Stereoselective synthesis of fluorinated aminoglycosyl phosphonates

Article abstract of DOI:10.1039/c4ob02317j

We describe the conjugate addition of lithiated difluoromethanephosphonates to a diverse range of nitroglycals as a convenient method for the highly stereoselective synthesis of fluorinated aminoglycosyl phosphonates. Our approach provides opportunities to produce hydrolytically stable mimics of biologically important aminoglycosyl phosphates.

Full text of DOI:10.1039/c4ob02317j

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Stereoselective synthesis of fluorinated
aminoglycosyl phosphonates†
Cite this: DOI: 10.1039/c4ob02317j
Received 31st October 2014,
Accepted 30th November 2014
Sanne Bouwman, Romano V. A. Orru and Eelco Ruijter*
DOI: 10.1039/c4ob02317j
www.rsc.org/obc
We describe the conjugate addition of lithiated difluoromethane-
phosphonates to a diverse range of nitroglycals as a convenient
method for the highly stereoselective synthesis of fluorinated amino-
glycosyl phosphonates. Our approach provides opportunities to
produce hydrolytically stable mimics of biologically important
aminoglycosyl phosphates.
The complexity of the wide variety of oligosaccharide struc-
tures found in nature is derived from a rational management
of various enzymes. The formation of glycosidic linkages is
typically achieved by glycosyl transferases via a controlled
transfer of monosaccharide structures from an activated donor
sugar substrate to acceptor substrates (e.g. oligosaccharides,
lipids and proteins).1,2 Donor sugar substrates are most com-
monly activated in the form of nucleoside diphosphate sugars
(e.g. UDP-Gal, UDP-GlcNAc, etc.), which are generally referred
to as Leloir donors.3 However, nucleoside monophosphate
sugars and lipid phosphates are also suitable for activation
(Fig. 1A). The inhibition of this transfer reaction provides an
excellent opportunity for the potential modulation of oligo-
saccharide biosynthesis and interference with biological signaling
and metabolism. Accordingly, significant effort has been
devoted to the synthesis of hydrolytically stable glycosyl phos-
phate mimics, such as C-glycoside analogs.4–6 In particular the
synthesis of 2-deoxy-2-acetamido derivatives is of special inter-
est because of the widespread biological activity of various
aminoglycoside phosphates.7 For instance, sugars such as
N-acetylglycosamine (GlcNAc) and N-acetyl-galactosamine
(GalNAc) are involved in the biosynthesis of N-linked glyco-
proteins which are the key intermediates of many important
cell–cell and cell-pathogen recognition phenomena.8 In
addition, GlcNAc 1-phosphate is a substrate for nucleotidyl
Fig. 1 (A) Structures of aminoglycosyl donors and a cell wall intermedi-
ate. (B) Generalized reaction scheme of glycosyl transferases and struc-
ture of non-hydrolyzable glycosyl donor mimic.
transferase enzymes in the assembly of the fungal cell wall to
form the chitin layer.9 Likewise, C55-isoprenyl diphosphate
MurNAc(GlcNAc) pentapeptide (Lipid II, Fig. 1A) is an impor-
tant intermediate in the biosynthesis of the rigid peptido-
glycan layer of bacteria.10,11 Several synthetic approaches toward
2-deoxy-2-acetamido glycosyl-CH2-phosphonates are known.5,12–15
However, such ground state analogues have shown to be
moderate inhibitors of the corresponding aminoglycosyl trans-
ferases under physiological conditions.13 In light of this obser-
vation, better bio-isosteres might be obtained by the
introduction of fluorine substituents on the α-carbon of the
phosphonate, because their electron-withdrawing properties
will maintain the electronegativity of the oxygen.16–18 In fact,
the CF2-methylene linkage closely resembles the oxygen
linkage in terms of size, charge distribution and, conse-
quently, the second dissociation constant of the phosphate
Department of Chemistry & Pharmaceutical Sciences and Amsterdam Institute
for Molecules, Medicines and Systems (AIMMS), VU University Amsterdam,
De Boelelaan 1083, 1081 HVAmsterdam, The Netherlands. E-mail: e.ruijter@vu.nl
†Electronic supplementary information (ESI) available: Detailed experimental
procedures, characterization data, and copies of 1H, 13C, 19F and 31P NMR
spectra for all new compounds. See DOI: 10.1039/c4ob02317j
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moiety (pKa ).19 Glycosyl CF2-phosphonates are therefore readily available by recently developed methods by Vankar
attractive synthetic targets as probes for glycosyl transferases et al.26,27 We started screening the reactivity of several
2
and related enzymes (Fig. 1B).
