Chemoselective Dimerization of Phosphates

Article abstract of DOI:10.1021/acs.orglett.6b01466

A methodology for the synthesis of oligophosphate conjugates using phosphordiamidites is described. This strategy facilitates the straightforward preparation of C₂-symmetric dinucleoside tri-, penta-, and heptaphosphates. Moreover, unsymmetric

Full text of DOI:10.1021/acs.orglett.6b01466

Letter
pubs.acs.org/OrgLett
Chemoselective Dimerization of Phosphates
Alexandre Hofer,† Elia Marques,† Nicole Kieliger,† Sarah-Kirsten N. Gatter,† Sara Jordi,† Elena Ferrari,‡
Manuel Hofmann,§ Teresa B. Fitzpatrick,§ Michael O. Hottiger,‡ and Henning J. Jessen*,∥
‡
†Department of Chemistry and Department of Molecular Mechanisms of Disease, University of Zurich, Winterthurerstrasse 190,
CH-8057 Zurich, Switzerland
§Plant Biochemistry & Physiology Laboratory, Department of Botany and Plant Biology, University of Geneva, Quai E. Ansermet 30,
1211 Geneva, Switzerland
∥Institute of Organic Chemistry, Albert-Ludwigs-University Freiburg, Albertstr. 21, 79104 Freiburg i. B., Germany
S
* Supporting Information
ABSTRACT: A methodology for the synthesis of oligophos-
phate conjugates using phosphordiamidites is described. This
strategy facilitates the straightforward preparation of C2-
symmetric dinucleoside tri-, penta-, and heptaphosphates.
Moreover, unsymmetric compounds such as thiamine
adenosine triphosphate and thiamine cytidine triphosphate
can be prepared. The material is used to study the inhibitory
activity of thiaminylated nucleotides against adenosine
diphosphate ribosyltransferases.
ribosyltransferases (formerly poly-ADP-ribose polymerases;
PARPs).
inucleoside polyphosphates (NpxNs) and thiaminylated
Dnucleotides are terminally modified oligophosphates. In
this paper, a rapid, general, and convenient synthesis of C2-
symmetric NpxNs with odd numbers of phosphate units and
thiaminylated nucleotides is presented, which is based on
chemoselective homologative homo- and heterodimerizations
of two phosphate monoesters with phosphordiamidites (P-
diamidites, Figure 1). Radioactivity-based and colorimetric
assays with Thp3A call into question the reported inhibitory
activity of this compound against adenosine diphosphate (ADP)
Conjugates of nucleotides containing phosphoric anhydrides
as bridging units have important and diverse functions in biology.
Dinucleoside polyphosphates, for example, are signaling
molecules involved in the regulation of blood pressure1,2 or
insulin and glucose levels.3,4 ADP-ribose polymers are involved
in DNA repair mechanisms, transcriptional regulations,
apoptosis, and necrosis.5,6 Adenosine thiamine triphosphate
(Thp3A) is the only known thiaminylated nucleotide to date7 and
is accumulated in E. coli under metabolic stress conditions.8 Its
inhibitory potential on ADP ribosyltransferase diphtheria toxin-
like 1 (ARTD1, formerly PARP-1) activity has been studied.9
However, its roles in a broader biological context remain to be
elucidated.
Accumulation of negative charge on the phosphoric anhydride
(P-anhydride) bond and the instability of the target compounds
impose significant challenges on their synthesis and isolation.
Even so, different strategies have been developed and
summarized.10−13
Chemoselective phosphate condensations to minimize
protecting group manipulations have been reported,14−24 but
such couplings often require long reaction times, tedious
purification procedures, and give variable yields.13,20
Application of PIII chemistry to synthesize P-anhydrides
enables very fast reactions.25−29 Recently, it was demonstrated
that phosphoramidites (P-amidites, Figure 1A) can be coupled to
the terminal phosphates of nucleotides,30,31 enabling the
Figure 1. (A) Chemoselective couplings of P-amidites with phosphates
for the iterative homologation of oligophosphate esters. (B) Proposed
block coupling strategy for the synthesis of oligophosphate-bridged P-
esters by homologative dimerization of two phosphate esters using P-
diamidites.
