Synthesis of the enantiomers of XYLNAc and LYXNAc: Comparison of β-N-acetylhexosaminidase inhibition by the 8 stereoisomers of 2-N-acetylamino-1,2,4-trideoxy-1,4-iminopentitols

Article abstract of DOI:10.1039/c4ob00097h

The enantiomers of XYLNAc (2-N-acetylamino-1,2,4-trideoxy-1,4-iminoxylitol) are prepared from the enantiomers of glucuronolactone; the synthesis of the enantiomers of LYXNAc (2-N-acetylamino-1,2,4-trideoxy-1,4-iminolyxitol) from an l-arabinono-δ-lactone and a d-ribono-δ-lactone is reported. A comparison is made of the inhibition of β-N-acetylhexosaminidases (HexNAcases) and α-N-acetylgalactosaminidase (α-GalNAcase) by 8 stereoisomeric 2-N-acetylamino-1,2,4-trideoxy-1,4-iminopentitols; their N-benzyl derivatives are better inhibitors than the parent compounds. Both XYLNAc and LABNAc are potent inhibitors against HexNAcases. None of the compounds show any inhibition of α-GalNAcase. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4ob00097h

Organic &
Biomolecular Chemistry
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Synthesis of the enantiomers of XYLNAc and
LYXNAc: comparison of β-N-acetylhexosaminidase
inhibition by the 8 stereoisomers of 2-N-
Cite this: Org. Biomol. Chem., 2014,
12, 3932
acetylamino-1,2,4-trideoxy-1,4-iminopentitols†
Elizabeth V. Crabtree,a,b R. Fernando Martínez,b,d Shinpei Nakagawa,c Isao Adachi,c
Terry D. Butters,a Atsushi Kato,*c George W. J. Fleeta,b and Andreas F. G. Glawar*a,b
The enantiomers of XYLNAc (2-N-acetylamino-1,2,4-trideoxy-1,4-iminoxylitol) are prepared from the
enantiomers of glucuronolactone; the synthesis of the enantiomers of LYXNAc (2-N-acetylamino-1,2,4-
trideoxy-1,4-iminolyxitol) from an L-arabinono-δ-lactone and a D-ribono-δ-lactone is reported. A com-
parison is made of the inhibition of β-N-acetylhexosaminidases (HexNAcases) and α-N-acetylgalactos-
aminidase (α-GalNAcase) by 8 stereoisomeric 2-N-acetylamino-1,2,4-trideoxy-1,4-iminopentitols; their
N-benzyl derivatives are better inhibitors than the parent compounds. Both XYLNAc and LABNAc are
potent inhibitors against HexNAcases. None of the compounds show any inhibition of α-GalNAcase.
Received 13th January 2014,
Accepted 1st April 2014
DOI: 10.1039/c4ob00097h
www.rsc.org/obc
Introduction
β-N-Acetylhexosaminidases (HexNAcases) and α-N-acetylgalactos-
aminidases (α-GalNAcases) respectively hydrolyze N-acetylglu-
cosamine (GlcNAc) and N-acetylgalactosamine (GalNAc)
residues of glycoconjugates and glycolipids. Control of the
levels of conjugation of proteins and lipids by GlcNAc and
GalNAc is crucial under a number of pathogenic conditions.1
Hexosaminidase inhibitors have chemotherapeutic potential
as pharmacological chaperones2 for lysosomal storage dis-
orders such as Tay–Sachs and Sandhoff diseases.3,4 α-GalNA-
case inhibition would be of interest for the treatment of
Schindler–Kanzaki5 disease and protection of the macrophage-
activating factor as cancer treatment.6,7
There are three iminosugar natural products (isolated from
bacterial broths) which are nanomolar inhibitors of HexNA-
cases (Fig. 1): siastatin B 1,8–10 pochonicine 2,11,12 and nagsta-
tin 3.13,14 Although hundreds of iminosugars15,16 have been
isolated from plants, none of them has as yet shown any
Fig. 1 Potent inhibitors of β-N-acetylhexosaminidases at µM level or
lower.
aOxford Glycobiology Institute, University of Oxford, South Parks Road, Oxford,
OX1 3QU, UK. Fax: +44 (0)1865 275216; Tel: +44 (0)1865 275342
bChemistry Research Laboratory, 12 Mansfield Road, Oxford, OX1 3TA, UK.
E-mail: Andreas.Glawar@chem.oxon.org; Fax: +44 (0)1865 285002;
Tel: +44 (0)1865 275645
cDepartment of Hospital Pharmacy, University of Toyama, 2630 Sugitani,
Toyama 930-0194, Japan. E-mail: kato@med.u-toyama.ac.uk
dDepartamento de Química Orgánica e Inorgánica, QUOREX Research Group,
Universidad de Extremadura, Avda. Elvas s/n, E-06006 Badajoz, Spain
†Electronic supplementary information (ESI) available: NMR spectra for 20–23,
29–33, 36 and 40–46. See DOI: 10.1039/c4ob00097h
inhibition of HexNAcases. There are a large number of synthetic
piperidine mimics of GlcNAc which are potent inhibitors,
such as PUGNAc (O-(2-acetamido-2-deoxy-D-glucopyranosyl-
idene) amino-N-phenylcarbamate) derivatives 4,17–19 GlcNAc-
thiazolines 5,20,21 and DNJNAc (2-acetamido-1,2-dideoxy-
D-gluco-nojirimycin) 6.22,23 The D-galacto-analogue DGJNAc 724
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is the only known potent inhibitor of α-GalNAcases but is also
a good inhibitor of HexNAcases.25 Both the amides of
hydroxylated pipecolic 826 and azetidine 927 carboxylic acids
are potent inhibitors of HexNAcases. ADMDP (1-acetamido-
1,2,5-trideoxy-2,5-imino-D-mannitol) 1028,29 is a rare example
of a potent pyrrolidine HexNAcase inhibitor. The pyrrolidine
LABNAc (2-acetamido-1,4-imino-1,2,4-trideoxy-L-arabinitol) 11L
is an iminopentitol which is a micromolar inhibitor of
HexNAcases.30
Whereas the inhibition by piperidine analogues of hexose
glycosidases is usually31 easy to rationalize in terms of the
structure of the corresponding sugar, pyrrolidine analogues of
pentoses give unexpected results. All of the D-1,4-iminopenti-
tols 12D–15D have been reported as natural products (Fig. 2).
The arabino isomer DAB (1,4-dideoxy-1,4-imino-D-arabini-
tol) 12D32–34 is a good – but the unnatural enantiomer LAB
(1,4-dideoxy-1,4-imino-L-arabinitol) 12L is a much more potent
and specific – inhibitor of α-D-glucosidases. The lyxo-isomer
13D34,35 causes potent inhibition of α-D-galactosidases, as do
other natural products with the same stereochemistry; its
enantiomer 13L inhibits the same enzyme weakly.36 The
iminoxylitol 14D37 is a weak α-D-glucosidase inhibitor whereas
the ribitol 15D33 does not inhibit any glucosidases at all.
None of the 1,4-dideoxy-1,4-iminohexitols has as yet been
isolated as a natural product. The D-mannitol derivative DIM
(1,4-dideoxy-1,4-imino-D-mannitol) 16D, with an additional
carbon in the side chain relative to the lyxitol 13D, is a potent
inhibitor of α-D-mannosidases;38,39 there is thus a change from
potent inhibition of α-D-mannosidase in the hexitol 16D to
potent α-D-galactosidase inhibition in the pentitol 13D. In con-
trast, L-DIM 16L is a potent inhibitor of α-L-rhamnosidase;40,41
the cut down of carbon side chains to 13L causes a loss of α-L-
rhamnosidase – and a gain in weak α-D-glucosidase – inhi-
bition. In contrast to the piperidines DNJNAc 6 and DGJNAc 7,
the pyrrolidine analogues of GlcNAc 1742 and GalNAc 1843
showed only weak HexNAcase inhibition.
Fig. 3 Summary of inhibition of Jack bean β-N-acetylhexosaminidase
by N-acetylamino-iminopentitols.
and 22L from an L-arabinono-δ-lactone and
a D-ribono-
δ-lactone, respectively. For the first time this allows a compari-
son of the HexNAcase inhibition profiles of all eight
stereoisomeric 2-N-acetylamino-1,2,4-trideoxy-1,4-iminopenti-
tols (Fig. 3); it has previously been shown that neither enantio-
mer of the ribose analogues 24D and 24L inhibits any
glycosidases.44 Both D-XYLNAc 20D and LABNAc 11L are
potent inhibitors. N-benzylation leads to better inhibitors than
the parent compounds. None of the compounds shows any
inhibition of α-GalNAcase.
Results and discussion
Synthesis of enantiomers of XYLNAc 20
The azidodiol 27L was the key intermediate for the synthesis
of XYLNAc 20D from the acetonide of glucuronolactone 26D
(Scheme 1); conversion of 27L to the ditriflate allowed efficient
formation of the pyrrolidine ring by a double nucleophilic dis-
placement at C6 and C3. The acetonide 26D which has only
C-5 OH unprotected allows introduction of azide with inver-
sion, reduction of the lactone at C6 and inversion of configur-
ation at C3 to afford the azido diol 27L in 70% yield on a
multigram scale.24,45 L-Glucuronolactone acetonide 26L46,47
was converted to the enantiomer L-XYLNAc 20L by an identical
procedure.
