Synthesis and polymerase incorporation of β,γ-modified α-L-threofuranosyl thymine triphosphate mimics

Article abstract of DOI:10.1016/j.bmcl.2016.07.008

Three β,γ-modified α-L-threofuranosyl nucleoside triphosphates were synthesized. The β,γ-modified tTTPs undergo a single incorporation event with HIV RT but undergo multiple incorporations to form full-length product with engineered thermophilic polymeras

Full text of DOI:10.1016/j.bmcl.2016.07.008

Bioorganic & Medicinal Chemistry Letters 26 (2016) 3958–3962
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and polymerase incorporation of b,
c-modified
a-L-threofuranosyl thymine triphosphate mimics
⇑
Zhe Chen, Kirsten N. Meek, Alexandra E. Rangel, Jennifer M. Heemstra
Department of Chemistry and the Center for Cell and Genome Science, University of Utah, Salt Lake City, UT 84112, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Three b,c-modified a-L-threofuranosyl nucleoside triphosphates were synthesized. The b,c-modified
Received 2 May 2016
Revised 30 June 2016
Accepted 2 July 2016
Available online 4 July 2016
tTTPs undergo a single incorporation event with HIV RT but undergo multiple incorporations to form
full-length product with engineered thermophilic polymerases.
Ó 2016 Elsevier Ltd. All rights reserved.
Keywords:
TNA
Nucleosides
Triphosphate mimics
Polymerases
Primer extension
Alpha-
L
-threofuranosyl nucleic acid (TNA) is an artificial genetic
show anti-HIV activity, presumably from incorporation by HIV
RT, resulting in chain termination.10
Given the encouraging results from these studies, we were curi-
ous to explore the biological activity of tTTP analogues having
polymer that has been proposed as a possible biological precursor
to DNA and RNA.1–3 In addition to its potential role in early forms
of life, TNA has received great interest due to its unique chemical
and biological properties. For example, the 20–30 backbone linkage
found in TNA makes it highly resistant to cleavage by nucleases,4
but still allows TNA to form stable Watson–Crick duplexes with
DNA or RNA.5 Furthermore, TNA can be transcribed from a DNA
template using engineered versions of the thermophilic poly-
merases Deep Vent (exo-), 9°N, and KOD, which are capable of
modifications at the b,
(Fig. 1). b, -Modified dNTPs have been widely used as chemical
probes to study the catalytic mechanisms of DNA polymerases,
as the leaving group capability of the b, -diphosphate unit can
be fine-tuned by changing the electron withdrawing capability of
the unnatural bridging group.11–13 Additionally, b,
-modified
c-bridging oxygen of the triphosphate
c
c
c
accepting
a
-
L
-threofuranosyl nucleoside triphosphates tNTPs as
dNTPs have been reported to serve as substrates for a variety of
DNA polymerases. For example, Krayevsky and coworkers reported
substrates.6–9 These polymerases also retain their ability to utilize
deoxyribose nucleoside triphosphates dNTPs, suggesting that the
mutations made to the polymerases serve to relax their substrate
specificity.
that dTTP having a CF2 or CHF group replacing the b,
c-bridging
oxygen are substrates for human DNA polymerases
a and b, as well
as Avian Myeloblastosis Virus (AMV) reverse transcriptase.14 Tak-
ing a slightly different approach, Wang and coworkers synthesized
and studied a series of modified AZT triphosphate mimics. Unsur-
Considering the ability of tNTPs to be selectively accepted by
polymerases having relaxed substrate specificity or lack of exonu-
clease editing ability, TNA provides a potentially useful scaffold for
the design of anti-viral therapeutics. In 2003, Chaput and Szostak
carried out DNA primer extension experiments using HIV reverse
transcriptase (HIV RT). Interestingly, they found that the enzyme
will incorporate two sequential tTTP monomers, but this appears
to then cause chain termination.6 In a subsequent study, Herdewijn
and coworkers demonstrated that 20-deoxythreosyl phosphonates
prisingly, they found that modification of
inhibition of HIV RT. However, an AZT analogue having a b,
modification and a non-bridging -BH3 modification showed a Ki
a,b-linkage led to poor
c-CF2
a
value comparable to that of AZT, but with significantly enhanced
stability in serum and cell extract.15
Despite these encouraging results, b,c-modified triphosphates
have yet to be widely explored as potential therapeutics, as nucle-
oside triphosphates are typically not capable of penetrating the cell
membrane. However, Dinh and coworkers have reported a prodrug
approach aimed at overcoming this limitation through acylation of
⇑
the
c
-phosphate with a fatty acid or cholesterol analogue.16,17
Corresponding author. Tel.: +1 801 581 4191; fax: +1 801 581 8433.
