Syntheses of novel azasugar-containing mimics of heparan sulfate fragments as potential heparanase inhibitors

Article abstract of DOI:10.1016/j.carres.2010.05.032

A series of eight novel, highly modified mono-, di- and trisaccharide derivatives, each containing an iminoalditol moiety, has been synthesized in the quest for potential heparanase inhibitors.

Full text of DOI:10.1016/j.carres.2010.05.032

Carbohydrate Research 345 (2010) 1831–1841
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Syntheses of novel azasugar-containing mimics of heparan sulfate fragments
as potential heparanase inhibitors
a
a
Kirsty A. Allen b, Rosemary L. Brown b, Gillian Norris b, Peter C. Tyler a, , Derek K. Watt , Olga V. Zubkova
*
a Carbohydrate Chemistry, Industrial Research Limited, Gracefield Rd, Lower Hutt, New Zealand
b Institute of Molecular Biosciences, Massey University, Private Bag 11222, Palmerston North, New Zealand
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of eight novel, highly modified mono-, di- and trisaccharide derivatives, each containing an
iminoalditol moiety, has been synthesized in the quest for potential heparanase inhibitors.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 2 February 2010
Received in revised form 25 May 2010
Accepted 29 May 2010
Available online 4 June 2010
Keywords:
Iminoalditol
Heparan sulfate
Heparanase
Inhibitor
1. Introduction
GlcN(NS,6S)-a-(1?4)-GlcA-b-(1?4)-GlcN(NS,6S) although the
minimum size required for recognition is a trisaccharide.11 Unfortu-
nately, no kinetic data are available for the enzymatic cleavage of
this compound due to extreme difficulty in obtaining inhibition
constants for heparanase. The finding that this tetrasaccharide is
hydrolyzed by the enzyme contradicts earlier studies which con-
cluded that a 2-O-sulfated uronic acid is required in heparanase
substrates. In particular, it was thought that a sulfated uronic acid
moiety is required two residues towards the reducing end of the glu-
curonosyl moiety that is hydrolyzed by the enzyme.12,13
Mammalian heparanase (heparanase 1 or Hpa1) is an endo-b-D-
glucuronidase that hydrolyzes heparan sulfate (HS) at specific
sites, and is thus involved in the degradation of the extracellular
matrix.1 The enzyme is over-expressed in many human tumours,
and this expression correlates with more aggressive and invasive
cancers and with poor patient prognoses. Heparanase degradation
of HS results in the release of factors that promote angiogenesis
and tumour vascularization and the enzyme is also involved in
metastasis and in tissue repair. Consequently, for several years
heparanase inhibitors have been targeted as potential anti-cancer
drugs.2–8 Until recently, the link between metastasis and heparan-
ase has been based mostly on circumstantial evidence because val-
idated heparanase inhibitors have also possessed other biological
activities. Recently, however, the link between heparanase and
metastasis of cancer in mice has been clearly demonstrated by
the use of a viral vector carrying antisense–heparanase DNA.9
HS is a linear polymer of repeating disaccharide units consisting
Heparanase is thought to act with a retaining hydrolytic mech-
anism and belongs to the clan A hydrolase family.14,15 The two glu-
tamic acid residues in the active site of the enzyme that are
presumed to act as the proton donor and nucleophile in the hydro-
lysis step have been identified by homology and confirmed by site-
directed mutagenesis.14 However, there is no detailed information
available on the nature of the transition state(s) of the enzymic
hydrolysis of HS.
It is expected that potent inhibitors of heparanase might be
attained by synthesizing stable mimics of the transition state.
Enzymes can catalyze reactions by lowering the energy of activation
of the reaction,16,17 often by factors of 1010–1015. Stable compounds
that mimic the transition state will convert a proportion of this
energy of activation into thermodynamic binding energy giving rise
to unusually stable enzyme inhibitor complexes.18 The transition
states of a number of N-ribosyltransferases have been determined
using kinetic isotope effects,19 and some exceedingly potent transi-
tion state analogue inhibitors have been designed and synthe-
sized.20–23 These compounds have employed a ribooxacarbenium
of
a-1,4-linked glucosamine and 1,4-linked b-D-gluc-(or a-L-id-)
uronic acid. The polymer is variously N- and O-sulfated and has a
block-like domain structure with regions of high sulfation, low sul-
fation and no sulfation.10 Heparanase hydrolyzes HS at particular
glucuronic acid residues within regions of the molecule that have
relatively high degrees of sulfation.11 The smallest heparanase sub-
strate known to date is the tetrasaccharide
DHexA-b-(1?4)-
* Corresponding author.
E-mail address: p.tyler@irl.cri.nz (P.C. Tyler).
0008-6215/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2010.05.032

1832
K. A. Allen et al. / Carbohydrate Research 345 (2010) 1831–1841
R2O
OR3
O
ion mimic as this is an important feature of the transition states of
these enzymes.
R1O
Many glycosidase inhibitors are known that are also oxacarbe-
nium ion mimics and this feature is thought to be important in
their inhibitory activity.24–26 We have postulated that it is likely
heparanase achieves catalysis via oxacarbenium ion transition
state(s) and so have designed potential inhibitors that will have
oxacarbenium ion character. This has led us to the choice of aza-
sugar analogues as potential inhibitors.
CbzHN
OMe
R2 R3
R1
TfO OTBDMS
O
11
12
H
H
H
H
i
vi
-CHPh-
ii
13 Bn -CHPh-
BnO
iii
iv
14 Bn
15 Bn
H
H
H
v
CbzHN
16
TBDMS
OMe
Together with the minimal structural requirements for hepa-
ranase recognition, the heparan sulfate trisaccharide mimic 46
was settled on as an inhibitor target. For comparative purposes
and ease of preparation, the closely related compounds 32 and
33 were also chosen as targets. It is of interest to note that the syn-
thesis of a GlcA-S-linked version of this trisaccharide has been de-
scribed recently; but no heparanase inhibition data were
reported.27
OTBDMS
O
OTBDMS
O
vii
NC
BnO
OHC
BnO
CbzHN
CbzHN
OMe
OMe
18
17
Scheme 2. Reagents and conditions: (i) (a) PhCH(OMe)2, TsOH, DMF, 33 °C, 1.5 h
(70%); (b) Et3N, evaporation, 60 °C (70%); (ii) BnBr, BaO, Ba(OH)2Á8H2O, DMF, 25 °C,
22 h (64%); (iii) MeOH, H+ resin, 65 °C, 1 h (84%); (iv) TBDMSCl, imidazole,
ClCH2CH2Cl, 50 °C, 1.4 h (98%); (v) Tf2O, CH2Cl2, py , 20 °C, 1 h (94%); (vi) Bu4NCN,
CH2Cl2, rt, 2d, 54% (from 16); (vii) DIBAL-H, CH2Cl2, À78 °C, 2.5 h (59%).
2. Results and discussion
This report describes the preparation of compounds 7–10, 24,
25, 32, 33 and 46 (Schemes 1, 3, 4 and 6) which are analogues of
mono-, di- and trisaccharides each containing functionalities con-
sidered likely to give rise to inhibition of heparanase.
CO2Me
OTBDMS
Initially, compounds 7–10 were chosen as relatively simple and
readily accessible targets. Since sugar-like pyrrolidines have good
glycosidase-inhibiting properties, it was considered possible that
these carboxy- and hydroxy-substituted pyrrolidine derivatives
may have useful bioactivities and that sulfate 10, in particular, might
prove to be a heparanase inhibitor. Standard manipulation of the
known chiral pyrrolidine 128 gave the primary alcohol 3. Oxidation
with pyridinium dichromate followed by methylation generated
the methyl ester 5 which on acid treatment provided the amine 6.
Saponification of 6 afforded the b-aminoacid 7 whereas reductive
amination of p-nitrobenzaldehyde and p-methoxybenzaldehyde
separately with 6 followed by saponification gave 8 and 9. The latter
was treated with SO3ÁNMe3 affording sulfate 10.
NH
O
i
OHC
BnO
+
CbzHN
OAc
6
OMe
18
OR3
O
CO2R5
N
R2O
OTBDMS
O
CO2Me
R1HN
OMe
OR4
R1 R2 R3
19 Cbz Bn TBDMS Ac Me
N
BnO
R4 R5
vii
vi
CbzHN
OMe
OH
25
ii
20 H TBDMS Ac Me
21 Ac Ac TBDMS Ac Me
22 Ac Ac Ac Me
23 Ac Ac SO3H Ac Me
H
iii
iv
v
In order to construct the eventual targets 32, 33 and 46, the aza-
sugar oxacarbenium ion mimics needed to be attached to the 4-po-
sition of a glucosamine residue. A stable methylene linkage was
chosen which led to the C-formyl pyranoside 18 (Scheme 2) as
H
OSO3H
CO2H
O
N
HO
AcHN
OMe
OH
24
R1
R2
H
Ac
Ac
Ac
Ac
Ac
R3
Scheme 3. Reagents and conditions: (i) NaBH(OAc)3, ClCH2CH2Cl , rt, 1.5 h (81%);
(ii) EtOH, NH4OH, Pd(OH)2/C (20%), H2, rt, 3 h (98%); (iii) py, 0 °C, Ac2O, rt, 3 h, (90%);
(iv) AcOH, H2O, 60 °C, 3 h, (94%); (v) (a) SO3ÁNMe3, DMF, 45 °C, 3 h; (b) MeOH,
15 min, rt (91%); (vi) NaOH, MeOH, H2O rt, 20 h (76%); (vii) NaOMe, MeOH, rt, 24 h
(79%).
1
2
3
4
5
6
Boc
Boc
Boc
Boc
Boc
H
CH2OH
CH2OTr
CH2OH
CO2H
CO2Me
CO2Me
R3
i
ii
NR1
iii
iv
v
OR2
an important intermediate. Reductive amination of this aldehyde
with the corresponding azasugars was expected to provide the de-
sired linkages.
vi
vii
viii
The galactosamine derivative 1129 was converted to the 4,6-
O-benzylidene compound 12 and O-benzylated under mild condi-
tions to give 13. Removal of the benzylidene and silylation of the
CO2H
CO2H
CO2H
NH
NCH2C6H4(p)OMe
NCH2C6H4(p)NO2
primary hydroxy group then afforded the
After conversion into the triflate 16 this material was treated with
tetrabutylammonium cyanide under argon in dichloromethane at
room temperature for two days to give the D-gluco-nitrile 17. That
D-galacto-alcohol 15.
OH
OR
ix
OH
R
H
9
7
8
10
SO3H
configurational inversion at C-4 occurred in this reaction was
established from the relevant vicinal 1H–1H NMR coupling con-
stants of compounds 16 and 17. The J3,4 and J4,5 values for the for-
mer are 2.5 and <1 Hz, respectively, indicating that H-4 is
Scheme 1. Reagents and conditions: (i) (a) TrCl, py, rt 48 h; (b) Ac2O, rt 4 h (88%);
ii) Pd(OH)2/C (20%), H2, 2 h, rt (97%); (iii) PyÁH2Cr2O7, DMF, rt 20 h (86%); (iv) MeI,
KHCO3, DMF, rt, 20 h (90%); (v) HCl, dioxane, CH2Cl2, rt, 2 h (99%); (vi) NaOH, MeOH,
H2O, rt, 20 h, (93%); (vii) (a) O2NÁC6H4ÁCHO(p), ClCH2CH2Cl; (b) NaBH(OAc)3, Ar, rt,
1.5 h; (c) NaOH, H2O, 5 h (73%); (viii) (a) MeOÁC6H4ÁCHO(p), ClCH2CH2Cl; (b)
NaBH(OAc)3, Ar, rt, 2 h; (c) NaOH, MeOH, H2O, rt 4 h (77%); (ix) (a) SO3ÁNMe3, DMF,
45 °C, 20 h; (b) MeOH, rt, 15 min (87%).
equatorially oriented as is required for
D-galactopyranoid com-
pounds in the 4C1 conformation. The J2,3 value of 10.5 Hz for

K. A. Allen et al. / Carbohydrate Research 345 (2010) 1831–1841
1833
OTBDMS
O
R2O
BnO
BnO
CO2Me
O
CO2Bn
NR1
i
N
26
+
39
N
BnO
3O
BnO
CbzHN
OMe
OH
25
OR3
OR7
CO2R4
R1
R2
Ac
i
OAc
40 Fmoc
O
N
O
ii
iii
R6O
R6O
41 H.(HOOC)2 Ac
R2O
O
BnO
BnO
42
H
H
R5
O
R
R2O
1OMe
R4 R5 R6 R7
N3
O
BnO
OC(NH)CCl3
R1
R2 R3
CO2Bn
BnO
iv
OR1
O
42 + 18
N
BnO
26
3O
CbzNH Bn TBDMS Me N3 Bn Ac
27
28
29
30
31
N
BnO
ii
CbzNH Bn
CbzNH Bn
H
H
Me N3 Bn Ac
iii
iv
v
H
H
N3 Bn
N3 Bn SO3H
NH2 SO3Na
H
R1
43 TBDMS
44
R2
CbzNH Bn SO3H
CbzHN
H
OMe
v
vi
NH2
H
SO3Na H
H
H
H
45 SO3NH4 SO3NH4
SO3Na
O
OSO3Na
O
CO2Na
N
NH4O3SO
vi
O
vii
HO
vii
O
HO2C
HO
HO
HO
OSO3NH4
O
HO
AcHN
O
O
AcHN
AcHN
N
OMe
HO
HO
32
46
SO3Na
O
AcHN
OMe
OSO3Na
O
CO2Na
N
O
HO
HO
Scheme 6. Reagents and conditions: (i) TBDMSOTf, CH2Cl2, sieves, À20 °C, 25 min
(64%); (ii) (a) morpholine, 18 °C, 35 min; (b) (CO2H)2Á2H2O (57% from compound
38); (iii) LiOH, H2O, THF, 4 °C, 17 h (84%); (iv) NaBH(OAc)3, ClCH2CH2Cl, rt, 18 h
(82%); (v) (a) HCO2H, THF, H2O, 60 °C, 3 h; (b) NH4OH, MeOH, 20 °C, 30 min (91%);
(vi) SO3ÁNMe3 DMF, 45 °C, 16 h (26%); (vii) (a) Pd(OH)2/C (20%), EtOH, H2O, NH4OH,
20 °C, 24 h (64% from 43); (b) Ac2O, H2O, MeOH, NaHCO3, rt, 18 h. (30%).
