N-Methylimidazolium chloride-catalyzed pyrophosphate formation: Application to the synthesis of Lipid i and NDP-sugar donors

Article abstract of DOI:10.1016/j.bmcl.2011.04.061

N-Methylimidazolium chloride is found to catalyze a coupling reaction between monophosphates and activated phosphorous-nitrogen intermediates such as a phosphorimidazolide and phosphoromorpholidate to form biologically important unsymmetrical pyrophosphat

Full text of DOI:10.1016/j.bmcl.2011.04.061

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5050–5053
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Bioorganic & Medicinal Chemistry Letters
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N-Methylimidazolium chloride-catalyzed pyrophosphate formation:
Application to the synthesis of Lipid I and NDP-sugar donors
⇑
Hirokazu Tsukamoto, Daniel Kahne
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
N-Methylimidazolium chloride is found to catalyze a coupling reaction between monophosphates and
activated phosphorous–nitrogen intermediates such as a phosphorimidazolide and phosphoromorpholi-
date to form biologically important unsymmetrical pyrophosphate diesters. The catalyst is much more
active, cheaper, and less explosive than 1H-tetrazole, known as the best catalyst for the pyrophosphate
formation over a decade. The mild and neutral reaction conditions are compatible with allylic pyrophos-
phate formation in Lipid I syntheisis. 31P NMR experiments suggest that the catalyst acts not only as an
acid but also as a nucleophile to form cationic and electrophilic phosphor-N-methylimidazolide interme-
diates in the pyrophosphate formation.
Received 28 March 2011
Accepted 13 April 2011
Available online 22 April 2011
Keywords:
N-Methylimidazolium chloride
Pyrophosphate formation
Phosphorimidazolide
Phosphoromorpholidate
Lipid I
Ó 2011 Elsevier Ltd. All rights reserved.
NDP-sugar donors
Attachment of a sugar to a protein or another sugar chain gives
the parent biomolecules diverse functions. Enzymes that catalyze
formation of glycosidic bonds to other molecules are known as gly-
cosyltransferases (Gtfs), and they typically use glycosyl donors
containing a nucleoside diphosphate (NDP) or lipid pyrophosphate
(LPP) on the anomeric carbon (Fig. 1).1 Synthetic methods to make
NDP- and LPP-sugar donors are required to elucidate glycosyltrans-
ferase function and to synthesize natural and unnatural oligosac-
charides chemoenzymatically.2 Our laboratory has been
searching for efficient methods to make bacterial LPP-sugars such
as Lipid I and Lipid II for studying enzymes involved in cell enve-
lope biosynthesis.3 Although synthetic routes to these molecules
have been reported, we have not been satisfied with the existing
approaches.3a–c,4,5 Here we describe an improved method to make
pyrophosphate bonds that is compatible with the formation of
both NDP- and LPP-sugars.
prepared in situ using excess carbonyl diimidazole (CDI) followed
by a methanol quench. These intermediates are sufficiently stable
that neither the corresponding symmetrical pyrophosphate nor
the methyl phosphodiester forms during activation and quenching.
Unfortunately, the desired unsymmetrical pyrophosphate products
also form very slowly (Scheme 1). Divalent metal ions such as Zn2+
,
Mg2+, Mn2+, Cd2+, and Sn2+ have been reported to promote pyro-
phosphate bond formation,9 but can promote decomposition of
allylic lipid pyrophosphates,10 which are both acid- and nucleo-
phile-sensitive.
One approach to the synthesis of Lipid I and Lipid II used 1H-tet-
razole as a Brönsted acid catalyst to promote condensation of a li-
pid phosphate and a sugar phosphorimidazolide, but the reaction
still took 4–7 days.4 Sekine and co-workers have shown that
Chemical strategies to make NDP- and LPP-sugars involve con-
densation of two phosphates, which requires activation of one of
them.2 Phosphoromorpholidates6 and phosphorimidazolides7 are
usually employed as activated intermediates in these condensa-
tions. Since nucleoside 50-monophosphoromorpholidates are com-
mercially available, phosphoromorpholidate intermediates are
commonly used to make NDP-sugars, and 1H-tetrazole is often
used to catalyze the condensation reactions.8 Because phosphorim-
idazolides are more reactive than phosphoromorpholidates, they
are preferred for making LPP-sugars.3a–c,4,5 They can be readily
OH
O
R2
O
O
R1 =
O
2
O
P
O-
O
P
O-
AcHN
4
O
O
OR1
X
X = -Ala-D-iGlu-Lys-D-Ala-D-Ala-OH
Heptaprenyl Lipid I : R2 = H
Heptaprenyl Lipid II: R2 = β-GlcNAc
O
O
Base
Sugar-1-O
P
O
P
O-
O
O
O-
NDP-Sugar
OH OH
⇑
Corresponding author.
E-mail address: kahne@chemistry.harvard.edu (D. Kahne).
Figure 1.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.04.061

