Human milk oligosaccharides: An enzymatic protection step simplifies the synthesis of 3′- and 6′-O-sialyllactose and their analogues

Article abstract of DOI:10.1016/S0008-6215(02)00016-2

We describe a chemo-enzymatic synthesis of 3′- and 6′-O-sialyllactose, two trisaccharides occurring in the 'acidic fraction' of the human milk oligosaccharides and endowed with potential antiadhesive activity. The key step is the highly regioselective 6′-

Full text of DOI:10.1016/S0008-6215(02)00016-2

Carbohydrate Research 337 (2002) 473–483
www.elsevier.com/locate/carres
Human milk oligosaccharides: an enzymatic protection step
simplifies the synthesis of 3%- and 6%-O-sialyllactose and their
analogues
Anna Rencurosi,a Laura Poletti,a Marco Guerrini,b Giovanni Russo,a Luigi Laya,*
aDepartment of Organic and Industrial Chemistry, Uni6ersity of Milan, 6ia G. Venezian, I-21-20133 Milan, Italy
bInstitute of Chemistry and Biochemistry ‘G. Ronzoni’, 6ia Colombo, I-81-20131, Milan, Italy
Received 6 November 2001; accepted 7 January 2002
Abstract
We describe a chemo-enzymatic synthesis of 3%- and 6%-O-sialyllactose, two trisaccharides occurring in the ‘acidic fraction’ of the
human milk oligosaccharides and endowed with potential antiadhesive activity. The key step is the highly regioselective
6%-O-acylation of benzyllactoside, which gave access to suitably protected lactose building blocks to be used as acceptors in the
sialylation reaction. Moreover, the synthesis of the carboxymethyl and sulfo analogues of the title compounds is reported. © 2002
Elsevier Science Ltd. All rights reserved.
Keywords: Human milk oligosaccharides; Enzymatic 6%-O-acylation; Benzyllactoside
1. Introduction
In this study, we turned our attention to sialylated
human milk oligosaccharides. It has been recently
demonstrated4 that whey glycoproteins, containing N-
acetylneuraminic acid, exert a potential inhibitory effect
on S-fimbriae mediated adhesion of E. coli.
The oligosaccharide fraction is the third most abun-
dant solid constituent (between 12 and 20 g/L) of
human milk and consists of more than one hundred
different structures. Despite many investigations1–3 that
have been reported on the subject, the physiological
role of these compounds is not completely understood.
The fact that human milk oligosaccharides bear struc-
tural homology to cell-surface glycoconjugates used as
receptors by pathogens may suggest their implication in
protecting breast-fed infants from infections. Concern-
ing this issue, evidence has emerged that these oligosac-
charides are able to inhibit the adhesion of
Streptococcus pneumoniae and Haemophilus influenzae
to human pharingeal or buccal epithelial cells.2 More-
over, the fucosylated fraction of small human milk
oligosaccharides inhibits the Escherichia coli adhesion
to uroepithelial cells.3
In order to assess the antiadhesive properties of
selected oligosaccharides present in human milk, we
describe herein the synthesis of 3%- and 6%-O-sialyllac-
toses and their corresponding simplified analogues
where the sialic acid unit is replaced by a negatively
charged group such as a sulfate and a carboxymethyl
group. Sulfate groups may serve as an effective sub-
stituent for sialic acid5 in some biological systems and
3%-O-sulfo-lactose has recently been found as an
oligosaccharide present in dog milk.6 Moreover the
carboxymethyl group in analogues of sialyl Lewis X
was shown to be useful for mimicking the negative
charge of sialic acid.7 In previous papers, we reported
on the use of lipase catalysed acylation as a tool to
prepare useful building blocks for oligosaccharide syn-
thesis.8–11 In the present paper, the enzymatically
catalysed 6%-O-acylation of lactose has been exploited
for designing a versatile chemo-enzymatically protected
lactose building block useful for the synthesis of both
3%- and 6%-O-sialyllactoses.
* Corresponding author. Fax: +39-02-58354061.
E-mail address: llay@mailserver.unimi.it (L. Lay).
0008-6215/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(02)00016-2

474
A. Rencurosi et al. / Carbohydrate Research 337 (2002) 473–483
as a 2:1 a/b mixture in 58% overall yield (Scheme 2).
Even though selectivity was not high, the two isomers
were completely separated by MP chromatography.
Anomeric configuration of the newly formed glycosidic
linkage for 10a and 10b was determined by comparison
of NMR data of the two compounds. In accordance
with the known 1H NMR empirical rules,14–16 l H-3%%eq
(a)=2.73\l H-3%%eq (b)=2.61, l H-4%% (a)=4.52Bl
H-4%% (b)=5.49, J7%%,8%% (a)=7.9 Hz\J7%%,8%% (b)=3.1 Hz.
Disappointingly, sialylation of acceptor 5 resulted in
a complex mixture of products from which most of the
acceptor was recovered unreacted and the supposed
3%-O-sialyllactose derivative was isolated in a very poor
yield. These poor results might be due to the steric
hindrance and to the presence of electron withdrawing
groups close to the reactive centre. Therefore, a new
and more reactive acceptor 8 was synthesised through
selective benzoylation17 of 3 followed by acidic hydroly-
sis of the isopropylidene acetal (Scheme 1).
Sialylation of this new acceptor in acetonitrile–THF
at −44 °C in the presence of TMSOTf as a promoter,
afforded the 3%-O-sialyllactoside 12 in 51% yield
(Scheme 2). This result is in line with that reported by
Lo¨nn et al.,18 who used a very similar acceptor employ-
ing a glycosyl xanthate as the donor. The (2%%“3%)
glycosylation regiochemistry was ascertained from the
observation of COSY cross-peaks between OH-2% (l
3.17) and H-2% (l 3.75) and OH-4% (l 2.38) and H-4% (l
3.63). Moreover, the HMBC19 experiment showed the
presence of the expected cross-peak between H-3% (l
4.09) and the quaternary carbon C-2%% (l 97.6). The a
configuration of the newly formed glycosidic linkage
was determined on the basis of the occurrence of the
Neu5Ac H-4 signal at l 4.97, as well as the J7%%,8%%=8.4
Hz; these data are in accordance with the reported
trend for similar compounds.18,20
Scheme 1. Reagents and conditions: (a) trifluoroethyl levuli-
nate, C. antarctica lipase, THF, 40 °C, 4 days, 83%; (b)
acetone, CSA, Sikkon, refl, 4 h, 78%; (c) BzCl, Py, DMAP,
CH2Cl2, 0 °C“rt, 48 h, 80%; (d) TFA (60%), CH2Cl2,
0 °C“rt, 1 h, 85%; (e) AcONH2NH3, EtOH–Et2O, rt, 20
min, 97%; (f) BzCN, TEA, THF, −25 °C, 17 h, 50%; (g)
TFA (60%), CH2Cl2, 0 °C“rt, 1.5 h, 92%.
2. Results and discussion
The selective protection of the 6%-OH position of
benzyllactoside 112 with
a levulinoyl group was
achieved in high yield through an enzymatic acylation11
catalysed by Candida antarctica lipase in THF (Scheme
1). The subsequent protection of 3%,4%-positions of 2 as
isopropylidene acetal afforded compound 3. Full ben-
zoylation of 3 gave the versatile building block 4 useful
for the synthesis of the target compounds. In fact,
orthogonal deprotection of isopropylidene or levulinoyl
groups afforded acceptors 5 and 6, respectively (Scheme
1), which were separately submitted to sialylation using
sialyl phospite 913 as a donor (Scheme 2).
The synthesis of 6%-O- and 3%-O-sulfated analogues of
the corresponding sialyllactoses was based on two dif-
ferent strategies. Lactoside 15 was obtained in three
Glycosylation of acceptor 6 in acetonitrile at −40 °C
with trimethylsilyltrifluoromethansulfonate (TMSOTf)
as a promoter, afforded the 6%-O-sialyllactoside 10a/b
Scheme 2. Reagents and conditions: (a) 9 (1.7 equiv), TMSOTf, CH3CN, −40 °C, 1 h, 58%, a/b 2:1; (b) TFA (60%), CH2Cl2;
(c) MeOH, MeONa; (d) NaOH, water, (e) H2, Pd/C, H2O; (f) 9 (2 equiv), TMSOTf, CH3CN–THF, −44 °C, 2 h, 51%.

A. Rencurosi et al. / Carbohydrate Research 337 (2002) 473–483
475
of the (alkoxycarbonyl) methyl group at 6%-position was
first attempted by treatment of 15 with ethyl bromoac-
etate in the presence of Ag2O and TBAI. Disappoint-
ingly, a complex mixture of products was obtained,
probably derived from acyl migration and/or hydroly-
sis. Much better results were achieved on the benzylated
compound 26,22 which was synthesised in three steps
from benzyllactoside 1 through regioselective 6%-O-sily-
lation,23 which afforded compound 25, followed by full
benzylation and final 6%-O-desilylation. Thus, alkylation
of 26 with tert-butyl-bromoacetate24 afforded com-
pound 27 in almost quantitative yield (Scheme 4).
Finally, compounds 10a, 12, 16, 18, 23 and 27 were
conventionally deprotected to afford the title com-
pounds 11, 13, 17, 19, 24, and 28, respectively. Full
deprotection of compound 10a was achieved by acidic
hydrolysis of the isopropylidene group, followed by
Zemple´n deacylation, saponification of the methylester
with NaOH, and hydrogenolysis of the anomeric benzyl
group, affording the 6%-O-sialyllactose 11 (Scheme 2).