HO
O
OMe
NaO3SHN
NaO3SHN
33
Scheme 4. Reagents and conditions: (i) TBDMSOTf, CH2Cl2, À20 °C, 25 min (65%);
(ii) HCO2H, THF, H2O, 60 °C, 5 h (78%); (iii) NaOH, H2O, MeOH, rt, 5 h (81%); (iv)
SO3ÁNMe3, DMF, 50 °C, 24 h (77%); (v) Pd(OH)2/C (20%), EtOH, NH4OH, H2, 17 h, rt
(76%); (vi) Ac2O, MeOH, H2O, rt, 3 h (78%); (vii) SO3ÁNMe3, Na2CO3, H2O, rt, 3 d
(66%).
showed that the pyranoside remained in the 4C1 conformation.
Desilylation of 27 followed by basic hydrolysis then gave carbox-
ylic acid diol 29. This was converted into the disulfate 30 which
was deprotected by hydrogenolysis to give the trisaccharide ana-
logue 31. The two amine groups were then separately acetylated
and sulfated to give the trisaccharide analogue targets 32 and 33.
The other sulfated trisaccharide analogue target 46 has an iso-
fagomine derivative as the oxacarbenium ion mimic. This required
the synthesis of the oxidized isofagomine derivative 39 (Scheme 5)
to be used as a glycosyl acceptor. The N-Boc-protected isofagomine
derivative 34 (itself derived from isofagomine31) was converted
into the 4,6-O-p-methoxybenzylidene derivative 35 followed by
benzylation of the remaining hydroxy group and hydrolysis of
the benzylidene acetal to give 37. Selective oxidation of the pri-
mary hydroxy group was followed by benzyl ester formation to
give 38. Attempted glycosylation of this N-Boc-protected acceptor
38 with the trichloroacetimidate donor 26 used previously
(Scheme 4) was unsuccessful due to loss of the Boc group. Replace-
ment of the Boc by the Fmoc-protecting group afforded the glyco-
syl acceptor 39 and glycosylation of this acceptor with the
trichloroacetimidate donor 26 was successful affording disaccha-
ride analogue 40 which was characterized after removal of the
compound 16 confirms this conclusion. Contrastingly, the J3,4 and
J4,5 values for cyano compound 17 are both 9.5 Hz indicating that
H-4 is axially oriented and that the compound has the D-gluco con-
figuration and exists in the 4C1 conformation. Selective reduction of
the nitrile to the corresponding imine was effected using DIBAH at
À78 °C and after hydrolytic workup the desired aldehyde 18 was
obtained.
Reductive amination of 18 with pyrrolidine 6 afforded the
disaccharide mimic 19 (Scheme 3) in good yield. Zemplen deacet-
ylation then gave the disaccharide glycosyl acceptor 25. In addi-
tion, compound 19 was converted into the sulfated disaccharide
mimic 24 on the basis that it might show heparanase inhibitor
properties. Hydrogenolysis of 19 followed by acetylation produced
the triacetate 21 which after desilylation, sulfation and then sapon-
ification afforded 24.
Condensation of the disaccharide acceptor 19 with the known
trichloroacetimidate donor 2630 gave the trisaccharide analogue
27 (Scheme 4). The
bond was determined from the small J1,2 coupling constant (3.5 Hz)
a anomeric configuration of the new glycosidic
while the large J2,3 and J3,4 values (9.0 and 9.2 Hz, respectively)
Fmoc group as the oxalic acid salt 41. The
a configuration of the
R4O
X
HO
HO
HO
BnO2C
HO
R3O
i
R2O
NR1
R4
NBoc
BnO
NFmoc
34
39
R1
R2
H
R3
X
35
36
37
38
Boc
Boc
Boc
Boc
CH.C6H4.OMe-(p) CH2
ii
iii
iv
Bn CH.C6H4.OMe-(p) CH2
v-vi
Bn
Bn
H
H
H
Bn
CH2
C=O
Scheme 5. Reagents and conditions: (i) (MeO)2HCÁC6H4ÁOMe-(p), TsOH, DMF, evaporation 33 °C, 1.5 h (83%); (ii) (a) BnBr, NaH, THF, (b) NaI, H2O, 65 °C, 2 h, (65%); (iii) H+
resin, MeOH, 20 °C, 2 h, (95%); (iv) (a) TEMPO, Bu4NBr, CH2Cl2, Ca(OCl)2, NaHCO3, H2O, 5 °C, 1.5 h; (b) BnBr, DMF, KHCO3, MeOH, 20 °C, 17 h (42%); (v) TFA, CH2Cl2, rt, 1 h; (vi)
FmocCl, CH2Cl2, H2O, Na2CO3, NaHCO3, rt, 16 h.

1834
K. A. Allen et al. / Carbohydrate Research 345 (2010) 1831–1841
new glycosidic bond in 41 was again established by the small J1,2
coupling constant (3.5 Hz) while the large J2,3 and J3,4 values (10
and 9 Hz, respectively) showed that the pyranoside remained in
the 4C1 conformation.
The desired trisaccharide structure was generated by the reduc-
tive coupling of free amine 42 with the branched aldehyde 18-
afforded trisaccharide analogue 43. Desilylation was effected with
formic acid followed by mild base treatment to remove traces of
formate esters affording diol 44. Then sulfation of the hydroxy
groups gave compound 45 which was globally deprotected by
hydrogenolysis and then N-acetylated to give the target 46. We
found that the corresponding N-sulfate of 46 proved too unstable
for characterization.
Chromatography (EtOAc/hexanes 1:4) gave trityl derivative 2
(3.05 g, 88%) as a white amorphous solid. 1H NMR (CDCl3) d
7.39–7.43 (m, 5H), 7.24–7.31 (m, 10H), 5.1–5.14 (m, 1H), 4.08–
4.15 (m, 2H), 3.23–3.64 (m, 2H), 3.05–3.13 (m, 2H), 2.48–2.52
(m, 1H), 2.02 (s, 3H), 1.44 (s, 9H); 13C NMR d 170.8, 154.7, 144.1,
129.0, 128.2, 127.5, 127.5, 87.2, 79.9, 75.6, 75.0, 63.1, 60.7, 51.3,
50.9, 47.4, 47.0, 44.9, 43.9, 28.9, 21.4. HRMS (ESI) calcd for
C
31H35NO5Na (M+Na)+ m/z 524.2413, found 524.2394.
5.1.2. (3R,4R)-3-Acetoxy-1-tert-butoxycarbonyl-4-(hydroxy-
methyl)pyrrolidine (3)
Trityl derivative (2) (3 g, 5.98 mmol) was dissolved in EtOH
(50 mL) and treated with palladium hydroxide on carbon
(200 mg, 20% Pd, moisture ca. 60%). The mixture was stirred for
2 h under hydrogen at rt and atmospheric pressure. The catalyst
was filtered off and washed with abs. EtOH (15 mL), and the filtrate
and washings were taken to dryness. Chromatography of the resi-
due (EtOAc/hexanes 1:2) furnished alcohol 3 (1.5 g, 97%) as a clear
syrup. 1H NMR (CDCl3) d 5.13–5.17 (m, 1H), 4.08–4.15 (m, 2H),
3.65–3.71 (m, 2H), 3.3–3.6 (m, 2H), 2.4–2.48 (m, 1H), 2.07 (s,
3H), 1.45 (s, 9H); 13C NMR d 171.2, 154.9, 80.1, 75.2, 74.3, 62.1,
60.8, 51.2, 50.6, 46.8, 46.6, 45.9, 28.8, 21.4. HRMS (ESI) calcd for
3. Biochemical results
Human heparanase was cloned and recombinant active enzyme
produced in cell culture and purified. The enzyme activity was
measured with and without the potential inhibitors using HPLC
analysis of a fluorescent substrate. Compounds 7–10, 24, 25, 32,
33 and 46 were tested but none demonstrated any observable inhi-
bition at a concentration of 500 lM. The known inhibitor suramin
was used as a control. Details are provided in Section 5.
C
12H21NO5Na (M+Na)+ m/z 282.1317, found 282.1306.
5.1.3. (3S,4R)-4-Acetoxy-1-(tert-butoxycarbonyl)pyrrolidine-3-
carboxylic acid (4)
4. Conclusions
Alcohol (3) (0.7 g, 2.7 mmol) in dry DMF (15 mL) was treated
with pyridinium dichromate (5 g, 13.2 mmol). Molecular sieves
(1 g) were added, the reaction mixture was stirred at rt for 20 h,
diluted with EtOAc and filtered through a pad of silica gel. Concen-
tration afforded acid 4, 0.634 g, (86%) as a syrup which was used in
the next reaction without further purification. 1H NMR (CDCl3) d
8.35–8.43 (m, 1H), 5.2–5.4 (m, 1H), 4.09–4.2 (m, 2H), 3.05–3.1
(m, 1H), 2.01 (s, 3H), 1.39 (s, 9H); 13C NMR d 173.8, 169.3, 153.4,
79.4, 73.5, 72.8, 59.5, 50.1, 49.6, 47.6, 46.7, 45.6, 27.4, 19.9. HRMS
We have synthesized mono- di- and trisaccharide azasugar
derivatives that mimic a putative oxacarbenium ion transition
state of the heparanase-catalyzed hydrolysis of HS. None showed
any inhibitory activity up to a concentration of 500 lM suggesting
that either the transition state of the reaction does not involve oxa-
carbenium ion character or the trisaccharide mimic is too small to
induce recognition by the enzyme.
(ESI) calcd for
296.1101.
C
12H19NO6Na (M+Na)+ m/z 296.1110, found
5. Experimental
5.1. General methods
5.1.4. Methyl (3S,4R)-4-acetoxy-(1-tert-butoxycarbonyl)pyrr-
olidine-3-carboxylate (5)
Melting points were measured on a Reichert hot stage micro-
scope and are uncorrected. Optical rotations were determined with
Acid 4 (0.634 g, 2.32 mmol) in dry DMF (25 mL) was treated
with MeI (0.433 mL, 6.96 mmol) and KHCO3 (0.697 g, 6.96 mmol).
The reaction mixture was stirred at rt for 20 h and concentrated
to dryness. The residue was dissolved in EtOAc, filtered through a
microfibre filter and the filtrate was concentrated to dryness. Chro-
matography (EtOAc/hexanes 1:4) gave methyl ester 5 (0.6 g, 90%)
as a clear syrup. 1H NMR (CDCl3) d 5.3–5.6 (m, 1H), 4.35–4.22
(m, 2H), 3.66 (s, 3H), 3.28–3.4 (m, 2H), 3.03–3.08 (m, 1H), 2.01
(s, 3H), 1.39 (s, 9H); 13C NMR d 170.4, 169.2, 153.1, 135.9, 78.9,
73.6, 72.8, 51.5, 50.8, 49.9, 49.4, 47.7, 46.7, 45.7, 45.5, 27.4, 19.9.
HRMS (ESI) calcd for C13H21NO6Na (M+Na)+ m/z 310.1291, found
310.1263.
a
Perkin–Elmer 214 polarimeter and are in units of
10À1 deg cm2 gÀ1 1H NMR spectra were obtained at either 300 or
.
