Synthetic potential of fucosyltransferase III for the synthesis of fluorescent-labeled milk oligosaccharides

Article abstract of DOI:10.1080/07328300500341965

Various fundamental biologic roles of milk oligosaccharides have been recognized; however, their structure-affinity relationship is still not fully revealed. Herein, we describe the synthesis of the fluorescent-labeled milk oligosaccharides 3-(5-dimethyla

Full text of DOI:10.1080/07328300500341965

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Synthetic Potential of Fucosyltransferase III
for the Synthesis of Fluorescent‐labeled Milk
Oligosaccharides
Said Rabbani a , Federica Compostella b , Laura Franchini b , Beatrice
Wagner a , Luigi Panza c & Beat Ernst a
a Institute of Molecular Pharmacy, Pharmacenter of the University of Basel ,
Basel, Switzerland
b Dipartimento di Chimica, Biochimica e Biotecnologie per la Medicina ,
Università di Milano , Milano, Italy
c Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e
Farmacologiche , Università del Piemonte Orientale , Novara, Italy
Published online: 02 Feb 2007.
To cite this article: Said Rabbani , Federica Compostella , Laura Franchini , Beatrice Wagner , Luigi Panza &
Beat Ernst (2005) Synthetic Potential of Fucosyltransferase III for the Synthesis of Fluorescent‐labeled Milk
Oligosaccharides, Journal of Carbohydrate Chemistry, 24:8-9, 789-807, DOI: 10.1080/07328300500341965
To link to this article: http://dx.doi.org/10.1080/07328300500341965
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Journal of Carbohydrate Chemistry, 24:789–807, 2005
Copyright # Taylor & Francis LLC
ISSN: 0732-8303 print 1532-2327 online
DOI: 10.1080/07328300500341965
Synthetic Potential of
Fucosyltransferase III for the
Synthesis of Fluorescent-
labeled Milk
Oligosaccharides
Said Rabbani
Institute of Molecular Pharmacy, Pharmacenter of the University of Basel, Basel,
Switzerland
Federica Compostella and Laura Franchini
`
Dipartimento di Chimica, Biochimica e Biotecnologie per la Medicina, Universita di
Milano, Milano, Italy
Beatrice Wagner
Institute of Molecular Pharmacy, Pharmacenter of the University of Basel, Basel,
Switzerland
Luigi Panza
Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e Farmacologiche,
`
Universita del Piemonte Orientale, Novara, Italy
Beat Ernst
Institute of Molecular Pharmacy, Pharmacenter of the University of Basel, Basel,
Switzerland
Received July 25, 2005; accepted September 1, 2005.
Address correspondence to Beat Ernst, Institute of Molecular Pharmacy, Pharmacenter
of the University of Basel, Klingelbergstrasse 50, CH-4056 Basel, Switzerland.
Fax: þ41-61-2671551; E-mail: beat.ernst@unibas.ch and Luigi Panza, Dipartimento
`
di Scienze Chimiche, Alimentari, Farmaceutiche e Farmacologiche, Universita del
Piemonte Orientale, Via Bovio 6, Novara 28100, Italy. Fax: þ39-321-375845; E-mail:
luigi.panza@pharm.unipmn.it
789