H. Tsukamoto, D. Kahne / Bioorg. Med. Chem. Lett. 21 (2011) 5050–5053
5051
O
P
O-
O
P
O-
O
P
O-
O
P
O-
1H-tetrazole is not effective for activation of the phosphorimidazo-
lide.9a Herein we report the use of N-methylimidazolium chloride
(NMIꢀHCl) as a catalyst for the phosphorimidazolide coupling reac-
tion in Lipid I synthesis. The catalyst is much more active, cheaper,
and less explosive than 1H-tetrazole. The reaction conditions are
mild, neutral, and compatible with the allylic pyrophosphate bond
formation. We show that this method also works well for NMP–
morpholidate coupling reactions with a sugar 1-phosphate. In
addition, we report a new synthetic route to make Lipid I that in-
volves introduction of the peptide after pyrophosphate bond for-
mation. The peptide is the variable portion in naturally occurring
Lipid I and II substrates,11 and our new approach allows more
flexibility in the synthesis of substrates containing different
sluggish!
N
R1O
N
HO
OR2
R1O
O
OR2
+
1 day to 1 week
1
2
3
Scheme 1.
O
O
P
O-
MeOH at 60
C
N
PhO
P
N
PhO
OMe
.
Imidazole HCl 3 h
ONa
.
Et3N HCl 12 h
4
5
72 h
Scheme 2.
OAc
O
OR1
O
R1O
Catalyst
AcO
O
O
O
O
P
O
P
O
P
AcHN
AcHN
DMF-THF
(1:1)
rt
+
O
P
X
H4NO
OR
O
O
OR
MeO2C
R2OC
.
OH NEt3
ONH4
8a, b
ONH4 ONH4
.
6: X = OH NEt3
9a, b: R1 = Ac, R2 = OMe
10a, b: R1 = H, R2 = OH
b
a
N
N
7: X =
a 6, CDI, DMF then MeOH
1 M LiOH H2O, THF-MeOH-H2O (3:1:1)
R =
2
b
.
a: n = 1
n
b: n = 4
Scheme 3.
Table 1
Effect of catalysts on pyrophsophate formation via Scheme 3
Entry
8
Catalyst (4 equiv)
pKa (H2O)
Time (d)
Yield of 10a (%)
1
2
3
4
5
6
7
8
9
10
8a
8a
8a
8a
8a
8a
8a
8a
8a
8b
—
—
7
7
1
1
0.5
0.5
0.5
0.5
1
69
71
69
75
77
72
75
68
63
54
Et3NꢀHCl
10.8
9.2
7.0
7.1
7.1
5.2
5.2
4.9
7.1
DMAPꢀHCl
ImidazoleꢀHCl
N-MethylimidazoleꢀHCl
N-MethylimidazoleꢀHOTf
PyridineꢀHCl
PyridineꢀHOTf
Tetrazole
N-MethylimidazoleꢀHCl
0.5
a
Yields were determined after removal of protecting groups.
OH
O
OH
O
HO
HO
O
a
O
O
P
O
P
O
P
O
P
AcHN
AcHN
O
O
OR
O
O
OR
HO2C
X-OC
ONH4 ONH4
ONH4 ONH4
10a, b
X = -Ala-D-iGlu-Lys-D-Ala-D-Ala
11a: R = C20H33
11b: R = C35 57
H
Scheme 4. Reagents: aH-Ala-
D
-iGlu(OMe)-Lys(Z)-
D
-Ala-D-Ala-OMeꢀHCl, DIEA, DMTMM, MeOH then 1 M LiOHꢀH2O, THF, H2O 61% for 11a, 67% for 11b.
CyHN
O
N
AcO
AcO
.
HO
HO
N
O
OAc
O
OH
O
NH
Cy+HN
a
O-
P
O
+
O
N3CH2COHN
N3CH2COHN
O
N
O
O
P
OH
.
O UDP
O
O
14
12
OH 2NEt3
13
OH OH
Scheme 5. Reagents and conditions: a(1) tetrazole, Py., 3 days or N-methylimidazoleꢀHCl, DMF, 12 h; (2) NaOMe, MeOH two steps 71% for tetrazole, 83% for N-
methylimidazoleꢀHCl.