500 MHz and are referenced to TMS. Coupling constants (J) are
reported to the nearest 0.5 Hz. 13C NMR spectra were recorded at
75 or 125 MHz. Electro-spray ionization (ESI) mass spectra were
recorded on a PerSeptive Biosystems Mariner time of flight mass
spectrometer run in either positive- or negative-ion mode (as indi-
cated by the charge on the charged species). Elemental analyses
were carried out at the Campbell Microanalytical Laboratory, Uni-
versity of Otago, Dunedin, New Zealand. TLC was performed on
aluminium-backed Silica Gel 60 F254 (E. Merck) with detection
by UV absorption and/or by heating after dipping in
(NH4)6Mo7O24Á6H2O (5 g) and Ce(SO4)2 (100 mg) in aqueous
H2SO4 (100 mL, 5%). Flash column chromatography was performed
5.1.5. Methyl (3S,4R)-4-acetoxypyrrolidine-3-carboxylate (6)
Methyl ester 5 (0.386 g, 1.344 mmol) in dry CH2Cl2 (5 mL) was
treated with HCl in dioxane (4 M, 3 mL). The reaction mixture
was stirred at rt for 2 h and concentrated to dryness. Chromatogra-
phy (EtOAc/MeOH 4:1) gave the amine 6, 0.249 g (99%) as a clear
syrup. 1H NMR (MeOH-d4) d 5.46–5.48 (m, 1H), 4.68 (s, 3H), 3.6–
3.66 (m, 2H), 3.25–3.49 (m, 3H), 2.02 (s, 3H); 13C NMR d 172.1,
171.8, 75.9, 53.9, 51.6, 48.9, 47.8, 21.2. HRMS (ESI) calcd for
C8H14NO4 (M+H)+ m/z 188.0923, found 188.0914.
on Scharlau silica gel (40–60
over MgSO4.
lm). Organic solutions were dried
5.1.1. (3R,4R)-3-Acetoxy-1-tert-butoxycarbonyl-4-(trityloxy-
methyl)pyrrolidine (2)
Boc-protected amine (1)28 (1.5 g, 6.9 mmol) in dry pyridine
(30 mL) was treated with recrystallized trityl chloride (3.85 g,
13.81 mmol). The reaction mixture was stirred at rt for 48 h,
Ac2O (0.846 g, 8.28 mmol) was added and after stirring for 4 h at
rt the solution was taken to dryness. EtOAc was added and the
mixture was washed with water (Â2), dried and concentrated.
5.1.6. (3S,4R)-4-Hydroxypyrrolidine-3-carboxylic acid (7)
Amine 6 (46 mg, 246
lmol) in MeOH (2 mL) and water (1 mL)
was cooled to 0 °C and treated with NaOH solution (aq, 2 M,

K. A. Allen et al. / Carbohydrate Research 345 (2010) 1831–1841
1835
300
l
L). The reaction mixture was stirred at rt for 20 h and concen-
der high vacuum at 60 °C. The product was dissolved in CH2Cl2
(750 mL) and washed with HCl (0.5 M, 750 mL) followed by NaH-
CO3 solution (satd, aq). The solution was dried and concentrated
and the product was crystallized from CH2Cl2/hexanes to give title
compound 12 (14.62 g, 70%) as a white solid: mp 194.5–195.0 °C;
trated to dryness. Chromatography (CH2Cl2/MeOH/NH4OH 5:4:1)
furnished acid 7 (33 mg, 93%) as a clear syrup. 1H NMR (D2O) d
3.56–3.59 (m, 3H), 3.37–3.43 (m, 2H), 2.99–3.04 (m, 1H); 13C
NMR
d 177.7, 73.5, 54.0, 52.1, 47.2. HRMS (ESI) calcd for
C5H10NO3 (M+H)+ m/z 132.0666, found 132.0652.
[a]
+118 (c 0.9, EtOAc). 1H NMR (CDCl3) d 7.30–7.55 (m, 10H),
D
5.69 (s, 1H), 5.10 (m, 3H), 4.85 (d, J = 3.5 Hz, 1H), 4.28 (dd,
J = 12.5, 1.5 Hz, 1H), 4.22 (d, J = 3 Hz, 1H), 4.16 (br dd, J = 3,
10 Hz, 1H), 4.07 (dd, J = 12.5, 1.5 Hz, 1H), 3.81 (dt, J = 11, 11,
3.5 Hz, 1H), 3.66 (m, J = 1.5,1.5 Hz, 1H), 3.38 (s, 3H), 2.58 (d,
J = 11 Hz, 1H); 13C NMR (CDCl3) d 157.3, 137.9, 136.7, 126.7–
129.6, 101.7, 100.1, 75.9, 69.7, 69.5, 67.5, 63.2, 55.9, 52.6. HRMS
(ESI) calcd for C22H26NO7 [M+H]+ m/z 416.1709, found 416.1714.
Anal. Calcd for C22H25NO7Á0.66H2O: C, 61.82; H, 6.21; N, 3.28.
Found: C, 61.59; H, 6.26; N, 3.22.
5.1.7. (3S,4R)-4-Hydroxy-1-(4-nitrobenzyl)pyrrolidine-3-
carboxylic acid (8)
Amine (6) (23 mg, 123
was treated with p-nitrobenzaldehyde (22 mg, 147
by NaBH(OAc)3 (33 mg, 142 mol), and the reaction mixture was
lmol) in dry 1,2-dichloroethane (2 mL)
l
mol) followed
l
stirred at rt under argon for 1.5 h. The reaction solution was diluted
with CH2Cl2 (13 mL), washed with water and satd NaHCO3 solu-
tion, dried and taken to dryness. The residue, dissolved in MeOH
(2 mL) and water (1 mL), was cooled to 0 °C and treated with NaOH
solution (2 M, 200
lL), stirred at rt for 5 h and concentrated to dry-
5.1.11. Methyl 3-O-benzyl-4,6-O-benzylidene-2-N-(benzyloxy-
ness. Chromatography [CH2Cl2/MeOH/NH4OH (26%) 7:2:0.5] fur-
nished nitrobenzyl derivative 8, (24 mg. 73%) as a clear syrup. 1H
NMR (DMSO-d6) d 8.22–8.26 (m, 2H), 7.62–7.66 (m, 2H), 4.4–
4.45 (m, 1H), 2.89–2.95 (m, 2H), 2.63–2.79 (m, 3H), 2.48–2.57
(m, 2H); 13C NMR d 175.2, 147.5, 146.8, 129.7, 123.7, 73.1, 62.4,
58.6, 55.9, 52.9. HRMS (ESI) calcd for C12H15N2O5 (M+H)+ m/z
267.0986, found 267.0931.
carbonyl)amino-2-deoxy-a-D-galactopyranoside (13)
Galactoside 12 (10 g, 20 mmol), BaO (20 g, 130 mmol),
Ba(OH)2Á8H2O (6.1 g, 20 mmol), benzyl bromide (4.8 mL, 40 mmol)
and dry DMF (240 mL) were combined and the mixture was stirred
under argon at 25 °C for 22 h. MeOH (50 mL) was added and the
mixture was stirred for 1 h after which CH2Cl2 (1 L) and H2O (1 L)
were added and the mixture was shaken and filtered. The organic
phase was washed twice with water and then dried. After removal
of the solvent, the product was crystallized from CH2Cl2/hexanes to
give the title compound 13 as a white solid (7.8 g, 64%): mp 214–
5.1.8. (3S,4R)-4-Hydroxy-1-(4-methoxybenzyl)pyrrolidine-3-
carboxylic acid (9)
Amine 6 (28 mg, 150
was treated with p-anisaldehyde (20 mg, 179
(40 mg, 190 mol) was added and the reaction mixture was stirred
l
mol) in dry 1,2-dichloroethane (2 mL)
215 °C; [a]
+158 (c 0.1, EtOAc). 1H NMR (CDCl3) d 7.2–7.6 (m,
D
lmol). NaBH(OAc)3
15H), 5.49 (s, 1H), 5.15 (d, J = 12.5 Hz, 1H), 5.06 (d, J = 12.5 Hz,
1H), 4.90 (br s, 1H), 4.80 (br d, J = 9.5 Hz, 1H), 4.68 (d, J = 12.5 Hz,
1H), 4.59 (d, J = 12.5 Hz, 1H), 4.44 (ddd, J = 11, 9.5, 3.5 Hz, 1H),
4.25 (dd, J = 12.5, 1.5 Hz, 1H), 4.20 (d, J = 3 Hz, 1H), 4.01 (dd,
J = 12.5, 1.5 Hz, 1H), 3.65 (dd, J = 3.5, 11 Hz, 1H), 3.54 (br s, 1H),
3.34 (s, 3H); 13C NMR (CDCl3), d 156.5, 138.7, 138.2, 137.0,
126.7–138.7, 101.3, 100.2, 75.0, 73.4, 71.1, 69.9, 67.2, 63.2, 55.9,
50.6; HRMS (ESI) calcd for C29H32NO7 [M+H]+ m/z 506.2179, found
506.2169. Anal. Calcd for C29H31NO7: C, 68.88; H, 6.18; N, 2.77.
Found: C, 68.88; H, 6.33; N, 2.77.
l
at rt under argon for 2 h. The reaction solution was diluted with
CH2Cl2 (15 mL), washed with water (5 mL) and NaHCO3 solution
(5 mL), dried and taken to dryness. The residue was dissolved in
MeOH (2 mL) and water (1 mL) and the solution was cooled to
0 °C, treated with NaOH solution (2 M, 300
for 4 h and concentrated to dryness. Chromatography (CH2Cl2/
MeOH/NH4OH 7:2:0.5) furnished methoxybenzyl derivative
lL) and stirred at rt
9
(29 mg, 77%) as a clear syrup. 1H NMR (MeOH-d4) d 7.32–7.35
(m, 2H), 6.86–6.89 (m, 2H), 5.51–5.53 (m, 1H), 4.15–4.30 (m,
2H), 3.7 (s, 3H), 3.45–3.50 (m, 2H), 3.3–3.36 (m, 2H), 2.88–3.09
(m, 1H); 13C NMR d 177.8, 162.6, 133.5, 124.8, 115.9, 74.9, 61.5,
60.5, 56.7, 56.2, 48.8. HRMS (ESI) calcd for C13H18NO4 (M+H)+ m/z
252.1239, found 252.1240.
5.1.12. Methyl 3-O-benzyl-2-N-(benzyloxycarbonyl)amino-2-
deoxy-a-D-galactopyranoside (14)
Galactoside 13 (6.9 g) was dissolved in MeOH (400 mL) and
Amberjet 1200 acid resin (Rohm and Haas, H+, 50 mL) was added.
The mixture was heated under reflux with stirring for 1 h, and the
resin was removed by filtration and washed with MeOH (150 mL).
The MeOH solutions were combined, Et3N (1 mL) was added and
the solution was concentrated to dryness. The product was crystal-
lized from CH2Cl2/hexanes to give the title compound 14 as a white
5.1.9. (3S,4R)-1-(4-Methoxybenzyl)-4-(sulfoxy)pyrrolidine-3-
carboxylic acid (10)
Methoxybenzyl derivative (9) (20 mg, 80 lmol) in dry DMF
(1.5 mL) was treated with SO3ÁNMe3 (55 mg, 0.4 mmol). The reac-
tion mixture was heated at 45 °C for 20 h, MeOH (0.5 mL) was
added and the mixture was stirred for 15 min and concentrated
to dryness. Chromatography [CH2Cl2/MeOH/NH4OH (26%) 7:2:1]
afforded sulfate 10 (23 mg, 87%) as a clear syrup. 1H NMR (D2O)
d 7.43–7.46 (d, J = 8.6 Hz, 2H), 7.03–7.06 (d, J = 8.6 Hz, 2H), 5.22–
5.23 (m, 1H), 4.34–4.43 (m, 2H), 3.83 (s, 3H), 3.59–3.66 (m, 2H),
3.34–3.42 (m, 2H), 3.23–3.32 (m, 1H); 13C NMR d 175.9, 160.6,
132.6, 122.7, 115.1, 79.7, 59.1, 58.1, 55.9, 55.0, 52.1. HRMS (ESI)
calcd for C13H18NO4 (MÀSO3+H)+ m/z 252.1236, found 252.1240;
calcd for C13H16NO7S (MÀH)À m/z 330.0642, found 330.0656.
solid (4.8 g, 84%): mp 157–158 °C; [a]
+107 (c 1, EtOAc). 1H NMR
D
(CDCl3) d 7.23–7.41 (m, 10H), 5.19 (d, J = 12 Hz, 1H), 5.07 (d,
J = 12 Hz, 1H), 4.81 (br d, J = 9.5 Hz, 1H), 4.76 (d, J = 3.5 Hz, 1H),
4.66 (d, J = 12 Hz, 1H), 4.52 (d, J = 12 Hz, 1H), 4.24 (br m, 1H),
4.07 (d, J = 2.5 Hz, 1H), 3.96 (dd, J = 6, 11.5 Hz, 1H), 3.81 (dd,
J = 4.5, 11 Hz, 1H), 3.72 (dd, J = 4.5, 6 Hz, 1H), 3.51 (dd, J = 3,
10.5 Hz, 1H), 3.33 (s, 3H), 2.67 (br s, 1H), 2.28 (br s, 1H); 13C
NMR (CDCl3) d 156.5, 137.8, 136.8, 128.1–128.9, 99.6, 76.7, 71.8,
69.9, 67.5, 67.3, 63.4, 55.7, 50.5. HRMS (ESI) calcd for C22H28NO7
[M+H]+ m/z 418.1866, found: 418.1884. Anal. Calcd for
C22H27NO7: C, 63.30; H, 6.52; N, 3.36. Found: C, 63.45; H, 6.62;
5.1.10. Methyl 4,6-O-benzylidene-2-N-(benzyloxycarbonyl)-
N, 3.33.
amino-2-deoxy-a-D-galactopyranoside (12)
To methyl 2-N-(benzyloxycarbonyl)amino-2-deoxy-
a-
D
-galac-
5.1.13. Methyl 3-O-benzyl-2-N-(benzyloxycarbonyl)amino-6-O-
tert-butyldimethylsily-4-C-cyano-2,4-dideoxy-a-D-glucopy-
ranoside (17)
Galactoside derivative 14 (4.60 g, 11.0 mmol) and imidazole
(3.68 g, 54 mmol) were dried together by the addition of dry MeCN
topyranoside29 (11, 16.4 g, 50 mmol), DMF (174 mL), benzaldehyde
dimethylacetal (32 mL, 213 mmol) and TsOH (0.56 g) were added.
