A Phosphoramidite Analogue of Cyclotriphosphate Enables Iterative Polyphosphorylations

Article abstract of DOI:10.1002/anie.201814366

An iterative polyphosphorylation approach is described, which is based on a phosphoramidite (P-amidite) derived reagent (c-PyPA) obtained from the cyclization of pyrophosphate with a reactive diisopropylaminodichlorophosphine. This type of reagent is unprecedented as it represents a reactive P-amidite without protecting groups. The reagent proved to be stable in solution over several weeks. Its utility is described in the context of iterative monodirectional and bidirectional polyphosphorylations. The ensuing functionalized cyclotriphosphate can be opened with a variety of nucleophiles providing ready access to diverse functionalized polyphosphate chains of defined length with several tags, including both P-N and P-O labels. Their interaction with exo- and endopolyphosphatases is described.

Full text of DOI:10.1002/anie.201814366

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
International Edition Chemie
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Accepted Article
Title: The cyclic phosphoramidite reagent c-PyPA enables iterative
polyphosphorylations
Authors: Jyoti Singh, Nicole Steck, Debaditya De, Alexandre Hofer,
Alexander Ripp, Ilya Captain, Manfred Keller, Paul Wender,
Rashna Bhandari, and Henning Jacob Jessen
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201814366
Angew. Chem. 10.1002/ange.201814366
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Angewandte Chemie International Edition
10.1002/anie.201814366
COMMUNICATION
A phosphoramidite analog of cyclotriphosphate enables iterative
polyphosphorylations
[a]
[a]
[b]
[a]
[a]
[a]
Jyoti Singh , Nicole Steck , Debaditya De , Alexandre Hofer , Alexander Ripp , Ilya Captain ,
[a]
[c]
[b]
[a]
Manfred Keller , Paul A. Wender , Rashna Bhandari , and Henning J. Jessen*
yield obtained (ca. 10%).[11] Very recently, a new approach for the
Abstract: An iterative polyphosphorylation approach is described,
which is based on a phosphoramidite (P-amidite) derived reagent (c-
PyPA) obtained from the cyclization of pyrophosphate with a reactive
diisopropylamino dichlorophosphine. This type of reagent is
unprecedented as it represents a reactive P-amidite without protecting
groups. The reagent proved to be stable in solution over several
weeks. Its utility is described in the context of iterative monodirectional
and bidirectional polyphosphorylations. The ensuing functionalized
cyclotriphosphate can be opened with a variety of nucleophiles
providing ready access to diverse functionalized polyphosphate chains
of defined length with several tags, including both P-N and P-O labels.
Their interaction with exo- and endopolyphosphatases is described.
activation of cyclotriphosphate has been developed by
[16]
Cummins. In a wider context, the development of a P-(III) based
triphosphorylation procedure including mixed oxidation states has
[17]
been pioneered by Ludwig and Eckstein and a phosphoramidite
(
P-amidite) for the introduction of methylene bisphosphonates has
[18]
recently been reported by Filippov.
reagents
However, no P-amidite
[19]
without protecting groups are available for
triphosphorylations.
Here, an approach is presented based on the development of
a P-amidite derived reagent, which we dubbed c-PyPA (cyclic
pyrophosphoryl P-amidite, Scheme 1) that proceeds through
P(III)-P(V) intermediates[ in a rapid, modular, and high-yielding
fashion. It does not require the use of protecting groups.
20]
Condensed phosphates, such as found in nucleoside
[
1]
triphosphates (NTPs), are of central importance in biology. In
addition, higher homologs, such as adenosine tetra- and
[
2]
pentaphosphates
have been identified and synthetically
modified nucleoside hexaphosphates are used in next generation
sequencing.[ The pervasiveness of this type of modification in
3,4]
[
5]
biology, however, extends far beyond nucleotides. For example,
inorganic polyphosphate (polyP) is a polydisperse biopolymer that
can be composed of tens to thousands of phosphate unit.[6] PolyP
has been well characterized in bacteria where it was shown to
serve crucial functions, e.g. during the bacterial stress response,
in biofilm formation, and as a phosphate storage molecule. In
yeast, polyP synthesis is regulated by inositol pyrophosphates[7]
via activation of the SPX domain of the vacuolar transporter
chaperone complex.[8] In mammals, polyP is involved in the
regulation of the blood-clotting cascade, but the enzymes
elusive.[
9]
Reports
suggest
generating
it
remain
Scheme 1. cyclic-pyrophosphoryl-P-amidite (c-PyPA) 3 enables the
synthesis of polyP with defined chain lengths.
polyphosphorylation as a new posttranslational modification of
proteins,[
10,11]
and cellular delivery of a polydisperse, labeled polyP
[
12]
has recently been achieved.
