Phospho-carboxylic anhydride of a homologated nucleoside leads to primer degradation in the presence of a polymerase

Article abstract of DOI:10.1016/j.bmcl.2014.04.042

Starting from thymidine, through a series of key synthetic transformations (e.g., Wittig reaction, hydroboration, Mitsunobu reaction and TEMPO oxidation) a nucleoside homologue bearing a phospho-carboxylic anhydride group at 6′ position was synthesized. The potential of polymerases to catalyze amide bond formation was investigated by using a modified primer with an amino group at 3′ position and the synthesized phosphoanhydro compound as substrate. Unfortunately, we did not observe the desired product either by gel electrophoresis or mass spectrometry. In contrast, the instability of the phosphoanhydro compound could lead to pyrophosphate formation and thus, to pyrophosphorolysis of the primer rather than to primer extension.

Full text of DOI:10.1016/j.bmcl.2014.04.042

Bioorganic & Medicinal Chemistry Letters 24 (2014) 2720–2723
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Phospho-carboxylic anhydride of a homologated nucleoside leads
to primer degradation in the presence of a polymerase
⇑
Dhuldeo Kachare, Xiao-Ping Song, Piet Herdewijn
Medicinal Chemistry, Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, 3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 20 February 2014
Revised 9 April 2014
Accepted 10 April 2014
Available online 19 April 2014
Starting from thymidine, through a series of key synthetic transformations (e.g., Wittig reaction, hydrob-
oration, Mitsunobu reaction and TEMPO oxidation) a nucleoside homologue bearing a phospho-carbox-
ylic anhydride group at 60 position was synthesized. The potential of polymerases to catalyze amide bond
formation was investigated by using a modified primer with an amino group at 30 position and the syn-
thesized phosphoanhydro compound as substrate. Unfortunately, we did not observe the desired product
either by gel electrophoresis or mass spectrometry. In contrast, the instability of the phosphoanhydro
compound could lead to pyrophosphate formation and thus, to pyrophosphorolysis of the primer rather
than to primer extension.
Keywords:
Phospho-carboxyl anhydride
Nucleosides
Ó 2014 Elsevier Ltd. All rights reserved.
Polymerases
Phosphorolysis
Homologated AZT
In coeval life, proteins and nucleic acids are intricately depen-
dent upon each other for a host of functions. Protein enzymes
(polymerases) are necessary for the synthesis of DNA and RNA,
while nucleic acids (ribosomes) are necessary for the synthesis of
proteins. According to the RNA world hypothesis, early life used
nucleic acids for both information storage and chemical catalysis
before the emergence of protein enzymes. However, it still remains
unclear how nucleic acids were able to assemble and replicate
before the advent of protein enzymes. This means that in contem-
porary life, proteins can catalyze phosphodiester bond formation
(nucleic acid synthesis) and nucleic acids can catalyze amide bond
formation (protein synthesis). Our motivation in this context
comes from the recently proposed ‘Molecular Midwife’ hypothesis1
and has the aim to investigate if polymerases can also catalyze
amide bond formation. Furthermore, investigations on alternative
information systems in the field of synthetic biology ask for a care-
ful mapping of the substrate specificity of natural enzymes, such as
polymerases.2
reaction and (b) the introduction of a positive charge in the oligo-
nucleotide (after incorporation reaction) to facilitate detection by
gel chromatography and mass spectrometry. Due to the expected
difficulties in obtaining a carboxylate activated, unprotected amino
acid, we decided to test the reaction with a 30-azido congener. The
azido group could then be reduced to an amino group after incor-
poration, if that would be necessary for analytic purposes.
The synthetic scheme started from commercially available thy-
midine 1, in which compounds 2 and 3 are synthesized by using
literature procedures reported by Tronchet et. al.3
Compound 3 was oxidized to 4 using Dess–Martin periodinane
(DMP) reagent. Wittig reaction was carried out on 4, using potas-
sium tert-butoxide and methyl triphenylphosphonium bromide
in THF to afford the vinyl compound 5. The vinyl compound 5
was reacted with benzylchloromethyl ether (BOM-Cl) to protect
the NH group in thymine ring to furnish compound 6. BOM pro-
tected compound 6 was reacted with 9-borabicyclo[3.3.1]nonane
(9-BBN) and subsequent treatment with hydrogen peroxide in
alkaline medium to give 7.4 The removal of BOM group by treat-
ment with Pd/C 10% wt. in MeOH in presence of cyclohexene under
reflux conditions, also lead to removal of the TBS group at the 30
position affording 8. Homologated thymidine 8 was protected at
the 60-O-position with 4,40-dimethoxytrityl chloride in pyridine
yielding compound 9. The subsequent synthesis of compound 11
involved an inversion of the 30-carbon atoms from S-configuration
to R-configuration which was accomplished in two steps. Cyclisa-
tion of compound 9 under Mitsunobu condition gave the O2,
30-anhydro derivative 10, which is a useful intermediate for
In a first effort to test the potential of polymerases to catalyze
an amide bond, we considered the synthesis of a modified nucleo-
side with a carboxylate group at 60 position and an amine group at
30 position. The carboxylate group needs to be activated (for exam-
ple as mixed anhydride with phosphate) before the compound can
be tested as substrate for polymerases. The selection of a 30-amino
group is based on (a) the possibility to get chain elongation
⇑
Corresponding author. Tel.: +32 16 337387; fax: +32 16 337340.
E-mail address: Piet.Hedewijn@rega.kuleuven.be (P. Herdewijn).
http://dx.doi.org/10.1016/j.bmcl.2014.04.042
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