E-mail address: heemstra@chem.utah.edu (J.M. Heemstra).
http://dx.doi.org/10.1016/j.bmcl.2016.07.008
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.

Z. Chen et al. / Bioorg. Med. Chem. Lett. 26 (2016) 3958–3962
3959
O
N
OH
O
N
H
HO
O
O
O
O
O
O
NH
NH
a,b
e
c
O
O
O
P
O
P
O
P
O
P
O
P
O
P
HO
OH
OH
OBz
HO
HO
HO
O
O
O
O
HO
X
O
O
O
2
3
L-ascorbic acid
OH OH OH
OH OH OH
OH
OH
TBDPSO
O
O
O
OH
OBz
d
O
f
OAc
tTTP, 1a
β,γ-modified tTTP analogues
1b
: X=CH2
: X=CHF
OBz
TBDPSO
OBz
TBDPSO
1c
6
4
5
1d: X=CF2
O
Figure 1. Chemical structure of tTTP 1a and b,
c
-modified tTTP analogues 1b–d.
O
NH
NH
N
O
N
O
Taking a similar approach, Meier and coworkers have recently
reported the successful cellular delivery of nucleoside triphos-
g
h
TBDPSO
HO
O
O
phates having a lipophilic acyloxybenzyl group at the c-phosphate,
OBz
OBz
which can be cleaved by intracellular esterases to reveal the nucle-
oside triphosphate therapeutic.18,19 These recent developments
suggest that viable prodrug approaches could be implemented
for b,c-modified nucleoside triphosphates, which encouraged us
to further explore their ability to be incorporated by DNA
7
8
Scheme 1. Synthesis of 20-O-Bz protected thymine nucleoside 8. Reagents and
conditions: (a) (i) 30% H2O2, CaCO3, H2O, 18 h, 0 °C-rt; (ii) active charcoal, 70 °C, 2 h,
80%; (b) 10% H2SO4 in H2O, pTsOH, CH3CN, 2 h, reflux, 90%; (c) BzCl, pyridine,
CH2Cl2; 0 °C, 0.5 h, 75%; (d) TBDPS-Cl, imidazole, DMAP (cat), CH3CN, 18 h, 0 °C-rt
86%.; (e) DIBAL-H, THF, ꢀ78 °C, 0.5 h; (f) Ac2O, DMAP, pyridine, CH2Cl2, ꢀ78 °C-rt,
1.5 h, 90% from 4; (g) (i) thymine, BSA, CH3CN, 60 °C, 15 min; (ii) TMSOTf, 60 °C, 2 h,
82%; (h) TBAF, THF, 0 °C, 1 h, 86%.
polymerases.