The synthesis of c-PyPA 3 comprises the reaction of
diisopropylamino dichlorophosphine 1 and pyrophosphate 2 as the
Therefore, access to modified polyphosphates is highly
desirable, yet the synthesis of such labile structures in
2
× tetrabutylammonium (TBA) salt in equimolar amounts in the
[
13]
monodisperse form is significantly understudied. Approaches to
analogs of polyP exist, but they rely on the biochemical synthesis
of polydisperse polyP followed by modification resulting in P-
amidate end-labeling.[14] An efficient chemical approach would
require being rapid, iterative, high-yielding and additionally enable
the introduction of orthogonal modifications. A recent report has
made use of the activation of cyclotriphosphate (also often
referred to as trimetaphosphate, tmp) as an N-methylimidazolium
salt according to Taylor[15] followed by nucleophilic displacement
and ring opening with an amine nucleophile to obtain a modified
presence of a base under strictly dry conditions. c-PyPA 3 can be
prepared both in DMF or MeCN and is stable over weeks in
31
solution if stored under argon at -20 °C. The P NMR of reagent
c-PyPA 3 is shown in figure 1 B (i) with a diagnostic shift of
3
1
30 ppm and a J P-P coupling (triplet) for the P(III) nucleus and -
20 ppm (doublet) for the P(V) nuclei with an integration of 1:2. The
decomposition product is the corresponding cyclic H-phosphonate
that forms by hydrolysis of the P-N bond (see scheme S6 in the
supporting information).
Next, in analogy to established P-amidite chemistry,[21] the
8
P in a one-flask operation. A drawback of this procedure is the low
activation of
3 with acidic activators was studied. 4,5-
[
22]
Dicyanoimidazole (DCI) and 5-(ethylthio)-1H-tetrazole (ETT)
proved to be efficient in this process. Initial studies were
conducted with phenylphosphate as readily available nucleophile,
which reacted immediately with activated c-PyPA 3 giving a 1-
deoxycyclotriphosphate 4 (for Hantzsch Widman Nomenclature
see the supporting information, table S1). This intermediate was
then oxidized using meta-chloroperbenzoic acid (mCPBA) to the
cyclotriphosphate 5, which can be traced due to its characteristic
[
a]
Jyoti Singh, Nicole Steck, Alexandre Hofer, Alexander Ripp, llya
Captain, Dr. M. Keller, Dr. Henning. J. Jessen
University of Freiburg, Institute of Organic Chemistry, Albertstrasse
21, 79104 Freiburg, Germany
henning.jessen@oc.uni-freiburg.de
Debaditya De, Dr. Rashna Bhandhari
Laboratory of Cell Signaling, CDFD, Hyderabad, India
rashna@cdfd.org.in
Dr. Paul A. Wender
Stanford University, Chemistry Department
[
[
b]
c]
3
1
P chemical shift at the trifurcation of -35 ppm and the coupling
pattern (doublet of triplet with identical J values) as shown in figure
B (iii). These structures can then be ring-opened with different
1
nucleophiles, and we investigated several conditions that would
be well suited for obtaining linearized oligophosphate chains with
different modifications. The hydrolysis of cyclotriphosphate 5
3
33 Campus Drive, Stanford, CA 94305-5080
Wenderp@stanford.edu
Supporting information for this article is given via a link at the end
of the document.
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Angewandte Chemie International Edition
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COMMUNICATION
2
+
2+
simply by addition of water is slow and even after three hours
nucleophiles in the presence of Mg or Zn . In all cases, the
addition of Mg2 gave better results. These reactions thus provide
access to modified polyPs, both with terminal P-N and P-O
modifications, making this approach especially versatile.