D. Kachare et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2720–2723
2721
O
N
O
N
O
N
O
N
NH
NH
NH
NH
O
O
O
O
b)
c)
HO
a)
TBSO
H
O
HO
d)
O
O
O
O
OTBS
OTBS
OTBS
OH
1
2
3
4
O
O
O
N
O
O
N
BOM
BOM
O
NH
NH
N
NH
N
DMTr
N
O
N
O
OH
OH
N
O
O
O
e)
f)
g)
h)
O
O
OH
O
O
O
OTBS
OTBS
OH
OTBS
5
6
7
8
9
O
O
N
O
O
NH
N
NH
NH
DMTr
DMTr
N
O
OH
O
O
O
N
O
OH
N
O
l)
k)
i)
j)
O
O
O
O
O
N3
13
N3
N3
12
10
11
Scheme 1. Reagents and conditions: (a) TBDMSCl, Py, added at rt for 1 h, then stirred at 60–70 °C, 22 h, 98.8%; (b) TFA/H2O (10:1, v/v), DCM, rt, 45 min then ice bath for
30 min, 80%; (c) Dess–Martin periodinane, MeCN, 80 °C; (d) methyltriphenylphosphonium bromide, potassium tert-butoxide, THF, added at 0 °C and stirred at rt, overnight,
53%; (e) BOM-Cl, DBU, MeCN, 0 °C to rt, 2 h, 80.6%; (f) 9-BBN, THF, added at 0 °C and stirred at rt, overnight; then added NaOH and H2O2 at 0 °C and stirred at rt, 6 h, 57.2%; (g)
Pd/C 10% wt., cyclohexene, MeOH, reflux, 57%; (h) DMTr-Cl, pyridine, rt, 70.7%; (i) PPh3, DIAD, MeCN, 0 °C, 12 h, 87.7%; (j) NaN3, DMF, 140 °C, 12 h, 85.3%; (k) 3%
trichloroacetic acid, DCM, rt, 66.7%; (l) TEMPO, bis(acetoxy)iodobenzene, MeCN/H2O (1:1, v/v), rt, overnight, 57.7%.
substitutions at the 30-position. Opening of the aza-eno–ether in 10
with sodium azide in DMF gave protected AZT derivative 11.5 Com-
pound 11 was deprotected with 3% trichloroacetic acid to furnish
homologated alcohol 12, which was subsequently oxidized at 60
position to afford the corresponding carboxylic acid 13 (Scheme 1).
Acyl monophosphates (14) are known as intermediates in bio-
synthesis, but unfortunately due to the low stability their synthesis
and purification are not much explored. As far as we know, there is
no literature dealing with the synthesis of mixed anhydrides (15)
in which the nucleoside moiety contains a carboxylic acid func-
tionality (Scheme 2).
Our first attempt to obtain anhydride 15 from compound 13 by
using bis(trimethylsilyl)-tributylstannyl phosphate was unsuc-
cessful.6 Further efforts for its synthesis by using diethyl chloro-
phosphate, diphenyl chlorophosphonate, and bis(tetraethyl
ammonium) ethyl phosphate were also unsuccessful.7,8 Therefore
we focused on a two-step reaction. The acid 13 was first reacted
with ethylchloroformate and tri-n-butylamine to furnish the corre-
sponding anhydride, then the obtained intermediate was reacted
with tri-n-butylammonium phosphate (0.5 M in DMF) affording
the desired mixed anhydride 15 (Scheme 2).9,10 The tri-n-butylam-
monium phosphate (0.5 M in DMF) reagent was synthesized by
using a reported procedure by El-Tayeb et al.11 Compound 15,
however, was unstable and was isolated by partitioning the reac-
tion mixture between ethyl acetate and cold water. The product
was collected in the aqueous layer and isolated by freeze drying.
Further purification of compound 15 was carried out by HPLC with
C18 column using a gradient elution buffer of triethylammonium
acetate (TEAA) and acetonitrile.
In the context of probing template-directed amide bond forma-
tion by polymerases, compound 15 bearing a phospho-carboxylic
acid anhydride function at 60-position of sugar moiety was used
as substrate, a modified primer (P1) (see Table 1) was installed
with 30-NH2-20,30-dideoxycytidine at 30 terminal and template T1
O
O
NH2
NH
N
N
NH
O
O
N
O
O
O P
OH
N
O
P
OH
O
N
O
N
i)
R
O
HO
O
O
HO
ii)
O
O
N3
13
N3
15
Mixed anhydride
HO
14
Acyl-AMP
OH
Scheme 2. Chemical structure of acyl phosphate (14) and mixed anhydride (15); (i) ethylchloroformate, n-Bu3N, 1,4-dioxane, 0 °C–rt; (ii) 0.5 M tri-n-butylammonium
phosphate, DMF.

