Synthesis of carbohydrate methyl phosphoramidates

Article abstract of DOI:10.1021/ol500894k

A two-step route for introducing methyl phosphoramidate moieties onto carbohydrates is reported. The approach uses methyl pivolyl H-phosphonate as the phosphorylating reagent to produce an isolable carbohydrate H-phosphonate intermediate that is then oxidized by a Todd-Atherton reaction. The stability of the product methyl phosphoramidates was subsequently evaluated using various deprotection strategies.

Full text of DOI:10.1021/ol500894k

Letter
pubs.acs.org/OrgLett
Synthesis of Carbohydrate Methyl Phosphoramidates
Roger A. Ashmus and Todd L. Lowary*
Alberta Glycomics Centre and Department of Chemistry, The University of Alberta, Gunning−Lumieux Chemistry Centre,
Edmonton, Alberta T6G 2G2, Canada
S
* Supporting Information
ABSTRACT: A two-step route for introducing methyl phosphoramidate moieties onto carbohydrates is reported. The approach
uses methyl pivolyl H-phosphonate as the phosphorylating reagent to produce an isolable carbohydrate H-phosphonate
intermediate that is then oxidized by a Todd−Atherton reaction. The stability of the product methyl phosphoramidates was
subsequently evaluated using various deprotection strategies.
Campylobacter jejuni is the leading cause of bacterial gastro-
enteritis worldwide.1 The outer surface of campylobacters is
functionalized with species-specific capsular polysaccharides
(CPS), the production of which is required for virulence.2 The
CPS produced by different C. jejuni strains express a broad
diversity of structures, due in part to phase-variable
modifications. Among these motifs are unique O-methyl
phosphoramidate (MeOPN) groups, which are found in over
70% of all C. jejuni strains.3 As examples, Figure 1 shows the
Due to the rarity of these MeOPN motifs, which exist as a
single although currently unknown stereochemistry on
phosphorus,3 their antigenicity could be explored for the
development of vaccines.5 In addition, access to MeOPN-
functionalized carbohydrates could help unravel the biosyn-
thetic pathway by which these groups are assembled and lead to
the future development of new therapeutic agents against C.
jejuni. However, such studies require a method for the synthesis
of MeOPN groups.
Previous reports have described the preparation of N-
protected alkyl/aryl phosphoramidate diesters6 and aryl
phosphoramidates on nucleotide analogues.7 However, to
date, the synthesis of these moieties possessing an unprotected
nitrogen has not been reported. Syntheses of phosphoramidates
often involve the use of phosphoryl chloride as the
phosphorylating reagent.8 We therefore initially investigated
the use of POCl3 with a model substrate, 1,2:3,4-di-O-
isopropylidene galactopyranose (1, Scheme 1). However,
Scheme 1. Initial Approaches for Introducing MeOPN
Motifs onto Carbohydrates
Figure 1. Repeating structures of CPS from (a) C. jejuni 11168H and
(b) C. jejuni 81−176.
several products were formed in this reaction, and purification
of the desired product was difficult. As an alternative, we
explored the use of diphenyl phosphite by double displacement
of the O-phenyl groups, first with 1 and then methanol,
followed by a Todd−Atherton reaction with a primary amine
(Scheme 1). Although the product is formed and can be
repeating core structures of two C. jejuni strains that possess
MeOPN motifs in their CPS. The role of these MeOPN
moieties remains unclear. However, recent studies suggest they
play roles in serum resistance and colonization3 and they have
also been shown to have insecticidal activity, although this latter
activity has been questioned.4
Received: March 25, 2014
Published: April 16, 2014
© 2014 American Chemical Society
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Letter
isolated with relative ease, the yields were low. We also
explored other commercially available phosphorylating reagents
including 2-cyanoethyl N,N-diisopropylchlorophosphoramidite
and methyl dichlorophosphate; however, the results obtained
were inferior to those obtained using diphenyl phosphite.
Given the modest, albeit positive, results obtained using
diphenyl phosphite, we explored other H-phosphonate-based
reagents (5−7, Scheme 2) to affect this transformation. The
Scheme 2. Synthesis of Phosphorylating Reagents 5−7
preparation of 5−7 involved first the conversion of methyl H−
phosphonate ammonium salt 39 into its tetra-N-butylammo-
nium counterpart (4).10 Reagent 5 can be synthesized by the
addition of pivaloyl chloride (PivCl) to 4 in pyridine.11
Treatment of a solution of 4 in 9:1 CH2Cl2−pyridine with
PivCl either in the presence or absence of 2-chloro-4-
nitrophenol12 led to 6 and 7, respectively.7c,8
In the protocol developed, reagents 5−7 were generated in
situ, 1 was added and the reaction was monitored by thin-layer
chromatography. Of the three reagents, we found 7 to be the
most successful in producing the desired methyl sugar H-
phosphonate 8 (Figure 2). The compound can easily be
converted to the benzyl-protected MeOPN-containing mono-
saccharide 2 in a subsequent Todd−Atherton reaction with
benzylamine.
Figure 2 shows a representative reaction sequence with
monitoring by 31P NMR spectroscopy. H-Phosphonate salt 4
was consumed within 20 min after addition of PivCl to generate
a new product with a 31P signal consistent with 7.7c,13 After the
formation of 7 was complete, 1 was added, and after being
stirred for 1 h, the mixture was worked up. 31P NMR
spectroscopy of the product showed two new signals in an ∼1:1