Additionally, the introduction of a non-hydrolysable terminal
methylene bisphosphonate was possible and afforded 14 in 47%
isolated yield enabling access to non-hydrolysable analogs. Why
these systems prefer linearization over branching is currently
unknown. One reason could be steric crowding at the trifurcation,
thereby repelling incoming nucleophiles.
+
significant amounts of cyclotriphosphate 5 were still detectable
figure 1 C (i)). Hydrolysis with NaOH was much faster and
(
required only 15 minutes almost exclusively resulting in the linear
phenyl tetraphosphate 6 (figure 1 C (ii). Hydrolysis with aqueous
HCl was equally rapid compared to the basic conditions (see the
supporting information, NMR page 14), but did not occur as
selectively. Treatment with aqueous ammonia led to linearization
and formation of a terminal phosphoramidate 7 with high
selectivity. Of note, such structures can be hydrolysed to the
terminal phosphates by simple adjustment of pH.[23] Apparently,
Furthermore, the application of P(III) chemistry provides a
platform to access sulfur-containing analogs as well: 1-
deoxycyclotriphosphate 4 was oxidized using Beaucage’s reagent
which resulted in the formation of 16 as judged by the chemical
shift and the coupling pattern (26 ppm, dt, see the supporting
information, NMR page 13). 1-Deoxy-1-thiocyclotriphosphate 16
was treated with propargyl amine resulting in the linear terminal
phosphoramidate 17 isolated in 40% yield. This strategy therefore
enables the extension of a phosphate-containing precursor by
three phosphate units using 3 followed by an additional extension
in the linearization step with the potential of further diversification
(
modified) amines react much faster than alcohols as nucleophiles
with the cyclotriphosphate 5, which was demonstrated by the
addition of ethanolamine, exclusively leading to P-amidate 8.
Instead of amines as nucleophiles, also phosphates can be used
scheme 2). The scope of this strategy is shown in table 1. For
example, prop-2-yn-1-yl phosphate worked well as nucleophile in
the reaction sequence when a divalent metal ion (e.g. Mg , Zn )
was used as additive, giving compound 11 after purification by
strong anion exchange chromatography (SAX) in 41% yield. Not
only phenylphosphate but also alkyne tagged phosphates such as
pent-4-yn-1-yl phosphate were successfully used in this
sequence. The introduction of three additional phosphate units by
linearization of 5 with cyclotriphosphate gave 15 in 35% isolated
yield. This demonstrates that it is also possible to linearize 5 by
addition of modified phosphate, pyrophosphate and triphosphate
(
2
+
2+
(e.g.
oligophosphate
introduction,
thiophosphorylation,
amidation). This part of the study revealed the usefulness of 3 as
a reagent for monodirectional phosphoric anhydride syntheses in
which one can introduce six phosphate units in a one-flask
operation using monophosphates as starting materials.
Figure 1: A) General scheme for NMR reactions. B) 31P{ H} NMR study monitoring the formation of a cyclotriphosphate intermediate 5 under
ambient conditions (for more details see supporting information) (i) c-PyPA 3 in reaction mixture. (ii) The 1-deoxycyclotriphosphate 4 (signal
at 101 ppm) formed after adding an equimolar amount of phenylphosphate TBA salt to c-PyPA 3. ETT was used as an activator, r.t., 10 min.
1
(iii) Addition of mCPBA at 0 ºC gave cyclotriphosphate 5, showing a characteristic peak doublet of triplet at -35 ppm. C) Ring opening reactions
under different conditions. (i) Treatment with water resulted in a slow and unselective reaction. (ii) Treatment with NaOH afforded the phenyl
tetraphosphate- δ-OH 6 almost exclusively. (iii) + (iv) Ring-opening under basic conditions delivered phenyl tetraphosphate-δ-amidates 7, 8
within 15 min. Furthermore the phenyl tetraphosphate-δ-amidates 7 were treated with 1 M HCl, which resulted in cleavage of P-N amidate
bond and afforded phenyl tetraphosphate 6 (see the supporting information, NMR page 16). Abbreviations: Ph: phenyl; tmp: tmp
(cyclotriphosphate); ETT: 5-(ethylthio)-1H-tetrazole; mCPBA: meta-chloroperbenzoic acid; TBA: tetrabutyl ammonium.