The solution was subjected to rotary evaporation at 33 °C for
90 min. Et3N (2 mL) was added and the solvent was evaporated un-

1836
K. A. Allen et al. / Carbohydrate Research 345 (2010) 1831–1841
followed by its evaporation. The mixture was dissolved under argon
in CH2ClCH2Cl (150 mL) and TBDMSCl (3.27 g, 21.7 mmol) was
added. The mixture was heated at 50 °C with stirring for 85 min
(reaction complete in 70 min, tlc), cooled and concentrated to a syr-
up. The product was taken up in CH2Cl2 (500 mL), washed with brine
(Â2), then water, dried (MgSO4) and purified by column chromatog-
raphy (EtOAc/hexanes, 2:3) to give methyl 3-O-benzyl-2-N-(benzyl-
gave title compound 18 as a white solid (0.531 g, 0.98 mmol,
59%). The product was recrystallized from a small volume of hex-
anes: mp 73–75 °C; [a]
+43.1 (c 1, CHCl3); 1H NMR (300 MHz,
D
CDCl3) d 9.79 (d, J = 2.5 Hz, 1H), 7.16–7.36 (m, 10H), 5.14 (d,
J = 12 Hz, 1H), 5.06 (d, J = 12 Hz, 1H), 4.95 (br d, J = 7 Hz, 1H),
4.68 (br s, 1H), 4.55 (d, J = 11 Hz, 1H), 4.48 (d, J = 11 Hz, 1H),
3.97–4.05 (m, 2H), 3.91 (ddd, J = 4.5, 5.5, 10.5 Hz, 1H), 3.74 (dd,
J = 10.5, 4.5 Hz, 1H), 3.66 (dd, J = 10.5, 5.5 Hz, 1H), 3.35 (s, 3H),
3.01 (br m, 1H), 0.88 (s, 9H), 0.05 (s, 3H), 0.04 (s, 3H); 13C NMR
(CDCl3) d 200.8, 156.3, 138.0, 136.7, 128.2–128.9, 99.7, 75.9, 74.4,
70.2, 67.4, 64.7, 57.7, 55.6, 55.2, 26.2, À5.2, À5.1. Anal. Calcd for
oxycarbonyl)amino-6-O-tert-butyldimethylsilyl-2-deoxy-a-D-gal-
actopyranoside (15) as a slightly impure syrup (5.73 g, 98%). 1H
NMR (CDCl3) d 7.2–7.4 (m, 10H), 5.17 (d, J = 12 Hz, 1H), 5.05 (d,
J = 12 Hz, 1H), 4.86 (d, J = 9.7 Hz, 1H), 4.70 (d, J = 3 Hz, 1H), 4.65
(d, J = 12 Hz, 1H), 4.52 (d, J = 12 Hz, 1H), 4.15–4.30 (dt, J = 3, 10,
10 Hz, 1H), 4.04 (d, J = 2.5 Hz, 1H), 3.78 (dd, J = 6, 10 Hz, 1H), 3.66
(t, J = 6 Hz, 1H), 3.48 (dd, J = 10.5, 3.5 Hz, 1H), 3.30 (s, 3H), 2.62 (br
s, 1H), 0.89 (s, 9H), 0.07 (s, 6 H); 13C NMR (CDCl3) d 156.6, 138.2,
137.0, 128–129, 99.5, 77.2, 71.7, 70.7, 67.2, 66.1, 62.9, 55.4, 50.7,
26.3, À5.0, À5.0. The slightly impure galactoside 15 (4 g, 9.6 mmol)
was dissolved in CH2Cl2 (67 mL) and pyridine (40 mL), the solution
was cooled and stirred in an ice/water/methanol bath at À10 °C. Tri-
flic anhydride (4.84 mL, 29 mmol) was added dropwise with stir-
ring over 10 min, the stirring was continued for 10 min at À10 °C
then for 1 h at 20 °C. The mixture was concentrated at 40 °C and
residual reagents were evaporated by azeotropic distillation with
toluene. CH2Cl2 (500 mL) was added to the residue and the solution
was washed with cold HCl (M, 500 mL), then with cold water fol-
lowed by cold NaHCO3 (cold, satd, 500 mL). The solution was dried
and concentrated. Crystallization of the residue from CH2Cl2/hex-
anes yielded methyl 3-O-benzyl-2-N-(benzyloxycarbonyl)amino-
6-O-tert-butyldimethylsilyl-2-deoxy-4-O-trifluoromethanesulfo-
C29H41NO7Si: C, 64.06; H, 7.60; N, 2.58. Found: C, 63.79; H, 7.61;
N, 2.59.
5.1.15. Methyl 4-C-[(3S,4R)-4-acetoxy-3-(methoxycarbonyl)-
pyrrolidin-1-yl]methyl-3-O-benzyl-2-(benzyloxycarbonyl)-
amino-6-O-tert-butyldimethylsilyl-2,4-dideoxy-a-D-gluco-
pyranoside (19)
Amine (6) (166 mg, 887
was treated with aldehyde 18 (530 mg, 975
H(OAc)3 (207 mg, 975 mol), and the reaction mixture was stirred at
l
mol) in dry 1,2-dichloroethane (10 mL)
lmol) followed by NaB-
l
rt under argon for 1.5 h. The solution was diluted with CH2Cl2,
washed with water and satd NaHCO3 solution, dried and concen-
trated. Chromatography (EtOAc/hexanes 1:4) furnished the N-Cbz-
protected disaccharide analogue 19 (564 mg, 81%) as a clear syrup.
1H NMR (CDCl3) d 7.15–7.22 (m, 10H), 5.26–5.28 (m, 1H), 4.95–
5.03 (m, 2H), 4.44–4.53 (m, 3H), 3.98–4.05 (m, 2H), 3.74 (s, 3H),
3.69–3.71 (m, 1H), 3.61–3.65 (m, 1H), 3.27 (s, 3H), 3.06–3.08 (m,
1H), 2.90–2.91 (m, 1H), 2.7–2.72 (m, 1H), 2.46–2.6 (m, 2H), 2.31–
2.41 (m, 1H), 2.14–2.2 (m, 2H), 1.96 (s, 3H), 1.81–1.93 (m, 1H),
0.83 (s, 9H), 0.0 (s, 6H); 13C NMR d 171.6, 169.5, 154.9, 137.2,
135.5, 127.5, 127.4, 127.3, 127.2, 127.1, 126.9, 126.7, 126.6, 98.2,
75.7, 74.8, 72.2, 69.4, 65.8, 63.7, 59.8, 58.0, 55.5, 53.9, 53.8, 52.1,
51.1, 49.1, 40.5, 24.9, 21.6, 0.0. HRMS (ESI) calcd for C37H55N2O10Si
(M+H)+ m/z 715.3631, found 715.3645.
nyl-a-D
-galactopyranoside (16) as a white solid (4.69 g, 94%). 1H
NMR (CDCl3) d 7.23–7.39 (m, 10H), 5.37 (d, J = 2.5 Hz, 1H), 5.11
(br s, 2H), 4.82 (d, J = 12 Hz, 1H), 4.74 (br s, 1H), 4.65 (d, J = 8 Hz,
1H), 4.44 (d, J = 12 Hz, 1H), 4.12–4.25 (br m, 1H), 3.83 (t, J = 7 Hz,
1H), 3.66–3.76 (m, 2H), 3.57 (dd, J = 2.5, 10.5 Hz, 1H), 3.30 (s,
3H), 0.90 (s, 9H), 0.08 (s, 6H). Dry CH2Cl2 (43 mL) was added to a
mixture of the crude triflate (16) (4.32 g) and dry tetrabutylammo-
nium cyanide (4.44 g, 16.5 mmol) under Ar. The resulting solution
was stirred for 2 d whereupon it was diluted with CH2Cl2 (400 mL)
and washed with water (Â3). After drying (MgSO4) and evapora-
tion of the solvent, the product was purified by flash column chro-
matography (EtOAc/hexanes 1:3 then 2:3) and crystallized from
5.1.16. Methyl 4-C-[(3S,4R)-4-acetoxy-3-(methoxycarbonyl)-
pyrrolidin-1-yl]methyl-2-amino-6-O-tert-butyldimethylsilyl-
2,4-dideoxy-a-D-glucopyranoside (20)
N-Cbz-protected disaccharide analogue 19 (170 mg, 238
lmol)
in EtOH (9 mL), containing NH4OH (26%, 1.5 mL), was treated with
Pd(OH)2 on carbon (20 mg, 20%). The reaction mixture was stirred
for 3 h under hydrogen at ambient temperature and pressure, the
catalyst was filtered off and washed with EtOH (2 mL), the filtrate
and washings were taken to dryness and chromatography of the
residue (EtOAc) gave amino-disaccharide analogue 20 (114 mg,
98%) as a clear syrup. 1H NMR (CDCl3) d 5.28–5.31 (m, 1H), 4.62
(d, J = 3.5 Hz, 1H), 3.66 (s, 3H), 3.6–3.65 (m, 2H), 3.31–3.34 (m,
1H), 3.29 (s, 3H), 3.0–3.06 (m, 2H), 2.93–2.98 (m, 1H), 2.62–2.92
(m, 4H), 2.52–2.61 (m, 2H), 1.99 (s, 3H), 1.85–1.91 (m, 1H), 0.84
(s, 9H), 0.01 (s, 6H); 13C NMR d 172.7, 170.7, 100.9, 76.3, 76.1,
70.8, 64.2, 60.6, 60.2, 57.5, 57.1, 56.7, 55.4, 52.7, 50.0, 40.9, 26.2,
EtOAc/hexanes to give title cyano-
3.58 mmol, 54% from 16) as a white solid: mp 98 °C; [
D
-gluco-compound 17 (1.9 g,
+69 (c
a
]
D
0.7, CHCl3); 1H NMR (CDCl3): d 7.25–7.35 (m, 10H), 5.14 (d,
J = 12 Hz, 1H), 5.07 (d, J = 12 Hz, 1H), 4.87 (d, J = 11 Hz, 1H), 4.81
(br d, J = 8.5 Hz, 1H), 4.67–4.71 (m, 2H), 3.77–3.94 (m, 5H), 3.35
(s, 3H), 2.99 (dd, J = 9.5, 9.5 Hz, 1H), 0.91 (s, 9H), 0.10 (s, 3H),
0.09 (s, 3H); 13C NMR (CDCl3) d 156.2, 137.5, 136.6, 128.4–129.0,
118.4, 99.7, 76.3, 69.8, 67.6, 63.5, 55.8, 54.8, 35.8, À5.1, À5.0.
HRMS (ESI) calcd for C29H41N2O6Si [M+H]+ m/z 541.2734, found
541.2731. Anal. Calcd for C29H40N2O6Si: C, 64.41; H, 7.46; N,
5.18. Found: C, 64.38; H, 7.58; N, 5.05.
21.2, 0.0. HRMS (ESI) calcd for
491.2789, found 491.2745.
C
22H43N2O8Si (M+H)+ m/z
5.1.14. Methyl 3-O-benzyl-2-N-(benzyloxycarbonyl)amino-6-O-
tert-butyldimethylsilyl-2,4-dideoxy-4-C-formyl-a-D-glucopy-
ranoside (18)
5.1.17. Methyl 2-acetamido-4-C-[(3S,4R)-4-acetoxy-3-(methoxy-
carbonyl)pyrrolidin-1-yl]methyl-3-O-acetyl-6-O-tert-butyldi-
methylsilyl-2,4-dideoxy-a-D-glucopyranoside (21)
The C-cyano compound 17 (0.89 g, 1.65 mmol) was dried azeo-
tropically with toluene and dissolved in dry CH2Cl2 (30 mL). After
the solution was cooled to À78 °C, DIBAL-H (20% solution in tolu-
ene, 3.43 mL, 4.1 mmol) was added over 10 min. The solution
was then stirred for 150 min after which potassium hydrogen tar-
trate buffer (M, pH 6.25, 60 mL) was added, and the mixture was
stirred at 20 °C for 15 min. CH2Cl2 (300 mL) was added and the or-
ganic phase was washed with NaHCO3 solution (satd, aq Â2) and
dried. Removal of the solvent and purification of the residue by
flash column chromatography (EtOAc/hexanes, 20:80 and 40:60)
A
solution of amino-disaccharide analogue 20 (113 mg,
230
lmol) in dry pyridine (2.5 mL) was cooled to 0 °C, Ac2O
(1 mL) was added and the reaction mixture was stirred at rt for
3 h. The resulting solution was concentrated in vacuo and co-evap-
orated with toluene to afford a syrup. Chromatography (EtOAc/
hexanes 1:1) gave tri-N,O,O-acetyl compound (21) (119 mg, 90%)
as a clear syrup. 1H NMR (CDCl3) d 5.60–5.63 (m, 1H), 5.25–5.27
(m, 1H), 4.92–4.99 (m, 1H), 4.62 (d, J = 3.5 Hz, 1H), 4.04–4.11

K. A. Allen et al. / Carbohydrate Research 345 (2010) 1831–1841
1837
(m, 2H), 3.88–3.93 (m, 2H), 3.69–3.74 (m, 1H), 3.64 (s, 3H), 3.46–
3.47 (m, 1H), 3.29 (s, 3H), 3.04–3.1 (m, 1H), 2.9–2.91 (m, 1H),
2.64–2.66 (m, 2H), 2.29–2.35 (m, 2H), 1.97 (s, 6H), 1.87 (s, 3H),
0.83 (s, 9H), 0.0 (s, 6H); 13C NMR d 172.9, 171.9, 170.8, 170.3,
98.8, 76.2, 72.8, 72.0, 64.2, 60.8, 57.0, 55.2, 54.4, 52.9, 52.5, 50.4,
40.2, 26.3, 23.6, 21.3, 21.2, 18.7, 14.5, À4.8. HRMS (ESI) calcd for
rt for 4 h. The solution was neutralized with Amberlyst-15 ion-ex-
change resin and concentrated to dryness. Chromatography
(EtOAc/petroleum ether 1:4) afforded alcohol 25 (0.372 g, 79%) as
a clear syrup. 1H NMR (CDCl3) d 7.15–7.23 (m, 10H), 4.95–5.03 (m,
2H), 4.63–4.64 (d, J = 3.5 Hz, 1H), 4.38–4.53 (m, 2H), 4.36–4.37 (m,
1H), 3.91–3.95 (m, 2H), 3.66–3.7 (m, 1H), 3.61 (s, 3H), 3.49–3.51
(m, 1H), 3.4–3.48 (m, 2H), 3.27 (s, 3H), 3.02–3.05 (m, 1H),
2.79–2.81 (m, 1H), 2.65–2.67 (m, 1H), 2.4–2.54 (m, 3H), 1.88–1.97
(m, 1H), 0.82 (s, 9H), 0.0 (s, 6H); 13C NMR d 172.5, 154.9, 137.2,
135.4, 127.5, 127.3, 127.2, 127.1, 126.9, 126.6, 98.2, 73.0, 72.3,
72.1, 65.9, 63.2, 61.8, 54.8, 54.1, 53.9, 52.2, 51.8, 50.9, 40.4, 24.9,
0.0. HRMS (ESI) calcd for C35H53N2O9Si (M+H)+ m/z 673.3525, found
673.3487.
