Diphosphate formation using cyanuric chloride or triisopropylbenzenesulfonyl chloride as the activating agents

Article abstract of DOI:10.1016/j.tetlet.2011.01.034

By using cyanuric chloride or 2,4,6-triisopropylbenzenesulfonyl chloride (TIPSCl) as the condensing agent and magnesium bromide as the promoter, the phosphate cross-coupling reactions are effectively carried out to give lipid-saccharide and pyrrolidine-gu

Full text of DOI:10.1016/j.tetlet.2011.01.034

Tetrahedron Letters 52 (2011) 2232–2234
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Diphosphate formation using cyanuric chloride or
triisopropylbenzenesulfonyl chloride as the activating agents
⇑
Teng-Chi Lin, Jim-Min Fang
Department of Chemistry, National Taiwan University, Taipei 106, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 14 January 2011
By using cyanuric chloride or 2,4,6-triisopropylbenzenesulfonyl chloride (TIPSCl) as the condensing agent
and magnesium bromide as the promoter, the phosphate cross-coupling reactions are effectively carried
out to give lipid–saccharide and pyrrolidine–guanosine diphosphates. Under optimized conditions, the
TIPSCl-facilitated cross-coupling reactions complete in a short reaction time (ꢀ5 h) without the compli-
cation of possible self-coupling reactions.
Keywords:
Diphosphate compounds
Cyanuric chloride
2,4,6-Triisopropylbenzenesulfonyl chloride
Phosphate coupling reaction
Ó 2011 Elsevier Ltd. All rights reserved.
Diphosphate (also called pyrophosphate) is an important func-
tional group that plays indispensible roles in many biological pro-
cesses. For example, sugar nucleoside diphosphates act as the
glycosyl donors of glycosyltransferases in the synthesis of oligosac-
charides and glycoconjugates.1 Consecutive condensation of iso-
prenyl pyrophosphates produces geranyl pyrophosphate (GPP),
farnesyl pyrophosphate, and many other essential derivatives.2
Lipid II containing an undecaprenyl pyrophosphate moiety is the
substrate of transglycosylases for bacteria to form the polymeric
peptidoglycan of the cell wall.3
Though the commercially available nucleoside diphosphates
can be utilized to react with appropriate electrophiles (e.g., glyco-
syl halides) to generate a plethora of diphosphate compounds,4 the
diphosphate group is generally constructed by the coupling of two
monophosphate components.5 However, the cross coupling of two
different monophosphates is often complicated by the self-
coupling reactions. Moreover, the synthesis and isolation of
diphosphate compounds must be performed in proper conditions
because the diphosphate bonds are easily degraded by acids, bases,
and nucleophiles.
In one approach, Moffatt, Khorana, and co-workers have acti-
vated nucleoside monophosphates to the corresponding phosp-
horomorpholidates for the subsequent coupling reactions with
other monophosphates,6 for example, sugar phosphates, to provide
a variety of nucleoside diphosphates (Scheme 1, where R1 is a
nucleoside and R2 is a saccharide). The coupling reaction is greatly
accelerated by using 1H-tetrazole as the synergistic acid and nucle-
ophilic catalyst.7 This method has been widely utilized for the syn-
thesis of diphosphate compounds, including a bacterial cell wall
precursor UDP-N-acetylmuramyl-pentapetide (Park nucleotide).8
However, this method may encounter problem in prior preparation
and isolation of the activated phosphoromorpholidates (1P Fig. 1).
In another approach, a monophosphate can be activated by 1,10-
carbonyldiimidazole (CDI).9–15 After generation of the imidazolide
intermediate 2P (Fig. 1), excess CDI is destroyed by methanol, and
1H-tetrazole can be added to promote the cross-coupling reaction
with a second monophosphate substrate. This CDI-activation pro-
cedure is usually performed in mild conditions that are suitable
for the synthesis of sugar nucleoside diphosphates9–11 and lipid II
analogs.12–15 These two types of coupling reactions, via phosphoro-
morpholidate or phosphorimidazolidate intermediates, usually
take days to form the desired diphosphate products, and the over-
all yields may be low in many cases.
Other reagents have also been reported for activating mono-
phosphates in attempts to facilitate the diphosphate bond forma-
tion.16–19 An alcohol compound (e.g., thymidine) is treated with
5-nitro-cycloSal phosphorochloridite (3),16 followed by oxidation,
to give 5-nitro-cycloSal phosphate (3P), which is an activated
monophosphate ready for the coupling reaction with sugar mono-
phosphate, giving the diphosphate compound after aqueous work-
up.
A nucleoside monophosphate is activated by diphenyl
chlorophosphonate (4) to form a phosphoric anhydride (4P),17
which reacts with sugar monophosphate by selective replacement
of the diphenylphosphoryl moiety to give the nucleoside–saccha-
ride diphosphate. A monophosphate is treated with trifluoroacetic
anhydride and N-methylimidazole sequentially to afford a phosp-
horo-N-methylimidazolidate (5P) as the active intermediate to re-
act with another monophosphate to form the diphosphate
product.18,19 However, the most popular methods for diphosphate
bond formation still reside on the coupling reactions of a mono-
phosphate with the prior prepared phosphoromorpholidate or
the in situ generated phosphorimidazolidate (CDI activation).
⇑
Corresponding author.
E-mail address: jmfang@ntu.edu.tw (J.-M. Fang).
0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.01.034