NMR data for 11 are in agreement with those previ-
ously reported for the natural compound.25 Compound
12 was converted into the known compound 13 by
Zemple´n deacylation, saponification with NaOH, and
hydrogenolysis of the anomeric benzyl ether. NMR
data for 13 are consistent with those reported for the
natural compound25 and by Ogawa.20 Sulfated lactose
derivatives 17 and 19 were quantitatively obtained from
16 and 18 by hydrogenolysis of the anomeric benzyl
ether. Zemple´n deacylation of 23, followed by one-pot
saponification with NaOH and hydrogenolysis, af-
forded compound 24, whereas 28 was obtained from 27
by acidic hydrolysis of the tert-butyl ester followed by
hydrogenolysis of the remaining benzyl groups. Final
desalting of the deprotected compounds was performed
by gel permeation chromatography through a Sephadex
G10 column, followed by freeze-drying.
Scheme 3. Reagents and conditions: (a) trifluoroethyl levuli-
nate, C. antarctica lipase, THF, 40 °C, 4 days, 83%; (b) Ac2O,
Py, 80%; (c) AcONH2NH3, EtOH–Et2O, rt, 2 h, 98%; (d)
SO3NMe3 1.5 equiv, DMF, 60 °C, 2 days; (e) MeONa,
MeOH, rt, 81%; (f) Bu2SnO (1.1 equiv), MeOH, refl, 16 h; (g)
SO3NMe3 (2 equiv), DMF, rt, 2 days, 75%; (h) H2, Pd/C,
MeOH–water, rt.
steps from benzyllactoside 1 through enzymatic 6%-O-
levulinoylation, followed by acetylation (“14) and re-
moval of the levulinoyl group with hydrazinium acetate
(Scheme 3). The sulfation of 15 (SO3NMe3 complex),
followed by deacetylation, provided the 6%-O-sulfated
lactoside 16. The 3%-O-sulfated lactoside 18 was ob-
tained in a one pot procedure,21 by treatment of benzyl-
lactoside 1 with Bu2SnO and SO3NMe3 complex
(Scheme 3).
The same approach was applied to the synthesis of
3%-O-(alkoxycarbonyl) methyllactoside (Scheme 4). In
this case, the alkylation of the stannylene acetal ob-
tained from benzyllactoside 1 produced compound 20
and the corresponding lactone 21. This mixture was
treated without separation with sodium methoxide (“
22), followed by acetylation to afford pure compound
23 in good yield. The acetylation step was required in
order to better purify the compound. The introduction
Scheme 4. Reagents and conditions: (a) Bu2SnO 1.1 equiv, MeOH, refl, 16 h; (b) BrCH2COOEt 5 equiv, TBAI, DMF, 40 °C, 48
h; (c) MeONa, MeOH, rt, 24 h; (d) Ac2O, Py, rt, 24 h, 56% from 1; (e) MeONa, MeOH, rt, 16 h, then water, 20 h; (f) H2, Pd/C,
91% from 23; (g) Bu2SnO 1.1 equiv, MeOH, refl, 16 h; (h) TDSCl 1.1 equiv, THF, rt, 18 h, 76%; (i) BnBr, NaH, DMF, rt, 16
h; (l) TBAF, THF, 0 °C“rt, 6 h, 66% from 25; (m) BrCH2COOtBu, NaOH, (Bu)4NHSO4, CH2Cl2, 4 h, 96%; (n) TFA (10%),
CH2Cl2, 30 min; (o) H2, Pd(OH)2/C, 72 h, 95%.

476
A. Rencurosi et al. / Carbohydrate Research 337 (2002) 473–483
Results of antiadhesion tests and BIACORE analysis
Hz, CHHPh), 4.64 (d, 1 H, CHHPh), 4.42 (d, 1 H, J1,2
will be reported elsewhere.
7.7 Hz, H-1), 4.37 (dd, 1 H, J6%a,6%b 12.0, J6%a,5% 3.2 Hz,
H-6%a), 4.33 (d, 1 H, J1%,2% 8.1 Hz, H-1%), 4.26 (dd, 1 H,
J6%b,5% 8.6 Hz, H-6%b), 4.12–4.05 (m, 3 H, H-4%, H-3%,
H-3), 3.85 (m, 2 H, H-6a, H-6b), 3.64–3.38 (m, 5 H,
H-2, H-4, H-5, H-2%, H-5%), 2.77–2.73 (m, 2 H, CH2
lev.), 2.62–2.58 (m, 2 H, CH2 lev.), 2.20 (s, 3 H, CH3
lev.), 1.49 (s, 3 H, CH3 isoprop.), 1.30 (s, 3 H, CH3
isoprop.); 13C NMR (CDCl3, 75.44 MHz): l 207.0 (CO
lev.), 172.6 (COO lev.), 110.6 (Cq isoprop.), 103.0,
101.8 (C-1, C-1%), 81.7, 79.3, 74.9, 74.3, 73.7, 73.2, 73.1,
71.5 (C-2, C-3, C-4, C-5, C-2%, C-3%, C-4%, C-5%), 71.3
(CH2Ph), 63.4, 62.2 (C-6, C-6%), 37.8 (CH2COCH3 lev.),
28.0 (CH3 lev.), 27.9 (CH2COO lev.), 26.2 (2 CH3
isoprop.). Anal. Calcd for C27H38O13 (570.583): C,
56.83, H, 6.71. Found: C, 56.95 H, 6.67.
3. Experimental
General methods.—1H and 13C NMR spectra were
recorded on Varian Gemini 200, Bruker AC 300,
Bruker Avance 400, Bruker Am 500 and Bruker 600
DRX spectrometers. The HMBC spectra were acquired
using 128 scans per series in 1K×256W data points
3
and optimised for the JCꢀH of 8 and 4 Hz. Melting
points were determined with a Bu¨chi apparatus and are
not corrected. Optical rotations were measured at rt
with a Perkin–Elmer 241 polarimeter. TLC was carried
out on E. Merck Silica-Gel 60 F254 plates (0.25 mm
thickness), and spots were visualised by spraying with a
solution containing H2SO4 (31 mL), ammonium molyb-
date (21 g) and Ce(SO4)2 (1 g) in 500 mL water,
followed by heating at 110 °C for 5 min. Column
chromatography was performed by the flash procedure
using E. Merck Silica-Gel 60 (230–400 mesh). Elemen-
tal analyses were performed using the Carlo–Erba ele-
mental analyser 1108. In the description of the 13C
spectra, signals corresponding to aromatic carbons were
omitted. Lipase from C. antarctica was purchased from
Roche Diagnostic (Chirazyme® L-2, c.f. C2 lyo). Sol-
vents were dried by standard procedures.
Benzyl 2-O-benzoyl-3,4-O-isopropylidene-6-O-le6uli-
noyl-i-
D
-galactopyranosyl-(1“4)-2,3,6-tri-O-benzoyl-
i- -glucopyranoside (4).—Compound 3 (272 mg, 0.48
D
mmol) was dissolved in dry CH2Cl2 (5 mL) under N2
atmosphere, then pyridine (410 mL, 5.1 mmol), DMAP
(cat) and benzoyl chloride (360 mL, 3.1 mmol) were
added at 0 °C and the reaction mixture was stirred 48 h
at rt. The reaction was quenched with MeOH and the
solution was concentrated under diminished pressure.
Purification by flash chromatography (6.5:3.5
petroleum ether–EtOAc) afforded 4 as white foam (378
mg, 80%): [h]2D0 +24.4° (c 1.3, CHCl3); 1H NMR
(CDCl3, 300 MHz): l 8.10–7.08 (m, 25 H, HAr), 5.63 (t,
1 H, J3,2=J3,4 9.3 Hz, H-3), 5.44 (t, 1 H, H-2), 5.09 (t,
1 H, J1%,2%=J2%,3% 7.4 Hz, H-2%), 4.80 (d, 1 H, J 12.6 Hz,
CHHPh), 4.68 (d, 1 H, J1,2 7.8 Hz, H-1), 4.64 (dd, 1 H,
J6a,5 2.8 Hz, H-6a), 4.62 (d, 1 H, J1%,2% 7.6 Hz, H-1%), 4.57
(d, 1 H, CHHPh), 4.50 (dd, 1 H, J6a,6b 11.8, J6b,5 4.8
Hz, H-6b), 4.24–4.18 (m, 2 H, H-4, H-3%), 4.05 (dd, 1
H, J4%,5% 1.7, J4%,3% 5.8 Hz, H-4%), 3.95 (dd, 1 H, J6%a,6%b
11.5, J6%a,5% 4.9 Hz, H-6%a), 3.82–3.73 (m, 2 H, H-5,
H-5%), 3.64 (dd, 1 H, J6%b,5% 7.0 Hz, H-6%b), 2.77–2.73 (m,
2 H, CH2 lev.), 2.56–2.51 (m, 2 H, CH2 lev.), 2.20 (s, 3
H, CH3 lev.), 1.48 (s, 3 H, CH3 isoprop.), 1.22 (s, 3 H,
CH3 isoprop.); 13C NMR (CDCl3, 50.29 MHz): l 206.3
(CO lev.), 172.2 (COO lev.), 165.9, 165.4, 165.1, 164.8
(COOBz), 110.7 (Cq isoprop.), 100.2, 99.1 (C-1, C-1%),
76.9, 75.5, 73.6, 73.2, 73.0, 72.7, 72.0, 70.9 (C-2, C-3,
C-4, C-5, C-2%, C-3%, C-4%, C-5%), 70.3 (CH2Ph), 62.7
(C-6, C-6%), 37.9 (CH2COCH3 lev.), 29.7 (CH3CO lev.),
27.9 (CH2COO lev.), 27.3, 26.0 (2 CH3 isoprop.); Anal.