This paper reports the synthesis of the enantiomers of
XYLNAc (2-N-acetylamino-1,2,4-trideoxy-1,4-iminoxylitol) 20D
and 20L from glucuronolactone, and the enantiomers of
LYXNAc (2-N-acetylamino-1,2,4-trideoxy-1,4-iminolyxitol) 22D
The conversion of the diol 27L to the corresponding ditri-
flate 28L followed by the double displacement with benzyl-
amine gave rise to the pyrrolidine 29L in good yield (87%,
Fig. 2 1,4-Dideoxy-1,4-iminoalditols.
Scheme 1 Strategy for the synthesis of xylo-enantiomers 20D and 20L.
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Scheme 3 Strategy for the synthesis of lyxo-enantiomers 22D and 22L.
Scheme 2 Reagents and conditions [yield for the enantiomer]: (i)
(CF3SO2)2O, pyridine, CH2Cl2, −40 °C, 1 h. (ii) NH2Bn, THF, rt, 3 h, 87%
over 2 steps [D-77%]. (iii) CF3COOH, H2O, rt, 5 h; then Ac2O, DIPEA,
DMAP, CH2Cl2, rt, 16 h, 84% over 2 steps [D-80%]. (iv) NaBH4, EtOH,
rt, 17 h; then Ac2O, DIPEA, DMAP, CH2Cl2, rt, 23 h, 66% over 2 steps
[D-85%]. (v) NaOMe, MeOH, rt, 2 h; then NaIO4, MeOH, H2O, rt, 2 h; then
NaBH4, H2O, rt, 4 h; then Ac2O, DIPEA, DMAP, CH2Cl2, rt, 18 h, 62%
from 31L [L-76%]. (vi) H2, Pd/C (10%), 1,4-dioxane, rt, 1 h; then Ac2O,
DIPEA, DMAP, CH2Cl2, rt, 18 h, 71% over 2 steps [L-66%]. (vii) NaOMe,
MeOH, rt, 2 h, 77% [L-77%]. (viii) H2, Pd black, 1,4-dioxane, H2O, rt, 4.5 h,
93% [L-100%].
Scheme 2). Hydrolysis of the acetonide of 29L with aqueous tri-
fluoroacetic acid and subsequent peracetylation gave 30L
(84%). The diacetate 30L underwent reduction by sodium
borohydride to afford the corresponding triol, which was con-
verted into the triacetate 31L (66% over two steps). Subsequent
deacetylation of 31L with sodium methoxide in methanol fol-
lowed by sodium periodate cleavage of the resulting diol,
reduction of the resulting aldehyde by sodium borohydride
and further acetylation led to the D-xylo pyrrolidine 32D in
62% yield from 31L. Hydrogenation of the azide 32D in the
presence of 10% palladium on carbon followed by acetylation
of the resulting amine gave the protected amide 33D (71%).
Removal of the esters in 33D with sodium methoxide gave the
N-benzyl xylo-pyrrolidine 21D (77%). Hydrogenolysis in the
presence of catalytic palladium black of the benzyl group in
21D afforded XYLNAc 20D in 93% yield (16% overall yield
from diol 27L; 20% for the L-xylo enantiomer 20L from 27D).
The structure of N-benzyl L-XYLNAc 21L was confirmed by
X-ray diffraction analysis.48
Scheme 4 Reagents and conditions [yield for the enantiomer]: (i)
(CF3SO2)2O, pyridine, CH2Cl2, −10 °C, 4 h.49 (ii) NaN3, DMF, rt, 18 h, 76%
over 2 steps. (iii) CF3COOH, H2O, 60 °C, 3.5 h, 82%. (iv) Ph2CN2, toluene,
reflux, 4 h, 75% [D-88%]. (v) DIBALH, CH2Cl2, −78 °C, 3 h; then NaBH4,
MeOH, rt, 4 h, 88% [D-62%]. (vi) Et3N, MsCl, CH2Cl2, 0 °C, 3 h, 100% [D-
98%]. (vii) BnNH2, toluene, 80 °C, 48 h, 90% [L-65%]. (viii) BF3·Et2O,
Ac2O, rt, 24 h, 70% [L-68%]. (ix) THF, AcOH, Ac2O, Zn, CuSO4, rt, 40 min,
80% [L-74%]. (x) NaOMe, MeOH, rt, 2 h, 81% [L-60%]. (xi) H2, Pd black,
1,4-dioxane, H2O, rt, 72 h, 100% [L-100%].
azide in DMF afforded the ribo-azidolactone 40L (76%). Treat-
ment of 40L with aqueous trifluoroacetic acid at 60 °C caused
removal of the acetonide protecting group and isomerization
of δ-lactone to form the γ-ribonolactone 36L (82%); 36L was
identical, save in its specific rotation, to a sample of 36D pre-
pared from D-ribonolactone 37D.50 Attempts to protect diol
36L under basic conditions gave an inseparable mixture of
C2-epimeric azides. Base catalysed epimerizations of α-azido-
lactones are common.52,53 Protection of base-sensitive alcohols
under neutral conditions by heating with diphenyldiazo-
methane gave the corresponding benzhydryl ethers in high
yields.54 Reaction of 36L with diphenyldiazomethane in
toluene gave the benzhydryl protected lactone 41L (75%).
Reduction of 41L by initial treatment with DIBAL at −78 °C fol-
lowed by sodium borohydride at rt gave the diol 42L (88%)
which on esterification with methanesulfonyl chloride in the
presence of pyridine afforded the dimesylate 43L in quantitat-
ive yield. Cyclization of the dimesylate with benzylamine via a
double SN2 displacement formed the D-lyxo pyrrolidine 44D
(90%). Reaction of 44D with acetic anhydride in the presence
of boron trifluoride-etherate caused exchange of the benz-
hydryl ethers to the more labile acetate groups to give 45D
(70%). Reduction of the azide in 45D by zinc/copper sulfate in
the presence of acetic anhydride formed the peracetylated
Synthesis of enantiomers of LYXNAc 22
The lyxo-enantiomers 22D and 22L were prepared from the
enantiomeric ribono-lactone azides 36L and 36D, respectively,
by formation of the pyrrolidine ring by joining C1 and C4 of
the lactone (Scheme 3). The L-azide 36L was prepared by intro-
duction of the azide with inversion of configuration at C2 of
the δ-arabinonolactone 35L, easily obtained from L-arabinose
34L.49 For the synthesis of L-LYXNAc 22L, ribonolactone 37D
was protected as the benzylidene δ-lactone 38D. Reaction of
the triflate of 38D with azide gave an efficient introduction
of azide with retention of configuration at C2; subsequent
hydrolysis formed 36D in 55% overall yield, as previously
described50,51 [60% yield of 36D from 38D; 55% from 37D].
Esterification of the arabino-alcohol 35L with triflic anhy-
dride gave the triflate 39L49 (Scheme 4) which with sodium
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amide 46D (80%).55 Removal of the acetate groups in 46D with inhibition and is not represented in Table 1. Both L-arabino
sodium methoxide gave the N-benzyl lyxo-pyrrolidine 23D (81%). 11L and D-xylo 20D are potent inhibitors of HexNAcases; a
Subsequent hydrogenolysis of the benzyl group in 23D in the modest inhibition is shown by the D-lyxo 22D isomer. None of
presence of palladium black gave LYXNAc 22D (100%) in an the compounds showed any inhibition of α-GalNAcase.
overall yield of 27% from azide 36L. The enantiomer L-LYXNAc
Alkylation of iminosugars56–58 can cause changes in both
22L was prepared by a similar procedure in 10% overall yield enzyme inhibition and cellular access.25 Benzylation of the
from azide 36D.
lyxo-pentitols 13D and 13L transforms α-D-galactosidase
inhibitors into potent inhibitors of α-L-rhamnosidase.35 Benzy-
lation of the 2-N-acetylamino-1,2,4-trideoxy-1,4-iminopentitols
Biological evaluation
The synthesis, in this paper, of the xylo-20 and lyxo-22 enantio- under investigation here in general causes an increase in the
meric iminopentitols allowed comparison of HexNAcase and potency of HexNAcase inhibition (Table 2); however the xylo-
α-GalNAcase inhibition by all eight of the 2-N-acetylamino- NH 20D [Ki = 0.29 μM] is a stronger inhibitor of the HL-60
1,2,4-trideoxy-1,4-iminopentitols (Table 1, Fig. 3). Neither derived enzyme than is the corresponding NBn derivative 21D
enantiomer of the ribo-pyrrolidine 24 showed any HexNAcase [Ki = 0.54 μM]. NBn-XYLNAc 21D was of comparably high
Table 1 Concentration of NH-pyrrolidine iminosugars in μM giving 50% inhibition of α-N-acetylgalactosaminidase and β-N-acetylhexosaminidases
Table 2 Concentration of NBn-pyrrolidine iminosugars in μM giving 50% inhibition of α-N-acetylgalactosaminidase and β-N-acetylhexosaminidases
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HexNAcase inhibition activity as NBn-LABNAc 19L, which has relationship has emerged as a universal structure–activity
been used to develop a cellular model system of Sandhoff and relationship for HexNAcase inhibitors. The simple and atom-
Tay–Sachs diseases.59
efficient pyrrolidines presented in this paper provide a signifi-
Besides α-GalNAcase and HexNAcases, compounds 20–23 cant extension to the range of HexNAcase inhibitors used as
were also screened against an extended set of glycosidase potential chemotherapeutic agents for a large number of
enzymes (see the ESI†): α-glucosidase (yeast), β-glucosidase diseases.