C
26H47N2O10Si (M+H)+ m/z 575.2995, found 575.2982.
5.1.18. Methyl 2-acetamido-4-C-[(3S,4R)-4-acetoxy-3-(methoxy-
carbonyl)pyrrolidin-1-yl]methyl-3-O-acetyl-2,4-dideoxy-a-D-
glucopyranoside (22)
Triacetyl derivative (21) (93 mg, 162 lmol) in AcOH (3 mL) and
water (0.7 mL) was heated at 60 °C for 3 h, the solution was diluted
with water and concentrated in vacuo. Chromatography (EtOAc/
MeOH 4:1) afforded hydroxy-derivative 22 (70 mg, 94%) as a clear
syrup. 1H NMR (MeOH-d4) d 5.2–5.22 (m, 1H), 4.92–4.99 (m, 1H),
4.56–4.57 (d, J = 3.5 Hz, 1H), 4.0–4.05 (m, 1H), 3.63–3.69 (m, 2H),
3.61 (s, 3H), 3.53–3.59 (m, 1H), 3.3 (s, 3H), 3.08–3.11 (m, 1H),
2.95–2.95 (m, 1H), 2.66–2.73 (m, 2H), 2.39–2.44 (m, 2H), 1.93 (s,
3H), 1.91 (s, 3H), 1.82 (s, 3H); 13C NMR d 174.6, 173.9, 173.1,
172.7, 100.5, 77.5, 73.9, 72.8, 64.8, 61.9, 61.5, 57.7, 56.0, 55.4,
54.4, 53.1, 51.5, 50.3, 43.3, 22.9, 21.4, 21.2. HRMS calcd for
5.1.22. Methyl 4-C-[(3S,4R)-4-{6-O-acetyl-2-azido-3,4-di-O-benzyl-
2-deoxy-
1-yl]methyl-3-O-benzyl-2-O-(benzyloxycarbonyl)amino-6-O-tert-
butyldimethylsilyl-2,4-dideoxy- -glucopyranoside (27)
The acceptor 25 (370 mg, 550 mol) and the glycosyl donor
26 (503 mg, 880
mol)30 were dried together in vacuo. Pow-
a-D-glucopyranosyl}oxy-3-(methoxycarbonyl)pyrrolidin-
a
-D
l
l
dered molecular sieves (4 Å, 300 mg) and CH2Cl2 (10 mL) were
added and the mixture was stirred for 45 min at rt. After the
C
20H33N2O10 (M+H)+ m/z 461.2140, found 461.2128.
mixture had been cooled to À20 °C TBDMS triflate (52
added and stirring was carried out at this temperature for
25 min. NaHCO3 (70 mg) and water (20 L) were added and
lL) was
l
5.1.19. Methyl 2-acetamido-4-C-[(3S,4R)-4-acetoxy-3-(methoxy-
carbonyl)pyrrolidin-1-yl]methyl-3-O-acetyl-2,4-dideoxy-6-O-
the mixture was allowed to warm to 20 °C. CH2Cl2 (20 mL)
was added and the mixture was filtered through a pad of Celite
and, after evaporation of the solvent, the residue was applied
directly to a column of silica for flash chromatography. Elution
with EtOAc/hexanes/CHCl3 (1:3:3) gave the TBDMS-protected
trisaccharide analogue 27 (387 mg, 65%) as a syrup. 1H NMR
(CDCl3) d 7.17–7.32 (m, 20H), 4.96–5.04 (m, 2H), 4.83–4.84
(m, 1H), 4.76–4.8 (m, 3H), 4.63–4.64 (d, J = 3.5 Hz, 1H), 4.49–
4.54 (m, 3H), 4.2–4.24 (m, 3H), 3.88–3.94 (m, 3H), 3.6 (s, 3H),
3.41–3.51 (m, 3H), 3.28 (s, 3H), 3.2–3.24 (m, 4H), 2.95–2.96
(m, 2H), 2.43–2.56 (m, 4H), 1.97 (s, 3H), 1.86–1.95 (m, 1H),
0.83 (s, 9H), 0.0 (s, 6H); 13C NMR d 174.5, 172.3, 157.7, 139.9,
139.5, 139.2, 138.2, 133.8, 133.6, 133.3, 132.9, 130.2, 130.1,
129.8, 129.3, 100.9, 98.8, 85.3, 82.7, 82.3, 81.8, 80.4, 77.1,
76.9, 75.0, 74.8, 71.3, 69.4, 68.6, 68.3, 67.9, 65.9, 64.8, 64.3,
61.8, 60.1, 58.2, 57.3, 56.9, 56.6, 55.3, 54.8, 53.8, 51.8, 43.1,
27.7, 22.5, 0.0. HRMS (ESI) calcd for C57H76N5O14Si (M+H)+ m/z
1082.5157, found 1082.5190.
sulfo-a-D-glucopyranoside (23)
Hydroxy-derivative 22 (64 mg, 139
lmol) was treated with
SO3ÁNMe3 complex (96 mg, 0.7 mmol) in dry DMF (3 mL). The reac-
tion mixture was heated at 45 °C for 3 h, MeOH (1 mL) was added
and the mixture was stirred for 15 min and concentrated in vacuo.
Chromatography (EtOAc/MeOH 4:1) afforded sulfate 23 (68 mg,
91%) as a clear syrup. 1H NMR (MeOH-d4) d 5.29–5.31 (m, 1H),
5.1–5.17 (m, 1H), 4.65–4.66 (d, J = 3.5 Hz, 1H), 4.33–4.37 (m, 1H),
4.13–4.19 (m, 2H), 4.09–4.11 (m, 1H), 3.72 (s, 3H), 3.4 (s, 3H),
3.31–3.35 (m, 2H), 3.3–3.31 (m, 1H), 3.1–3.3 (m, 1H), 2.8–2.81
(m, 2H), 2.51–2.53 (m, 2H), 2.04 (s, 3H), 2.0 (s, 3H), 1.92 (s, 3H);
13C NMR d 174.8, 173.9, 173.2, 172.9, 100.4, 77.7, 73.1, 71.3, 69.6,
61.9, 61.6, 58.1, 56.1, 55.5, 55.0, 54.3, 53.1, 51.6, 42.3, 22.9, 21.5,
21.3. HRMS (ESI) calcd for C20H31N2O13S (MÀH)À m/z 539.1542,
found 539.1549.
5.1.20. Methyl 2-O-acetamido-2,4-dideoxy-4-C-[(3S,4R)-4-
hydroxy-3-(carboxy)pyrrolidin-1-yl]methyl-6-O-sulfo-a-D-
glucopyranoside (24)
5.1.23. Methyl 4-C-[(3S,4R)-4-{6-O-acetyl-2-azido-3,4-di-O-benzyl-
Sulfate 23 (40 mg, 74
was cooled to 0 °C and treated with NaOH solution (2 M aq,
150 L). The reaction mixture was stirred at rt for 20 h and concen-
l
mol) in MeOH (2 mL) and water (1 mL)
2-deoxy-
1-yl]methyl-3-O-benzyl-2-O-(benzyloxycarbonyl)amino-2,4-dide-
oxy- -glucopyranoside (28)
TBDMS-protected trisaccharide analogue 27 (190 mg,
176 mol) in THF (6 mL) was dissolved in formic acid (3 mL) and
a-D-glucopyranosyl}oxy-3-(methoxycarbonyl)pyrrolidin-
l
a-D
trated to dryness. Chromatography [CH2Cl2/MeOH/NH4OH (26%)
6:3:0.5] furnished acid 24 (25 mg, 76%) as a clear syrup. 1H NMR
(D2O) d 4.88–4.89 (d, J = 3.5 Hz, 1H), 4.81–4.82 (m, 1H), 4.42–
4.45 (m, 2H), 4.26–4.3 (m, 2H), 4.04–4.11 (m, 2H), 3.93–3.99 (m,
3H), 3.8–3.87 (m, 4H), 3.7–3.75 (m, 4H), 3.64–3.68 (m, 1H), 3.6–
3.63 (m, 2H), 3.38 (s, 3H), 3.15–3.25 (m, 1H), 2.45–2.59 (m, 2H),
2.02 (s, 3H); 13C NMR d 176.5, 175.1, 101.0, 73.9, 72.4, 70.2, 68.2,
67.9, 67.1, 65.7, 56.0, 55.1, 54.3, 47.9, 22.3. HRMS (ESI) calcd for
l
water (1 mL) and the solution was heated at 60 °C for 5 h, diluted
with water and concentrated in vacuo. Chromatography (EtOAc/
petroleum ether 1:1) afforded hydroxy-derivative 28 (133 mg,
78%) as a syrup. 1H NMR (CDCl3) d 7.17–7.32 (m, 20H), 4.95–5.08
(m, 2H), 4.82–4.84 (d, 1H, J = 3.7 Hz), 4.75–4.8 (m, 3H), 4.56–4.61
(m, 2H), 4.51–4.53 (d, J = 3.5 Hz, 1H), 4.38–4.47 (m, 1H), 4.16–
4.21 (m, 3H), 3.86–3.95 (m, 3H), 3.61 (s, 3H), 3.41–3.49 (m, 3H),
3.26 (s, 3H), 3.2–3.25 (m, 4H), 3.23–3.24 (m, 1H), 3.02–3.04 (m,
2H), 2.48–2.59 (m, 3H), 1.96 (s, 3H), 1.87–1.95 (m, 1H); 13C NMR
d 171.3, 169.5, 154.8, 136.7, 136.5, 135.3, 127.5, 127.4, 127.17,
127.0, 126.9, 126.7, 98.3, 96.3, 78.9, 77.7, 76.9, 75.6, 74.4, 72.8,
72.2, 68.7, 65.9, 64.0, 62.0, 61.6, 59.3, 58.5, 54.5, 54.2, 54.0, 52.7,
51.3, 48.7, 43.5, 28.6. HRMS (ESI) calcd for C51H62N5O14 (M+H)+
m/z 968.4298, found 968.4242.
C
15H26N2O11S (MÀH)À m/z 442.1252, found: 442.1248.