T.-C. Lin, J.-M. Fang / Tetrahedron Letters 52 (2011) 2232–2234
2233
O
P
O
AcO
DCC
O
R1
+
O
N
P
O
R1
AcO
AcO
O
6 or 7
O
NH
O
O
P O
R1
O
P
O
+
O
O
AcHN
(promoters)
O
OH
OH
HNEt3
Monophosphate-1
Activating agent
Activated monophosphate
O
HNEt3
8a R1 = (CH2)5CH3
9
8b R1 = (CH2)9CH3
O
R2
O
P
O
8c R1 = (CH2)2-2-naphthyl
O
O
P
O
Monophosphate-2
YO
R2
O
O
P
O
R1
YO
YO
AcHN
O
+
Self-coupling products
1H-tetrazole
O
P
O
O
O
O
O
P
O
R1
Diphosphate
OH OH
10a, 10b, 10c Y = Ac
11a, 11b, 11c Y = H
Scheme 1. Formation of diphosphate compounds by the cross-coupling reaction of
two monophosphate.
MeONa
Scheme 2. Formation of aliphatic–saccharide diphosphates using cyanuric chloride
or 2,4,6-triisopropylbenzenesulfonyl chloride as the condensing agents.
We have previously devised a fluorescent lipid II-based sub-
strate for the assay of transglycosylation.15 The diphosphate com-
pound was successfully synthesized, albeit in low yield (<20%), by
the conjugation of a pentapeptide–disaccharyl phosphate with a
dansyl–lipid phosphate using the in situ CDI-activation method.
We also propose the possible fucosyltransferase inhibitors, such
as compound 14 (Scheme 3), which contain a moiety of guanosine
diphosphate (GDP) as the main binding component and a pyrroli-
dine group to mimic the oxonium transition state in transglycosy-
lation. In this Letter, we report the use of cyanuric chloride (CC, 6)
and 2,4,6-triisopropylbenzenesulfonyl chloride (TIPSCl, 7) as suit-
able condensing agents to facilitate the diphosphate bond forma-
tion using 10a–c and 14 as the model compounds.
CC and its analog, 2-chloro-4,6-dimethoxy-1,3,5-triazines
(CDMT), have been utilized to activate carboxylic acids for the
reactions with alcohols and amines to form esters and amides.20
To test the feasibility of CC in the promotion of diphosphate bond
formation, a solution of hexyl monophosphate in CH3CN was trea-
ted with CC (0.33 equiv) in the presence of pyridine for 2 h at room
temperature. The 31P NMR spectrum showed that the phosphorus
signal of hexyl monophosphate at dP 0.62 disappeared, and a new
signal at dP ꢁ13 was attributable to P1,P2-dihexyl diphosphate,
the self-coupling product, which was confirmed by the MS
analysis.
Encouraged by this result, we further investigated the cross-
coupling reaction of peracetyl GlcNAc monophosphate (9) with
hexyl monophosphate (8a, Scheme 2). In one experiment, 8a (as
the triethylammonium salt) was added slowly to a solution of CC
and pyridine in CH3CN at 0 °C, followed by the addition of GlcNAc
monophosphate 9 (as the triethylammonium salt). After stirring
for 24 h, the desired cross-coupling product 10a predominated in
the reaction mixture as shown by the 31P NMR and MS analyses,
though some self-coupling product of dihexyl diphosphate was
also observed. The crude product of 10a was then treated with so-
dium methoxide in MeOH to give 11a, which showed the molecu-
lar ions at m/z 464.1084 (single charged) and 231.5407 (double
charged) in the MS spectrum. The undesired self-coupling reaction
could not be eliminated even at a low temperature (ꢁ30 °C). Metal
ions, such as Mg2+, Zn2+, and lanthanides, having good affinity to
phosphate ion, were examined to see if they could promote the
phosphate coupling reactions.21 It was found that the addition of
MgBr2 (1 equiv) did facilitate the cross-coupling reaction to a great
degree. CC appeared to be a better condensing agent than CDMT in
promoting diphosphate bond formation. Another experiment using
a reversed procedure, that is, activation of GlcNAc monophosphate
9 with CC prior to the addition of hexyl monophosphate gave 10a
along with two self-coupling products derived from hexyl phos-
phate and 9, respectively.
Activating agent
Activated monophosphate
O
O
N
N
P
O
O
R1
R1
O
NH
O
1
1P
O
O
P
N
N
N
2P
2 (CDI)
O
N
N
O2N
O2N
O
3
3P
R1
O
O
O
P
P
O
O
Cl
By the activation of decyl monophosphate (8b) with CC, the
cross-coupling reaction with GlcNAc monophosphate 9 was carried
O
P
O
P
O
PhO
Cl
PhO
O
P
O
R1
4
4P
OPh
OPh
O
O
O
P
(CF3CO)2O /
N
N
O
R1
N
N
N
NH
O
5P
O
H3C
H3C
5
N
O
P
N
NH2
O
P
OH
X
N
X
X
+
N
O
HO
OH
O
O
N
N
N
OH
O
R1
t-BuO
O
Cl
N
X
P
O
13
12
OH OH
N
6 (CC, X = Cl)
O
6P
O
6' (CDMT, X = MeO)
6'P
N
NH
NH2
TIPSCl
O
O
S
O
P
N
O
P
O
N
S
Cl
O
O
R1
pyr, MgBr2
P
O
O
O
O
OH
O
O
O
N
Boc
O
O
14
7 (TIPSCl)
7P
OH
Figure 1. Some representative agents for activating monophosphate and the
Scheme 3. Synthesis of GDP–pyrrolidine derivative as a possible inhibitor of
activated intermediates for the subsequent cross-coupling reactions.
fucosyltransferases.