(almond), α-galactosidase (coffee beans), β-galactosidase
(bovine liver), α-mannosidase (Jack bean), β-mannosidase
(snail) and α-L-rhamnosidase (P. decumbens). As none of the
compounds showed inhibition against any of the enzymes in
this extended panel the HexNAcase inhibitors described in
Experimental
Chemical experimental
All commercial reagents were used as supplied. DMF was pur-
chased dry from the Aldrich Chemical Company in sure-seal
bottles. Methanol was purchased dry from Alfa Aesar in sure-
seal bottles and pyridine was purchased dry from the Fluka
Chemical Company in sure-seal bottles over molecular sieves.
All other solvents were used as supplied (analytical or HPLC
grade) without prior purification. Thin layer chromatography
(TLC) was performed on aluminium sheets coated with 60 F254
silica. Plates were visualised using a spray of 1% w/v potassium
permanganate in 1 M sodium hydroxide, or a 0.2% w/v cerium(IV)
sulphate and 5% ammonium molybdate solution in 2 M sul-
phuric acid. Flash column chromatography was performed on
Sorbsil C60 40/60 silica. Melting points were recorded on a
Kofler hot block and are uncorrected. Optical rotations were
recorded on a Perkin-Elmer 241 polarimeter with a path length
of 1 dm. Concentrations are quoted in g 100 mL−1. Infrared
spectra were recorded on a Perkin-Elmer 1750 IR Fourier
Transform spectrophotometer using thin films on a diamond
ATR surface or on NaCl/Ge plates unless otherwise stated.
Only the characteristic peaks are quoted. High resolution
mass spectra were recorded (HRMS) on a Bruker microTOF
mass analyser. Nuclear magnetic resonance (NMR) spectra
were recorded on Bruker AVIII 400 HD nanobay and Bruker
DQX 400 spectrometers (1H: 400 MHz and 13C: 100.6 MHz)
in the deuterated solvent stated if residual signals were used
as internal references; however, if D2O was used as the solvent,
acetonitrile was used as an internal reference. All chemical
shifts (δ) are quoted in ppm and coupling constants (J) in Hz,
and compounds were assigned using 2D COSY and HSQC
spectra.
this paper are shown to be highly selective.
It is noteworthy that the two powerful inhibitors L-arabino
11L and D-xylo 20D are pentitol analogues of galacto- 1742 and
gluco- 1843 iminohexitols; the loss of a carbon atom in the side
chain changes the pyrrolidines from weak to potent inhibitors.
The general structure–activity relationship, namely that an
increased flexibility through the addition of an additional
CH2OH unit to the side chain is counterproductive to enzy-
matic interaction and hence leads to decreased inhibition
activity, has been frequently observed for iminosugars.37,60,61
As a further structure–activity relationship it can be noted
that relative to D-xylo 21D, epimerization at C2 leads to a more
than 10-fold loss in activity as can be seen for NBn-LYXNAc
23D [Ki = 18.6 μM]. At the same time epimerization at C3 is
detrimental to activity levels as the D-ribo derivative 24D was
completely inactive against the same enzyme.44 However, epi-
merization at C4 has little influence on activity levels taking us
to the already mentioned NBn-LABNAc 19L [Ki = 2.0 μM for
human placenta]. xylo-NH 20D and xylo-NBn 21D are signifi-
cantly better inhibitors of both HexNAcases from Aspergillus
oryzae than are the L-arabino isomers 11L and 19L, respecti-
vely.
It can be concluded that while the C4 CH2OH configuration
does not influence potency (L-arabino 19L vs. D-xylo 21D) the
C3-OH up and C2-NAc down configurations are very crucial for
enzyme recognition. This trans-motif is universally shown by
potent piperidine HexNAcase inhibitors like 1, 3, 4, 6 and 7
(Fig. 1). Similarly if we recognize a methyl-amide as a bioiso-
stere of the NAc group, then the same trans-relationship of the
C3 and C2 residues can be found in both piperidine 8 and aze-
tidine 9.
XYLNAc synthesis
5-Azido-N-benzyl-3,5,6-trideoxy-3,6-imino-1,2-O-isopropyl-
idene-β-L-idofuranose 29L. Anhydrous pyridine (1.12 mL,
13.8 mmol), trifluoromethanesulfonyl anhydride (1.16 mL,
Conclusions
Easy access to the xylo- 20 and lyxo- 22 NAc pyrrolidines from 6.91 mmol) and dichloromethane (5 mL) were pre-mixed at
sugar lactones is reported and this allowed the comparison −40 °C. The diol 27L (564 mg, 2.30 mmol) was then added
with the previously synthesized arabino- 11 and ribo- 24 deriva- dropwise in dichloromethane (15 mL). The temperature was
tives. This paper is the first direct comparison of all eight then maintained at −25 °C. After 1 h, TLC analysis (ethyl
stereoisomeric 2-NAc-1,4-iminopentitols and their N-benzyl acetate–cyclohexane, 2 : 1) indicated complete conversion of
derivatives against a range of glycosidase enzymes – especially the starting material (Rf 0.24) to the product (Rf 0.84). The
HexNAcases and α-GalNAcase. Both the L-arabino 11L and 19L mixture was washed with HCl (2 M, 10 mL). The aqueous layer
and D-xylo 20D and 21D isomers have emerged as potent and was extracted using dichloromethane (2 × 20 mL). The com-
highly selective inhibitors in the micromolar range of a bined organic layers were dried (MgSO4), filtered and concen-
number of HexNAcases. The up-C3-OH–down-C2-NAc trans- trated in vacuo. Benzylamine (0.75 mL, 6.91 mmol) was added
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to a suspension of the crude ditriflate 28L in THF (15 mL). 5 × ArCH); δC (100 MHz, CDCl3) major anomer: 20.1, 20.5
After 3 h, LRMS indicated product formation by the presence (2 × CO2CH3), 55.5 (C-6, CH2Ph), 63.3 (C-5), 67.5 (C-3), 72.4
of a peak corresponding to a mass of 316 and no starting (C-2), 86.0 (C-4), 95.8 (C-1), 127.0, 128.0, 128.2 (ArCH), 138.6
material peak of mass 509. The mixture was concentrated and (Arquat), 169.0, 169.3 (2 × CO2CH3); minor anomer: 20.4, 20.9 (2
purified by flash column chromatography (cyclohexane–ethyl × CO2CH3), 56.1 (C-6), 57.0 (CH2Ph), 65.3 (C-5), 71.0 (C-3), 79.3
acetate, 3 : 1) to afford the pyrrolidine 29L (635 mg, 87%) as a (C-2), 90.1 (C-4), 101.5 (C-1), 127.0–138.8 (Ar), 169.0, 169.3 (2 ×
+
yellow oil. [α]D26 −11.8 (c, 1.62 in CHCl3); νmax (NaCl)/cm−1 2103 CO2CH3); HRMS (ESI+): calcd for C17H20N4O5Na ([M + Na]+):
(N3); δH (400 MHz, CDCl3) 1.33, 1.51 (2 × 3H, 2 × s, C(CH3)2), 383.1326, found: 383.1326.
2.34 (1H, dd, J6a,6b 10.4, J6a,5 6.6, H-6a), 3.30 (1H, dd, J6b,6a
Following a similar procedure, enantiomer 30D (1.61 g,
10.4, J6b,5 6.7, H-6b), 3.39 (1H, a-d, J3,4 5.1, H-3), 3.53 (1H, d, 80%) was obtained from 29D (1.75 g, 5.54 mmol). [α]2D5 −91.1
Jgem 13.3, CH2Ph), 3.96 (1H, dt, J5,6a+6b 6.6, J5,4 1.4, H-5), 4.15 (c, 1.21 in CHCl3).