Calcd for C55H54O17 (987.007): C, 66.93; H, 5.51.
Found: C, 66.99; H, 5.55.
Benzyl 6-O-le6ulinoyl-i-
D
-galactopyranosyl-(1“4)-
i- -glucopyranoside (2).—Benzyl b-
D
D
-lactoside (1)12
(700 mg, 1.62 mmol) was suspended in dry THF (70
mL). Trifluoroethyl levulinate8 (22 g, 113 mmol) and C.
antarctica lipase (2.1 g) were added, the suspension was
shaken for 4 days at 40 °C and monitored by TLC
(8:1.5:0.5 EtOAc–MeOH–water). The enzyme was
filtered, and the solvent was removed under diminished
pressure. Purification by flash chromatography (10:1
EtOAc–MeOH) afforded compound 2 as a white foam
(713 mg, 83%). Optical rotation value and NMR data
are in agreement with those reported by us in a previ-
ous publication.8
Benzyl
3,4-O-isopropylidene-6-O-le6ulinoyl-i-
D-
galactopyranosyl-(1“4)-i-
D
-glucopyranoside (3).—
Compound 2 (53 mg, 0.1 mmol) was dissolved in
acetone (3 mL) in an inert atmosphere, sikkon (180 mg)
was added and the mixture was stirred for 30 min. Then
a catalytic amount of camphorsulfonic acid (CSA) was
added and the suspension was refluxed for 4 h while
monitoring the reaction by TLC (9.5:0.5 EtOAc–
MeOH). After cooling to rt, the mixture was neu-
tralised with TEA, sikkon was filtered off and the
solvent was removed under diminished pressure. Purifi-
cation by flash chromatography (9:1 EtOAc–MeOH)
afforded compound 3 as a white foam (44 mg, 78%):
[h]2D0 +9.8° (c 1.7, MeOH); 1H NMR (CDCl3, 300
MHz): l 7.35–7.20 (m, 5 H, HAr), 4.87 (d, 1 H, J 11.8
Benzyl 2-O-benzoyl-6-O-le6ulinoyl-i-D-galactopyran-
osyl-(1“4)-2,3,6-tri-O-benzoyl-i- -glucopyranoside
D
(5).—Compound 4 (400 mg, 0.41 mmol), was dissolved
in CH2Cl2 (20 mL) and cooled to 0 °C. A 60% aq
CF3COOH (4 mL) was added under vigorous stirring
(TLC 1:1 petroleum ether–EtOAc). After 30 min at
0 °C and 30 min at rt, the reaction mixture was diluted

A. Rencurosi et al. / Carbohydrate Research 337 (2002) 473–483
477
with CH2Cl2, neutralised with NaHCO3 and partitioned
between water and CH2Cl2. The organic phase was
dried over Na2SO4, filtered and concentrated under
diminished pressure. Purification by flash chromatogra-
phy (1:4 petroleum ether–EtOAc), afforded 6 (325 mg,
85%) as an amorphous white solid: [h]2D0 +19.7° (c 1.0,
BzCN (204 mg, 1.56 mmol) in THF (2.5 mL) was
added dropwise. The reaction mixture was stirred for
17 h (TLC 1:9 MeOH–EtOAc), diluted with EtOAc,
quenched with MeOH (0.3 mL) and allowed to warm at
rt. After concentration under diminished pressure, se-
quential purification by flash and MP chromatography
(8:1.5 toluene–acetone) afforded 7 (203 mg, 50%), as a
white foam. [h]D20 −15.4° (c 1.0, CHCl3); 1H NMR
(CDCl3, 600 MHz): l 8.11–7.15 (m, 15 H, HAr), 5.21
(dd, 1 H, J2,3 9.4 Hz, H-2), 4.87 (2d overlapped, 2 H,
H-6a, CHHPh), 4.70 (d, 1 H, J 12.6 Hz, CHHPh), 4.60
(d, 1 H, J1,2 8.1 Hz, H-1), 4.55 (dd, 1 H, J6a,6b 12.0, J6b,5
5.2 Hz, H-6b), 4.39 (dd, 1 H, J6%a,6%b 12.1, J6%a,5% 2.8 Hz,
H-6%a), 4.30 (d, 1 H, J1%,2% 8.3 Hz, H-1%), 4.25 (dd, 1 H,
J6%b,5% 9.4 Hz, H-6%b), 4.10–4.06 (m, 3 H, H-3%, H-4%,
H-5%), 3.87 (t, 1 H, J3.4 9.2 Hz, H-3), 3.69 (br dd, 1 H,
J4,5 10.0 Hz, H-5), 3.63 (br dd, 1 H, J2,3 6.3 Hz, H-2%),
3.61 (t, 1 H, H-4), 2.54–2.35 (m, 4 H, 2 CH2 lev.), 2.02
(s, 3 H, CH3 lev.), 1.51 (s, 3 H, CH3 isoprop.), 1.32 (s,
3 H, CH3 isoprop.); 13C NMR (CDCl3, 150.86 MHz): l
206.8 (CO lev.), 171.6, 166.8, 165.3 (CO lev., Bz), 110.7
(Cq isoprop.), 103.4 (C-1%), 98.9 (C-1), 82.2 (C-4), 79.1
(C-3% or C-4%), 73.7 (C-3), 73.6 (C-5), 73.4 (C-2%), 73.1
(C-2), 73.0 (C-3% or C-4%), 71.5 (C-5%), 70.3 (CH2Ph),
63.9 (C-6), 63.2 (C-6%), 37.6 (CH3 lev.), 29.6, 28.0 (2
CH2 lev.), 27.8, 26.2 (2 CH3 isoprop.). Anal. Calcd for
C41H46O15 (778.795): C, 63.23; H, 5.95. Found: C,
63.20; H, 5.89.
1
CHCl3); H NMR (CDCl3, 200 MHz): l 8.09–7.08 (m,
25 H, HAr), 5.56 (t, 1 H, J2,3=J3,4 9.5 Hz, H-3), 5.46
(dd, 1 H, J1,2 7.4 Hz, H-2), 5.31 (t, 1 H, J1%,2%=J2%,3% 8.5
Hz, H-2%), 4.80 (d, 1 H, J 12.5 Hz, CHHPh), 4.65 (d, 1
H, J1,2 7.5 Hz, H-1), 4.60–4.47 (m, 4 H, H-6a, H-6b,
H-1%, CHHPh), 4.13 (t, 1 H, J3,4=J4,5 9.4 Hz, H-4),
3.82–3.35 (2 m, 8 H, H-5, H-3%, H-4%, H-5%, H-6%a,
H-6%b, 2 OH), 2.74–2.67 (m, 2 H, CH2 lev.), 2.51–2.43
(m, 2 H, CH2 lev.), 2.16 (s, 3 H, CH3 lev.); 13C NMR
(CDCl3, 300 MHz): l 206.7 (CO lev.), 172.4 (COO
lev.), 166.1, 166.0, 165.3 (COOBz), 101.1, 98.8 (C-1,
C-1%), 76.4, 75.5, 73.5, 73.1, 72.4, 72.3, 71.7, 68.6 (C-2,
C-3, C-4, C-5, C-2%, C-3%, C-4%, C-5%), 70.3 (CH2Ph),
62.8, 61.8 (C-6, C-6%), 37.9 (CH2COCH3 lev.), 29.8
(CH3CO lev.), 27.7 (CH2COO lev.). Anal. Calcd for
C52H50O17 (946.943): C, 65.96; H, 5.32. Found: C,
65.92; H, 5.34.
Benzyl 2-O-benzoyl-3,4-O-isopropylidene-i-
D-galac-
topyranosyl-(1“4)-2,3,6-tri-O-benzoyl-i- -glucopy-
D
ranoside (6).—Compound 4 (356 mg, 0.36 mmol) was
dissolved in 1:1 EtOH–Et2O (8 mL), then a 1 M
solution of AcONH3NH2 in EtOH (0.4 mL) was
dropped at rt. After 20 min, the solvents were removed
under diminished pressure and purification by flash
chromatography (3:1 toluene–EtOAc) afforded com-
pound 5 as a white solid (309 mg, 97%): mp 202–
Benzyl 6-O-le6ulinoyl-i-
D
-galactopyranosyl-(1“4)-
(8).—Com-
2,6-di-O-benzoyl-i- -glucopyranoside
D
pound 7 (564 mg, 0.72 mmol) was submitted to the
same conditions described for the preparation of 5.