2722
D. Kachare et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2720–2723
Figure 1. (A) incorporation study of compound 15 as substrate; (B) enzymatic reaction using pyrophosphate (PPi) as substrate; blank: no substrate in the reaction.
[P1T1] = 125 lM; [Therminator™ DNA pol] = 0.05 U/lL; time points: 10, 30 and 90 min.
Table 1
the abundant salts in the reaction were removed by Illustra™
Microspin™ G-25 column. Unfortunately, no desired product was
detected.
Overview of primer–template complexes used in the enzymatic polymerization
Primer 1 (P1)
Primer 2 (P2)
Template 1 (T1)
50-CAGGAAACAGCTATGACNH2-30
50-CAGGAAACAGCTATGAC-30
30-GTCCTTTGTCGATACTGACTGC-50
Enzymatic DNA polymerization is a reversible reaction.12 The
presence of high concentration of pyrophosphate in the reaction
mixture can lead to pyrophosphorolysis of primer or template to
form nucleoside triphosphates.12 Interestingly, as shown in
Figure 1, similar patterns were obtained for the reaction when
using either compound 15 (Fig. 1A) or pyrophosphate (Fig. 1B) as
substrate. The above observation demonstrates that the presence
of compound 15 can lead to pyrophosphorolysis of primer and/or
template in the presence of Therminator DNA polymerase. Most
probably, pyrophosphate is formed in the reaction mixture by
reaction of organic phosphate with 15. The necessary phosphate
could be originated in the partial degradation of compound 15 in
the reaction medium, which is an unstable compound. No primer
degradation or extension was observed when the reaction was per-
formed without enzyme (picture not shown). Primer degradation
was significantly suppressed in the presence of pyrophosphatase
(PPase) (Fig. 2A). Additionally, when using the unmodified oligonu-
cleotide as primer (P2) in the Therminator™ DNA polymerase
catalyzed reaction, compound 15 can cause similar primer degra-
dation pattern as showed in Figure 2B. The above experiments
show that compound 15 could induce similar primer degradation
as pyrophosphate in the presence of Therminator™ DNA polymer-
ase, due to pyrophosphate generation in the reaction mixture. The
observed primer extension (Fig. 2) might be explained by the
incorporation of dNMP’s (natural deoxynucleoside monophos-
phate), which were formed during degradation (as nucleoside tri-
phosphates) of primer and/or template by pyrophosphorolysis in
the presence of Therminator™ DNA polymerase.2
was employed as primer–template duplex. Initially, the substrate
properties of compound 15 were evaluated by four enzymes
(HIV-1 RT, Therminator, Taq and VentRÒ (exo-) DNA polymerase)
from different families. Unfortunately, using HIV-1 RT, Taq, and
VentRÒ (exo-) DNA polymerase as catalyst, no primer extension
was obtained. A small amount of primer extension (P+1, P+2)
was observed using Therminator™ DNA polymerase as catalyst,
and also several bands were detected below the primer baseline
(Fig. 1A).
Gel electrophoresis is a popular tool used to separate nucleic
acids based on both their molecular weight and charges. In our
case, when compound 15 would be successfully incorporated into
the primer (P1) the extended oligonucleotide will keep the same
number of negative charges and an increase in molecular weight
of 277 m/z. Regarding the minor difference between primer P1
and the formed product, protonation of the amine group of P1
might be a feasible way to change the gel mobility difference of
P1 and [P1+13]. Analytical gels of the Therminator™ DNA polymer-
ase catalyzed reaction mixture at two different pH values (gel 1
pH = 6.25, gel 2 pH = 8.3) were performed in order to detect the
small difference between P1 and the primer extended product.
However, no significant difference was detected for the gel picture
obtained at lower pH value comparing to the gel showed in
Figure 1A (pH = 8.3).
Furthermore, investigation to detect the incorporated product
was performed by mass spectrometry analysis. The sample was
prepared with unlabeled primer P1 and template T1. After reaction
The synthesis of the 50-one carbon extended nucleoside
homologue was readily accomplished from commercially available