(1H, d, Jgem 13.3, CH2Ph), 4.49 (1H, a-d, J2,1 3.6, H-2), 4.71 (1H,
3,5,6-Tri-O-acetyl-2-azido-N-benzyl-1,2,4-trideoxy-1,4-imino-L-
dd, J4,3 5.1, J4,5 1.6, H-4), 5.94 (1H, d, J1,2 3.6, H-1), 7.27–7.36 iditol 31L. The protected lactol 30L (977 mg, 2.17 mmol) was
(5H, m, 5 × ArCH); δC (100 MHz, CDCl3) 26.7, 27.4 (C(CH3)2), suspended in ethanol (20 mL) and cooled to 0 °C. Sodium
58.0 (C-6), 58.2 (CH2Ph), 64.7 (C-5), 72.7 (C-3), 83.3 (C-2), borohydride (308 mg, 8.14 mmol) was added and the reaction
88.0 (C-4), 107.0 (C-1), 112.2 (C(CH3)2), 127.5–137.7 (Ar); mixture was stirred for 17 h, after which LRMS analysis indi-
HRMS (FI+): calcd for C16H20N4O3 (M+): 316.1535, found: cated product formation by the presence of a peak corres-
+
316.1532.
ponding to a mass of 278. The reaction was quenched with
Following a similar procedure, enantiomer 29D (1.77 g, glacial acetic acid and concentrated, co-evaporating with
77%) was obtained from 27D (1.78 g, 7.27 mmol). [α]2D5 +5.3 (c, methanol five times. The crude residue was suspended in
1.26 in CHCl3).
DCM (10 mL) and stirred with DIPEA (1.89 mL, 10.8 mmol),
1,2-Di-O-acetyl-5-azido-N-benzyl-3,5,6-trideoxy-3,6-imino-L- DMAP (66 mg, 0.54 mmol) and acetic anhydride (1.54 mL,
idofuranose 30L. Pyrrolidine 29L (1.40 g, 4.43 mmol) was sus- 16.3 mmol) for 23 h. TLC analysis (cyclohexane–ethyl acetate,
pended in a mixture of trifluoroacetic acid (11.2 mL) and water 1 : 1) indicated formation of the product (Rf 0.60). The reaction
(2.8 mL) and stirred at room temperature. After 5 h, TLC ana- mixture was concentrated and purified by flash column chromato-
lysis (cyclohexane–ethyl acetate, 1 : 1) indicated complete con- graphy (cyclohexane–ethyl acetate, 4 : 1) to afford the triacetate
sumption of the starting material (Rf 0.79) and formation of 31L (726 mg, 66% over 2 steps) as a yellow oil. [α]2D5 −8.3
the product (Rf 0.10). The reaction mixture was concentrated, (c, 0.42 in CHCl3); νmax (NaCl)/cm−1 2107 (N3), 1745 (CvO);
co-evaporating with toluene, and the residue was suspended in δH (400 MHz, CD3CN) 1.99, 2.01, 2.09 (3 × 3H, 3 × s,
dichloromethane (30 mL). The product was washed with a 3 × CO2CH3), 2.34 (1H, dd, J1a,1b 10.1, J1a,2 7.6, H-1a), 3.18 (1H,
saturated aqueous solution of sodium bicarbonate (30 mL) dd, J1b,1a 10.2, J1b,2 6.6, H-1b), 3.42 (1H, dd, J 7.6, J 5.2, H-4),
and extracted with dichloromethane (3 × 30 mL). Flash 3.62 (1H, d, Jgem 13.4, CH2Ph), 4.03 (1H, d, Jgem 13.4, CH2Ph),
column chromatography of the residue (cyclohexane–ethyl 4.13 (1H, a-q, J 6.7, H-2), 4.23 (1H, dd, J6a,6b 11.7, J6a,5 8.2, H-6a),
acetate, 1 : 1 → 1 : 3) afforded the lactol (1.22 g, quant.) as a 4.46 (1H, dd, J6b,6a 11.8, J6b,5 3.4, H-6b), 5.15–5.20 (2H, m, H-3,
yellow oil. The crude lactol (805 mg, 2.92 mmol) was sus- H-5), 7.28–7.36 (5H, m, 5 × ArCH); δC (100 MHz, CD3CN) 20.4,
pended in dichloromethane (8 mL) and mixed with DIPEA 20.6, 20.6 (3 × CO2CH3), 54.7 (C-1), 60.5 (CH2Ph), 63.1 (C-4),
(2.03 mL, 11.68 mmol), DMAP (71 mg, 0.58 mmol) and acetic 63.3 (C-6), 63.8 (C-2), 71.2, 77.0 (C-3, C-5), 127.6–139.1 (ArCH),
anhydride (1.65 mL, 17.5 mmol). After 16 h, TLC analysis 170.5, 170.6, 170.9 (3 × CO2CH3); HRMS (ESI+): calcd for
(cyclohexane–ethyl acetate, 1 : 1) indicated complete conver- C19H24N4O6Na + ([M + Na]+): 427.1588, found: 427.1586.
sion of the starting material (Rf 0.10) to the product (Rf 0.79).
Following a similar procedure, enantiomer 31D (1.48 g,
The mixture was concentrated in vacuo and purified by flash 85%) was obtained from 30D (1.56 g, 4.33 mmol). [α]2D5 +12.4
column chromatography (cyclohexane–ethyl acetate, 4 : 1) to (c, 0.94 in CHCl3).
afford the diacetate 30L (977 mg, 84%) as a colorless oil in an
3,5-Di-O-acetyl-2-azido-N-benzyl-1,2,4-trideoxy-1,4-imino-
anomeric ratio of 1 : 3. [α]2D6 +75.3 (c, 0.91 in CHCl3); D-xylitol 32D. The triacetate 31L (199 mg, 0.49 mmol) was
νmax (NaCl)/cm−1 2106 (N3), 1755 (CvO); δH (400 MHz, CDCl3) stirred with sodium methoxide (8 mg, 0.15 mmol) in methanol
major anomer: 1.92, 2.01 (2 × 3H, 2 × s, 2 × CO2CH3), 2.72 (1H, (2 mL). After 2 h, TLC analysis (cyclohexane–ethyl acetate, 1 : 1)
dd, J6a,6b 10.5, J6a,5 4.2, H-6a), 3.04 (1H, dd, J6b,6a 10.5, J6b,5 5.5, indicated complete conversion of the starting material
H-6b), 3.65 (1H, d, Jgem 13.2, CH2Ph), 3.77 (1H, m, H-3), 3.78 (Rf 0.64) to the product (Rf 0.08). The mixture was concentrated
(1H, d, Jgem 13.2, CH2Ph), 3.80 (1H, m, H-5), 4.68 (1H, a-d, in vacuo and re-suspended in methanol–water 3 : 1 (1 mL).
J 6.9, H-4), 5.11 (1H, a-t, J2,1+3 4.4, H-2), 6.35 (1H, d, J1,2 4.2, Sodium periodate (126 mg, 0.59 mmol) was added and the
H-1), 7.21–7.30 (5H, m, 5 × ArCH); minor anomer: 2.04, 2.10 mixture was stirred vigorously. After 1 h a further portion of
(2 × 3H, 2 × s, 2 × CO2CH3), 2.32 (1H, a-t, J6a,6b+5 9.0, H-6a), sodium periodate (42 mg, 0.20 mmol) was added (as TLC ana-
3.20 (1H, dd, J6b,6a 9.1, J6b,5 6.9, H-6b), 3.37 (1H, d, J3,4 7.4, lysis had indicated remaining starting material). After a
H-3), 3.51 (1H, d, Jgem 13.6, CH2Ph), 4.11 (1H, d, Jgem 13.6, further 1 h, TLC analysis (ethyl acetate–methanol, 4 : 1) indi-
CH2Ph), 4.15 (1H, m, H-5), 4.71 (1H, dd, J4,3 7.1, J4,5 3.9, H-4), cated conversion of the starting material (Rf 0.79) to the
5.04 (1H, a-s, H-2), 6.20 (1H, a-s, H-1), 7.21–7.30 (5H, m, product (Rf 0.68). Sodium borohydride (46 mg, 1.23 mmol)
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was added to the reaction mixture with water (0.25 mL). After HRMS (ESI+): calcd for C18H25N2O5 ([M + H]+): 349.1758,
4 h, LRMS indicated formation of the product (mass 248) and found: 349.1751.
+
no remaining starting material (mass 246). The reaction was
Following a similar procedure, enantiomer 33L (82 mg,
quenched with glacial acetic acid and concentrated, co-evapor- 66%) was obtained from 32L (118 mg, 0.36 mmol). Mp 66 °C;
ating with methanol five times. The residue was suspended in [α]2D5 +35.2 (c, 1.03 in MeOH).