Here we report the synthesis and study of three b,
c-modified
tNTP analogues, which represents the first example of b,
c
-modifi-
cations in the context of a non-native carbohydrate scaffold. We
synthesized ‘wild-type’ tTTP 1a and tTTP analogues having a CH2,
CHF, or CF2 group replacing the b,c-bridging oxygen (1b–d), and
compared their incorporation efficiencies in primer extension reac-
tions using a variety of polymerases. Excitingly, we find that the b,
O
P
O
P
O
P
O
P
O
P
O
P
F
F
H
O
O
O
O
O
O
a
O
O
O
O
O
O
H
F
H
c
-modified tTTPs are substrates for HIV RT, and unlike 1a, which
11
10
9
requires two incorporations before chain termination, 1b–d show
no further elongation after only a single incorporation event. Addi-
tionally, we demonstrate that a mutant KOD polymerase is capable
of generating full length product in a primer extension experiment
b
b
O
P
O
O
P
O
P
F
F
using b,c-CF2 tTTP 1d. Together, these experiments explore new
OH
P
OH
HO
HO
chemical space by combining carbohydrate modifications with
triphosphate modifications, and demonstrate that this approach
can lead to enhanced inhibition of a viral reverse transcriptase.
To synthesize tTTPs 1a–d, we began by following a recently
HO
OH
HO
OH
H
F
12
13
Scheme 2. Synthesis of modified diphosphate building blocks 12 and 13. Reagents
and conditions: (a) (i) NaH, THF, DMF, 0 °C-rt 1 h; (ii) Selectfluor, rt, 3 h; (iii) NH4Cl,
H2O, 18% (10) and 30% (11); (b) CH2Cl2, TMSBr, rt, 48 h, 70–85%.
published procedure for generating 20-O-Bz protected
a-
L-threose
nucleoside 8 (Scheme 1).20 Starting from
L
-ascorbic acid, we were
able to synthesize 8 in high yield over 8 steps, providing the
mono-protected nucleoside precursor necessary for elaboration
into tTTP 1a and analogues 1b–d.
intermediate 14 when applied to the sterically hindered 30 hydro-
xyl of threose nucleoside 8. To compensate for the increased steric
hindrance of 8, we investigated the use of a variety of organic bases
in conjunction with the POCl3 and PO(OMe)3, and observed that
this significantly improved the efficiency of the phosphorylation
reaction. Specifically, we screened a series of nitrogen-containing
organic bases including pyridine, Et3N, Bu3N, DMAP, and proton
sponge (1,8-bis(dimethylamino)natpthalene), and found that pro-
ton sponge provided the highest yield of 14, with 80% conversion
observed by HPLC. Moreover, the hydrophobicity of the proton
sponge base enabled it to be easily removed at the conclusion of
the reaction sequence via extraction with Et2O under weakly basic
aqueous conditions. Having optimized conditions for generating
intermediate 14, we next turned to investigating the remainder
In parallel, we synthesized pyrophosphate analogues (fluo-
romethylene)bisphosphonic acid 12 and (difluoromethylene)-bis-
phosphonic acid 13 starting from the commercially available
tetraisopropyl methylenediphosphonate (9) according to a previ-
ously reported procedure (Scheme 2).11 While synthesis of
deoxyribose nucleoside 50 triphosphates is relatively straightfor-
ward, synthesis of threose nucleoside 30 triphosphates (tNTPs) is
much more difficult, owing to the increased steric hindrance of
the 3-hydroxyl group.21 Thus, once all of our key intermediates
were in hand, we chose to explore both one-pot and stepwise syn-
thetic routes to access the target compounds tTTP 1a and b,
c-
bridging oxygen modified tTTP analogues 1b–d.