application of PIII chemistry on unprotected substrates.32
A
Received: May 20, 2016
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.6b01466
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Letter
Figure 2. (A) Reaction sequence for the synthesis of C2-symmetric dinucleoside triphosphates via homologative dimerization of two NMPs. (B−F)
Monitoring of the reaction sequence for the synthesis of diuridine triphosphate (Up3U) by 31P[1H] NMR with the proposed assignment of the signals
after each step. iPr, isopropyl; Cy, cyclohexyl; Fm, 9-fluorenylmethyl; NMP, nucleoside monophosphate; TBA, tetrabutylammonium; ETT, 5-ethylthio-
1H-tetrazole; DMF, dimethylformamide; mCPBA, meta-chloroperbenzoic acid; UMP, uridine monophosphate.
major advance in the synthesis of modified condensed
phosphates would allow using a similar approach to simulta-
neously homologate and bridge two unprotected nonactivated
nucleotides (Figure 1B).16,33−35
It is known that P-diamidites can be reacted with alcohols to
yield P-amidites.36,37 In analogy, coupling to a phosphate would
yield a terminal mixed phosphoric anhydride P-amidite (Figure
2A, I) as an unstable intermediate. Reaction with another
phosphate would result in a PV−PIII−PV (II) bridge, which could
then be oxidized to the triphosphate (III). Deprotection would
then yield product IV.
Herein, it is shown that this strategy enables a highly
convenient and rapid synthesis of various symmetric NpxNs by
homologative homodimerization. Additionally, homologative
heterodimerization allowed the convenient synthesis of Thp3A
and facilitated the first synthesis of an analogue, Thp3C. The
obtained highly pure thiamine derivatives showed no inhibition
of ARDT1, neither in colorimetric nor radioactivity-based assays,
thus calling into question the previously reported inhibitory
effects of Thp3A on ARDT1.
Initially, homodimerization of unprotected UMP was studied.
Two equivalents of UMP TBA salt was treated in DMF with a
substoichiometric amount of 9-fluorenylmethyl-N,N,N′,N′-
tetraisopropyl P-diamidite (Figure 2A, 1) in the presence of
excess ETT. 31P NMR of the reaction mixture (Figure 2C)
showed that 1 (δ(31P): 120 ppm) was rapidly consumed, giving
rise to two new PIII species. Three new signals attributed to PV
nuclei appeared in addition to the remaining UMP (δ(31P): −1
ppm). Based on the chemical shifts and integrals observed, the
products of a single coupling (I, (δ(31P): 137 and −9 ppm) and
of a subsequent coupling (II, (δ(31P): 117, −10, and −11 ppm)
are postulated. Interestingly, peak splitting due to homonuclear
P−P coupling was not observed for the PIII−PV anhydride bonds.
This is consistent with the observations made for the coupling of
P-monoamidites with phosphates.30,32
After a few minutes, the signals assigned to I and UMP
disappeared in the 31P NMR (Figure 2D). The remaining set of
signals can be assigned to the PV−PIII−PV anhydride
intermediate II. Prochirality of the PIII atom results in two
distinct signals of the diastereotopic PV nuclei. Oxidation with
mCPBA gave Fm-protected diuridine triphosphate (III) after
precipitation. 31P NMR of the precipitated crude material
confirmed the presence of a single species (Figure 2E). The
intermediate was then deprotected with piperidine and
precipitated. The precipitate was confirmed to be a piperidinium
salt of diuridine triphosphate (Up3U) with a purity of 85% (see
Figure 2F for a 31P NMR spectrum of the crude precipitate) and
in 87% yield, obtained in less than 15 min reaction time under
ambient conditions. Purification by strong anion exchange
chromatography (SAX) gave pure Up3U in 60% yield. A slightly
modified protocol was then applied to the synthesis of symmetric
dinucleoside triphosphates from three other unprotected NMPs.
The results were comparable (see Table 1, entries 1−4). For the
synthesis of dicytidine triphosphate (Cp3C) and diadenosine
triphosphate (Ap3A), better results were obtained using P-
diamidite 2 (Figure 2A).