ratio, indicative of the two diastereomers present in the methyl
sugar H-phosphonate 8.14 Without further purification, 8 was
converted to 2 via a Todd−Atherton reaction with benzylamine
and bromotrichloromethane in the presence of triethylamine
for 4 h. Following workup, 31P NMR spectroscopy of the
product revealed two new signals for 2, corresponding to an
∼1:1 ratio of diastereomers.
Figure 2. (a) PivCl (3.6 equiv), 9:1 CH2Cl2−pyridine, 20 min, (b)
sugar substrate (1 equiv), 1 h followed by basic workup, (c) Bn-NH2
(1.5 equiv), TEA (3 equiv), CBrCl3 (10 equiv), DCM, 4 h followed by
basic workup. 31P NMR of two-step method for synthesizing the
MeOPN-containing saccharide.
in the substrates. These groups include benzylidene acetals,
benzyl ethers, benzoates and acetate esters, isopropylidene
ketals, and silyl ethers. In each case, the product was produced
as an ∼1:1 mixture of diastereomers, which in most cases is
inseparable. Two substrates, 49 and 50 (Figure 3), were,
however, found to be incompatible. In the reaction of N-acetate
49 with 7, a product of unknown structure was formed that was
resistant to the Todd−Atherton reaction. In the case of the
azide containing substrate 50, treatment with 7 presumably
leads to a Staudinger-type reaction leading to a methyl sugar
phosphate derivative.
With these protected MeOPN-containing saccharides in
hand, we studied the stability of this motif using various
deprotection strategies (Table 1). We first studied removal of
the nitrogen protecting group on the MeOPN motif. We found
that N-benzyl and N-p-methoxybenzyl groups on the MeOPN
can be removed oxidatively using sodium bromate/sodium
thiosulfite15 and ceric ammonium nitrate, respectively. We
found these groups could not be removed using standard
hydrogenolytic conditions (H2, Pd−C); no consumption of the
starting material was observed. The o-nitrobenzyl groups (18
and 21) could be removed by photolysis in apolar protic
solvents.
With these optimized conditions developed, a panel of
substrates was evaluated (Table 1). Entries 1 and 2 show that
the method can be used with benzylamine, p-methoxybenzyl-
amine, or o-nitrobenzylamine as the oxidant in the Todd−
Atherton reaction. In addition, the method is successful with
both primary and secondary hydroxyl groups on glucose and
galactose analogues in either in the pyranose or furanose ring
configurations. Notably, the method was capable of producing
protected precursors (28−30) of the motifs found in the CPS
of C. jejuni strains 11168H and 81−176 (see Figure 1).
The examples in Table 1 reveal that the conditions are
sufficiently mild that a variety of protecting groups can be used
With regard to the protecting groups on oxygen, benzylidene
acetals, and benzyl ethers can be reductively cleaved by
hydrogenation in the presence of Raney nickel (entries 4 and
5). Hydrogenation in the presence of a palladium catalyst led to
incomplete cleavage of the benzylidene acetals even after long
reaction times and the use of high pressure. In addition,
benzylidene acetals can be opened oxidatively to produce a
benzoate ester.15 Entry 8 demonstrates the simultaneous
removal of N-benzyl group and regioselective oxidative ring-
opening of the benzylidene acetal to produce 44 in good yield.
Benzoyl and acetyl esters can also be removed using a 7:2:1
mixture of methanol−water−triethylamine (entries 3, 6, 8, and
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Table 1. Introduction of MeOPN Moieties onto Carbohydrates Followed by Deprotection
*
a
**
ONB = o-nitrobenzyl. Used 5 and 6 equiv of phosphorylating reagent and PivCl, respectively. Products without structures can be found in the
Supporting Information.
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Letter
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(often complete) cleavage of the phosphoramidate from the
carbohydrate. Silyl ethers can be removed using HF·pyridine
(entries 6 and 7) and isopropylidene acetals can be removed
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benzylidene acetals led to loss of the phosphoramidate motif.
In conclusion, an efficient route for synthesizing MeOPN-
containing carbohydrates was developed using a two-step
protocol involving phosphorylation with methyl pivaloyl H-
phosphonate (7) followed by a Todd−Atherton reaction. The
method is compatible with secondary and primary hydroxyl
groups in the presence of a variety of protecting groups. In
addition, various deprotection strategies could be used without
affecting the MeOPN moiety. This method should be readily
applied to more complex glycans containing this unusual motif.
Currently under investigation is the development of methods
for installing these groups as a single diastereomer on
phosphorus as are explorations of the pathway by which the
MeOPN group is biosynthesized by campylobacters.
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ASSOCIATED CONTENT
■
phosphorus signals.
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S
* Supporting Information
1
Experimental data and H, 13C, and 31P NMR spectra for all
previously unreported compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*E-mail: tlowary@ualberta.ca.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the Natural Sciences and
Engineering Research Council of Canada and the Alberta
Glycomics Centre. R.A.A. thanks Alberta Innovates Technology
Futures for a graduate student scholarship.
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