To investigate the possibility of a one-pot synthesis of tTTP 1a
and analogues 1b–d from nucleoside intermediate 8, we first
focused on the synthesis of dichlorophosphoridate 14, which we
envisioned as a key intermediate (Scheme 3). The widely used
method for synthesizing nucleoside 50 triphosphates involves for-
mation of a dichlorophosphoridate intermediate via reaction of
the 50 hydroxyl with POCl3 in PO(OMe)3 using Yoshikawa’s
procedure.22 However, this approach failed to yield the desired
of the one-pot synthetic method. To couple the b,c-diphosphate
unit, we added to the reaction mixture tributylamine and either
tributylammonium pyrophosphate, methylenediphosphonic acid,
12, or 13, which yielded the benzoyl protected precursors to 1a–
d, respectively. The benzoyl group was then removed using con-
centrated ammonium hydroxide, and 1a–d were each purified
via aqueous extraction followed by reverse phase HPLC using a

3960
Z. Chen et al. / Bioorg. Med. Chem. Lett. 26 (2016) 3958–3962
One-pot synthesis:
O
N
O
N
O
NH
NH
NH
O
O
P
Cl
O
P
O
P
O
P
O
N
O
Cl
O
b
a
HO
X
O
O
O
HO
O
O
OH OH OH
OBz
OH
OBz
1a: X=O
14
8
1b: X=CH2
1c: X=CHF
1d: X=CF2
Stepwise Hoard-Ott synthesis:
O
O
N
O
N
NH
NH
NH
O
O
P
O
P
O
P
O
P
O
N
O
HO
X
O
O
O
c
d
HO
O
HO
O
O
OH OH OH
OH
OH
OBz
OBz
1a: X=O
8
15
1b: X=CH2
1c: X=CHF
1d: X=CF2
Scheme 3. Synthesis of tTTP 1a and analogues 1b–d using a one-pot or stepwise method. Reagents and conditions: (a) POCl3, PO(OMe)3, proton sponge; (b) (i)
tributylammonium pyrophosphate, methylenediphosphonic acid, 12, or 13, Bu3N, DMF; (ii) 1 M TEAB buffer, 15 min; (iii) NH4OH, rt, 4–6 h. Yields for 1a–d ꢁ 5%. (c) (i) POCl3,
PO(OMe)3, proton sponge, 0 °C, 2–4 h, 80%; (d) (i) CDI, DMF, rt, 4–6 h; (ii) tributylammonium pyrophosphate, methylenediphosphonic acid, 12, or 13, Bu3N, DMF, rt, 16–48 h;
(iii) 1 M TEAB buffer, 15 min; (iv) NH4OH, rt, 4–6 min. Yields from 8: 46% for 1a; 41% for 1b; 36% for 1c, and 30% for 1d.
C18 column. Together, the one-pot synthesis provided the desired
nucleoside triphosphates 1a–d, but the isolated yield of each pro-
duct was lower than expected at approximately 5%. We hypothe-
size that this low yield can be attributed to the high sensitivity
of dichlorophosphoridate intermediate 14 to trace water, as this
may have led to hydrolysis or other unwanted byproducts.
investigate the activity of our b,c-modified tTTP analogues 1b–d.
To survey primer extension efficiency, we utilized a previously
described assay format24 in which a DNA primer labeled with a
50 fluorescein (FAM) was annealed to a template having the com-
plement of the primer sequence followed by an A7 sequence. This
duplex was then combined with 50
lM of either dTTP, tTTP 1a, or
Although the one-pot synthetic method described above did not
produce the desired triphosphates in high yield, our observation
that proton sponge could efficiently catalyze the mono-phosphory-
lation reaction was useful toward developing a stepwise synthetic
route. Our stepwise route was inspired by the Hoard-Ott method,
in which a nucleoside monophosphate is prepared, then activated
using carbonyldiimidizole (CDI) to promote subsequent coupling
with pyrophosphate.23 As shown in Scheme 3, we utilized the same
conditions from our one-pot method to generate 14 in situ, but this
was hydrolyzed to nucleoside 30 monophosphate 15 via aqueous
workup under basic conditions. Monophosphate 15 was then acti-
vated using CDI, and reacted with either tributylammonium
pyrophosphate, methylenediphosphonic acid, 12, or 13, providing
the benzoyl protected precursors to 1a–d. Similar to our one-pot
method, the benzoyl group was deprotected using ammonium
hydroxide. The crude tTTPs were then precipitated from ethanol,
purified by reverse phase HPLC using a C18 column, and character-
ized by HPLC, 1H NMR, 19F NMR, 31P NMR and mass spectrometry.