Two examples of dinucleoside triphosphate analogues with a
β-thiophosphate moiety were also synthesized. In the oxidation
step, mCPBA was replaced by S8, giving sulfurized β-P(S)
triphosphates after deprotection and precipitation (Table 1,
entries 5 and 6). These results demonstrate that P-diamidite-
based dimerizations are a powerful new synthetic concept for the
generation of modified condensed phosphates.
In comparison with previously developed procedures,35,38−40
the presented methodology offers several advantages. The
reactions can be run under ambient conditions and are very
fast. Good-quality C2-symmetric dinucleoside triphosphates can
be isolated in high yields by simple precipitation. The
methodology is applicable to all canonical NMPs, demonstrating
its robustness, and without the need to protect or activate the
B
DOI: 10.1021/acs.orglett.6b01466
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a
Table 1. Synthesized Dinucleoside Triphosphates and
Analogues
Scheme 2. Synthesis of Thp3A and Thp3C
c
coupling
precipitated
purified
b
time
(min)
yield purity
yield
(%)
a
entry
product
Ap3A
P-diamidite
(%)
(%)
1
2
3
4
5
6
2
1
2
1
1
1
5
5
3
7
3
4
71
64
70
87
70
60
70
65
85
85
85
80
60
45
60
68
Gp3G
Cp3C
Up3U
Ap-p(S)-pA
Up-p(S)-pU
a
BArF, tetrakis[(3,5-trifluoromethyl)phenyl]borate; DMF, dimethyl-
formamide; NMP, nucleoside monophosphate; TBA, tetrabutylam-
monium; ETT, 5-ethylthio-1H-tetrazole; mCPBA, meta-chloroperben-
zoic acid; TFA, trifluoroacetic acid.
a
b
P-diamidite used for the homologative dimerization. Purity
c
according to 31P and H NMR. Yield after purification.
1
nucleotides. Finally, synthesis of thiophosphate analogues is
feasible by using S8 in the oxidation step.
To facilitate sequential couplings, a new P-diamidite (Scheme
2, 3) featuring two different amine moieties was developed. The
different reactivity toward acidic activation of the amine
substituents should improve the selectivity of the first reaction,
as the dimethylamine moiety would be replaced rapidly. To
synthesize thiamine derivatives, the P-diamidite protecting
strategy had to be changed, as decomposition under basic
conditions was observed, likely due to deprotonation of the
thiamine moiety. The Fm group was replaced with a p-
methoxybenzyl (PMB) group, which can be cleaved under
acidic conditions. Finally, thiamine monophosphate (ThMP)
had to be solubilized in DMF, which was eventually achieved by
conversion into its tetrakis[(3,5-trifluoromethyl)phenyl]borate
(BArF) salt.
Different methods have been used to synthesize Ap5A. They
involve either the opening of trimetaphosphate intermedi-
ates15,41 or the coupling of activated phosphates42 and are
sluggish. In the case of Ap7A, treatment of a mixture of ATP and
adenosine tetraphosphate with a carbodiimide for 24 h resulted
in formation of the product that was isolated after three
chromatographic steps in unspecified yield.43,44 Therefore,
homologative dimerization of ADP to give Ap5A and ATP to
Ap7A is an interesting alternative. However, the intermediates
were much more water-sensitive, and no products were formed
with the developed method under ambient conditions. Running
the coupling reaction under exclusion of water with predried
solvents and nucleotide salts for only 30 s (Scheme 1) and with
The ThMP BArF salt was treated with the asymmetric P-
diamidite 3 (Scheme 2). The doubly protonated phosphate ester
of ThMP was expected to both activate the P-diamidite and form
the dimethylammonium salt by protonation of the amine. Two
equivalents of UMP TBA salts were then added for the second
coupling, followed by ETT to complete the reaction, as the TBA
salt does not have the two protons required for conversion. After
oxidation and acidic cleavage of the PMB group, the crude
product was precipitated. Formation of the two symmetric
triphosphates Ap3A and Thp3Th could not be prevented
completely, and thus, a purification step was required. Pure
product in 20% yield was obtained after only a single
semipreparative ion pair HPLC. The same procedure was
applied to synthesize the first described structural analogue
featuring a pyrimidine nucleoside, namely, thiamine cytidine
triphosphate (Thp3C), in 24% yield.