preformed metalated difluoromethanephosphonates toward
Although several interesting methods have been developed benzylated nitroglycal.28 The first set of experiments (Table 1,
to replace the anomeric oxygen atom of carbohydrates by a entries 1–5) did not provide satisfactory results. Only the
difluoromethylene group,20,21 reports on the synthesis of lithiated reagent was able to undergo reasonable conjugate
2-deoxy-2-amino-CF2-glycosides are relatively scarce. This might addition to the nitroglycal, whereas all other cases gave no or
be explained by the difficulties associated with the incompat- only trace amounts of product while the remainder of the start-
ibility of neighboring nitrogen-based functional groups. More- ing material could be recovered. Pleasingly, the use of LDA in
over, the installation of a glycosyl acceptor with the correct combination with difluoromethanephosphonate significantly
relative stereochemistry has proven difficult. The most increased the yield from 47% to 84% (entry 8). A lower temp-
common approach toward the synthesis of 2-deoxy-2-acet- erature (−78 °C) is required as a result of the thermal instabil-
amido-C-glycosides is to employ other pyranose starting ity of lithiated difluoromethanephosphonates.29 Additional
materials and install the acetamido group after the C–C(F2) optimization revealed that the use of 1.2 equivalents of
bond formation. These approaches typically require many phosphonate is sufficient and that the reaction proceeds
steps and proceed in low overall yield.22
to completion within 30 minutes. Fluorinated C-glycosyl
The poor control over stereochemistry and the long reaction phosphonate 2a was obtained in very high diastereomeric
sequences prompted us to develop an efficient procedure excess. We assigned the 1,2-trans configuration to the major
toward 2-deoxy-2-acetamido-CF2-glycosyl phosphonates. Herein isomer based on NMR analysis. The large H-1/H-2 and H-2/H-3
1
we report a novel approach to difluoromethanephosphonate coupling constants (J1,2 and J2,3 ∼ 10 Hz) observed in the H
C-glycoside bond formation based on conjugate addition to NMR spectrum of 2a indicated a diaxial relationship between
readily available 2-nitroglycals. We will discuss and illustrate the hydrogen atoms, which is consistent with a 4C1 confor-
our approach as a convenient method for the highly stereo- mation of the glycosyl phosphonate. Since crucial signals in
selective synthesis of several fluorinated aminoglycosyl phos- the 1H NMR spectra of 2a overlapped, which prevented us
phonates. 2-Nitroglycals have been recognized as important from collecting useful data from 2D-NMR experiments, the
building blocks in carbohydrate chemistry because glycosyl β-configuration was confirmed by NOESY correlation between
acceptors can easily be installed at the anomeric centre via H-1 and H-5 after debenzylation of 2a.
conjugate addition, often proceeding with high diastereo-
After having defined the optimal conditions, the feasibility
selectivity.23–25 We envisioned the conjugate addition of and protecting group compatibility of the reaction was
difluoromethanephosphonate to these readily available explored and a series of fluorinated glycosyl phosphonates was
2-nitroglycals could provide access to difluorophosphonate C-gly- synthesized (Scheme 1). Differentially protected nitroglycals
cosides. Subsequent chemical manipulation of the 2-nitro were reacted with diethyl lithiodifluoromethanephosphonate
group would afford the 2-acetamido group.
to furnish 2a–2c in good yields and with high diastereomeric
The proposed reaction was initially evaluated with 3,4,6-tri- ratios. Apparently silyl, methyl and benzyl ethers are tolerated,
O-benzyl-2-nitro-D-glucal as the model substrate, which is whereas tri-O-acetyl nitroglycal (1d) gave no product formation,
Table 1 Optimization of the reaction conditions
Time
(min)
Yielda
(%)
Entry
X
Reagent
T (°C)
[M]
Equivalents
1
2
3
SiMe3
Br
Br
Br
ZnBr
Br
H
H
H
H
CsF
iPrMgCl
Cd
rt
rt
rt
rt
Cs
2
2
2
2
2
2
2
2
120
120
120
120
120
120
120
120
120
120
30
6
MgCl
CdBr
ZnBr
CuZnBr2
Li
Na
Li
Li
Li
Li
—
—
—
—
47
—
84
62
83
83c
4
Zn
5b
6
CuBr
nBuLi
NaHMDS
LDA
LDA
LDA
rt
−78
−78
−78
−78
−78
−78
7
8
9
10
11
1
1.2
1.2
H
LDA
a Determined by 19F NMR using α,α,α-trifluorotoluene as an internal standard. b DMF was used as solvent. c Diastereomeric ratio: 89 : 7 : 3 : 1.
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Scheme 1 Scope of fluorinated glycosyl phosphonates. Yields deter-
mined by 19F NMR using α,α,α-trifluorotoluene as an internal standard.
Scheme 2 (A) Rationalization of observed stereoselectivity; (B) Reduced
equilibration in the formation of 2j.