Purification by flash chromatography (EtOAc) afforded
8 (490 mg, 92%) as an amorphous glassy solid: [h]D20
1
203 °C; [h]2D0 +26.7° (c 1.0, CHCl3); H NMR (CDCl3,
200 MHz): l 8.05–7.15 (m, 25 H, HAr), 5.62 (t, 1 H,
1
J3,4=J2,3 8.8 Hz, H-3), 5.49 (dd, 1 H, J1,2 7.3 Hz, H-2),
−37.0° (c 1.1, CHCl3); H NMR (CDCl3, 300 MHz): l
5.14 (t, 1 H, J2%,3% 7.3 Hz, H-2%), 4.81 (d, 1 H, J 12.5 Hz,
CHHPh), 4.71 (d, 1 H, H-1), 4.64 (dd, 1 H, J6a,6b 12.2,
J6a,5 2.2 Hz, H-6a), 4.61 (d, 1 H, J1%,2% 7.5 Hz, H-1%), 4.58
(d, 1 H, CHHPh), 4.47 (dd, 1 H, J6b,5 4.7 Hz, H-6b),
4.25–4.16 (m, 2 H, H-4, H-3%), 4.00 (dd, 1 H, J4%3% 5.65,
J4%,5% 2.01 Hz, H-4%), 3.8 (ddd, 1 H, H-5), 3.56 (m, 1 H,
H-5%), 3.43–3.17 (m, 2 H, H-6%a, H-6%b), 1.51 (s, 3 H,
CH3 isoprop.), 1.28 (s, 3 H, CH3 isoprop.); 13C NMR
(CDCl3, 50.29 MHz): l 165.9, 165.5, 165.2, 164.9
(COOBz), 110.7 (Cq isoprop.), 100.5, 99.0 (C-1, C-1%),
77.0, 75.8, 73.9, 73.4, 73.3, 73.0, 71.8 (C-2, C-3, C-4,
C-5, C-2%, C-3%, C-4%, C-5%), 70.3 (CH2Ph), 62.8, 61.8
(C-6, C-6%), 27.4, 26.1 (2 CH3 isoprop.). Anal. Calcd for
C50H48O15 (888.907): C, 67.56; H, 5.44. Found: C,
67.62; H, 5.51.
8.15–7.11 (m, 15 H, HAr), 5.19 (t, 1 H, J2,3 8.7 Hz,
H-2), 4.85 (d, 1 H, H-6a), 4.81 (d, 1 H, CHHPh), 4.62
(d, 1 H, J1,2 7.2 Hz, H-1), 4.59 (d, 1 H, J 11.3 Hz,
CHHPh), 4.54 (dd, 1 H, J6b,5 5.6, J6a,6b 11.9 Hz, H-6b),
4.44 (br s, 1 H, OH), 4.34 (d, 1 H, J1,2 7.8 Hz, H-1%),
4.28 (dd, 1 H, J6%a,5% 3.3 Hz, H-6%a), 4.24 (dd, 1 H, J6%b,5%
4.4, J6%a,6%b 11.8 Hz, H-6%b), 4.01 (br s, 1 H, OH),
3.90–3.63 (m, 7 H, H-3, H-4, H-2%, H-3%, H-4%, H-5%,
OH), 3.54 (m, 1 H, H-5), 3.31 (br s, 1 H, OH),
2.57–2.40 (m, 4 H, 2 CH2 lev.), 2.03 (s, 3 H, CH3 lev.);
13C NMR (CDCl3, 50.29 MHz, 50 °C): l 207.2 (CO
lev.), 173.0, 167.1, 165.7 (COO lev., Bz), 104.3 (C-1%),
99.5 (C-1), 81.8, 73.8, 73.3, 71.3, 68.7 (C-2, C-3, C-4,
C-5, C-2%, C-3%, C-4%, C-5%), 70.6 (CH2Ph), 64.2, 63.6
(C-6, C-6%), 37.9 (CH2COCH3 lev.), 29.2 (CH3 lev.),
28.1 (CH2COO lev.). Anal. Calcd for C38H42O15
(738.731): C, 61.68; H, 5.73. Found: C, 61.63; H, 5.77.
Benzyl (methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-
Benzyl
3,4-O-isopropylidene-6-O-le6ulinoyl-i-
D-
galactopyranosyl-(1“4)-2,6-di-O-benzoyl-i-
D
-gluco-
pyranoside (7).—To a solution of 3 (300 mg, 0.53
mmol) in dry THF (7.5 mL), TEA (0.37 mL, 2.65
mmol) was added under Ar atmosphere. The reaction
mixture was cooled to −25 °C, and a solution of
3,5-dideoxy-
onate)-(2“6)-2-O-benzoyl-3,4-O-isopropylidene-i-
galactopyranosyl-(1“4)-2,3,6-tri-O-benzoyl-i- -gluco-
D-glycero-h-D-galacto-non-2-ulopyranosyl-
D
-
D

478
A. Rencurosi et al. / Carbohydrate Research 337 (2002) 473–483
pyranoside (10a) and its isomer (10b).—Compound 5
(390 mg, 0.44 mmol) and phosphite donor 913 (468 mg,
0.76 mmol) were dissolved in dry CH3CN (4 mL), and
cooled to −40 °C. A 0.5 M solution of trimethylsilyl-
trifluoromethansulfonate (TMSOTf) in CH3CN (150
mL, 0.075 mmol), was dropped under vigorous stirring.
After 1 h, the reaction mixture was neutralised with
TEA and concentrated. Flash chromatography purifica-
tion (95:5 toluene–EtOH) of the crude afforded a mix-
ture of 10a, 10b and glycal26 which was further purified
by MP chromatography to give 10a (236 mg, 39%) and
10b (115 mg, 19%) as white amorphous solids: 10a, [h]D20
H-5), 3.40 (s, 3 H, COOCH3), 2.61 (dd, 1 H, J3%%eq,3%%ax
12.9, J3%%eq,4%% 4.9 Hz, H-3%%eq), 1.92 (s, 3 H, CH3Ac), 1.85
(s, 3 H, CH3Ac), 1.82 (t, 1 H, J3%%ax,4%% 11.5 Hz, H-3%%ax),
1.71 (s, 3 H, CH3Ac), 1.65 (s, 3 H, CH3 isoprop.), 1.59
(s, 3 H, CH3Ac), 1.55 (s, 3 H, HNCOCH3), 1.38 (s, 3
H, CH3 isoprop.); 13C NMR (C6D6, 125.72 MHz): l
170.8, 170.4, 170.3, 169.6, 167.3, 166.1, 165.5, 165.2
(COOAc, NHAc, Bz, COOCH3), 110.9 (Cq isoprop.),
100.7 (C-1%), 99.8 (C-1), 99.19 (C-2%%), 77.1 (C-3%), 76.5
(C-4), 73.9 (C-2%), 73.6 (C-3), 73.5 (C-8%%), 73.4 (C-4%%),
73.3 (C-2) 73.1 (C-5), 72.9 (C-6%%), 71.9 (C-5%), 70.4
(CH2Ph), 69.0 (C-7%%), 69.0 (C-4%%), 63.6 (C-6), 63.2
(C-9%%), 62.5 (C-6%%), 52.4 (COOCH3), 49.9 (C-5%%), 38.0
(C-3%%), 27.7 (CH3 isoprop.), 26.7 (CH3 isoprop.), 22.9
(HNCOCH3), 20.8, 20.6, 20.48, 20.4 (4 CH3Ac), Anal.
Calcd for C70H75NO27 (1362.335): C, 61.71; H, 5.55; N,
1.03. Found: C, 61.78; H, 5.54; N, 0.96.
1
+6.8° (c 1.0, CHCl3); H NMR (C6D6, 500 MHz): l
8.30–6.91 (2 m, 25 H, HAr), 5.92 (ddd, 1 H, J8%%,9%%b 7.7
Hz, H-8%%), 5.87 (m, 2 H, H-2, H-3), 5.60 (t, 1 H, J2%,3%
7.2 Hz, H-2%), 5.44 (dd, 1 H, J6%%,7%% 2.3, J7%%,8%% 7.9 Hz,
H-7%%), 4.89 (dd, 1 H, J6a,6b 11.8, J6a,5 6.2 Hz, H-6a),
4.80 (m, 3 H, H 6b, H-4%%, H 1%), 4.70 (dd, 1 H, J8%%,9%%a
2.9, J9%%a,9%%b 12.2 Hz, H-9%%a), 4.63 (d, 1 H, J 12.7 Hz,
CHHPh), 4.52 (m, 1 H, H-4), 4.44 (d, 1 H, CHHPh),
4.41 (m, 2 H, H-1, H-5%%), 4.29 (dd, 1 H, J8%%,9%%b 7.7 Hz,
H-9%%b), 4.10 (dd, 1 H, J6%%,7%% 2.3, J5%%,6%% 10.8 Hz, H-6%%),
4.01 (dd, 1 H, J4%,5% 2.0, J3%,4% 5.4 Hz, H-4%), 3.96–3.87
(m, 3 H, H-3%, H-6%a, NH), 3.86 (br t, 1 H, H-5%), 3.59
(t, 1 H, J6%a,6%b 9.4, J6%b,5% 8.3 Hz, H-6%b), 3.42 (s, 3 H,
COOCH3), 3.34 (br t, 1 H, H-5), 2.73, (dd, 1 H,
J3%%eq,3%%ax 12.8, J3%%eq,4%% 4.7 Hz, H-3%%eq), 2.20 (s, 3 H,
CH3Ac), 2.07 (s, 3 H, CH3Ac), 1.96 (t, 1 H, J3%%ax,4%% 12.0
Hz, H-3%%ax), 1.92 (s, 3 H, CH3Ac), 1.58 (s, 3 H,
CH3Ac), 1.56 (s, 3 H, NHCOCH3), 1.54 (s, 3 H, CH3
isoprop.), 1.40 (s, 3 H, CH3 isoprop.); 13C NMR
(CDCl3, 100.62 MHz): l 171.3, 171.1, 170.7, 170.4,
170.3, 168.3, 166.4, 165.8, 165.6, 165.4 (COOAc,
NHAc, Bz, COOCH3), 110.7 (Cq isoprop.), 100.2, 99.4,
99.4 (C-1, C-1%, C-2%%), 77.4, 77.6, 74.3, 73.7, 73.2, 72.6,
71.7, 69.3, 69.0, 68.0 (C-2, C-3, C-4, C-5, C-2%, C-3%,
C-4%, C-5%, C-4%%, C-6%%, C-7%%, C-8%%), 70.7 (CH2Ph), 63.5,
63.1, 62.9 (C-6, C-6%, C-9%%), 53.2 (COOCH3), 49.9
(C-5%%), 38.1 (C-3%%), 27.9, 26.6 (2 CH3, isoprop.), 23.6
(NHCOCH3), 21.4–21.2 (4 CH3Ac). Anal. Calcd for
C70H75NO27 (1362.335): C, 61.71; H, 5.55; N, 1.03.