Both Thp3A and Thp3C were tested for their inhibitory activity
on human ARTD1/PARP-1, as a complete inhibition of the
enzyme had been reported at 10 μM Thp3A concentration with a
commercial colorimetric assay.9 Our radioactivity-based assay
did not confirm any inhibitory activity on ARTD1 or ARTD2
(formerly PARP-2) at concentrations up to 20 μM of either
Thp3A or Thp3C (see Figures S1−S4). To confirm these results,
the commercial colorimetric assay that had been used in the
initial report was repeated. Again, no inhibition of Thp3A (or
Thp3C) on ARTD1 could be detected up to 20 μM (see Figure
S5). It is likely that the inhibition was caused by an impurity, as
unfortunately no analytical data were provided.9
a
Scheme 1. Synthesis of Ap5A and Ap7A
a
TBA, tetrabutylammonium; ETT, 5-ethylthio-1H-tetrazole; DMF,
dimethylformamide; mCPBA, meta-chloroperbenzoic acid.
P-amidite 2 solved this issue. After oxidation and deprotection
followed by SAX, Ap5A and Ap7A were isolated in 50 and 18%
yield (Scheme 1), respectively. Homologative dimerization
strategy with P-diamidites enabled the preparation of oligophos-
phates with five and even seven phosphate units by direct
bridging of ADP or ATP.
31P NMR monitoring of the reaction sequence suggested that
the P-diamidite was generally converted very rapidly to
intermediate I. Rapid consumption of the P-diamidite and
accumulation of I (Figure 2C) indicated that the first coupling is
faster than the second one. Therefore, it should be possible to
couple two different phosphate esters sequentially to a P-
diamidite to synthesize asymmetric oligophosphate esters such as
Thp3A or any dinucleoside polyphosphate with regards to chain
length and nucleotide composition.
Synthesis of thiamine triphosphate derivatives is a challenging
task, underlined by the low yield (2.7%) and difficult purification
procedure in the previously reported synthesis of Thp3A.7,45
Nevertheless, recent results indicated that Thp3A is an inhibitor
of PARP1, and it was therefore perceived as an interesting target
for the extension of the methodology presented herein.
In summary, these results show that a homologative
dimerization of phosphate esters using P-diamidites is feasible.
The strategy allows for homo- and heterodimerizations and
facilitated the synthesis of highly challenging zwitterionic
thiaminylated nucleotides, thus demonstrating the generality
C
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and corresponding pentaphosphates and heptaphosphates.
Thus, this strategy represents a novel and broadly applicable
approach in the synthesis of condensed natural oligophosphate
conjugates and their analogues with a huge potential to simplify
current synthetic approaches.46
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.orglett.6b01466.
■
S
(25) Jessen, H. J.; Schulz, T.; Balzarini, J.; Meier, C. Angew. Chem., Int.
Ed. 2008, 47 (45), 8719.
Supplementary figures, experimental procedures, and
1
NMR spectra (31P reaction monitoring, H, 31P, and 13C
́
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AUTHOR INFORMATION
■
(28) Capolicchio, S.; Wang, H.; Thakor, D. T.; Shears, S. B.; Jessen, H.
J. Angew. Chem., Int. Ed. 2014, 53 (36), 9508.
(29) Kistemaker, H. A. V.; Lameijer, L. N.; Meeuwenoord, N. J.;
Overkleeft, H. S.; van der Marel, G. A.; Filippov, D. V. Angew. Chem., Int.
Ed. 2015, 54 (16), 4915.
(30) Cremosnik, G. S.; Hofer, A.; Jessen, H. J. Angew. Chem., Int. Ed.
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(31) Jessen, H. J.; Ahmed, N.; Hofer, A. Org. Biomol. Chem. 2014, 12
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Corresponding Author
*E-mail: henning.jessen@oc.uni-freiburg.de.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the SNSF (PP00P2_157607 to
H.J.J.). Research in the laboratory of M.O.H. is supported by the
Kanton of Zurich and the Swiss National Science Foundation
[310030_157019 to M.O.H.].
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