D. Kachare et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2720–2723
2723
Figure 2. (A) incorporation study of compound 15 using P1T1 as primer/template duplex in absence and presence of pyrophosphates, time points: 10, 20, 30 60, 120 min; (B)
incorporation study of compound 15, in absence and presence of pyrophosphatase using P2T1 as primer/template duplex, time points: 10, 30 and 90 min. [P1T1] = 125 M,
[P2T1] = 125 M, [Therminator™ DNA pol] = 0.05 U/ L, [PPase] = 0.01 U/ L, [compound 15] = 500 M, [dTTP] = 50 M.
l
l
l
l
l
l
thymidine. The key steps in the synthesis were the Wittig reaction
and the hydroboration of the thymidine precursor with 9-BBN to
furnish the corresponding homologated alcohol 12. The inversion
of configuration and introduction of the azide group at 30 position
was achieved by Mitsunobu reaction. Oxidation of compound 12
using TEMPO leads to the corresponding acid 13. Finally, a two-
step reaction was explored in order to obtain the mixed anhydride
15. Compound 13 was first activated by ethylchloroformate, then
the activated nucleoside carboxylic acid was reacted with tri-n-
butylammonium phosphate (0.5 M in DMF) to give the desired
product.
agreement n8 ERC-2012-ADG_20120216/320683. XNA = xenobi-
otic nucleic acids. Dr. D.K. received a research Grant from Federaal
Wetenschapsbeleid. Dr. X.-P.S. a research associate of the Belgian
National Fund for Scientific Research (FWO).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.
04.042.
The potential of polymerases to catalyze the amide bond forma-
tion between the 30-amino group of the modified primer and the
50-carboxylate group of compound 15 on a template directed man-
ner was investigated by a series of enzymes from different families.
Unfortunately, no product formation was detected either by mass
spectrometry or gel electrophoresis. One possible explanation
could be that compound 15 has not the appropriate geometry to
be recognized by the active site of polymerases. On the other hand,
the instability of the phospho-anhydride group of the mixed anhy-
dride 15 may lead to the formation of a significant amount of pyro-
phosphate and, thus, rather to primer degradation by enzymatic
pyrophosphorolyis than to primer extension.
References and notes
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Acknowledgments
10. Bonnaffe, D.; Dupraz, B.; Ughettomonfrin, J.; Namane, A.; Dinh, T. H.
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The research leading to these results has received funding from
the European Research Council under the European Union’s
Seventh Framework Program (FP7/2007-2013)/ERC Grant