 10.1021/jacs.8b03447.
7] Mei, H.; Liao, J.-Y.; Jimenez, R. M.; Wang, Y.; Bala, S.; McCloskey, C.; Switzer, C.;
Chaput, J. C. Synthesis and Evolution of a Threose Nucleic Acid Aptamer Bearing
7-Deaza-7-Substituted Guanosine Residues. J. Am. Chem. Soc. 2018, 140, 5706–5713.
DOI: 10.1021/jacs.7b13031.
[
8] Bala, S.; Liao, J.-Y.; Zhang, L.; Tran, C. N.; Chim, N.; Chaput, J. C. Synthesis
0
of 2 -Deoxy-a-l-threofuranosyl Nucleoside Triphosphates. J. Org. Chem. 2018, 83,
8840–8850. DOI: 10.1021/acs.joc.8b00875.
[
9] Arangundy-Franklin, S.; Taylor, A. I.; Porebski, B. T.; Genna, V.; Peak-Chew, S.;
Vaisman, A.; Woodgate, R.; Orozco, M.; Holliger, P. A Synthetic Genetic Polymer
with an Uncharged Backbone Chemistry Based on Alkyl Phosphonate Nucleic acids.
Nat. Chem. 2019, 11, 533–542. DOI: 10.1038/s41557-019-0255-4.
[
[
10] Bose, T.; Kumar, V. A. Simple Molecular Engineering of Glycol Nucleic Acid:
progression from self-pairing to cross-pairing with cDNA and RNA. Bioorg. Med.
Chem. 2014, 22, 6227–6232. DOI: 10.1016/j.bmc.2014.08.022.
11] Horhota, A. T.; Szostak, J. W.; McLaughlin, L. W. Glycerol Nucleoside Triphosphates:
synthesis and Polymerase Substrate activities. Org. Lett. 2006, 8, 5345–5347. DOI: 10.
1021/ol062232u.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
13
[
[
[
[
[
[
[
[
12] Chen, J. J.; Tsai, C.-H.; Cai, X.; Horhota, A. T.; McLaughlin, L. W.; Szostak, J. W.
Enzymatic Primer-Extension with Glycerol-Nucleoside Triphosphates on DNA Templates.
PLoS one 2009, 4, e4949. DOI: 10.1371/journal.pone.0004949.
13] Zou, K.; Horhota, A.; Yu, B.; Szostak, J. W.; McLaughlin, L. W. Synthesis of alpha-
L-threofuranosyl nucleoside triphosphates (tNTPs)). Org. Lett. 2005, 7, 1485–1487.
DOI: 10.1021/ol050081þ.
0
14] Kaiser, A.; Spies, S.; Lommel, T.; Richert, C. Template-directed synthesis in 3 - and
0
5
-direction with reversible termination. Angew. Chem. Int. Ed. Engl. 2012, 51,
299–8303. DOI: 10.1002/anie.201203859.
8
15] Blain, J. C.; Ricardo, A.; Szostak, J. W. Synthesis and Nonenzymatic template-
0
0
directed polymerization of 2 -amino-2 -deoxythreose nucleotides. J. Am. Chem. Soc.
014, 136, 2033–2039. DOI: 10.1021/ja411950n.
2
0
0
16] Zhang, S.; Zhang, N.; Blain, J. C.; Szostak, J. W. Synthesis of N3 -P5 -linked
phosphoramidate DNA by nonenzymatic template-directed primer extension. J. Am.
Chem. Soc. 2013, 135, 924–932. DOI: 10.1021/ja311164j.
17] Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O. Sequence-Selective Recognition
of DNA by Strand Displacement with a Thymine-Substituted Polyamide. Science
1
991, 254, 1497–1500. DOI: 10.1126/science.1962210.
18] Martinez, C. I.; Ansari, M. A.; Gibbs, R.; Burgess, K. Acyclic Nucleoside
Triphosphate Analogs as Terminators in Biocatalytic DNA Replication. Bioorg. Med.
Chem. Lett. 1997, 7, 3013–3016. DOI: 10.1016/S0960-894X(97)10135-4.
19] Martinez, C. I.; Thoresen, L. H.; Burgess, K.; Gibbs, R. A. An allylic/acyclic
adenosine nucleoside triphosphate for termination of DNA synthesis by DNA
template-dependent polymerases . Nucleic Acids Res. 1999, 27, 1271–1274. DOI: 10.
1093/nar/27.5.1271.
[
20] Diederichsen, U. Oligomers with Intercalating Cytosine-Cytosineþ Base Pairs and
Peptide Backbone: DNA i-Motif Analogues. Angew. Chem. Int. Ed. Engl. 1998, 37,
2273–2276. DOI: 10.1002/(SICI)1521-3773(19980904)37:16<2273::AID-ANIE2273>3.0.