Found: C, 61.66; H, 5.52; N, 0.97. 10b, [h]2D0 +23.5° (c
Sodium
galacto-non-2-ulopyranosylonate-(2“6)-i-
pyranosyl-(1“4)- -glucopyranose (11).—To a solution
5-acetamido-3,5-dideoxy-
D
-glycero-h-
D-
D
-galacto-
D
of 10a (235 mg, 0.17 mmol) in CH2Cl2, a 60% aq soln
of CF3COOH was added at 0 °C, and the reaction
mixture was stirred at this temperature for 3 h (TLC 9:1
toluene–EtOH). After dilution with water, neutralisa-
tion with Na2CO3 and extraction with CH2Cl2, the
organic phase was separated, dried over Na2SO4 and
concentrated to dryness. The obtained residue was then
dissolved in MeOH (4 mL) and treated with a 0.5 M
solution of MeONa in MeOH (110 mL) for 48 h at rt
(TLC 3:1:0.25 CHCl3–MeOH–water). The reaction
mixture was neutralised with Amberlite IR-120 resin
(H+ form), concentrated under diminished pressure,
dissolved in water (3 mL) and freeze-dried. The ob-
tained compound was dissolved in water (3 mL),
treated with 1 M aq NaOH (180 mL) for 24 h and
freeze-dried. The residue was dissolved in 1:4 MeOH–
water (6 mL) and submitted to hydrogenolysis in the
presence of 10% Pd/C for 24 h (TLC 6:3:1 acetone–
BuOH–water); the mixture was filtered through Celite
and freeze-dried. The residue was loaded onto a Sep-
hadex G 10 column (V0=150 mL, Vt=300 mL) and
eluted with 1:9 EtOH–water. The fractions containing
the product were collected and freeze-dried giving 11
1
1.0, CHCl3); H NMR (C6D6, 500 MHz): l 8.19–6.98
1
(2 m, 25 H, H), 5.98 (t, 1 H, H-3), 5.88 (dd, 1 H, J6%%,7%%
2.0, J7%%,8%% 3.1 Hz, H-7%%), 5.83 (t, 1 H, J2,3 8.8 Hz, H-2),
5.63 (t, 1 H, H-2%), 5.60 (ddd, 1 H, H-8%%), 5.49 (ddd, 1
H, J4%%,5%% 10.1 Hz, H-4%%), 5.26 (dd, 1 H, J9%%a,9%%b 12.4,
J9%%a,8%% 2.5 Hz, H-9%%a), 4.96 (d, 1 H, J5%%,NH 9.5 Hz, NH),
4.79 (d, 1 H, J1%,2% 7.9 Hz, H-1%), 4.77 (dd, 1 H, J6a,5 2.5,
J6a,6b 11.8 Hz, H-6a), 4.74 (dd, 1 H, J6b,5 5.1 Hz, H-6b),
4.69 (d, 1 H, J 12.7 Hz, CHHPh), 4.63 (d, 1 H, J1,2 7.2
Hz, H-1), 4.51 (dd, 1 H, J9%%b,8%% 8.1 Hz, H-9%%b), 4.47–
4.43 (m, 3 H, CHHPh, H-6%%, H-5%%), 4.28 (dd, 1 H, J3,4
8.4, J4,5 9.4 Hz, H-4), 4.17 (dd, 1 H, J3%,4% 5.5, J4%,5% 1.7
Hz, H-4%), 4.07 (m, 1 H, H-6%a), 3.97 (dd, 1 H, J2,3 7.3
Hz, H-3%), 3.88 (m, 2 H, H-5%, H-6%b), 3.61 (ddd, 1 H,
(82 mg, 74%) as a white foam. H and 13C NMR data
for this compound are in agreement with those reported
in the literature.25
Benzyl (methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-
3,5-dideoxy-
onate)-(2“3)-6-le6ulinoyl-i-
2,6-di-O-benzoyl-i- -glucopyranoside
D
-glycero-h-
D
-galacto-non-2-ulopyranosyl-
-galactopyranosyl-(1“4)-
(12).—Com-
D
D
pound 8 (100 mg, 0.13 mmol) and phosphite donor 913
(160 mg, 0.26 mmol) were dissolved in 2:1 CH3CN–
THF (1.5 mL), and cooled to −44 °C. A 0.5 M
TMSOTf solution in CH3CN (50 mL, 0.025 mmol) was
dropped under vigorous stirring. After 2 h, the reaction
mixture was neutralised with TEA and concentrated.

A. Rencurosi et al. / Carbohydrate Research 337 (2002) 473–483
479
Flash chromatography purification (95:5 CHCl3–
MeOH) of the crude mixture afforded unreacted 8 (22
mg, 22%) and a mixture of 12, glycal26 and other
unidentified compounds (presumably isomers of 12),
which was further purified by MP chromatography (3:2
toluene–acetone) to give 12 (81 mg, 51%) as a white
amorphous solid: [h]2D0 −14.6° (c 0.7, CHCl3); 1H
NMR (CDCl3, 600 MHz): l 8.09–7.10 (m, 15 H, HAr),
5.31 (td, 1 H, J7%%,8%%=J8%%,9%%a 8.4, J8%%.9%%b 2.5 Hz, H-8%%),
5.27 (br d, 1 H, H-7%%), 5.25 (t, 1 H, J1,2=J2,3 9.5 Hz,
H-2), 5.22 (d, 1 H, J5%%,NH 9.6 Hz, NH), 4.97 (td, 1 H,
J3%%ax,4%%=J4%%,5%% 10.5, J3%%eq,4%% 4.6 Hz, H-4%%), 4.93 (br d, 1
H, J6a,6b 10.7 Hz, H-6a), 4.82 (d, 1 H, J 12.6 Hz,
CHHPh), 4.61 (d, 1 H, CHHPh), 4.60 (d, 1 H, J1,2 7.8
Hz, H-1), 4.53 (dd, 1 H, J6b,5 5.6 Hz, H-6b), 4.52 (d, 1
H, J1%,2% 7.9 Hz, H-1%), 4.42 (s, 1 H, OH), 4.30 (dd, 1 H,
J9%%a,9%%b 12.4 Hz, H-9%%a), 4.27 (m, 2 H, H-6%a, H-6%b),
4.10 (br d, 1 H, J6%%,5%% 9.2 Hz, H-6%%), 4.08 (1 H, dd, J2%,3%
9.1, J3%,4% 3.4 Hz, H-3%), 3.93 (m, 2 H, H-5%%, H-9%%b), 3.84
(br t, 1 H, J2,3=J3,4 8.8 Hz, H-3), 3.82–3.68 (m, 4 H,
H-4, H-5, H-2%, H-5%), 3.80 (s, 3 H, COOCH3), 3.63 (br
s, 1 H, H-4%), 3.17 (d, 1 H, J 2.0 Hz, OH), 2.66 (dd, 1
H, J3%%eq,3%%ax 13.0 Hz, H-3%%eq), 2.63–2.43 (m, 4 H, 2 CH2
lev.), 2.38 (d, 1 H, J4%,OH 3.0 Hz, OH), 2.13 (s, 3 H, CH3
lev.), 2.05 (s, 3 H, CH3Ac), 2.02 (s, 3 H, CH3Ac), 2.01
(s, 3 H, CH3Ac), 2.00 (t, 1 H, H-3%%ax), 1.96 (s, 3 H,
CH3Ac), 1.88 (s, 3 H, HNCOCH3); 13C NMR (CDCl3,
150.86 MHz): l 206.9 (CO lev.), 172.6, 170.8, 170.5,
170.2, 170.1, 168.1, 166.2, 165.4 (9 COOAc, NHAc, Bz,
COOCH3, lev.), 104.2 (C-1%), 99.0 (C-1), 97.6 (C-2%%),
82.3 (C-5), 76.5 (C-3%), 73.6 (C-3), 73.2 (C-7%%), 73.0
(C-6%%), 72.9 (C-4), 72.4 (C-5%), 70.1 (CH2Ph), 69.0 (C-
2%), 68.8 (C-8%%), 68.2 (C-4%), 67.9 (C-4%%), 67.0 (C-2), 63.6
(C-6), 63.2 (C-6%), 62.4 (C-9%%), 53.3 (COOCH3), 49.8
(C-5%%), 37.7, 37.6 (C-3%%, CH2COCH3 lev.), 30.9 (CH3
lev.), 27.9 (CH2COO lev.), 23.1 (HNCOCH3), 21.1–
20.6 (4 CH3Ac). Anal. Calcd for C58H69NO27
(1212.159): C, 57.47; H, 5.74; N, 1.16. Found: C, 57.54;
H, 6.64; N, 1.19.