The stepwise synthetic route provided 1a–d with overall reaction
yields of 30–46% from nucleoside intermediate 8, representing a
significant improvement over the one-pot method.
one of the b, -modified tTTP analogues 1b-d, along with the HIV RT
c
enzyme. After incubating for 30 min at 37 °C, each primer exten-
sion reaction was quenched by the addition of denaturing gel load-
ing buffer, and run on a 20% denaturing PAGE gel. The gel was
imaged for FAM emission using a laser gel scanner to determine
the length of the primer sequence after primer extension using
each nucleoside triphosphate.
As shown in Figure 2 (lanes 2–6), all of the nucleoside triphos-
phates tested were capable of incorporation by HIV RT. As antici-
pated, dTTP provided the full length primer extension product
(lane 2), indicating processive incorporation, and tTTP provided a
primer extension product having two nucleotides added (lane 3),
consistent with the previous observation from Chaput and
Szostak.6 However, we observed that each of the b,
analogues undergo a single incorporation event, then block further
polymerization (lanes 4–6). Of the three tTTP analogues, b, -CH2-
c-modified tTTP
c
tTTP (1b, lane 4) appears to be the best potential inhibitor of HIV
OH3'
5'
primer
PBS
Once we had tTTP 1a and b,c-modified analogues 1b–d in hand,
AAAAAAA5'
3'
we set out to investigate their ability to serve as substrates for
polymerase enzymes. Given that DNA polymerases vary signifi-
cantly in their discrimination of modified nucleoside triphosphate
substrates, we chose to examine four polymerases across two cat-
egories: (1) HIV reverse transcriptase, for which tTTP had previ-
ously been shown to stop chain elongation, and (2) Kod RI, 9°N
RI, and Therminator, which are each able to polymerize TNA on a
DNA template using ‘wild type’ tNTPs.
lane
triphosphate
1
-
2
3
4
5
6
7
-
1a 1b 1c 1d
dTTP
+7
+2
+1
primer
Considering the previously demonstrated ability of tTTP and b,
-modified AZT analogues to inhibit HIV RT, we were curious to
Figure 2. Primer extension assay using HIV reverse transcriptase to analyze chain
termination by 1a–d. PBS = primer binding site.
c

Z. Chen et al. / Bioorg. Med. Chem. Lett. 26 (2016) 3958–3962
3961
5'
3'
OH3'
primer
PBS
AACACCAAACAAACCAACTACCATAACATACAACATATCA 5'
1
1a
2
-
3
4
5
6
7
-
8
1b
9
1c
10 11 12 13 14 15 16
1d 1a 1b 1c 1d
lane
tTTP
1b 1c 1d 1a
-
PC
9oN RI
Therminator
KOD RI
Figure 3. Primer extension assay using engineered thermophilic polymerases to analyze incorporation of 1a–d. PBS = primer binding site; PC = primer control.
RT, as incorporation to give the +1 primer extension product is
nearly quantitative, but no +2 product is observed on the gel. In
In conclusion, we report the synthesis and polymerase compat-
ibility of three novel b, -modified tTTP analogues 1b–d. The mod-
c
comparison, b,
ration, with a significant amount of unreacted primer remaining,
and b, -CF2-tTTP (1d, lane 6) gives primarily the +1 product, but
a small amount +2 product can also be observed. Together, these
results demonstrate that b, -modification can enhance the ability
c-CHF-tTTP (1c, lane 5) shows less efficient incorpo-
ified 30 nucleoside triphosphates were prepared in high yield using
the Hoard–Ott method, and were fully characterized by HPLC, 1H
NMR, 19F NMR, 31P NMR and mass spectrometry. We envision that
this synthetic approach can be easily extended to enable the future
c
c
synthesis of b,c-modified analogues of tATP, tGTP and tCTP. Primer
of unnatural nucleoside triphosphates to stop chain elongation,
extension assays using HIV RT revealed that 1b–d are more effi-
and the magnitude of this effect can be tailored by changing the
cient than ‘wild type’ tTTP 1a at stopping chain elongation, as ter-
structure of the bridging group at the b,
In addition to testing the activity of 1b–d with HIV RT, we were
curious to explore whether the b, -modified tTTP analogues could
c
-position.
mination is observed after
a
single incorporation event.