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COMMUNICATION
Monodirectional approach 
A) Monodirectional approach 
Scheme 2. Overview of the monodirectional triphosphorylation using
c-PyPA 3. Intermediate 5 can be diversified into several polyphosphate
conjugates. All reactions were conducted in MeCN or DMF.
B) 31P{ H} NMR of intermediate 20
1
Table 1: Conditions and yields obtained using monodirectional approach I
Entry
Monophosphate[a]
Conditions[b][c]
Nucleophile (equiv.)
NaOH
Yield
Product
1
2
3
4
5
6
7
8
9
Phenyl-1.0 TBA
Phenyl-1.0 TBA
Phenyl-1.0 TBA
Phenyl-1.0 TBA
Phenyl-1.2 TBA
Phenyl-1.0 TBA
Phenyl-1.2 TBA
Benzyl-1.5 TBA
MeCN/2/1.5
MeCN/2/1.5
MeCN/2/1.5
MeCN/2/1.5
MeCN/2/1.5
MeCN/2/1.5
MeCN/2/1.2
MeCN/2/1.5
MeCN/5/1.5
MeCN/5/1.5
MeCN/5/1.5
MeCN/2/1.2
99%[e]
98%[e]
95%[e]
91%[e]
80%[f]
65%[f]
41%[f]
29%[f]
6
7
NH
4
OH
CH
NH
2
2
CH
2
3
OH
8
NH(CH
2
CH
)
2
9
HC≡CCH
O (1)[d]
HC≡CCH
2
NH
2
(3.5)
10
6
Figure 2: A) Synthesis of polyP
21 by using monodirectional approach II:
5
H
2
c-PyPA 3 was selectively coupled to pyrophosphate 2 which results in
intermediate 20. Intermediate 20 was reacted with propargylamine and thus
2
-
(1.5)[d]
11
12
13
14
15
17
2
-PO
PO
(1.5) [d]
4
2
-
(1.5) [d]
transformed into polyP
21 used as a starting substrate for the second extension and ring opening with
9-(aminomethyl)anthracene.This sequence gave modified polyP 23. ATP 24
5 8 5
21. Synthesis of the tagged polyP 23 involved polyP
HC≡C(CH
2
)
3
4
Pentynyl-2CyNH+
2-
2 7
P O
44%[f]
Pentynyl-2CyNH+
PCP (1.5) [d]
47%[f]
8
10
11
12
Pentynyl-2CyNH+
104- (1.5) [d]
3
P O
35%[f]
40%[f]
was selectively coupled to 3 and the ring opening was performed with
propargylamine resulting in 25. B) 31P{ H} analysis of reactive intermediate 20
is shown.
1
Pentynyl-1.0 TBA
2 2
HC≡CCH NH (3.5)
note that it is also possible to initiate a selective monodirectional
extension even on an unmodified pyrophosphate by using
reduced amounts of c-PyPA 3 (figure 2). In this case, P NMR
[
a] Monophosphates were used as TBA or cyclohexylammonium salt. [b] 1 - 1.2
equiv. of c-PyPA 3 were added to the reaction mixtures. [c] The entries are given
as: solvent/ equiv. of ETT/ equiv. of oxidant (mCPBA) only in entry 12 use of
31
Beaucage’s reagent for the oxidation. [d] All reactions were treated with MgCl
1.5 equiv) except for the first five entries where the ring opening was performed
2
analysis of the intermediate 20 after oxidation indicated the
presence of a trifurcation (-36 ppm), a terminal phosphate (-13
ppm) and internal phosphoric anhydrides (-25 ppm, two signals)
in a 1:1:1:2 ratio. Therefore, it is also possible to introduce
selectively only one equivalent of c-PyPA 3 to an unprotected
pyrophosphate 2. Intermediate 20 was then linearized by the
addition of propargylamine. The use of propargylamine afforded
product 21 in 65% isolated yield. This strategy enables the
selective synthesis of polyP modified on one end only that can
then be extended again on the remaining unmodified other end.
For example, alkyne tagged 21 was extended again using c-PyPA
(
under basic conditions. [e] Yield after precipitation; compounds were essentially
pure and did not require further workup. [f] Yield after ion exchange
chromatography on Q-Sepharose using an aqueous solution of ammonium
bicarbonate for gradient eluent. Abbreviations: MeCN: acetonitrile, mCPBA:
meta-chloroperbenzoic
+
acid,
TBA:
tetrabutylammonium,
CyNH :
cyclohexylammonium salt.