(V0=17 mL, Vt=23 mL) and eluted with 1:9 EtOH–
water. The fractions containing the product were col-
lected and freeze-dried giving 13 (16 mg, 84%) as a
1
white powder. H and 13C NMR data for this com-
pound are in agreement with those reported in the
literature.20,25
Benzyl 2,3,4-tri-O-acetyl-6-O-le6ulinoyl-i-
D-galacto-
pyranosyl-(1“4)-2,3,6-tri-O-acetyl-i- -glucopyran-
D
oside (14).—To compound 2 (110 mg, 0.21 mmol) in
pyridine (4 mL), Ac2O (2 mL) was added and the
mixture was stirred at rt for 24 h. The reaction mixture
was quenched with MeOH and concentrated. The
residue was diluted with EtOAc and washed with 5%
HCl, then with water. The organic layer was dried over
Na2SO4, concentrated and purified by flash chromatog-
raphy (4:1 EtOAc–petroleum ether) affording 14 (130
mg, 80%) as a white foam: [h]2D0 −28.7° (c 1.1, CHCl3);
1H NMR (CDCl3, 200 MHz): l 7.42–7.23 (m, 5 H,
HAr), 5.33 (br d, 1 H, J3%,4% 3.0 Hz, H-4%), 5.17 (t, 1 H,
J2,3=J3.4 9.2 Hz, H-3), 5.10 (dd, 1 H, J1,2 7.6 Hz, H-2),
4.95 (m, 2 H, H-3%, H-4%), 4.85 (d, 1 H, J 12.3 Hz,
CHHPh), 4.60 (d, 1 H, CHHPh), 4.49 (m, 3 H, H-1,
H-1%, H-6a), 4.20–4.03 (m, 3 H, H-6b, H-6%a, H-6%b),
3.84 (m, 2 H, H-4, H-5%), 3.60 (ddd, 1 H, J5,6a 2.0, J5,6b
4.9, J4,5 9.8 Hz, H-5), 2.74 (m, 2 H, CH2 lev.), 2.53 (m,
2 H, CH2 lev.), 2.21, 2.16, 2.05, 2.00, 1.94 (5 s, 21 H,
CH3Ac, lev.); 13C NMR (CDCl3, 50.29 MHz): l 206.1
(CO lev.), 172.1, 170.2, 170.0, 169.7, 169.4, 168.9 (6
COOAc), 100.9, 99.0 (C-1, C-1%), 76.1, 72.7, 71.8, 70.9,
70.5, 69.6, 66.7 (C-2, C-3, C-4, C-5, C-2%, C-3%, C-4%,
C-5%), 70.6 (CH2Ph), 62.0, 60.9 (C-6, C-6%), 37.7
(CH2COCH3 lev.), 29.5 (CH3 lev.), 27.7 (CH2COO
lev.), 20.7–20.5 (6 CH3Ac). Anal. Calcd for C36H46O19
(782.739): C, 55.24; H, 5.92. Found: C, 55.29; H, 5.96.
Benzyl 2,3,4-tri-O-acetyl-i-
D
-galactopyranosyl-(1“
4)-2,3,6-tri-O-acetyl-i- -glucopyranoside (15).—Com-
D
pound 14 (95 mg, 0.12 mmol) was dissolved in 1:1
EtOH–Et2O (2 mL) and AcONH3NH2 (13 mg, 0.14
mmol) was added at rt. After 2 h, the mixture was
concentrated and purified by flash chromatography (9:1
toluene–acetone) affording 15 (81 mg, 98%) as a white
Sodium
galacto-non-2-ulopyranosylonate-(2“3)-i-
pyranosyl-(1“4)- -glucopyranose (13).—To a solution
5-acetamido-3,5-dideoxy-
D
-glycero-h-
D-
1
foam: [h]2D0 −13.3° (c 0.8, CHCl3); H NMR (CDCl3,
D
-galacto-
D
200 MHz): l 7.35–7.13 (m, 5 H, HAr), 5.34 (d, 1 H, J3%,4%
3.3 Hz, H-4%), 5.17 (t, 1 H, J3,4=J2,3 9.2 Hz, H-3), 5.14
(dd, 1 H, J1%,2% 7.6, J2%,3% 10.4 Hz, H-2%), 4.98 (dd, 1 H,
H-3%), 4.96 (dd, 1 H, J1,2 7.4 Hz, H-2), 4.86 (d, 1 H, J
12.2 Hz, CHHPh), 4.62–4.51 (m, 4 H, H-1, H-1%, H-6a,
CHHPh), 4.09 (dd, 1 H, J6a,6b 11.9, J6b,5 5.2 Hz, H-6b),
3.86 (br t, 1 H, J4,5 9.6 Hz, H-4), 3.70–3.48 (m, 4 H,
H-5, H-5%, H-6%a, H-6%b), 2.17–1.95 (6 s, 18 H, CH3Ac);
13C NMR (CDCl3, 75.44 MHz): l 170.9, 170.4, 170.1,
169.9, 169.6, 169.1 (6 COOAc), 101.1, 99.0 (C-1, C-1%),
76.2, 74.1, 73.5, 72.6, 71.7, 71.0, 69.5, 67.7 (C-2, C-3,
C-4, C-5, C-2%, C-3%, C-4%, C-5%), 70.0 (CH2Ph), 62.1,
60.8 (C-6, C-6%), 20.9–20.7 (6 CH3Ac). Anal. Calcd for
C31H40O17 (684.639): C, 54.38; H, 5.89. Found: C,
54.36; H, 5.82.
of 12 (35 mg, 0.029 mmol) in MeOH (2 mL), a 0.5 M
solution of MeONa in MeOH (12 mL) was added and
the reaction mixture was stirred for 48 h at rt (TLC
3:1:0.35 CHCl3–MeOH–water). The solution was neu-
tralised by Amberlite IR 120 resin (H+ form), filtered,
concentrated under diminished pressure and, after dilu-
tion in water, freeze-dried. The resulting product was
dissolved in water (1 mL), treated with 1 M aq NaOH
(80 mL) for 24 h and freeze-dried. The residue was
dissolved in 1:4 MeOH–water (3 mL) and hy-
drogenolysed in the presence of 10% Pd/C for 24 h
(TLC 6:3:1 acetone–BuOH–water). The mixture was
filtered through Celite and freeze-dried. Finally the
residue was loaded onto a Sephadex G 10 column

480
A. Rencurosi et al. / Carbohydrate Research 337 (2002) 473–483
Benzyl 6-O-sulfo-i-
D
-galactopyranosyl-(1“4)-i-
D
-
(C-6a), 61.0 (C-6b). Anal. Calcd for C12H21NaO14S
(444.342): C, 32.44; H, 4.76. Found: C, 32.49; H, 4.72.
glucopyranoside sodium salt (16).—Compound 15 (186
mg, 0.27 mmol), was dissolved in dry DMF (3 mL) in
Ar atmosphere, the SO3NMe3 complex (56 mg, 0.40
mmol) was added and the reaction mixture was stirred
at 60 °C (TLC 4:1 CH2Cl2–MeOH). After 48 h, the
reaction was quenched with a few drops of MeOH and
concentrated. Flash chromatography purification
(CHCl3, then 10:1 CHCl3–MeOH, and 5:1 CHCl3–
MeOH) afforded a white amorphous solid (203 mg).
The resulting product was dissolved in dry MeOH (8
mL) under nitrogen; after cooling to 0 °C a 1 M
solution of MeONa in MeOH (400 mL) was added and
the reaction mixture was stirred at rt for 7 h. The
solution was percolated on a column (1×10 cm) filled
with Dowex 50 WX8 (H+ form) resin and the eluate
was concentrated and purified by flash chromatography
(5:8:0.7 MeOH–CHCl3–water). The fractions contain-
ing the product were concentrated to a volume of 3 mL
and percolated on a Dowex 50 WX8 (Na+ form)
column (1×10 cm). The resulting eluate was finally
concentrated to dryness affording 16 (117 mg, 81%), as
a white amorphous solid: [h]2D0 −11.5° (c 0.6, water);
1H NMR (D2O, 500 MHz): l 7.51–7.42 (m, 5 H, HAr),
4.94 (d, 1 H, J 11.7 Hz, CHHPh), 4.77 (d, 1 H,
CHHPh), 4.57 (d, 1 H, J1,2 8.1 Hz, H-1), 4.48 (d, 1 H,
J1%,2% 7.4 Hz, H-1%), 4.21 (appearing as a d, 2 H, J 6.2
Hz, H-6%a, H-6%b), 4.00 (dd, 1 H, J4%,5%B2 Hz, H-4%),
3.99–3.98 (m, 2 H, H-6a, H-5%), 3.81 (dd, 1 H, J5,6b 5.1,
J6a,6b 12.4 Hz, H-6b), 3.69 (dd, 1 H, J2%,3% 10.0, J3%,4% 3.5
Hz, H-3%), 3.64 (m, 3 H, H-3, H-4, H-5), 3.56 (dd, 1 H,
H-2%), 3.38 (t, 1 H, J2,3 9.4 Hz, H-2); 13C NMR (D2O,
125.72 MHz): l 105.9 (C-1%), 103.9 (C-1), 82.3 (C-4),
77.5 (C-5), 77.3 (C-3), 75.6 (C-2), 75.7 (C-5%), 75.2
(C-3%), 74.3 (CH2Ph), 73.5 (C-2%), 71.1 (C-4%), 70.0 (C-
6%), 63.1 (C-6). Anal. Calcd for C19H27NaO14S
(534.465): C, 42.70; H, 5.09. Found: C, 42.75; H, 5.17.