2234
T.-C. Lin, J.-M. Fang / Tetrahedron Letters 52 (2011) 2232–2234
out in THF solution to give 10b accompanied by some P1,P2-didecyl
diphosphate. Saponification of 10b afforded 11b, which has been
used as a substrate for the assay of bacterial galactosyltransfer-
ase.22 In agreement with the previous report,22 diphosphate 11b
exhibited two phosphorus signals at dP ꢁ10 and ꢁ12.
ꢀ50% yield by HPLC on a C18 column, and characterized by the
MS, 1H, and 31P NMR spectra. In addition to the desired proton res-
onance at d 5.93 (1H, d, J = 5.9 Hz) for H-8 of the guanine moiety,
this sample might be contaminated with an unidentified impurity
showing a blur signal nearby d 5.95 (see Supplementary NMR spec-
trum). In a comparison experiment, diphosphate 14 was also syn-
thesized via the GMP–morpholidate intermediate in 52%
conversion yield after a prolonged reaction time (70 h) as shown
by the 31P NMR analysis (Supplementary Fig. S4).
In summary, we have found that CC and TIPSCl are proper re-
agents for the activation of monophosphate. The cross-coupling
reactions for the formation of diphosphate compounds, for exam-
ple, 10a–c and 14, are best conducted in the presence of MgBr2.
The cross-coupling reactions using TIPSCl/MgBr2 promoters pro-
ceed rapidly in hours compared to days in the conventional meth-
ods via phosphoromorpholidate or phosphorimidazolidate
intermediates.
It was difficult to isolate and quantify P1-GlcNAc-P2-alkyl
diphosphates 10a,b and 11a,b due to lack of chromophore. Thus,
the cross-coupling reaction with phosphoric acid mono(2-naph-
thylethyl) ester (8c), a UV active compound, was further investi-
gated. The cross-coupling reaction of 8c and the GlcNAc
monophosphate 9 (both as the triethylammonium salts) was con-
ducted in CH3CN solution using CC, pyridine, and MgBr2 as promot-
ers. After stirring at room temperature for 48 h, the reaction
mixture was shown by HPLC analysis to contain 44% of diphos-
phate 10c in addition to the starting materials (Supplementary
Fig. S1). No apparent peak corresponding to the symmetric diphos-
phate derived from 8c or 9 was detected. The diphosphate product
10c was isolated in 25% yield by HPLC on a reversed-phase C18-A
column, and fully characterized by the MS, 1H, 13C, and 31P NMR
analyses. The relatively low isolation yield of diphosphate 10c
(25%), compared with 44% conversion yield, might be attributable
to the susceptibility of the glycosidic and diphosphate bonds to
hydrolytic cleavage during the separation process.
Acknowledgment
We thank the National Science Council for financial support.
Supplementary data
In another approach, TIPSCl was explored to promote the
diphosphate bond formation. Activation of monophosphate with
TIPSCl gives an intermediate of phosphate–sulfonate mixed anhy-
dride (7P),23–25 which can be selectively attacked by nucleophiles
at the phosphorus atom presumably due to the steric effect and
high leaving potency of the triisopropylsulfonate group. This acti-
vation method is first suggested by Khorana for the formation of
the C30–C50 internucleotide linkage,23 and has been applied to
the synthesis of monophosphate di- and triesters.24,25
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2011.01.034.
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