CO;2-P.
[21] Diederichsen, U.; Weicherding, D.; Diezemann, N. Side chain homologation of
alanyl peptide nucleic acids: pairing selectivity and stacking. Org. Biomol. Chem.
2
005, 3, 1058–1066. DOI: 10.1039/b411545g.
[
22] Talukder, P.; Dedkova, L. M.; Ellington, A. D.; Yakovchuk, P.; Lim, J.; Anslyn,
E. V.; Hecht, S. M. Synthesis of Alanyl Nucleobase Amino Acids and Their
Incorporation into proteins. Bioorg. Med. Chem. 2016, 24, 4177–4187. DOI: 10.
1016/j.bmc.2016.07.008.
[
[
[
23] Gade, C. R.; Dixit, M.; Sharma, N. K. Dideoxy Nucleoside Triphosphate (ddNTP)
Analogues: Synthesis and Polymerase Substrate Activities of Pyrrolidinyl Nucleoside
Triphosphates (prNTPs). Bioorg. Med. Chem. 2016, 24, 4016–4022. DOI: 10.1016/j.
bmc.2016.06.043.
24] Ludwig, J.; Eckstein, F. Rapid and Efficient Synthesis of Nucleoside
0
0
0
0
5
-0-(1-Thiotriphosphates), 5 -Triphosphates and 2 ,3 -Cyclophosphorothioates
Using 2-Chloro-4H-1,3,2-Benzodioxaphosphorin-4-One. J. Org. Chem. 1989,
4, 631–635. DOI: 10.1021/jo00264a024.
5
25] Patwa, A. N.; Gonnade, R. G.; Kumar, V. A.; Bhadbhade, M. M.; Ganesh, K. N.
Ferrocene-bis(thymine/uracil) conjugates: base pairing directed, spacer dependent
self-assembly and supramolecular packing. J. Org. Chem. 2010, 75, 8705–8708. DOI:
10.1021/jo101813z.

1
4
C. R. GADE AND N. K. SHARMA
[
26] Patwa, A. N.; Gupta, S.; Gonnade, R. G.; Kumar, V. A.; Bhadbhade, M. M.; Ganesh,
K. N. Ferrocene-linked thymine/uracil conjugates: base pairing directed self-
assembly and supramolecular packing. J. Org. Chem. 2008, 73, 1508–1515. DOI: 10.
1021/jo7023416.
[
27] Verma, S.; Mishra, A. K.; Kumar, J. The Many Facets of Adenine: coordination,
Crystal Patterns, and catalysis. Acc. Chem. Res. 2010, 43, 79–91. DOI: 10.1021/
ar9001334.
[28] Sivakova, S.; Rowan, S. J. Nucleobases as Supramolecular motifs. Chem. Soc. Rev.
2
005, 34, 9–21. DOI: 10.1039/b304608g.
[29] Perumalla, S. R.; Pedireddi, V. R.; Sun, C. C. Protonation of Cytosine: Cytosinium
vs Hemicytosinium Duplexes. Crystal Growth Design 2013, 13, 429–432. DOI: 10.
1021/cg3014915.
[
30] Pedireddi, V.; Ranganathan, A.; Ganesh, K. N. Cyanurate Mimics of hydrogen-
bonding patterns of nucleic bases: crystal structure of a 1:1 molecular complex
of 9-ethyladenine and N-methylcyanuric acid. Org. Lett. 2001, 3, 99–102. DOI: 10.
1021/ol006811p.
[
[
[
[
31] Sanjayan, G. J.; Pedireddi, V.; Ganesh, K. N. Cyanuryl-PNA Monomer:
synthesis and Crystal structure. Org. Lett. 2000, 2, 2825–2828. DOI: 10.1021/
ol006257j.
32] Gade, C. R.; Sharma, N. K. Helical supramolecular self-assembly by prolamide thy-
midine/uridine analogues. Supramol Chem 2018, 30, 965–969. DOI: 10.1080/
10610278.2018.1498500.
33] Sanger, F.; Nicklen, S.; Coulson, A. R. DNA Sequencing with Chain-Terminating
Inhibitors. Proc. Nat. Acad. Sci. USA 1977, 74, 5463–5467. DOI: 10.1073/pnas.74.12.
5463.
34] Hu, G. DNA polymerase-catalyzed addition of nontemplated extra nucleotides
0
to the 3 end of a DNA fragment . DNA Cell Biol. 1993, 12, 763–770. DOI: 10.1089/
dna.1993.12.763.