Reaction conditions:
1 (0.50 mmol), HCF2PO(OR)2 (0.60 mmol,
1.2 equiv.), LDA (0.60 mmol, 1.2 equiv.), THF, −78 °C.
diastereomeric excess should be attributed to the direct
presumably due to the incompatibility of esters with the addition of the lithiated phosphonate. Thus, we hypothesized
lithiated phosphonate. Also the tribenzylated galactal deriva- that the formation of the thermodynamically more stable 1,2-
tive 1e reacted smoothly under these conditions. We then diequatorial 2-nitrophosphonate likely results from a ring
turned our attention to the rhamnal and arabinal derivatives opening/ring closure event (Scheme 2A), as observed pre-
1f and 1g, which were also readily converted to the corres- viously.33 We anticipated that the introduction of more rigidity
ponding desired products 2f and 2g in good yield and with by means of a (tBu)2Si protecting group creating a trans-fused
high dr’s.
bicyclic system would provide insight in the mechanism. The
In order to introduce a set of orthogonal protection groups 5H4 conformer is unattainable in case of the 4,6-O-di-tert-butyl-
in the products, the difluoromethanephosphonate tolerance silylene protected glycal 1j, since the ring-fused bonds cannot
was examined next. For this reason, allyl and benzyl protected adopt the axial positions. Consequently, attack from the α-face
4
difluoromethanephosphonates were constructed by means of on the half-chair H5 is highly preferred, as it proceeds via a
a Michaelis–Becker reaction between the corresponding phos- chair-like transition state, while β-attack will give a twist-boat-
phites and chlorodifluoromethane.30 Unfortunately, reactions like transition state which is much higher in energy. Thus, we
employing these phosphonates yielded the corresponding anticipated to increase the preference for the (potentially bio-
glycosyl derivatives 2h and 2i in poor to modest yield. The logically more relevant) α-anomer of 2j.
driving factor in the reduced coupling efficiency is most likely
To this end, the addition reaction was performed with 1j
the instability of these lithiated phosphonates due to the good and indeed the stereoselectivity of the phosphonate addition
leaving group capacity of benzylic and allylic alkoxides. This was found to be time dependent (Scheme 2B). Thus, the α/β
was supported by the formation of a significant amount of ratio of 2j, being 5 : 1 after 10 min, decreased to a 5 : 3 ratio
2-deoxy-2-nitro-1O-benzylglycoside as a side product in the after 120 min. This supports our hypothesis that the initially
reaction of 1a with dibenzyl difluoromethanephosphonate.
formed α-isomer I equilibrates to the β-isomer III via inter-
Subsequently, we set out to rationalize the observed stereo- mediate II under the basic reaction conditions.
selectivity. Glycals are known to be conformationally flexible
Finally, we explored the conversion of 2-deoxy-2-nitroglycosyl
and can exist either in the normal half chair 4H5 or the phosphonates to fluorinated aminoglycosyl phosphonates
inverted conformer 5H4, with the predominant conformation (Scheme 3). Reduction of the nitro group of 2a with Zn/HCl fol-
being highly dependent on hydroxyl substitution.31,32 Taking lowed by acetylation of the resulting amine with Ac2O/pyridine
into account that this would affect the stereoselectivity of afforded 3 in 71% yield over two steps. Selective cleavage of the
the conjugate reaction, it is not likely that the high value of benzyl protective groups by catalytic hydrogenation provided
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Scheme 3 Synthetic elaboration toward fluorinated aminoglycosyl
phosphonates. a Determined by 19F NMR using α,α,α-trifluorotoluene as
the internal standard.
the partially deprotected aminoglycosyl phosphonate 5. This
compound could also be obtained when 2a was first subjected
to hydrogenation with Pd(OH)2/C followed by N-acetylation in
90% yield over two steps. Treatment of glycosyl phosphonate 3
with TMSI provided the target aminoglycosyl phosphonate 6.
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Conclusions
We have established a highly efficient and stereoselective route
to fluorinated aminoglycosyl phosphonates starting from
readily available nitroglycals.34 We have shown that introdu-
cing rigidity in the carbohydrate framework could provide
access to the biologically important α-anomer. The use of
orthogonal protection strategies provides opportunities for
post-modification of the carbohydrates or further functionali-
zation of the phosphonates to obtain mimics of nucleotide
diphosphate derivatives (e.g. UDP-GlcNAc) as competitive
inhibitors of glycosyl transferases.
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Acknowledgements
This work was financially supported by the Priority Medicines
Program of the Netherlands Organization for Health research 28 We chose to report yields and product distribution deter-
and Development (ZonMW).
mined by 19F NMR with an internal standard to provide an
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accurate representation of the actual conversion and 31 A. Nowacki, D. Walczak and B. Liberek, Carbohydr. Res.,
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ally as single diastereomers after chromatographic purifi- 34 While our manuscript was under review,
a
similar
cation (cf. 3–7).
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