5052
H. Tsukamoto, D. Kahne / Bioorg. Med. Chem. Lett. 21 (2011) 5050–5053
oligopeptides for mechanistic investigations of peptidoglycan bio-
synthetic enzymes.
The MurNAc polyprenyl pyrophosphates 10a, b were used to
make the corresponding Lipid I analogs via a new route in which
the protected pentapeptide was coupled following formation of
the diphosphate (Scheme 4). DMTMM14 was the most effective re-
agent for condensation between 10a, b and a small excess of pro-
tected pentapeptide.15 Subsequent hydrolysis afforded tetra- and
hepta-prenyl Lipid I 11a, b in good yields. Both can be quantita-
tively converted to the corresponding Lipid II analogs chemoenzy-
matically for studies of peptidoglycan biosynthetic enzymes and
their inhibitors.3b
In the condensation of two phosphates counter ions play impor-
tant roles, but little attention has been paid to them. In fact, in
chemical schemes the counter cations of activated phosphate 1
and phosphate monoester 2 are often omitted (Scheme 1). Trialky-
lammonium salts of 1 and 2 are frequently used for pyrophosphate
formation because they increase the solubility of both 1 and 2 in
organic solvents; they also increase the nucleophilicity of 2. How-
ever, the trialkyammonium salts are not acidic enough (aqueous
pKEt3NH+ 10.8) to effectively protonate 1, as required for P–N bond
dissociation, which may account for sluggish reactivity in the for-
mation of pyrophosphate bonds. Cramer and co-workers showed
that methanolysis of sodium phosphorimidazolide 4 is much faster
in the presence of imidazolium chloride (pKimidazoleꢀH+ 7.0) than tri-
ethylammonium chloride (Scheme 2).7b We wondered whether
adding a salt such as imidazolium or pyridinium chloride, which
comprise a weaker base than a trialkylamine and a stronger acid
than a phosphate, to a mixture of 1 and 2 would lead to counter
ion exchange to produce more a electrophilic imidazolium or
pyridinium phosphorimidazolide 1 in situ.
Phosphorimidazolide 7, prepared from the parent triethylam-
monium phosphate 612 and CDI, was used to make the pyrophos-
phates of polyprenyl monophosphate diammonium salts 8a and
b in anhydrous THF–DMF (1:1) solvent (Scheme 3).13 The complete
conversion of tetraprenyl phosphate 8a to the corresponding pyro-
phosphate 9a took one week in the absence of catalyst (Table 1, en-
try 1). Addition of weaker amine hydrochlorides (entries 2, 3, 4,
and 7) promoted the reaction much more effectively than triethyl-
amine, which did not appear to accelerate coupling. N-methylimi-
dazolium chloride (NMIꢀHCl, pKBH+ 7.1) was more effective than
1H-imidazole chloride (ImꢀHCl, pKBH+ 7.0) (entry 4 vs 5) and com-
parable to pyridinium chloride (PyꢀHCl, pKBH+ 5.2) (entry 5 vs 7).
Use of trifluoromethanesulfonic acid salts (NMIꢀHOTf and PyꢀHOTf)
slightly reduced the yields of pyrophosphate 10a (entry 5 vs 6, 7 vs
8). While 1H-tetrazole accelerated pyrophosphate formation with
phosphorimidazolide 7, the reaction was slower and the yield re-
duced compared with NMIꢀHCl and PyꢀHCl (entry 5, 7 vs 9).
NMIꢀHCl transformed the longer lipid phosphpate, heptaprenyl
8b, into the corresponding pyrophosphate 9b in good yield within
half a day (entry 10).
We also investigated the use of the NMIꢀHCl catalyst for NDP-
sugar synthesis. Khorana’s morpholidate method, which involves
coupling commercially available nucleoside 50-monophosphoro-
morpholidate
4-morpholine-N,N0-dicyclohexyl-carboxamidine
salts, is the most widely used approach for NDP-sugar synthesis.6
These NMP-morpholidate intermediates have a poor leaving group
(pKmorpholineꢀH+ 8.4) and a basic counter cation (pKguanidineꢀH+ 11.9).
Condensation of sugar 1-phosphate 1216 and UMP-morpholidate
13 took 3 days even in the presence of tetrazole, long considered
the best catalyst for these reactions (Scheme 5).8,17 In contrast,
our NMIꢀHCl catalyst in DMF greatly improved the reaction rate
(12 h) and yield,18 giving the desired UDP-GalNAz 14 product after
removal of the acetate protecting groups.
The difference between ImꢀHCl and NMIꢀHCl as catalysts sug-
gests that these compounds act not only acids but also as nucleo-
philes in pyrophosphate formation as shown in Scheme 6.
Electronically neutral phosphorimidazolide and phosphoromor-
pholidate 15a, b could be substituted by N-methylimidazole to
form a cationic phosphor-N-methylimidazolide intermediate 18,
which would be more susceptible to nucleophilic displacement
with a monophosphate anion 2 to give pyrophosphate 3. Consis-
tent with the formation of a new, cationic intermediate, we ob-
serve the appearance of downfield-shifted 31P resonances in the
31P NMR spectra of phosphorimidazolide 7 and UMP-morpholidate
13 after the addition of NMIꢀHCl.19 In the case of 13, the downfield
31P resonance that appeared upon addition of excess NMI-HCl had
a chemical shift similar to that of authentic TFA-protected UMP-
N-methylimidazolide (ꢁ10.6 vs ꢁ10.9 ppm).20 Although phos-
phor-N-methylimidazolide is a very reactive intermediate, its
preparation requires multiple steps including treatment with
acidic trifluoroacetic anhydride, during which care must be given
O
P
O-
O
P
O-
R1O
O
OR2
3
N
.
Cl- +HN
= NMI HCl
O
P
O
P
R1O
X
HO
OR2
.
O- R3NH+
R3N HCl
NMI H O-
+
.
15a, b
2
O
O
P
N
N+
a: X =
b: X =
N
N
R1O
P
O-
N
R1O
X
O- NMI H
+
.
O
18
16a, b
O
P
HX
R1O
X
OH NMI
17a, b
Scheme 6.