5.1.21. Methyl 4-C-[(3S,4R)-3-O-benzyl-2-O-(benzyloxycarbonyl)*
amino-6-O-tert-butyldimethylsilyl-2,4-dideoxy-4 C-[4-hydroxy-
3-(methoxycarbonyl)pyrrolidin-1-yl]methyl-a-D-glucopy-
ranoside (25)
N-Cbz-protected disaccharide analogue (19) (0.5 g, 0.699 mmol)
in dry MeOH (7 mL) was stirred with NaOMe solution (1%, 150 L) at
l

1838
K. A. Allen et al. / Carbohydrate Research 345 (2010) 1831–1841
5.1.24. Methyl 4-C-[(3S,4R)-4-{2-azido-3,4-di-O-benzyl-2-deoxy-
-glucopyranosyl}oxy-3-(carboxy)pyrrolidin-1-yl]methyl-2-O-
(benzyloxycarbonyl)amino-3-O-benzyl-2,4-dideoxy-a-D-gluco-
56.21, 55.4, 53.37, 42.18, 23.33, 23.12. HRMS (ESI) calcd for
C
a-D
23H38N3O19S2 (MÀH+)À m/z 724.1546, found 724.1549.
pyranoside (29)
5.1.27. Methyl 4-C-{(3S,4R)-4-[2-deoxy-6-O-sulfo-2-sulfoamino-
a-D-glucopyranosyl}oxy-3-(carboxy)pyrrolidin-1-yl]methyl}-
Hydroxy derivative 28 (110 mg, 114
MeOH (2 mL) and water (0.5 mL), and the solution was cooled to
0 °C and treated with NaOH solution (2 M, 120 mol) with stirring
l
mol) was dissolved in
2,4-dideoxy-6-O-sulfo-2-sulfoamino-
a
-D
-glucopyranoside pen-
l
tasodium salt (33)
at rt for 5 h and concentrated to dryness. Chromatography (EtOAc)
furnished dihydroxy acid 29 (84 mg, 81%) as a clear syrup. 1H NMR
(CDCl3) d7.16–7.47 (m, 20H), 5.06–5.26 (m, 2H), 4.86–5.02 (m, 3H),
4.75–4.76 (d, 1H, J = 3.5 Hz), 4.53–4.71 (m, 3H), 4.51–4.52 (d,
J = 3.5 Hz, 1H), 4.37–4.52 (m, 4H), 4.05–4.03 (m, 2H), 3.88–3.96
(m, 1 H), 3.82–3.85 (m, 4H), 3.65–3.77 (m, 2H), 3.50 (t, J = 10 Hz,
1H), 3.23 (s, 3H), 3.15–3.23 (m, 2H), 2.67–2.88 (m, 1H), 2.54–
2.58 (m, 2H), 2.39–2.45 (m, 3H), 1.87–1.95 (m, 1H); 13C NMR d
156.2, 138.3, 138.2, 138.0, 136.7, 128.9, 128.8, 128.7, 128.6,
128.5, 128.4, 128.3, 99.6, 98.3, 80.33, 79.2, 75.7, 75.5, 74.3, 73.1,
72.5, 67.4, 64.8, 63.5, 61.96, 58.9, 57.3, 55.6, 54.6, 51.1, 45.0. HRMS
(ESI) calcd for C48H56N5O13 (MÀH)À m/z 910.3870, found 910.3870.
Di-amino-trisaccharide (31) (40 mg, 62.3 lmol) was dissolved
in water (2.5 mL) at rt, treated with SO3ÁNMe3 (100 mg) and
Na2CO3 (100 mg), and the reaction mixture was stirred at rt for
3 d. The resulting solution was applied on a column of silica gel
and eluted with CH2Cl2/MeOH/NH4OH (5:4:0.5) to afford di-N-sul-
fate (33), (33 mg, 66%) as a syrup. NMR (D2O) d 5.26–5.27 (d, 1H,
J = 3.5 Hz), 5.03–5.04 (d, 1H, J = 3.5 Hz), 4.37–4.44 (m, 3H), 4.32–
4.4.35 (m, 4H), 4.19–4.29 (m, 4H), 4.01–4.15 (m, 2H), 3.86–3.93
(m, 4H), 3.77–3.83 (m, 3H), 3.62–3.73 (m, 3H), 3.43–3.59 (m,
3H), 3.33 (s, 3H), 2.41–2.59 (m, 1H); 13C NMR d 175.36, 101.5,
99.36, 71.25, 70.77, 70.31, 69.92, 69.73, 68.72, 68.33, 67.09,
65.77, 59.42, 58.58, 57.81, 52.27, 49.37, 47.38, 39.03. HRMS (ESI)
calcd for C19H31N3O23S4 (MÀ4H)À4, Z = À4, m/z 199.2556, found
199.2556; for C19H32N3O23S4 (MÀ3H)À3, Z = À3, m/z 266.0103,
found 266.0119; for C19H33N3O23S4 (MÀ2H)À2, Z = À2, 399.5194,
found 399.5204.
5.1.25. Methyl 4-C-[(3S,4R)-4-{2-amino-2-deoxy-6-O-sulfo-
glucopyranosyl}oxy-3-(carboxy)pyrrolidin-1-yl]methyl-2-amino-
2,4-dideoxy-6-O-sulfo- -glucopyranoside trisodium salt (31)
Acid (29) (150 mg, 164 mol) in dry DMF (3 mL) was treated
a-D-
a-D
l
with SO3ÁNMe3 (137 mg, 1 mmol) and the mixture was heated at
50 °C under argon for 24 h. MeOH (1 mL) was added and the mix-
ture was stirred for 15 min and concentrated in vacuo. Chromatog-
raphy (CH2Cl2/MeOH/NH4OH (26%), 7:2:0.5) furnished disulfate 30
(135 mg, 77%) as a syrup which was not formally characterized.
5.1.28. (3R,4R,5R)-N-tert-Butoxycarbonyl-3,4-dihydroxy-5-
hydroxymethyl-piperidine (34)
The dihydroxy-hydroxymethylpiperidine isofagomine31 (2.08 g,
14.2 mmol) was dissolved in H2O (100 mL) and MeOH (50 mL).
Et3N (3 mL) and di-tert-butyl dicarbonate (6.17 g, 28.3 mmol) were
added and the solution was stirred for 16 h at 20 °C. Concentration
followed by dry flash column chromatography (MeOH/CH2Cl2,
1:15, 2:15) and recrystallization of the residue from Me2CO, Et2O
and hexanes gave title compound 34 as a white solid (1.85 g,
This disulfate 30 (60 mg, 56 lmol) in EtOH (5 mL) and NH4OH
(26%, 1.5 mL) was treated with palladium hydroxide on carbon
(20%, 30 mg). The reaction mixture was stirred for 17 h under
hydrogen at rt and ambient pressure, the catalyst was filtered off
and washed with EtOH (1 mL), and the filtrate and washings were
concentrated to dryness. Chromatography of the residue [CH2Cl2/
MeOH/NH4OH (26%), 5:4:0.5] gave di-amino-trisaccharide disul-
fate analogue 31 (34 mg, 76%) as a syrup. 1H NMR (D2O) d 5.32–
5.33 (d, J = 3.5 Hz, 1H), 5.14–5.15 (d, J = 3.5 Hz, 1H), 4.62 (dd,
J = 5, 12 Hz, 1H), 4.24–4.29 (m, 4H), 4.18–4.19 (m, 3H), 4.14–4.15
(m, 3H), 4.07–4.11 (br d, J = 11 Hz, 1H), 3.98–4.04 (m, 3H), 3.93–
3.95 (m, 2H), 3.87–3.89 (m, 3H), 3.81–3.83 (br d, J = 11 Hz, 1H),
3.74–3.78 (m, 2H), 3.69–3.71 (m, 2H), 3.59 (t, J = 10 Hz, 1H), 3.28
(s, 3H), 13C NMR d 175.3, 99.2, 96.1, 79.1, 71.6, 71.3, 70.8, 70.0,
69.2, 68.7, 67.0, 66.8, 66.3, 65.5, 56.2, 55.3, 53.9, 52.6, 47.1. HRMS
7.5 mmol, 53%): mp 99–101 °C; [a]
+13.2 (c 2, MeOH); 1H NMR
D
(CDCl3) d 4.71 (br s, 1H), 4.18 (d, J = 12 Hz, 1H), 4.02 (br s, 1H),
3.78 (dd, J = 10.5, 4.5 Hz, 1H), 3.70 (dd, J = 10.5, 4 Hz, 1H), 3.42–
3.51 (m, 2H), 2.50–2.68 (m, 2H), 1.69 (br m, 1H), 1.45 (s, 9H). 13C
NMR (CDCl3) d 155.4, 80.9, 76.4, 71.9, 62.5, 48.3, 45.1, 43.8, 28.8.
Anal. Calcd for C11H21NO5: C, 53.43; H, 8.56; N, 5.66. Found: C,
53.12; H, 8.48; N, 5.69.
5.1.29. (3R,4R,5R)-N-tert-Butoxycarbonyl-3,4-dihydroxy-5-
hydroxymethyl-4,5’-O-p-methoxybenzylidene-piperidine (35)
The piperidine amide 34 (14.14 g, 57 mmol) was dissolved in
dry DMF (160 mL) and p-anisaldehyde dimethyl acetal (11.8 mL,
69 mmol), methyl orange (25 mg) and TsOH (533 mg) were
added. The solution was subjected to rotary evaporation at
33 °C for 90 min, then NaHCO3 solution (satd, aq, 40 mL), Na2CO3
solution (aq, M, 20 mL) and H2O (100 mL) were added. The mix-
ture was concentrated to a small volume under high vacuum,
the residue was extracted with Et2O (1 L) and the solution was
washed with brine (700 mL). The aqueous layer was back-
extracted with diethyl ether (Â2). The ether solutions were com-
bined, dried and concentrated and the residue was purified by
flash column chromatography (EtOAc/hexanes 4:6) to give im-
pure title compound 35 which was recrystallized from diethyl
ether/hexanes to give a white solid (17.3 g, 47 mmol, 83%): mp
(ESI) calcd for
641.1429.
C
19H34N3O17S2 (MÀH)À m/z 641.1403, found
5.1.26. Methyl 4-C-{(3S,4R)-4-[2-acetamido-2-deoxy-6-O-sulfo-
-glucopyranosyl}oxy-3-(carboxy)pyrrolidin-1-yl]methyl}-2-
a
-D
acetamido-2,4-dideoxy-6-O-sulfo-a-D-glucopyranoside
trisodium salt (32)
Di-amino-trisaccharide analogue (31) (30 mg, 43.8
dissolved in MeOH (2 mL) and water (200 L) and cooled to 0 °C.
Acetic anhydride (400 L) was added and the reaction mixture
lmol) was
l
l
was stirred at rt for 3 h. The resulting solution was concentrated
in vacuo and co-evaporated with toluene to afford a syrup.
Chromatography [CH2Cl2/MeOH/NH4OH (26%), 5:4:0.5] gave di-
N-acetate (32), 27 mg (78%) as a clear syrup. NMR (MeOH-d4) d
4.94–4.95 (d, J = 3.5 Hz, 1H), 4.69–4.7 (d, J = 3.5 Hz, 1H), 4.64 (br
s, 1H), 4.3–4.34 (m, 2H), 4.25–4.29 (m, 3H), 4.2–4.24 (m, 2H),
4.14–4.18 (m, 2H), 4.0 (dd, J = 2, 12 Hz, 1H), 3.98–3.99 (m, 2H),
3.95–3.96 (m, 2H), 3.94 (t, J = 10 Hz, 1H), 3.87–3.88 (m, 1H),
3.74–3.87 (m, 2H), 3.69 (t, J = 9 Hz, 1H), 3.55–3.59 (m, 2H), 3.4–
3.47 (m, 2H), 3.3 (s, 3H), 2.2–2.3 (m, 1H), 2.05 (s, 3H), 1.99 (s,
3H); 13C NMR d 176.45, 174.4, 100.61, 98.73, 80.59, 73.13, 72.77,
72.48, 72.3, 69.26, 68.87, 68.54, 61.72, 58.15, 57.97, 57.34, 56.42,
112.5–113 °C; [a]
+3.6 (c 2, EtOAc); 1H NMR d 7.41 (d, J = 9 Hz,
D
1H), 6.89 (d, J = 9 Hz, 1H), 5.52 (s, 1H), 4.37 (br s, 1H), 4.15 (dd,
J = 4.5, 11 Hz, 1H), 3.99 (br s, 1H), 3.80 (s, 3H), 3.66–3.75 (m,
1H), 3.57 (t, J = 11 Hz, 1H), 3.43 (t, J = 10 Hz, 1H), 2.68 (d,
J = 2.5 Hz, 1H), 2.64 (dd, J = 11.5, 11.5 Hz, 1H), 2.38 (br t,
J = 12 Hz, 1H), 1.87–2.01 (m, 1H), 1.46 (s, 9H). 13C NMR (CDCl3)
d 160.6, 154.9, 130.7, 128.0, 114.1, 102.3, 85.5, 80.8, 68.8, 68.7,
55.7, 48.4, 43.5, 37.4, 28.8. Anal. Calcd for C19H27NO6: C, 62.45;
H, 7.45; N, 3.83. Found: C, 62.57; H, 7.42; N, 3.78.

K. A. Allen et al. / Carbohydrate Research 345 (2010) 1831–1841
1839
5.1.30. (3R,4R,5R)-3-Benzyloxy-N-tert-butoxycarbonyl-4-
hydroxy-5-hydroxymethyl-4,5’-p-methoxybenzylidene-
piperidine (36)
to give a white solid that was recrystallized from ether/hexanes
to give the title ester 38 (274 mg, 0.62 mmol, 42%): mp 114–
115 °C; [
a]
À1.5 (c 1, EtOAc); 1H NMR (CDCl3) d 7.24–7.40 (m,
D
Cyclic acetal 35 (2.17 g, 5.94 mmol) was dissolved in dry THF
(100 mL) containing BnBr (1.38 mL, 11.9 mmol). NaH (60% in oil,
3.6 g, 89 mmol) was slowly added followed by H2O (dropwise,
0.32 mL, 18 mmol) and NaI (50 mg). The mixture was heated under
reflux for 2 h, after which it was cooled to 40 °C and MeOH (40 mL)
was added. Stirring was applied for 30 min, H2O (500 mL) and
CH2Cl2 (500 mL) were added and the mixture was shaken. The
resulting organic layer was washed again with water and dried
(MgSO4). Removal of the solvent and flash column chromatography
(EtOAc/hexanes 3:7) gave crude product which was recrystallized
from Et2O/hexanes to give title compound 36 (1.77 g, 3.89 mmol,
10H) 5.18 (t, J = 13 Hz, 2H), 5.13 (d, J = 13 Hz, 1H), 4.70 (d,
J = 11.5 Hz, 1H), 4.63 (d, J = 11.5 Hz, 1H), 4.26 (br m, 1H), 3.89 (dt
J = 2, 9.5 Hz, 1H), 3.24–3.31 (m, 1H), 2.99 (s, 1H), 2.80 (br s, 1H),
2.51–2.63 (m, 2H), 1.44 (s, 9H). 13C NMR (CDCl3) d 171.5, 154.6,
138.4, 135.9, 128.2–129.0, 80.9, 78.5, 74.4, 72.8, 67.2, 48.6, 45.7,
44.7, 28.7. Anal. Calcd for C25H31NO6: C, 68.01; H, 7.08; N, 3.17.