DCM (1 mL), and DIPEA (0.34 mL, 1.96 mmol) was added fol-
2-Acetamido-N-benzyl-1,2,4-trideoxy-1,4-imino-D-xylitol 21D.
lowed by DMAP (12 mg, 0.10 mmol) and acetic anhydride The diacetate 33D (69 mg, 0.20 mmol) was stirred with sodium
(0.28 mL, 2.94 mmol). After stirring for 18 h, TLC analysis methoxide (2 mg, 0.04 mmol) in methanol (1 mL) for 2 h. TLC
(cyclohexane–ethyl acetate, 1 : 1) indicated formation of the analysis (ethyl acetate–methanol, 4 : 1) indicated the product
product (Rf 0.55), and the mixture was pre-adsorbed onto silica (Rf 0.31) and no starting material (Rf 0.60). The mixture was
and purified by flash column chromatography (cyclohexane– concentrated and purified by flash column chromatography
ethyl acetate, 5 : 1) to afford the diacetate 32D (101 mg, 62%) (acetone) to afford the N-benzyl amide 21D (41 mg, 77%) as a
as a colourless oil. [α]2D5 −39.6 (c, 0.82 in CHCl3); νmax (NaCl)/ pale yellow solid. Mp 114–117 °C; [α]2D5 −30.3 (c, 0.85 in
cm−1 2106 (N3), 1743 (CvO); δH (400 MHz, CDCl3) 2.05, 2.12 MeOH); νmax (NaCl)/cm−1 3282 (OH), 1651 (CvO amide I),
(2 × CH3, 2 × s, 2 × CO2CH3), 2.29 (1H, dd, J1a,1b 9.8, J1a,2 7.9, 1557 (CvO amide II); δH (400 MHz, MeOD) 1.93 (3H, s,
H-1a), 3.19–3.26 (2H, m, H-1b, H-4), 3.51 (1H, d, Jgem 13.2, NHCOCH3), 2.12 (1H, a-t, J 9.0, H-1a), 2.82–2.86 (1H, m, H-4),
CH2Ph), 3.96–4.01 (1H, m, H-2), 4.03 (1H, d, Jgem 13.4, CH2Ph), 3.17 (1H, dd, J1b,1a 9.4, J1b,2 7.0, H-1b), 3.46 (1H, d, Jgem 13.0,
4.11 (1H, dd, J5a,5b 11.4, J5a,4 7.0, H-5a), 4.19 (1H, dd, J5b,5a 11.4, CH2Ph), 3.71 (1H, dd, J5a,5b 11.4, J5a,4 4.2, H-5a), 3.82 (1H, dd,
J5b,4 4.6, H-5b), 5.19 (1H, dd, J 6.9, J 5.5, H-3), 7.27–7.35 (5H, m, J5b,5a 11.3, J5b,4 5.9, H-5b), 4.06 (1H, d, Jgem 12.9, CH2Ph),
5 × ArCH); δC (100 MHz, CDCl3) 20.8, 20.9 (2 × CO2CH3), 4.09–4.15 (2H, m, H-2, H-3), 7.23–7.36 (5H, m, 5 × ArCH); δC
55.2 (C-1), 58.8 (CH2Ph), 62.0 (C-5), 62.2 (C-4), 63.4 (C-2), 77.3 (100 MHz, MeOD) 21.5 (NHCOCH3), 55.9 (C-1), 56.6 (C-2), 59.0
(C-3), 127.4–137.7 (Ar), 170.3, 170.6 (2 × CO2CH3); HRMS (ESI+): (CH2Ph), 60.4 (C-5), 66.9 (C-4), 76.8 (C-3), 127.3–138.4 (ArCH),
+
calcd for C16H20N4O4Na+ ([M
355.1370.
+
Na]+): 355.1377, found: 172.6 (NHCOCH3); HRMS (ESI+): calcd for C14H21N2O3
([M + H]+): 265.1547, found: 265.1545.
Following a similar procedure, enantiomer 32L (118 mg,
Following a similar procedure, enantiomer 21L (41 mg,
76%) was obtained from 31D (191 mg, 0.47 mmol). [α]2D5 +30.5 77%) was obtained from 33L (71 mg, 0.20 mmol). Mp
(c, 1.06 in CHCl3).
123–126 °C; [α]2D5 +39.9 (c, 0.99 in MeOH).
2-Acetamido-3,5-di-O-acetyl-N-benzyl-1,2,4-trideoxy-1,4-imino-
2-Acetamido-1,2,4-trideoxy-1,4-imino-D-xylitol (XYLNAc) 20D.
D-xylitol 33D. Palladized carbon (10%, 29 mg, 0.03 mmol) was Palladium black (8 mg) was added to a solution of the
added to a solution of the azide 32D (89 mg, 0.27 mmol) in N-benzyl pyrrolidine 21D (41 mg, 0.17 mmol) in 1,4-dioxane :
1,4-dioxane (2 mL). The flask was degassed and flushed with water 1 : 1 (1 mL). The flask was degassed and flushed with
hydrogen and stirred under a hydrogen atmosphere for 1 h. hydrogen. After stirring for 4.5 h under a hydrogen atmos-
TLC analysis (cyclohexane–ethyl acetate, 1 : 1) indicated a base- phere, analysis by TLC (ethyl acetate–methanol, 4 : 1) indicated
line product and no remaining starting material (Rf 0.69). The complete transformation of the starting material (Rf 0.22) to
mixture was filtered through Celite® eluting with 1,4-dioxane the baseline product. The mixture was filtered through
and concentrated in vacuo.
Celite®, eluting with 1,4-dioxane and water. Concentration
The resulting crude amine (83 mg, quant.) was suspended in vacuo afforded XYLNAc 20D (25 mg, 93%) as a pale yellow
in dichloromethane (0.8 mL) and stirred with DIPEA (0.09 mL, solid. Mp 162–170 °C, decomposed. [α]2D5 −6.9 (c, 1.33 in H2O);
0.54 mmol), DMAP (7 mg, 0.05 mmol) and acetic anhydride νmax (Ge)/cm−1 3274 (OH, NH), 1645 (CvO amide I), 1542
(0.08 mL, 0.81 mmol). After 18 h, TLC analysis (ethyl acetate) (CvO amide II); δH (400 MHz, D2O) 1.87 (3H, s, NHCOCH3),
indicated complete conversion of baseline starting material to 2.57 (1H, dd, J1a,1b 12.3, J1a,2 4.7, H-1a), 3.09 (1H, a-dd, J 11.2,
the product (Rf 0.15). The mixture was concentrated and puri- J 6.3, H-4), 3.27 (1H, dd, J1b,1a 12.3, J1b,2 7.1, H-1b), 3.56 (1H,
fied by flash column chromatography (ethyl acetate–cyclo- dd, J5a,5b 11.3, J5a,4 6.7, H-5a), 3.67 (1H, dd, J5b,5a 11.3, J5b,4 4.7,
hexane, 12 : 1) to afford the amide 33D (67 mg, 71%) as a H-5b), 3.95–3.99 (1H, m, H-2), 4.05 (1H, dd, J 4.6, J 2.6, H-3); δC
yellow solid. Mp 72–75 °C; [α]2D5 −35.4 (c, 0.70 in MeOH); νmax (100 MHz, D2O) 22.2 (NHCOCH3), 48.8 (C-1), 58.1 (C-2), 60.3
(NaCl)/cm−1 3280 (NH), 1741 (CvO ester), 1656 (CvO amide (C-5), 61.6 (C-4), 76.3 (C-3), 174.3 (NHCOCH3); HRMS (ESI+):
I), 1550 (CvO amide II); δH (400 MHz, MeOD) 1.91 (3H, s, calcd for C7H15N2O3+ ([M + H]+): 175.1077, found: 175.1077.
NHCOCH3), 2.03, 2.07 (2 × 3H, 2 × s, 2 × CO2CH3), 2.19 (1H,
Following a similar procedure, enantiomer 20L (13 mg,
a-t, J1a,1b+2 9.3, H-1a), 3.14 (1H, dd, J1b,1a 9.2, J1b,2 7.2, H-1b), 100%) was obtained from 21L (19 mg, 0.08 mmol). Mp 160 °C,
3.20 (1H, a-dt, J4,3 7.2, J4,5 4.7, H-4), 3.51 (1H, d, Jgem 13.1, decomposed; [α]2D5 +5.3 (c, 0.61 in H2O).
CH2Ph), 4.03 (1H, d, Jgem 13.1, CH2Ph), 4.10–4.20 (2H, m, 2 ×
LYXNAc synthesis
H-5), 4.29 (1H, a-dt, J2,1a 9.2, J2,3 6.8, H-2), 5.23 (1H, dd, J3,4
7.7, J3,2 6.5, H-3), 7.24–7.33 (5H, m, 5 × ArCH); δC (100 MHz,
MeOD) 19.8, 19.8 (CO2CH3), 21.5 (NHCOCH3), 53.4 (C-2), 55.4 fluoromethanesulfonyl anhydride (3.97 mL, 23.9 mmol) was
(C-1), 58.6 (CH2Ph), 62.1 (C-5), 62.5 (C-4), 77.0 (C-3), added dropwise to stirred solution of 35L (2.81 g,
127.4–138.1 (Ar), 171.1, 171.4, 172.3 (2 × CO2CH3, NHCOCH3); 14.9 mmol) in pyridine (35 mL) at −10 °C under nitrogen.