Benzyl (3-O-sulfo-i-
-glucopyranoside sodium salt (18).—Compound 1 (218
D
-galactopyranosyl)-(1“4)-i-
D
mg, 0.50 mmol) and Bu2SnO (135 mg, 0.54 mmol) were
stirred in refluxing dry MeOH (4 mL) for 2 h under
nitrogen. The reaction mixture was concentrated to
dryness and the dry dibutylstannylene intermediate was
treated with SO3NMe3 complex (144 mg, 1.0 mmol) in
dry dioxane (4 mL) at rt. After 48 h, the reaction was
diluted with MeOH (3 mL), filtered and concentrated.
The residue was then purified by flash chromatography
(5:8:1 MeOH–CHCl3–water); the fractions containing
the product were concentrated, dissolved in MeOH and
percolated into a cation exchange resin column (Dowex
50 WX8, H+ form, 1×10 cm). The eluate was concen-
trated and percolated on a Dowex 50 column (Na+
form, 1×10 cm) affording, after removal of the sol-
vents, 18 (200 mg, 75%) as a white amorphous solid:
[h]2D0 −17.2° (c 1.0, MeOH); 1H NMR (D2O, 500
MHz): l 4.95 (d, 1 H, J 11.4 Hz, CHHPh), 4.78 (d, 1
H, CHHPh), 4.58 (d, 1 H, J1%,2% 7.9 Hz, H-1%), 4.57 (d,
1 H, J1,2 8.0 Hz, H-1), 4.34 (dd, 1 H, J2%,3% 9.8, J3%,4% 3.3
Hz, H-3%), 4.30 (d, 1 H, J4%,5%B1 Hz, H-4%), 4.01 (dd, 1
H, J5,6a 2.3, J6a,6b 12.4 Hz, H-6a), 3.84 (dd, 1 H, J5,6b
5.2 Hz, H-6b), 3.78 (m, 1 H, H-5%), 3.75 (m, 2 H, H-6%a,
H-6%b), 3.70 (t, 1 H, J4,5 9.7 Hz, H-4), 3.70 (t, 1 H,
H-2%), 3.64 (t, 1 H, J3,4 8.9 Hz, H-3), 3.59 (ddd, 1 H,
H-5), 3.37 (t, 1 H, J2,3 9.2 Hz, H-2); 13C NMR (D2O,
125.72 MHz): l 105.4 (C-1%), 103.9 (C-1), 82.9 (C-3%),
81.3 (C-4), 77.7 (C-5%), 77.6 (C-5), 77.2 (C-3), 75.8
(C-2), 74.3 (CH2Ph), 71.9 (C-2%), 69.6 (C-4%), 63.7 (C-
6%), 62.9 (C-6). Anal. Calcd for C19H27NaO14S
(534.465): C, 42.70; H, 5.09. Found: C, 42.77; H, 5.12.
3-O-Sulfo-i-
D
-galactopyranosyl-(1“4)- D-glucopy-
ranose sodium salt (19).—Compound 18 (40 mg, 0.075
mmol) was submitted to the same procedure described
for 17, affording 19 (32 mg, 96%) as a white powder:
6-O-Sulfo-i-
D
-galactopyranosyl-(1“4)-D-glucopy-
1
ranose sodium salt (17).—A solution of 16 (25 mg,
0.046 mmol) in 3:1 MeOH–water (4 mL) was hy-
drogenolysed in the presence of 10% Pd/C for 16 h,
(TLC 6:3:1 acetone–BuOH–water). The mixture was
filtered through Celite and freeze-dried. The residue was
loaded onto a Sephadex G 10 column (V0=17 mL,
Vt=23 mL) and eluted with 1:9 EtOH–water. The
fraction containing the product were collected and
freeze-dried affording 17 (18 mg, 88%) as a white
[h]2D0 +40.7° (c 1.1, water). H and 13C NMR data are
in agreement with those reported in the literature for
the natural compound.6
Benzyl
methyl-i-
2,4,6-O-tri-O-acetyl-3-O-methoxycarbonyl-
-galactopyranosyl-(1“4)-2,3,6-tri-O-ace-
D
tyl-i- -glucopyranoside (23).—Compound 1 (200 mg,
D
0.46 mmol) and Bu2SnO (125 mg, 0.50 mmol) were
stirred in refluxing dry MeOH (4 mL) under N2 for 16
h. The reaction mixture was concentrated to dryness
and the dibutylstannylene intermediate, after coevapo-
ration with toluene, was dissolved in dry DMF (4 mL)
under Ar and treated with ethyl bromoacetate (255 mL,
2.3 mmol) and TBAI (cat.) at 40 °C for 48 h (TLC 8:1.5
CH2Cl2–MeOH). The reaction mixture was concen-
trated under diminished pressure and chromatographed
to give a pale yellow syrup (181 mg) containing two
compounds, namely the expected product 20 and the
corresponding lactone derivative 21. The mixture of
1
powder: [h]2D0 +29.4° (c 1.0, water); H NMR (D2O,
200 MHz): l 5.28 (d, 0.28 H, J1d,2d 3.4 Hz, H-1a), 4.72
(d, 0.7 H, J1e,2e 7.9 Hz, H-1b), 4.57 (d, 1 H, J1%,2% 7.7 Hz,
H-1%), 4.27 (appearing as a d, 2 H, H-6%a, H-6%b),
4.06–3.56 (m, 9.3 H, H-2a, H-3, H-4, H-5, H-6a, H-6b,
H-2%, H-3%, H-4%, H-5%), 3.36 (br t, 0.7 H, J
9.1 Hz,
2,3
H-2b); 13C NMR (CDCl3, 75.44 MHz): 103.9 (C-1%),
96.5 (C-1b), 92.6 (C-1a), 80.4, 80.2, 75.5, 75.2, 74.6,
73.7, 73.2, 72.3, 72.0, 71.6, 70.9, 69.1, 68.2 (C-6%), 61.1

A. Rencurosi et al. / Carbohydrate Research 337 (2002) 473–483
481
these compounds was dissolved in dry MeOH (4 mL)
and treated with a 1 M solution of MeONa (30 mL) in
MeOH. After the complete conversion of the faster
moving compound into the second (TLC 4:1 CH2Cl2–
MeOH), the mixture was neutralised with Amberlite IR
120 (H+ form) and concentrated leading to crude com-
pound 22. The residue was dissolved in pyridine (4 mL)
and treated with Ac2O (2 mL). After one night, the
reaction mixture was concentrated, diluted with CH2Cl2
and washed with water. The organic layer was sepa-
rated, dried over Na2SO4 and concentrated. Purification
by flash chromatography (1:1 EtOAc–petroleum ether)
afforded 23 as a white foam (193 mg, 56% from 1): [h]D20
(C-2, C-3, C-4, C-5, C-2%, C-3%, C-4%,C-5% a and b), 69.3
(OCH2COO−), 61.1 (C-6%), 60.9 (C-6b), 60.8 (C-6a).
Anal. Calcd for C14H23NaO13 (422.314): C, 39.82; H,
5.49. Found: C, 39.80; H, 5.57.
Benzyl 6-O-thexyldimethylsilyl-i-D-galactopyranosyl-
(1“4)-i- -glucopyranoside (25).—Compound 1 (200
D
mg, 0.46 mmol) and Bu2SnO (125 mg, 0.50 mmol) were
stirred in refluxing dry MeOH (4 mL) for 16 h. The
reaction mixture was concentrated to dryness and the
dibutylstannylene intermediate was dissolved in dry
THF (3 mL) and treated with TDSCl (102 mL, 0.52
mmol) at rt for 18 h. After adding some drops of TEA,
the solution was concentrated under diminished pres-
sure. Purification by flash chromatography (95:5 then
9:1 CH2Cl2–MeOH) afforded 25 (201 mg, 76%). In
order to allow an unambiguous identification of the
product, a portion of 25 (50 mg) was submitted to
standard acetylation (Ac2O, Py) and characterised. [h]D20
1
−9.6° (c 1.1, CHCl3); H NMR (CDCl3, 200 MHz): l
7.40–7.21 (m, 5 H, HAr), 5.39 (br d, 1 H, J3%,4% 3.1 Hz,
H-4%), 5.15 (t, 1 H, J3,4=J2,3 9.0 Hz, H-3), 5.00 (t, 1 H,
J2%,3% 7.9 Hz, H-2%), 4.95 (t, 1 H, J2,3 7.8 Hz, H-2), 4.86
(d, 1 H, J 12.3 Hz, CHHPh), 4.59 (d, 1 H, CHHPh),
4.54 (dd overlapping signal, 1 H, H-6a), 4.52 (d, 1 H,
J1,2 8.0 Hz, H-1), 4.46 (d, 1 H, J1%,2% 8.1 Hz, H-1%),
4.19–4.08 (m, 5 H, H-6b, H-6a%, H-6b%, OCH2COO),
3.80 (t, 1 H, J3,4=J4,5 9.8 Hz, H-4), 3.75 (br t, 1 H,
J4%,5%=J5%,6% 7.0 Hz, H-5%), 3.72 (s, 3 H, COOCH3),
3.63–3.58 (m, 2 H, H-5, H-3%), 2.13 (s, 9 H, 3 CH3Ac),
2.08, 2.04, 1.98 (3 s, 9 H, 3 CH3Ac); 13C NMR (CDCl3,
50.29 MHz): l 170.3, 170.2, 170.0, 169.6 (6 COOAc, 2
overlapping signals), 101.0, 98.9 (C-1, C-1%), 78.3, 76.2,
72.9, 72.7, 71.7, 70.6, 70.5, 65.2 (C-2, C-3, C-4, C-5,
C-2%, C-3%, C-4%, C-5%), 70.6 (CH2Ph), 66.0, 62.1, 61.2
(C-6, C-6%, OCH2COO), 51.7 (COOCH3), 20.70–20.59
(CH3Ac). Anal. Calcd for C34H44O19 (756.702): C,
53.97; H, 5.86, Found: C, 54.01; H, 5.81.