Additionally, we found that the modified tTTPs could serve as sub-
strates for the KOD RI, 9°N RI, or Therminator polymerases that
have been previously reported for DNA-dependent TNA polymer-
ization. In this case, we find that the modified triphosphates are
less efficiently incorporated than tTTP. However, full length primer
c
serve as substrates for the engineered thermophilic polymerases
that have been shown to transcribe TNA polymers from DNA tem-
plates. For these experiments, we utilized a mixed-sequence DNA
template composed of nucleotides T, C, and A (Fig. 3). This tem-
plate sequence was chosen because TNA polymerization on G-rich
templates is known to be challenging, and the use of a template
having >50% A would provide a rigorous test for the enzymatic
polymerization of our modified tTTP analogues. To survey primer
extension efficiency, we annealed the template with a FAM-labeled
extension product can be formed using b,c-CF2 triphosphate 1d in
conjunction with the KOD RI polymerase. Together, these studies
are the first to explore the synthesis and enzymatic activity of
nucleoside triphosphates having both a non-ribose sugar and mod-
ifications to the triphosphate unit. We anticipate future expansion
of this chemical space to investigate additional b,
and nucleobases, as well as prodrug modifications to the b,c
ified triphosphates. We envision that this will provide additional
insight into the substrate preferences of polymerase enzymes
and further promising leads for anti-viral drug development.
c
-modifications
-mod-
primer in a 2:1 molar ratio in 1ꢂ Thermopol buffer with 1
MnCl2 and KOD RI, 9°N RI, or Therminator polymerase.9 We then
added to each reaction mixture 100 M of tTTP 1a or b, -modified
tTTP 1b–d, along with 100 M each of tATP, tGTP, and tCTP. The
lM
l
c
l
reaction mixtures were incubated for 8 hours at 55 °C, then
quenched with stop buffer (1ꢂ TBE, 20 mM EDTA, 7 M urea) and
analyzed via 12% denaturing PAGE gel. For comparison, control
reactions were performed having no tTTP (lanes 2, 7, and 12). As
Acknowledgments
This work was supported by the DARPA Folded Non-Natural
Polymers with Biological Function (Fold F(x)) Program under
award number N66001-14-2-4054. Any opinions, findings, and
conclusions or recommendations expressed in this publication
are those of the authors and do not necessarily reflect the views
of DARPA. We are thankful to Prof. John C. Chaput and laboratory
for the generous gift of plasmids for KOD RI and 9°N RI, and for
many helpful conversations.
shown in Figure 3, all of the b,c-modified tTTP analogues provide
some degree of primer extension compared to the control lanes
having tTTP omitted, although with lower efficiency compared to
‘wild type’ tTTP 1a. Of the three modified tTTP analogues, incorpo-
ration efficiency follows the trend of 1d > 1c > 1b, which is not sur-
prising given the increased leaving group ability of the
diphosphates having electron withdrawing fluorine atoms. We also
observed that primer extension efficiency followed the trend of
KOD RI > 9°N RI > Therminator with regard to enzyme identity.
While these results show that analogues 1b–d are less efficient
than 1a for transcription of TNA polymers, we are encouraged that
the combination of CF2-modified analogue 1d with KOD RI poly-
merase does provide some full length product in the primer exten-
sion reaction (lane 5). The differences in incorporation efficiency of
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.07.
008.
the b,c-modified triphosphate mimics using engineered ther-
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the pyrophosphate as increasingly electronegative atoms are
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