2-Azido-3,4-O-isopropylidene-L-ribono-1,5-lactone 40L. Tri-
a
3938 | Org. Biomol. Chem., 2014, 12, 3932–3943
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After 4 h at −10 °C, TLC analysis (ethyl acetate–cyclohexane, 1 : 50 → 1 : 10) to yield the benzhydryl ether 41L (2.18 g, 75%)
1 : 1) showed the formation of a single product. The reaction as a yellow solid. Mp 116–118 °C; [α]2D6 −42.0 (c, 1.14 in CHCl3);
mixture was diluted with dichloromethane (80 mL), washed νmax (film)/cm−1 2115 (N3), 1786 (CvO); δH (400 MHz, CDCl3)
with 2 M HCl (50 mL) and the aqueous layer was further 3.39 (1H, dd, J5a,5b 10.8, J5a,4 2.4, H-5a), 3.64 (1H, dd,
extracted with dichloromethane (2 × 100 mL). The combined J5b,5a 10.8, J5b,4 2.8, H-5b), 4.30 (1H, d, J2,3 6.0, H-2), 4.33 (1H,
organic extracts were dried (MgSO4) and concentrated in vacuo d, J3,2 6.0, H-3), 4.59 (1H, t, J4,5b/J4,5a 2.8, H-4), 5.27, 5.59 (2 ×
to give the triflate 39L as a pale yellow solid (3.50 g, 73%). 1H, s, CHPh2), 7.16–7.35 (20H, m, ArCH); δC (100 MHz, CDCl3)
Sodium azide (0.97 g, 15.0 mmol) was added to a stirred solu- 59.7 (C-2), 67.7 (C-5), 75.6 (C-3), 82.7 (C-4), 83.8, 84.8 (2 ×
tion of the triflate 39L (3.00 g, 9.4 mmol) in DMF (42 mL) CHPh2), 126.7–140.8 (Ar), 171.6 (C-1); HRMS (ESI+): calcd for
+
under nitrogen at r.t. The reaction mixture was stirred for 18 h, C31H27N3NaO4 ([M
+
Na]+): 528.1894, found: 528.1903;
after which time TLC analysis (ethyl acetate–cyclohexane, 2 : 1) elemental analysis calcd (%) for C31H27N3O4: C 73.65, H 5.38,
indicated no remaining starting material and the formation of N 8.31, found: C 73.58, H 5.37, N 8.27.
a product (Rf 0.5). The reaction mixture was concentrated
Following a similar procedure, enantiomer 41D (1.64 g,
in vacuo and the residue was partitioned between dichloro- 88%) was obtained from 36D (638 mg, 3.69 mmol). [α]2D5 +32.6
methane (60 mL) and water (60 mL). The aqueous layer was (c, 0.56 in CHCl3).
further extracted with dichloromethane (3 × 60 mL). The com-
2-Azido-3,5-di-O-benzhydryl-2-deoxy-L-ribitol 42L. Diisobutyl-
bined organic extracts were dried with MgSO4, filtered and aluminum hydride solution (25 wt% in toluene, 0.48 mL,
concentrated in vacuo to yield a yellow oil which was purified 0.71 mmol, 1.2 equiv.) was added to a stirred solution of the
using column chromatography (ethyl acetate–cyclohexane, protected lactone 41L (0.3 g, 0.6 mmol) in dichloromethane
2 : 1) to yield the azido L-ribono-lactone 40L (1.51 g, 76%) as a (2.5 mL) at −78 °C. After 3 hours, analysis by TLC (ethyl
white solid. Mp 80–82 °C; [α]2D0 +165.4 (c, 0.47 in CHCl3); νmax acetate–cyclohexane, 1 : 3) showed complete conversion to a
(film)/cm−1 2114 (N3), 1756 (CvO); δH (400 MHz, CDCl3) 1.36, single product (Rf 0.33). The crude mixture was diluted with
1.47 (2 × 3H, 2 × s, C(CH3)2), 3.81 (1H, d, J2,3 3.2, H-2), 4.19 dichloromethane (10 mL), sodium potassium tartrate (13 mL)
(1H, d, J5a,5b 13.2, H-5a), 4.44 (1H, d, J5b,5a 13.2, H-5b), 4.58 was added and the mixture was stirred overnight. The aqueous
(1H, d, J4,3 8.0, H-4), 4.84 (1H, dd, J3,2 3.2, J3,4 8.0, H-3); δC layer was washed with dichloromethane (2 × 25 mL), the com-
(100 MHz, CDCl3) 24.3, 25.8 (C(CH3)2), 59.2 (C-2), 67.4 (C-5), bined organic layers were dried and the resulting mixture was
72.4 (C-4), 75.5 (C-3), 111.0 (C(CH3)2), 166.6 (C-1); HRMS concentrated in vacuo to yield a colourless oil to which metha-
+
(ESI+): calcd for C8H11N3NaO4 ([M + Na]+): 236.0642, found: nol (2.3 mL) and NaBH4 (34 mg, 0.89 mmol, 1.5 eq.) were
236.0652.
added under an atmosphere of N2. After 4 hours, analysis by
2-Azido-2-deoxy-L-ribono-1,4-lactone 36L. The δ-lactone 40L TLC (ethyl acetate–cyclohexane, 1 : 2) showed no residual start-
(1.51 g, 7.1 mmol) was dissolved in a mixture of trifluoroacetic ing material and a major spot due to the product (Rf 0.45).
acid (9.6 mL) and water (4.8 mL) and heated to 60 °C. After Glacial acetic acid was added dropwise to adjust to neutral pH.
3.5 h, TLC analysis (ethyl acetate–cyclohexane, 3 : 2) indicated The solvents were removed in vacuo and the remaining residue
no remaining starting material and a new product (Rf 0.16). was purified by flash chromatography (ethyl acetate–cyclo-
The reaction mixture was concentrated in vacuo and the hexane, 1 : 3) to yield the diol 42L as a colourless oil (264 mg,
residue was purified by column chromatography (ethyl 88% over 2 steps). [α]2D6 +32.2 (c, 0.49 in CHCl3); νmax (film)/
acetate–cyclohexane, 2 : 1) to the azido γ-lactone 36L (1.0 g, cm−1 3442 (OH), 2099 (N3); δH (400 MHz, CD3CN) 3.15 (1H, t,
82%) as a colourless oil. [α]2D0 −44.2 (c, 1.02 in ethyl acetate), J 5.6, OH-1), 3.37 (1H, dd, J5a,5b 10.0, J5a,4 6.0, H-5a), 3.42 (1H,
[Lit.50 for enantiomer [α]D20 +54.2 (c, 0.28 in ethyl acetate)]; d, J 5.6, OH-4), 3.56 (1H, dd, J5b,5a 10.0, J5b,4 4.0, H-5b), 3.61
−1
νmax (film)/cm
2119 (N3), 1771 (CvO); δH (400 MHz, (1H, m, J1a,1b 11.6, J1a,OH 5.6, J1a,2 3.2, H-1a), 3.65 (1H, dd,
(CD3)2CO) 3.05 (1H, br s, OH), 3.85 (2H, s, H-5a and H-5b), 4.45 J3,4 6.0, J3,2 3.2, H-3), 3.79 (1H, ddd, J1b,1a 11.6, J1b,OH 6.0, J1b,2
(1H, d, J2,3 3.6, H-2), 4.50 (1H, d, J4,5 2.0, H-4), 4.62 (1H, d, J3,2 1.2, H-1b), 3.84 (1H, m, H-2), 3.99 (1H, m, H-4), 5.35, 5.73
4.0, H-3), 5.24 (1H, br s, OH); δC (100 MHz, (CD3)2CO) 61.7 (2 × 1H, s, CHPh2), 7.24–7.39 (20H, m, ArCH); δC (100 MHz,
(C-2), 61.9 (C-5), 72.1 (C-3), 87.6 (C-4), 172.9 (C-1); HRMS (ESI+): CD3CN) 62.1 (C-1), 65.4 (C-2), 70.2 (C-4), 70.5 (C-5), 77.7 (C-3),
calcd for C5H7N3NaO4+ ([M + Na]+): 196.0329, found: 196.0334.
82.7, 83.9 (2 × CHPh2), 127.1–143.1 (Ar); HRMS (ESI+): calcd
2-Azido-3,5-di-O-benzhydryl-2-deoxy-L-ribono-1,4-lactone 41L. for C31H31N3NaO4+ ([M + Na]+): 532.2207, found: 532.2211.