1
−29.4° (c 1.3, CHCl3); H NMR (CDCl3, 200 MHz): l
7.38–7.22 (m, 5 H, HAr), 5.39 (br d, 1 H, J3%,4% 3.1 Hz,
H-4%), 5.14 (t, 1 H, J3,4=J2,3 9.3 Hz, H-3), 5.08–4.91
(m, 3 H, H-2, H-2%, H-3%), 4.85 (d, 1 H, J 12.3 Hz,
CHHPh), 4.59 (d, 1 H, CHHPh), 4.53–4.45 (m, 3 H,
H-1, H-1%, H-6a), 4.10 (dd, 1 H, J6a,6b 11.8, J5,6b 4.8 Hz,
H-6b), 3.82 (t, 1 H, J3,4=J4,5 9.5 Hz, H-4), 3.70–3.50
(m, 4 H, H-5, H-5%, H-6%a, H-6%b), 2.12, 2.11, 2.03, 2.02,
1.99, 1.95 (6 s, 18 H, 6 CH3Ac), 1.60 (dq, 1 H, H TDS),
0.91–0.79 (m, 12 H, 4 CH3, TDS), 0.07, 0.04 (2 s, 6 H,
2 CH3Si TDS).
Benzyl 2,3,4-tri-O-benzyl-i-
D
-galactopyranosyl-(1“
4)-2,3,6-tri-O-benzyl-i- -glucopyranoside (26).—To a
D
solution of 25 (165 mg, 0.29 mmol) in dry DMF (2
mL), BnBr (415 mL, 3.49 mmol) and NaH (60% suspen-
sion in mineral oil 104 mg, 2.60 mmol) were added, and
the reaction mixture was stirred at rt overnight (TLC
1:9 EtOAc–petroleum ether). After quenching the ex-
cess of NaH with MeOH, the reaction mixture was
concentrated, the residue was diluted with CH2Cl2 and
the organic phase was washed with water. The organic
layer was separated, dried over Na2SO4 and concen-
trated. The residue was purified by flash chromatogra-
3-O-Carboxymethyl-i-
D
-galactopyranosyl-(1“4)-D-
glucopyranose sodium salt (24).—To a solution of com-
pound 23 (20 mg, 0.026 mmol) in MeOH (3 mL), a 0.5
M solution of MeONa in MeOH (30 mL) was added
and the reaction mixture was stirred at rt for 16 h. Then
water (2 mL) and 0.5 M solution of MeONa in MeOH
(30 mL) were added, the mixture was stirred for addi-
tional 20 h at rt, then concentrated under diminished
pressure. The residue was dissolved in 1:1 MeOH–wa-
ter and hydrogenolysed in the presence of 10% Pd/C for
16 h, (TLC 6:3:1 acetone–BuOH–water). The mixture
was filtered through Celite and freeze-dried. The residue
was dissolved in 0.1 M NaOH (0.5 mL), loaded onto a
Sephadex G 10 column (V0=17 mL, Vt=23 mL) and
eluted with 1:9 EtOH–water. The fraction containing
the product were collected and freeze-dried giving 24 (9
mg, 91%) as a white powder: [h]2D0 +59.1° (c 0.5,
phy (0.6:10 EtOAc–petroleum ether) affording
a
colourless syrup (300 mg). The resulting compound was
dissolved in THF (10 mL) and, after cooling the solu-
tion to −12 °C, treated with a 1 M TBAF solution
(540 mL, 0.54 mmol). The reaction mixture was stirred
at rt for 6 h (TLC 2:3 EtOAc–petroleum ether), then
the solution was diluted with EtOAc, and washed with
satd NH4Cl. The organic layer was separated, dried
over Na2SO4, filtered and concentrated. The crude com-
pound was purified by flash chromatography (3:7 then
2:3 EtOAc–petroleum ether) affording 26 (185 mg,
66%) as a white foam: [h]2D0 −12.6° (c 1, CHCl3), lit.22
−14° (c 1.29, CHCl3). NMR data for this compound
are in agreement with those reported in the literature.22
1
water); H NMR (D2O, 300 MHz): l 5.26 (d, H 0.3,
J1a,2 3.8 Hz, H-1a), 4.70 (d, H 0.7, J1b,2 7.9 Hz, H-1b),
4.52 (d, 1 H, J1%,2% 7.8 Hz, H-1%), 4.11 (br s, 3 H),
4.06–3.56 (m, 10.3 H), 3.32 (bt, 0.70 H, J 8.3 Hz,
H-2b); 13C NMR (D2O, 75.44 MHz): 179.2 (COO−),
103.6 (C-1%), 96.6 (C-1b), 92.6 (C-1a), 82.7, 79.1, 79.0,
75.9, 75.6, 75.1, 74.6, 73.5, 72.2, 72.0, 70.9, 70.7, 66.2

482
A. Rencurosi et al. / Carbohydrate Research 337 (2002) 473–483
Benzyl
methyl-i-
zyl-i- -glucopyranoside (27).—To a solution of com-
2,3,4-tri-O-benzyl-6-O-tert-butoxycarboxy-
-galactopyranosyl-(1“4)-2,3,6-tri-O-ben-
Anal. Calcd for C14H23NaO13 (422.314): C, 39.82; H,
5.49. Found: C, 39.88; H, 5.56.
D
D
pound 26 (120 mg, 0.12 mmol) in CH2Cl2 (0.2 mL),
tert-butyl bromoacetate (377 mL, 1.47 mmol),
Bu4NHSO4 (51 mg, 0.16 mmol) and 33% aq NaOH (0.8
mL) were added and the reaction mixture was stirred at
rt for 4 h (TLC 2:3 EtOAc–petroleum ether). The
reaction mixture was diluted with water and extracted
with CH2Cl2, the organic layer was separated, dried
over Na2SO4, filtered and concentrated. The crude com-
pound was purified by flash chromatography (1:9 then
1:4 EtOAc–petroleum ether) affording 27 (129 mg,
96%) as a colourless syrup: [h]2D0 +3.2° (c 1.1, CHCl3);
1H NMR (CDCl3, 300 MHz): l 5.00 (d, 1 H, J 10.7 Hz,
CHHPh), 4.98 (d, 1 H, J 11.8 Hz, CHHPh), 4.94 (d, 1
H, J 12.1 Hz, CHHPh), 4.90 (d, 1 H, J 10.9 Hz,
CHHPh), 4.80 (d, 1 H, J 11.2 Hz, CHHPh), 4.75 (d, 1
H, CHHPh), 4.74 (d, 1 H, CHHPh), 4.73 (d, 1 H, J
11.8 Hz, CHHPh), 4.69 (d, 1 H, CHHPh), 4.65 (d, 1 H,
CHHPh), 4.63 (d, 1 H, CHHPh), 4.55 (d, 1 H, J 12.1
Hz, CHHPh), 4.48 (d, J 7.3 Hz, H-1 or H-1%), 4.42 (d,
J 6.9 Hz, H-1 or H-1%), 4.41 (d, 1 H, CHHPh), 4.15–
3.90 (m, 2 H), 3.83–3.70 (m, 4 H), 3.65 (d, 1 H, J 16.4
Hz, OCHHCOO), 3.57–3.29 (m, 8 H), 1.44 (s, 9 H, 3
CH3t-Bu); 13C NMR (CDCl3, 75.44 MHz): l 169.4
(COOt-Bu), 102.8, 102.5 (C-1, C-1%), 83.0, 82.5, 81.9,
80.0, 76.7, 75.2, 73.5, 73.0 (C-2, C-3, C-4, C-5, C-2%,
C-3%, C-4%, C-5%), 75.3, 75.2, 75.0, 64.7, 73.2, 72.6, 70.9,
69.3, 69.0, 68.3 (7 CH2Ph, C-6, C-6%, OCH2COOt-Bu),
60.3 (Cq t-Bu), 28.1 (CH3t-Bu). Anal. Calcd for
C67H74O13 (1087.297): C, 74.01; H, 6.86. Found: C,
74.09; H, 6.89.
Acknowledgements
A.R. is grateful to Professor R.R. Schmidt for help-
ful discussion on the sialyllactosides synthesis. This
work was supported by EU-NOFA project (grant
FAIR CT973142), MURST (Ministero dell’Universita`
e della Ricerca Scientifica e Tecnologica) and CNR
(Centro di Studio sulle Sostanze Organiche Naturali).
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[h]2D0 +24.2° (c 0.6, water); H NMR (D2O, 300 MHz):
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(d, 1 H, J1%,2% 7.8 Hz, H-1%), 4.17–3.65 (m, 13.3 H), 3.42
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l 178.7 (COO−), 103.8 (C-1%), 96.6 (C-1b), 92.7 (C-1a),
79.8, 79.7, 75.7, 75.3, 74.7, 74.2, 72.3, 72.1, 71.8, 69.6
(C-2, C-3, C-4, C-5, C-1%, C-2%, C-3%, C-4%, C-5% a and b),
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