Found C, 67.88; H, 7.04; N, 3.07.
5.1.33. Benzyl (3R,4R,5S)-3-benzyloxy-N-[(9-fluorenylmethyloxy)-
carbonyl]-4-hydroxy-piperidine-5-carboxylate (39)
The benzyl ester 38 (1.47 g, 3.33 mmol) was dissolved in CH2Cl2
(125 mL) and TFA (12.5 mL) was added. After 1 h at 20 °C, toluene
(80 mL) was added and the solution was concentrated. Toluene
addition and evaporation were repeated twice more. The syrupy
product was then dissolved in CH2Cl2 (90 mL) and 9-fluorenylm-
ethyl chloroformate (1.97 g, 7.61 mmol), aqueous Na2CO3 (1 M,
45 mL) and aqueous NaHCO3 (satd, 45 mL) were added while the
mixture was stirred in an ice/water bath. Stirring of the two-phase
mixture was continued for 30 min at 0 °C and then at 20 °C for
16 h. After this time, additional CH2Cl2 (100 mL) was added and
the organic layer was washed with water (200 mL) and dried. Flash
column chromatography (EtOAc/hexanes 1:3) gave the Fmoc-pro-
65.4%) as a white solid: mp 91.5–92 °C; [a] +26.3 (c 1, EtOAc);
D
1H NMR (CDCl3) d 7.43 (d, J = 9 Hz, 2H), 7.23–7.37 (m, 5H), 6.90
(d, J = 9 Hz, 2H), 5.58 (s, 1H), 4.84 (t, J = 12 Hz, 1H), 4.70 (t,
J = 12 Hz, 1H), 4.20–4.55 (br s, 1H), 4.15 (dd, J = 11, 4.5 Hz 1H),
3.85–4.00 (br s, 1H), 3.80 (s, 3H), 3.53–3.70 (m, 3H), 2.66 (dd,
J = 11, 12.5 Hz, 1H), 2.37 (br m, 1H), 1.89–2.02 (m, 1H), 1.44 (s,
9H). 13C NMR d 160.4, 154.9, 138.9, 131.1, 127.1–128.8, 114.0,
101.8, 85.3, 80.7, 75.6, 73.5, 68.7, 55.7, 47.3, 43.4, 38.1, 28.7. Anal.
Calcd for C26H33NO6: C, 68.55; H, 7.30; N, 3.07. Found: C, 68.55; H,
7.25; N, 3.07.
5.1.31. (3R,4R,5R)-3-Benzyloxy-N-tert-butoxycarbonyl-4-
hydroxy-5-hydroxymethyl-piperidine (37)
tected 39 as a colourless syrup (1.82 g, 3.23 mmol, 97%); [
a
]
D À18.4
(c 1, EtOAc); 1H NMR (300 MHz, CDCl3) d 7.71 (d, J = 7 Hz, 2H), 7.53
(d, 2H), 7.17–7.45 (m, 14H), 5.16 (s, 2H), 4.24–4.75 (br m, 5H), 4.19
(t, J = 6 Hz, 1H), 3.94–4.15 (br m, 1H), 3.83 (br m, 1H), 2.95–3.30 (br
m, 1H), 2.70–2.95 (m, 2H), 2.50 (s, br, 2H). 13C NMR (CDCl3) d 171.3,
155.1, 144.2, 141.8, 138.3, 135.9, 127.5–129.0, 125.2, 120.4, 78.5,
78.3, 74.2, 72.8, 67.9, 67.8, 67.3, 48.3, 47.8, 45.9, 44.6. Anal. Calcd
for C35H33NO6: C, 74.58; H, 5.90; N, 2.49. Found C, 74.49; H, 5.93;
N, 2.48.
The methoxybenzylidene piperidine derivative 36 (3.70 g,
8.12 mmol) and Amberjet 1200 (H+) (15 g) were added to MeOH
(250 mL). The mixture was stirred at 20 °C for 2 h when the resin
was removed by filtration and washed with methanol. The filtrate
and washings were combined and taken to pH 7 with Na2CO3 (aq,
M, ca. 0.5 mL). The resulting solution was concentrated and puri-
fied by flash column chromatography (EtOAc/hexanes 7:3) to yield
a white solid (2.61 g, 7.73 mmol, 95%) that was recrystallized from
Et2O/hexanes to give title compound 37: mp 90.5–91 °C; [
a]
D +12.7
5.1.34. Benzyl (3R,4R,5S)-4-(6-O-acetyl-2-azido-3,4-di-O-benzyl-
(c 1.6, EtOAc); 1H NMR (CDCl3) d 7.26–7.39 (m, 5H), 4.71 (d,
J = 11.5 Hz, 1H), 4.57 (d, J = 11.5 Hz, 1H), 4.1–4.7 (br s, 1H), 4.00
(d, J = 12 Hz, 1H), 3.68 (t, J = 5 Hz, 2H), 3.53 (dd, J = 9, 9.5 Hz, 1H),
3.24–3.32 (m, 1H), 3.14 (s, 1H), 2.78 (br m, 1H), 2.52 (t, J = 12 Hz,
2H), 1.70–1.83 (m, 1H), 1.45 (s, 9H). 13C NMR (CDCl3) d 155.0,
138.4, 128.2–129.0, 80.6, 79.4, 76.3, 72.5, 63.6, 45.6, 44.8, 43.6,
28.7. Anal. Calcd for C18H27NO5: C, 64.07; H, 8.07; N, 4.15. Found:
C, 64.19; H, 8.06; N, 4.09.
2-deoxy-a-D-glucopyranosyl)oxy-3-benzyloxy-piperidine-5-
carboxylate. Mono-oxalic acid salt (41)
The piperidine alcohol 39 (2.00 g, 3.55 mmol) and 6-O-acetyl-2-
azido-3,4-di-O-benzyl-2-deoxy-a-D-glucopyranosyl trichloroace-
timidate (3.78 g, 6.61 mmol) were dried together by benzene
azeotroping. Powdered molecular sieves (4 Å, 1.6 g) and CH2Cl2
(20 mL) were added and the mixture was stirred for 45 min at
20 °C and cooled to À20 °C. TBDMSOTf (122
added, and the mixture was stirred at this temperature for
25 min. NaHCO3 (700 mg) and H2O (100 L) were added, and the
lL, 0.53 mmol) was
5.1.32. Benzyl (3R,4R,5S)-3-benzyloxy-N-tert-butoxycarbonyl-4-
hydroxy-piperidine-5-carboxylate (38)
l
mixture was allowed to warm to 20 °C. CH2Cl2 (100 mL) was added
and the mixture was filtered through a pad of Celite and, after
evaporation of the solvent, the residue was applied directly to a
column of silica gel for flash chromatography. Elution with
EtOAc/hexanes/CHCl3 (25:65:10) produced partly purified product
which was re-chromatographed in the same way to give com-
pound 40 as an impure syrup which was dissolved in morpholine
(80 mL) and stirred at 18 °C for 35 min whereupon the resulting
solution was poured onto ice cold water (800 mL) and extracted
(Â3) with Et2O (400 mL, 80 mL and 80 mL). The ether extracts were
combined, benzene (120 mL) was added and the solution was con-
centrated to a syrup. Flash column chromatography (EtOAc/hex-
anes/CHCl3/Et3N, 7:2:1:0.05) gave a syrup (1.70 g, 2.26 mmol,
64%) from which mono-oxalate 41 (1.71 g, 2.03 mmol, 57% from
39) was obtained as a white solid, after the addition of oxalic acid
dihydrate (0.29 g, 2.30 mmol), followed by crystallization from
EtOH (40 mL), by addition of iPrOH, Et2O and hexanes; mp 131–
The BOC-protected piperidine 37 (500 mg, 1.48 mmol), TEMPO
(7.5 mg) and Bu4NBr (50 mg) were dissolved in CH2Cl2 (15 mL).
While the solution was stirred vigorously in an ice/water bath, a
suspension of Ca(OCl)2 (463 mg, 3.23 mmol) in NaHCO3 solution
(aq, 0.28 M, 15 mL) was added over 15 min such that the reaction
temperature remained below 5 °C. The biphasic mixture was stir-
red on ice for a further 70 min when EtOH (10 mL) was added,
and the mixture was dried by rotary evaporation at 40 °C to give
a solid residue which was extracted by stirring with acetone
(50 mL). The remaining solids were removed by filtration and
washed with additional Me2CO (50 mL). The acetone solutions
were combined and the solvent was removed to give a crude resi-
due (600 mg). This was added to DMF (25 mL), and KHCO3
(350 mg), MeOH (0.60 mL) and BnBr (0.54 mL, 4.5 mmol) were
added, and the suspension was then stirred for 17 h at 20 °C. The
resulting mixture was concentrated to a syrup under high vacuum,
taken up in CH2Cl2 (100 mL) and washed with H2O (100 mL). The
organic layer was dried and concentrated and the residue was
purified by flash column chromatography (EtOAc/hexanes 25:75)
132° ; [
a]
+31.4 (c 1.5, EtOAc); 1H NMR (300 MHz, CDCl3) d
D
7.13–7.39 (m, 20H), 5.09 (d, J = 3.5 Hz, 1H), 5.01 (d, J = 12 Hz,
1H), 4.80–4.92 (m, 4H), 4.60 (d, J = 11.5 Hz, 1H), 4.57

1840
K. A. Allen et al. / Carbohydrate Research 345 (2010) 1831–1841
(d, J = 11 Hz, 1H), 4.44 (d, J = 11.5 Hz, 1H), 4.33 (t, J = 4.5 Hz, 1H),
4.24 (dd, J = 2, 12 Hz, 1H), 4.13 (dd, J = 5, 12 Hz, 1H), 3.93 (dd,
J = 9, 10 Hz, 1H), 3.83–3.89 (m, 1H), 3.78–3.83 (m, 1H), 3.35–3.65
(m, 4H), 3.49 (dd, J = 9, 9.5 Hz, 1H), 3.39 (dd, J = 3.5, 10 Hz, 1H),
3.05 (br d, J = 4 Hz, 1H), 1.92 (s, 3H); 13C NMR (75 MHz, CDCl3) d
170.5, 169.4, 163.3, 137.5, 137.2, 136.3, 134.6, 128.7–128.0, 98.3.
80.1, 77.8, 75.7, 75.2, 72.6, 72.2, 71.3, 70.6, 68.0, 63.4, 62.6, 43.0,
41.8. 40.8, 20.6. Anal. Calcd for C44H48N4O13: C, 62.85; H, 5.75; N,
6.66. Found C, 62.85; H, 5.79; N, 6.62. HRMS (ESI) calcd for
up was dissolved in dry MeOH (96 mL) and methanolic NH4OH
(7 M, 16 mL) was added. After 30 min at 20 °C the sample was con-
centrated and flash column chromatography (EtOAc/hexanes/
CHCl3/Et3N 5:2:1:0.05) gave the diol 44 (0.662 g, 0.59 mmol, 91%)
as
a
colourless syrup;
[a]
+45.9 (c 0.5, EtOAc); 1H NMR
D
(500 MHz, CDCl3) d 7.19–7.36 (m, 30H), 5.36 (br s, 1H), 5.15 (d,
J = 12 Hz, 1H), 5.09 (d, J = 12 Hz, 1H), 5.01–5.07 (m, 3H), 4.80–
4.85 (m, 3H), 4.69 (d, J = 2.5 Hz, 1H), 4.64 (d, J = 11 Hz, 1H), 4.56
(d, J = 11.5 Hz, 1H), 4.50 (d, J = 11.5 Hz, 1H), 4.42 (d, J = 11 Hz,
1H), 4.13 (t, J = 6.5 Hz, 1H), 4.05 (dt, J = 2.5, 9.5 Hz, 1H), 3.86 (dd,
J = 8.5, 10.5 Hz, 1H), 3.52–3.82 (m, 7H), 3.45–3.50 (m, 1H), 3.32
(s, 3H), 3.22–3.30 (br m, 1H), 3.24 (dd, J = 3.5, 10 Hz, 1H), 2.96
(br s, 1H), 2.76 (dt, J = 8.5, 4 Hz, 1H), 2.72 (br s, 1H), 2.49 (d,
J = 12.5 Hz, 1H), 2.34 (br s, 2H), 2.18 (dd, J = 9.5, 12.5 Hz, 1H),
2.18 (dd, J = 9.5, 12.5 Hz, 1H), 1.82–2.00 (m, 3H); 13C NMR
(75 MHz, CDCl3) d 171.6, 156.2, 138.2, 138.0, 136.7, 135.4, 127.9–
128.9, 99.8, 98.0, 80.2, 78.3, 77.7, 76.1, 75.6, 75.4, 74.5, 73.9,
72.4, 72.2, 67.6, 67.3, 64.1, 63.8, 61.7, 55.9, 55.8, 55.7, 55.4, 53.1,
46.6, 42.4. Anal. Calcd for C63H71N5O14: C, 67.42; H, 6.38; N, 6.24.