Conceptually, such an extension as described above should
also work in bidirectional fashion, if the starting
a
phosphate/pyrophosphate units were unmodified. In this
scenario, the addition of at least two equivalents of 3 would enable
the rapid generation of even longer modified polyP chains in a
one-flask procedure. To study this potential reactivity, unmodified
pyrophosphate 2 was reacted with an excess of 3. The use of 3.5
equivalents of c-PyPA 3 indeed facilitated complete consumption
of pyrophosphate 2 and, after oxidation, led to the bidirectionally
extended intermediate 18 that was then linearized using amine
nucleophiles (scheme 3). In a recent report, this structure had
been obtained using P(V) chemistry, albeit in a low yield (ca.
3
followed by linearization with the amine nucleophile 9-
(
aminomethyl)anthracene leading to polyP 23 in 11% isolated
8
yield with different tags at both termini (figure 2). Due to solubility
issues of 21 in organic solvents, the second extension generally
suffered from lower yields. In addition, 3 was applied to the
extension of ATP by three phosphate units and introduce an
alkyne tag at the terminal phosphate in a one-flask procedure.
This directly resulted in a modified nucleoside hexaphosphate in
37% yield after SAX. Such nucleotides are used in single
molecule real time sequencing.[
0%).[ c-PyPA 3 enabled the synthesis of compound 19 in 40%
11]
1
3]
isolated yield.
Bidirectional approach
Scheme 3. Reagents and intermediates involved in the synthesis of polyP
modified on both termini by applying 3.5 eq of c-PyPA 3.
8
19
Using the bidirectional approach, one will always obtain polyP
with identical modifications at both termini. In this context, it is of
Scheme 4. Alkyne tagged polyPs 19, 21 can be transformed into fluorescein
labeled polyPs 26 and 27 using click chemistry.
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Angewandte Chemie International Edition
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COMMUNICATION
P-N and P-O end labels. This novel reagent opens new
possibilities to target densely charged and unstable modified
polyphosphates, as exemplified by mono- and bidirectional
extensions followed by selective nucleophilic ring-opening. The
obtained polyP analogs were studied in the context of their
interaction with phosphatases.
Acknowledgements
We gratefully acknowledge financial support from the HFSP
grant (Human Frontier Science Program RGP0025/2016). We
thank MagRes of the University of Freiburg for significant amount
of time for NMR spectroscopy. We thank Prof. G. M. Blackburn
for helpful discussions concerning nomenclature.
Keywords: Cyclic phosphoramidite, Triphosphorylation,
Monodisperse polyP, Phosphoric anhydrides.
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5 8
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(
8
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8
19 is protected from digestion by
[
8
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8
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5
21 and bis alkyne
8
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exopolyphosphatases (PPX)[25] that processively cleave polyPs
[
either internally or externally (Figure 3). FAM labelled P
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activity (figure 3A). Presence of an alkyne group at both ends of
19 is sufficient to prevent ScPPX digestion (figure 3B).
However, endophosphatases like ScDDP1 can digest bis-alkyne
modified P 19 while bis-FAM-P 27 resists ScDDP1 activity
figure 3C). The bulky FAM groups at both ends of 27 may block
5
26
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8
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(
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In summary, a novel triphosphorylation reagent 3 has been
developed. Its usefulness in the context of one-flask
polyphosphorylation reactions has been described, culminating in
the synthesis of several modified polyphosphates with or without
[
[
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Entry for the Table of Contents
Layout 2:
COMMUNICATION
[
a]
[a]
Jyoti Singh
Debaditya
, Nicole Steck ,
[b]
Going long
De ,Alexandre
[a]
[a]
Hofer , Alexander Ripp , Ilya
A triphosphorylation reagent was developed,
Captain[a],Manfred Keller[a]
Paul A. Wender , Rashna
,
[c]
which
enables
access
to
defined
[
b]
Bhandari
, and Henning J.
polyphosphates in
a
one-flask operation.
[a]
Jessen*
These polyphosphate chains are modified
on either one end or both ends with different
tags.
Page No. – Page No.
A
phosphoramidite analog of
cyclotriphosphate enables
iterative polyphosphorylations
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