Diphenyldiazomethane (3.93 g, 20.2 mmol) was added to a
Following a similar procedure, enantiomer 42D (1.02 g,
refluxing stirred solution of the unprotected lactone 36L 62%) was obtained from 41D (1.64 g, 3.24 mmol). [α]2D5 −32.5
(1.00 g, 5.8 mmol) in toluene (140 mL). After 3 hours, TLC ana- (c, 0.73 in CHCl3).
lysis (ethyl acetate–cyclohexane, 1 : 3) showed that the starting
2-Azido-3,5-di-O-benzhydryl-2-deoxy-1,4-di-O-methanesulfonyl-
material (Rf 0.15) remained, so one further equivalent of diazo- L-ribitol 43L. Triethylamine (0.76 mL) was added to a stirred
methane was added. The mixture was kept under heating for solution of the diol 42L (696 mg, 1.37 mmol) in dichloro-
one hour, after which TLC analysis indicated no remaining methane (4 mL) at 0 °C under an atmosphere of N2. After
starting material and a new product (Rf 0.6). The reaction 20 min, methanesulfonyl chloride (0.42 mL, 5.46 mmol,
mixture was concentrated in vacuo and the residue was puri- 4.0 equiv.) was added dropwise. After 3 hours, analysis by TLC
fied by column chromatography (ethyl acetate–cyclohexane, (ethyl acetate–cyclohexane, 1 : 1) showed no residual starting
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material and a major spot due to the product (Rf 0.73). The sol- 2.17 (2 × 3H, s, 2 × CH3), 2.61 (1H, dd, J1a,1b 11.0, J1a,2 5.4, H-1a),
vents were removed in vacuo and the remaining residue was 3.02 (1H, dd, J1b,1a 11.0, J1b,2 3.0, H-1b), 3.20 (1H, m, J4,3 7.6,
purified by flash chromatography (ethyl acetate–cyclohexane, H-4), 3.53 (1H, d, J 13.4, CH2Ph), 3.97 (1H, m, J2,3 5.6, J2,1a 5.4,
1 : 3) to yield the dimesylate 43L as a colourless oil (910 mg, J2,1b 3.0, H-2), 4.09 (1H, d, J 13.4, CH2Ph), 4.25 (2H, m, H-5,
quant.). [α]2D6 +21.4 (c, 0.99 in CHCl3); νmax (film)/cm−1 2107 H-5′), 5.42 (1H, dd, J3,4 7.6, J3,2 5.6, H-3), 7.27–7.33 (5H, m,
(N3), 1359, 1177 (CvO); δH (400 MHz, CDCl3) 2.92 (3H, s, ArCH); δC (100 MHz, CDCl3) 20.5, 20.8 (2C, 2 × CH3), 55.0 (C-1),
CH3), 2.96 (3H, s, CH3), 3.65 (1H, dd, J5a,5b 11.2, J5a,4 7.2, 58.8 (CH2Ph), 59.8 (C-2), 61.6 (C-4), 62.9 (C-5), 73.5 (C-3),
H-5a), 3.76 (1H, dd, J5b,5a 11.2, J5b,4 4.0, H-5b), 3.85 (1H, dd, 127.4–137.7 (Ar), 170.2, 170.7 (2C, 2 × CvO); HRMS (ESI+): calcd
J3,2 5.6 Hz, J3,4 3.6, H-3), 3.94 (1H, ddd, J2,1a 8.0, J2,3 5.6, J2,1b for C16H20N4NaO4+ ([M + Na]+): 355.1377, found: 355.1378.
3.2, H-2), 4.07 (1H, dd, J1a,1b 10.8, J1a,2 8.0, H-1a), 4.44 (1H, dd,
Following a similar procedure, enantiomer 45L (43 mg,
J1b,1a 10.8, J1b,2 3.2, H-1b), 5.09 (1H, m, J4,5a 6.8, J4,5b 4.0, J4,3 68%) was obtained from 44L (110 mg, 0.19 mmol). [α]2D5 +63.2
3.6, H-4), 5.33, 5.70 (2 × 1H, s, CHPh2), 7.27–7.34 (20H, m, (c, 1.31 in CHCl3).
ArCH); δC (100 MHz, CDCl3) 37.5, 38.7 (2 × CH3), 61.1 (C-2),
67.2 (C-5), 68.5 (C-1), 75.1 (C-3), 80.4 (C-4), 83.6, 84.5 D-lyxitol 46D. Zinc dust (0.659 g, 10.08 mmol) was added to a
(2
CHPh2), 126.8–140.9 (Ar); HRMS (ESI+): calcd for stirred solution of the azide 45D (0.167 g, 0.50 mmol) in
C33H35N3NaO8S2+ ([M + Na]+): 688.1758, found: 688.1744.
5.6 mL of THF–AcOH–Ac2O (3 : 2 : 1); 1.25 mL of saturated
2-Acetamido-3,5-di-O-acetyl-N-benzyl-1,2,4-trideoxy-1,4-imino-
×
Following a similar procedure, enantiomer 43D (1.30 g, aqueous CuSO4 was added in order to activate the zinc. After
98%) was obtained from 42D (1.02 g, 2.00 mmol). [α]2D5 −22.6 40 min, analysis by TLC (cyclohexane–ethyl acetate, 1 : 1)
(c, 0.83 in CHCl3).
showed no residual starting material. The reaction mixture was
2-Azido-3,5-di-O-benzhydryl-N-benzyl-1,2,4-trideoxy-1,4-imino- filtered through Celite and the filtrate was concentrated
D-lyxitol 44D. The dimesylate 43L was stirred in benzylamine in vacuo by co-evaporation with toluene. Purification by flash
(1.4 mL) and toluene (6.4 mL) at 80 °C under an atmosphere chromatography eluting with 4 : 1 cyclohexane–ethyl acetate
of N2. After 48 hours, analysis by TLC (ethyl acetate–cyclo- and 1 : 4 methanol–ethyl acetate gave the protected iminolyxi-
hexane, 1 : 1) showed no residual starting material and a major tol 46D (0.14 g, 80%) as a brown oil (Rf baseline): [α]2D0 −14.7 (c,
spot due to the product (Rf 0.8). The solvents were removed 0.97 in CHCl3); νmax (film)/cm−1 1741 (CvO ester), 1659 (CvO
in vacuo and the resulting yellow residue was purified by flash amide I), 1539 (N–H amide II); δH (400 MHz, CDCl3) 1.99 (3H,
chromatography (ethyl acetate–cyclohexane, 1 : 5) to yield the s, NHCOCH3), 2.08, 2.09 (2 × 3H, s, 2 × CH3), 2.64 (1H, dd,
N-benzyl lyxitol 44D as a yellow oil (445 mg, 90%). [α]2D6 −4.1 J1a,1b 10.4, J1a,2 6.0, H-1a), 2.81 (1H, dd, J1b,1a 10.0, J1b,2 3.2,
(c, 1.02 in CHCl3); νmax (film)/cm−1 2101 (N3), 1494, 1452 H-1b), 3.16 (1H, m, J4,3 7.6, J4,5 6.0, H-4), 3.51 (1H, d, J 12.8,
(arom); δH (400 MHz, CDCl3) 2.37 (1H, dd, J5a,5b 10.8, J5a,4 4.8, CH2Ph), 4.03 (1H, d, J 12.8, CH2Ph), 4.15 (2H, d, J5,4 6.0, H-5a
H-5a), 2.95 (1H, m, H-5b), 3.12 (1H, m, H-2), 3.42 (1H, d, and H-5b), 4.62 (1H, m, H-2), 5.46 (1H, dd, J3,4 7.6, J3,2 6.0,
J 13.4, CH2Ph), 3.61 (1H, m, H-4), 3.64 (1H, dd, J1a,1b 10.0, J1a,2 H-3), 5.89 (1H, bs, NHCOCH3), 7.28–7.36 (5H, m, ArCH);
5.6, H-1a), 3.96 (1H, dd, J1b,1a 10.0, J1b,2 6.0, H-1b), 4.16 (1H, d, δC (100 MHz, CDCl3) 20.7, 20.9 (2C,
2 × CH3), 23.3
J 13.4, CH2Ph), 4.22 (1H, dd, J3,2 8.0, J3,4 5.6, H-3), 5.32, 5.57 (CH3CONH), 49.1 (C-2), 57.0 (C-1), 58.9 (CH2Ph), 62.0 (C-5),
(2 × 1H, s, CHPh2), 7.22–7.37 (25H, m, ArCH); δC (100 MHz, 62.1 (C-4), 72.2 (C-3), 127.5–128.8 (Ar), 169.6, 169.8, 170.5 (3C,
+
CDCl3) 55.4 (C-5), 59.0 (CH2Ph), 60.3 (C-4), 64.1 (C-2), 68.8 3 × CvO); HRMS (ESI+): calcd for C18H24N2NaO5 ([M + Na]+):
(C-1), 77.2 (C-3), 82.8, 84.3 (2 × CHPh2), 126.8–142.3 (Ar); 371.1577, found: 371.1579.
HRMS (ESI+): calcd for C38H37N4O2 ([M + H]+): 581.2911,
found: 581.2910.
Following a similar procedure, enantiomer 46L (164 mg,
74%) was obtained from 45L (211 mg, 0.64 mmol). [α]2D5 +16.7
+
Following a similar procedure, enantiomer 44L (743 mg, (c, 1.15 in CHCl3).
65%) was obtained from 43D (1.30 g, 1.96 mmol). [α]2D5 +1.6
(c, 0.95 in CHCl3).
2-Acetamido-N-benzyl-1,2,4-trideoxy-1,4-imino-D-lyxitol 23D.