Found: C, 67.59; H, 6.53; N, 6.22.
C
42H47N4O9 [MÀCOOHCOOÀ]+ m/z 751.3338; found 751.3344.
5.1.35. Benzyl (3R,4R,5S)-4-(2-azido-3,4-di-O-benzyl-2-deoxy-
-glucopyranosyl)oxy-3-benzyloxy-piperidine-5-carboxylate
(42)
a-
D
The piperidine salt 41 (1.51 g, 1.80 mmol) was dissolved in cold
THF (200 mL) and water (46 mL). LiOH (aq, M, 10 mL) was added
and the mixture was stirred for 17 h at 4 °C. It was then shaken
with CH2Cl2 (800 mL) and brine (50%, 1L), and the brine was
back-extracted twice with CH2Cl2 (2 Â 100 mL). The organic phases
were combined and washed again with dilute brine, which was
again back-extracted with CH2Cl2 (100 mL). The organic phase
was dried and flash column chromatography (EtOAc/hexanes/
CHCl3/Et3N, 7:2:1:0.05) followed by (EtOAc/Et3N, 99:1) gave alco-
5.1.37. Methyl 2-acetamido-4-C-[(3R,4R,5S)-4-(2-acetamido-2-
deoxy-6-O-sulfo-a-D-glucopyranosyl)-5-carboxy-3-hydroxy-
piperidin-1-yl)methyl]-2,4-dideoxy-6-O-sulfo-a-D-glucopyran-
oside diammonium salt (46)
hol 42 (1.07 g, 1.51 mmol, 84%) as a colourless syrup: [a] +12.6
D
(c 0.5, EtOAc); 1H NMR (300 MHz, CDCl3) d 7.23–7.37 (m, 20H),
5.46 (d, 4 Hz, 1H), 5.07 (d, J = 12.5 Hz, 1H), 4.99 (d, J = 12.5 Hz,
1H), 4.81–4.85 (m, 3H), 4.54–4.67 (m, 3H), 4.22 (dd, J = 7.5,
7.5 Hz, 1H), 3.92 (dd, J = 8.5, 10.5 Hz, 1H), 3.82 (dd, J = 2, 11.5 Hz,
1H), 3.46–3.70 (m, 4H), 3.19–3.28 (m, 1H), 3.23 (dd, J = 4,
10.5 Hz, 1H), 3.11 (dd, J = 4, 13 Hz, 1H), 2.87 (dd, J = 8.5, 13 Hz,
1H), 2.56–2.70 (m, 2H). 13C NMR (75 MHz, CDCl3) d 172.3, 138.4,
138.3, 135.8, 128.0–129, 98.4, 80.2, 79.2, 78.5, 77.0, 75.7, 72.4,
72.1, 67.3, 61.8, 48.5, 47.9, 46.7. Anal. Calcd for C40H44N4O8: C,
67.78; H, 6.26; N, 7.90. Found C: 67.95; H, 6.56; N 7.60.
The trisaccharide analogue 44 (75 mg, 0.067 mmol) was dried
azeotropically with benzene then Me3NÁSO3 (185 mg, 1.33 mmol)
and dry DMF (6 mL) were added, and the mixture was stirred for
16 h at 45 °C under Ar. It was concentrated under high vacuum
at 40 °C and then dissolved in MeOH. Silica gel was added and
the solvent was evaporated under reduced pressure. The resulting
silica was transferred to the top of a flash silica gel column, and
elution with CH2Cl2/MeOH/NH4OH (aq, 26%) (7:2:0.5) gave disul-
fate diammonium salt 45 as an impure solid (95 mg, 88%). The
crude material was dissolved in EtOH/H2O (11 mL, 7.5:1) and
NH4OH (28%, 0.28 mL), Pd(OH)2 on carbon (260 mg, ca. 20%) were
added, and the mixture was stirred for 24 h at 20 °C under an
atmosphere of hydrogen. The product was filtered through Celite
and the filtrate was concentrated at 37 °C. The resulting material
5.1.36. Methyl 4-C-[(3R, 4R, 5S)-4-(2-azido-3,4-di-O-benzyl-2-
deoxy-
piperidin-1-yl)methyl]-3-O-benzyl-2-(benzyloxycarbonyl)-
amino-2,4-dideoxy- -glucopyranoside (44)
a-D-glucopyranosyl)-3-benzyloxy-5-benzyloxycarbonyl-
a-D
The piperidine glycoside 42 (0.56 g, 0.79 mmol) and aldehyde
18 (0.45 g, 0.82 mmol) were mixed and dried by addition and re-
moval of benzene. NaBH(OAc)3 (0.69 g, 3.3 mmol) was added fol-
lowed by (CH2)2Cl2 (13 mL), and the mixture was stirred for 18 h.
CH2Cl2 (200 mL) was then added and the organic solution was
washed with a mixture of Na2CO3 (aq, M) and NaHCO3 (satd, aq)
1:1 (200 mL). The aqueous phase was back-extracted twice with
CH2Cl2 (40 mL). The organic solutions were combined and dried
and flash column chromatography (EtOAc/hexanes/Et3N 3:7:0.05)
was stirred with water, MeOH (5.0 mL, 2:3), Ac2O (600
NaHCO3 (500 mg) for 3 h. Additional NaHCO3 (300 mg) and Ac2O
(600 L) were added and the mixture was stirred for 18 h at
lL) and
l
20 °C and adsorbed onto flash column silica gel and applied to a
i
chromatographic column containing pure gel. Elution with PrOH/
MeOH/NH4OH (28%, aq), 4:4:3 gave crude 46. This was re-col-
umned in the same solvent system to give 46 as a glassy solid
(22 mg, 64% from compound 44). 1H NMR (500 MHz, D2O) d 5.31
(s, br, 1H), 4.90 (d, J = 3.5 Hz, 1H), 3.90–4.50 (m, 12 H), 3.86 (t,
J = 10.0 Hz, 1H), 3.67 (dd, J = 9.0, 10.0 Hz, 1H), 3.66 (s, br, 1H),
3.47 (s, 3H), 3.20–3.55 (br, m, 4H), 2.92 (br, s, 1H), 2.46 (br m,
1H), 2.12 (s, 6H). 13C NMR (125 MHz, CDCl3) d 176.8, 175.7,
175.3, 99.5, 97.8, 72.3, 71.5, 71.4, 70.4, 68.5, 68.2, 67.6, 58.9,
56.9, 56.3, 55.2, 54.6, 39.6, 23.0, 22.9. HRMS (ESI) Calcd for
gave the adduct 43 as
a slightly impure, colourless syrup
(0.801 g, 0.65 mmol, 82%). 1H NMR (500 MHz, CDCl3) d 7.20–7.35
(m, 30H), 5.61 (d, J = 4 Hz, 1H), 4.78–5.16 (m, 5H), 4.69 (d,
J = 3 Hz, 1H), 4.43–4.66 (m, 5H), 3.94–4.05 (m, 3H), 3.87 (dd,
J = 8, 10 Hz, 1H), 3.82 (br d, J = 10.5 Hz, 1H), 3.47–3.68, m, 6H),
3.34 (s, 3H), 3.31 (t, J = 10 Hz, 1H), 3.19 (dd, J = 4, 10.5 Hz, 1H),
2.97–3.04 (m, 2H), 2.75 (ddd, J = 4, 10.5, 11 Hz, 1H), 2.43 (d,
J = 12.5 Hz, 1H), 2.16–2.29 (m, 1H), 1.87–2.05 (m, 2H), 1.65–1.80
(m, 2H), 0.89 (s, 9H), 0.064 (s, 3H), 0.057 (s, 3H); 13C NMR
(75 MHz, CDCl3) d 172.7, 156.3, 138.5, 138.4, 136.8, 135.5, 128.1–
129.0, 99.5, 98.1, 80.8, 80.3, 78.6, 78.1, 75.7, 75.5, 74.6, 74.4,
72.5, 72.0, 67.7, 67.4, 64.8, 63.8, 61.9, 57.5, 56.6, 55.9, 55.2, 54.6,
48.0, 41.8, 26.4, À4.6. This silyl ether of diol 44 (0.801 g,
0.65 mmol) was dissolved in THF/HCOOH/H2O (6:3:1, 120 mL)
and heated at 60 °C for 3 h. The mixture was concentrated, dis-
solved in acetone, water (40 mL, 1:1) and again concentrated. This
addition/concentration was repeated and finally the sample was
taken up in acetone and concentrated to a syrup. The resulting syr-
C
24H47N5O20S2 [MÀ2NH4]2À m/z 376.5790. Found 376.5788.
6. FITC-labelled heparan sulfate degradation assay
6.1. Heparanase expression in COS-7 cells and purification
COS-7 (African green monkey kidney, ATCC) cells stably trans-
fected with human heparanase cDNA were cultured and passaged
at confluence into T-175 triple flasks. Cells from three clones were
harvested with 1 mL 0.25% trypsin (Medica Pacifica), centrifuged
and washed in PBS. Preparation of the cell lysate and isolation of
heparanase were carried out as reported.32 Cell extracts were

K. A. Allen et al. / Carbohydrate Research 345 (2010) 1831–1841
1841
sonicated briefly then centrifuged at 12,000g for 10 min at 4 °C to
tested at high substrate concentration (30
l
g/mL) and then with
remove insoluble debris. Soluble fractions were stored at À80 °C in
1 mL aliquots. The presence of Heparanase in the purified fractions
was confirmed using western blotting (anti-heparanase polyclonal
antibodies, Insight, Ins-AB-04002) and its activity tested by ELISA
assay (Heparan Degrading Enzyme Assay Kit (Takara)) and an HPLC
assay.
lower concentration (0.33 lg/mL). Control reactions with substrate
only and substrate plus semi-purified extracts from transfected
COS-7 cells without inhibitor were prepared in duplicate with each
experiment, together with a suramin control reaction. No inhibi-
tion was seen with any of the compounds at 500 lV whereas the
IC50 for suramin under these conditions was approximately 25 lM.
6.2. Substrate preparation
Acknowledgements
FITC-labelled heparan sulfate (HS) was prepared by adding FITC
(fluorescein-5-isothiocyanate) to HS from bovine kidney (Sigma)
dissolved in 0.1 M Na2CO3 (pH 9.35) in a 1:2 ratio (weight), respec-
tively, and the labelling reaction was stirred overnight at 4 °C in
the dark. The labelled material was purified using a G-25 spin col-
umn (GE Healthcare) to remove excess FITC and then fractionated
on a sephacryl-300 10/60 gel filtration column (GE Healthcare).
The labelled substrate was collected in 1 mL fractions, and 80 frac-
tions were assayed in a multiwell format with a fluorescent plate
reader (excitation 492, emission 524). The labelled substrate was
present in two peaks over 40 fractions. To maximize peak resolu-
tion, each fraction was concentrated separately and material from
the first peak (largest molecular weight) was used for the inhibitor
studies. The concentration of FITC-labelled heparan sulfate (frac-
tion 37) was determined to be 0.33 mg/mL using the Blyscan glu-
cosaminoglycan assay. The labelled substrate was diluted 100
We thank Herbert Wong of Industrial Research Limited and
Oleg A. Zubkov of Victoria University, Wellington, for providing
the NMR and mass spectrometric data, respectively. We are grate-
ful to Professor R. J. Ferrier for assistance with preparation of the
manuscript.
Reference
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fold and 20
lL was used in 50
lL reactions, resulting in a final sub-
strate concentration of 1.3
lg/mL (65 ng per reaction).
6.3. HPLC assay
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An HPLC assay was developed to monitor the degradation of la-
belled HS by heparanase. A variety of buffers were tested and it
was found that buffers containing 0.2 mg/mL BSA, pH 4.5, with
10% glycerol and 0.1% Triton X-100 added were the most suitable
in terms of best enzyme activity and minimal column blockage.
The introduction of a pre-column in-line filter unit and an extra
column filter was necessary to prevent column blockages.
Assay experiments were prepared with and without 5 lL of
semi-purified heparanase in reaction buffer (25 mM sodium dim-
ethylglutarate, 0.1 M NaCl, 0.1% Triton X-100, 10% glycerol, pH
4.5). After incubation at 37 °C for 90 min, each assay was quenched
by the addition of 5
60 °C for 5 min, before being filtered through a 0.22-
aration was effected by loading 25 L of each assay experiment
ll of 1.5 M Tris–HCl, pH 8.8, then heated to
l
m filter. Sep-
l
onto a Superdex 200 column connected to a fluorescence detector
and eluting with the buffer at a flow rate of 0.3 ml/min. The sub-
strate peak (retention time 35 min) was degraded in the presence
of heparanase to about one third the original peak height, with a
prominent product peak appearing with a retention time of 55 min.
In control experiments, extracts containing equivalent protein
amounts from wild-type COS-7 cells not expressing heparanase
did not degrade the substrate.
6.4. Inhibitor studies
Compounds 7–10, 24, 25, 32, 33 and 46 were tested for efficacy
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as inhibitors at 500
lM concentration using the same reaction con-
ditions as mentioned above. The potential inhibitors were initially