H. Tsukamoto, D. Kahne / Bioorg. Med. Chem. Lett. 21 (2011) 5050–5053
5053
C.-H.; Chang, Y.-F.; Wang, J.-T.; Shih, H.-W.; Hsu, Y.-F.; Chen, C.-W.; Chen, S.-K.;
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Lett. 2010, 12, 1608.
to its moisture sensitivity. In contrast, adding NMI ꢀ HCl as a cata-
lyst to less reactive intermediates allows simple manipulation and
ready availability of starting materials while being compatible
with acid-sensitive functional groups.
N-Methylimidazolium chloride was found to be superior in
activity, cost, and safety to 1H-tetrazole, long considered the best
catalyst for pyrophosphate bond formation. Our new method com-
bines availability and stability of phosphorimidazolide and
phosphoromorpholidate with high reactivity of phosphor-
N-methylimidazolide. The mild and neutral reaction conditions
are compatible with allylic pyrophosphate formation in Lipid I.
5. (a) Schwartz, B.; Markwalder, J. A.; Wang, Y. J. Am. Chem. Soc. 2001, 123, 11638;
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Acknowledgment
This research was supported by the National Institutes of
Health (R01 GM076710 and R01 GM066174).
Supplementary data
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Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.04.061.
13. Removal of MeOH and DMF from 7 and counter cation exchange of 8a, b with a
trialkylamine are omitted in our method.
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