Sodium methoxide (8 mg) was added to a stirred solution of
2-Azido-3,5-di-O-acetyl-N-benzyl-1,2,4-trideoxy-1,4-imino-D-lyxitol diester 46D (102.0 mg, 0.29 mmol) in MeOH (1.1 mL) at room
45D. Boron trifluoride diethyl etherate (0.60 mL, 6.5 equiv.) temperature under an atmosphere of N2. After 2 h, analysis by
was added to a stirred solution of the benzhydryl protected TLC (ethyl acetate–propan-2-ol, 7 : 2) indicated no remaining
pyrrolidine 44D (418 mg, 0.72 mmol) in acetic anhydride starting material. The solvents were removed in vacuo, and the
(3.5 mL) at 0 °C under an atmosphere of N2. After 24 h, ana- resulting residue was purified by flash chromatography (ethyl
lysis by TLC (ethyl acetate–cyclohexane, 1 : 4) showed no acetate–propan-2-ol, 7 : 2) to yield the N-benzylpyrrolidine 23D
residual starting material and a spot due to the product on the (62.5 mg, 81%) as a white solid (Rf 0.29). Mp 68–70 °C;
baseline. The solvent was removed in vacuo and the remaining [α]2D0 −62.0 (c, 1.45 in MeOH); νmax (film)/cm−1 3279 (OH),
residue was dissolved in ethyl acetate (60 mL), washed with 1651 (CvO amide I), 1530 (N–H amide II); δH (400 MHz,
saturated aqueous NaHCO3 (60 mL), dried (MgSO4), and con- MeOD) 1.97 (3H, s, NHCOCH3), 2.78 (1H, dd, J1a,1b 8.4, J1a,2
centrated in vacuo. The residue was purified by flash chromato- 6.0, H-1a), 2.86 (1H, dd, J1b,1a 8.4, J1b,2 4.4, H-1b), 2.95 (1H, m,
graphy (ethyl acetate–cyclohexane, 1 : 5) to yield diacetate 45D as J4,3 8.4, J4,5 4.4, H-4), 3.60 (1H, d, J 10.4, CH2Ph), 3.73 (1H, dd,
a yellow oil (168 mg, 70%). [α]D20 −69.3 (c, 1.06 in CHCl3); νmax J5a,4 3.6, J5a,5b 9.2, H-5a), 3.82 (1H, dd, J5b,4 4.8, J5b,5a 8.8,
(film)/cm−1 2106 (N3), 1743 (CvO); δH (400 MHz, CDCl3) 2.04, H-5b), 4.08 (1H, d, J 10.4, CH2Ph), 4.31 (1H, m, H-2), 4.33 (1H,
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Paper
dd, J3,4 8.4, J3,2 4.4, H-3), 7.26–7.40 (5H, m, ArCH); δC Co.) as the substrate, which was dissolved at 4 mM concen-
(100 MHz, MeOD) 22.7 (CH3CONH), 52.1 (C-2), 56.8 (C-1), 60.6 tration in a 50 mM citrate/citric acid buffer at pH 5. The presence
(CH2Ph), 60.8 (C-5), 69.5 (C-4), 72.6 (C-3), 128.5–130.4 (Ar), of sufficient enzyme activity was confirmed prior to each per-
+
173.3 (CvO); HRMS (ESI+): calcd for C14H20N2NaO3
formed Ki measurement. Materials for β-N-acetylhexosaminidase
activity from human placenta were the same as above. Method:
([M + Na]+): 287.1366, found: 287.1362.
Following a similar procedure, enantiomer 23L (71 mg, enzyme (5 µL) and an inhibitor solution (5 µL) were added to a
60%) was obtained from 46L (156 mg, 0.45 mmol). Mp 96-well microtitre plate which had been pre-incubated at 37 °C
58–61 °C; [α]2D5 +60.5 (c, 0.91 in MeOH).
for 5 min before starting the reaction by the addition of 40 μL of
2-Acetamido-1,2,4-trideoxy-1,4-imino-D-lyxitol (LYXNAc) 22D. the substrate solution. After a further 40 min of incubation at
Palladium black (9 mg) was added to a stirred solution of 37 °C the reaction was quenched by the addition of 200 μL of
N-benzyl lyxitol 23D (44.3 mg, 0.17 mmol) in 1,4-dioxane 0.5 M sodium carbonate (aq.). For HL-60 derived enzyme, fluo-
(0.55 mL) and H2O (0.55 mL) at room temperature under an rescence was measured using a microtitre plate reader (Molecular
atmosphere of H2. After 72 h, analysis by TLC (ethyl acetate– Devices SPECTRAmax M5 Rom v2.1.35, ex. 355 nm, em. 460 nm,
propan-2-ol, 7 : 2) showed no residual starting material cutoff 455 nm). For human placenta derived enzyme the released
(Rf 0.29) and a spot due to the product on the baseline. The Pd p-nitrophenol was measured as described above. Data sets in
black was filtered off over Celite® and washed with MeOH. triplicate were subjected, in Prism 4.0a, to a Lineweaver–Burk
The solvents were removed in vacuo to yield LYXNAc 22D analysis (1/rate against 1/[substrate concentration]) using a suit-
(29.8 mg, quant.) as a yellow oil. [α]2D5 +32.2 (c, 1.06 in MeOH); able range of substrate solutions and inhibitor concentrations.
νmax (film)/cm−1 3318 (OH), 1647 (CvO amide I), 1533 (N–H The obtained slopes were plotted against the inhibitor concen-
amide II); δH (400 MHz, MeOD) 2.00 (3H, s, NHCOCH3), 2.89 tration and the Ki obtained from the X-axis intercept.
(1H, dd, J1a,1b 10.7, J1a,2 9.5, H-1a), 3.24 (1H, dd, J1b,1a 11.0,
J1b,2 8.5, H-1b), 3.33 (1H, m, H-4), 3.72 (1H, dd, J5a,4 6.6, J5a,5b
11.0, H-5a), 3.84 (1H, dd, J5b,4 5.8, J5b,5a 11.0, H-5b), 4.21 (1H,
t, J3,2+4 3.8, H-3), 4.35 (1H, a-dd, J2,1b 8.4, J2,3 4.0, J2,1a 9.2, H-2);
Glossary of abbreviations
Abbreviation Term
δC (100 MHz, MeOD) 21.6 (CH3CONH), 47.4 (C-1), 53.6 (C-2),
α-GalNAcases α-N-Acetylgalactosaminidases
60.4 (C-5), 63.5 (C-4), 70.6 (C-3), 172.4 (CvO); HRMS (ESI+):
ADMDP
1-Acetamido-1,2,5-trideoxy-2,5-imino-
calcd for C7H15N2O3+ ([M + H]+): 175.1076, found: 175.1077.
Following a similar procedure, enantiomer 22L (30 mg,
100%) was obtained from 23L (44 mg, 0.17 mmol). [α]2D5 −25.8
(c, 0.81 in MeOH).
D-mannitol
DAB
1,4-Dideoxy-1,4-imino-D-arabinitol
2-Acetamido-1,2-dideoxy-D-galacto-nojirimycin
2-Acetamido-1,2-dideoxy-D-gluco-nojirimycin
N-Acetylgalactosamine
N-Acetylglucosamine
β-N-Acetylhexosaminidases
1,4-Dideoxy-1,4-imino-L-arabinitol
2-Acetamido-1,4-imino-1,2,4-trideoxy-
L-arabinitol
DGJNAc
DNJNAc
GalNAc
GlcNAc
HexNAcases
LAB
Biological experimental
IC50 determinations: the enzymes β-N-acetylhexosaminidases
(from human placenta, bovine kidney, Jack bean, and Aspergil-
lus oryzae), α-N-acetylgalactosaminidase (from chicken liver)
and p-nitrophenyl glycosides were purchased from Sigma-
Aldrich Co. The cell lysate of the human acute amyloid leuke-
mia cell line HL-60 (RBRC) was used as the source of β-N-acetyl-
hexosaminidase. For this purpose, cells were cultured in RPMI
1640 medium containing 10% fetal calf serum, 100 units mL−1 of
penicillin and 100 μg mL−1 streptomycin (Invitrogen) at 37 °C
under 5% CO2. On completion, cells were washed and centri-
fuged into a pellet from phosphate buffered saline. The cell pellet
was then lysed in deionized H2O (1.5 mL) using a glass dounce
homogenizer, and after centrifugation at 500g for 5 min at 4 °C,
the supernatant was removed and used as an enzyme source. The
glycosidase activities were determined using an appropriate p-
nitrophenyl glycoside as the substrate at the optimum pH of each
enzyme [the HL-60 cell lysate assay was performed at pH 4.5]. The
reaction mixture (1 mL) contained 2 mM of the substrate and an
appropriate amount of the enzyme. The reaction was stopped by
adding 2 mL of 400 mM Na2CO3. The released p-nitrophenol was
measured spectrometrically at 400 nm.
LABNAc
LYXNAc
PUGNAc
2-N-Acetylamino-1,2,4-trideoxy-1,4-iminolyxitol
O-(2-Acetamido-2-deoxy-D-glucopyranosylidene)-
amino-N-phenylcarbamate
XYLNAc
2-N-Acetylamino-1,2,4-trideoxy-1,4-iminoxylitol
Acknowledgements
This work was supported by EPSRC and an Oxford Glyco-
biology Scholarship (EVC), Cancer Research UK (AFGG), Junta
de Extremadura, Ministerio de Educación y Ciencia and
FEDER (CTQ2010-18938/BQU) (RFM), Grant-in-Aid for Scienti-
fic Research (C) (no. 23590127) (AK) from the Japanese Society
for the Promotion of Science (JSPS), and the Leverhulme Trust
(GWJF).
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