S. Rabbani et al.
790
Various fundamental biologic roles of milk oligosaccharides have been recognized;
however, their structure-affinity relationship is still not fully revealed. Herein,
we describe the synthesis of the fluorescent-labeled milk oligosaccharides 3-(5-dimethy-
laminonaphthalene-1-sulfonylamino)propyl b-D-galactopyranosyl-(1 ! 3)-[a-L-fucopyr-
anosyl-(1 ! 4)]-2-acetamido-2-deoxy-b-D-glucopyranoside (1) and 3-(5-dimethylamino-
naphthalene-1-sulfonylamino)propyl b-D-galactopyranosyl-(1 ! 3)-[a-L-fucopyranosyl-
(1 ! 4)]-b-D-glucopyranoside (2) as useful tools for synthetic, analytic, and biologic
applications. For the fucosylation of lactose and lacto-N-biose, the chemical and the
enzymatic syntheses using fucosyltransferase III were compared.
Keywords Chemo-enzymatic synthesis, Fucosyltransferase III, Fluorescent-labeled
milk oligosaccharides
INTRODUCTION
In human milk a great structural variety of oligosaccharides exists, which
ranges from simple lactose to complex neutral or charged oligosaccharides con-
taining lactose, lactosamine, and lacto-N-biose disaccharides, which may be
connected in various ways, fucosylated, or sialylated. So far, more than a
hundred different structures have been isolated and characterized.[1]
Although it has been traditionally observed that breastfeeding of infants is
advantageous for their healthy development, only recently the biologic import-
ance of milk oligosaccharides has been recognized.[1a] Among numerous other
biologic roles, it has been demonstrated that they can inhibit bacterial adhesion
to epithelial cells, and thus exert a protective action against infections.[1a,2]
However, the oligosaccharidic structures responsible for these processes are
yet to be identified and characterized.
Within a project devoted to the characterization of human milk
oligosaccharides, different structures have been synthesized by chemical and
chemo-enzymatic methods.[3] There, hydrolases have been exploited for the
selective protection of specific hydroxyl groups in different building blocks,
thus facilitating their manipulation for the subsequent chemical glycosylation
reactions.
A second class of enzymes, which proved to be extremely useful for
oligosaccharide preparation, are the glycosyltransferases devoted to the syn-
thesis of glycosidic linkages in vivo. Scope and limitations of these enzymes
have been extensively explored.[4] In particular, it has been demonstrated
that some fucosyltransferases not only efficiently process natural substra-
tes,[4a] but also are quite tolerant toward structural variations of the
acceptor molecules. In addition, they also exhibit a broad tolerance toward
modifications of the nucleotide sugar donor.[4b] This raised the question of
whether fucosyltransferases could also be applied to the enzymatic fucosyla-
tion of fluorescent-labeled milk oligosaccharides.

Fluorescent-labeled Milk Oligosaccharides
791
Fluorescent labels are an extremely useful tool, because they allow the
monitoring of low concentration of the chemical species of interest involved
in biochemical processes. Fluorescence is commonly used for analytical
purposes to visualize oligosaccharides after cleavage from glycoconjugates, to
facilitate identification of particular oligosaccharides,[5] or to allow monitoring
the progress of enzymatic reactions.[6] Fluorescent labeling has also been
widely used for the determination of the cellular distribution of bioactive com-
pounds, including carbohydrates,[7] and in the present case could be exploited
to obtain information on adhesion phenomena between sugars and pathogenic
microorganisms. In addition, fluorescent-labeled milk oligosaccharides would
allow the elucidation of their role in cell recognition and to gain further infor-
mation on their biodistribution. These findings could prove to be extremely
useful for future applications in drug targeting.[8]
RESULTS AND DISCUSSION
As a consequence of its reported high substrate tolerance,[4] fucosyltransferase
III (FucT III, EC2.4.1.65)[For the cDNA of FucT III see Ref.[9]] was selected for
the enzymatic synthesis of fluorescent-labeled milk oligosaccharides, that is,
the Lewisa and Lewisx derivatives 1 and 2 (Sch. 2). For this approach, the
fluorescent-labeled disaccharides 3 and 4 (Sch. 1) were chemically synthesized
and subsequently enzymatically or chemically fucosylated.[4] The N-acetyl-
glucosamine derivative 6, which can easily be obtained from peracetylated glu-
cosamine 5,[10] was used as starting material for the synthesis of lacto-N-biose
derivative 3. Deacetylation and benzylidene formation[11] afforded compound 7
in a state ready for glycosylation with O-(2,3,4,6-tetra-O-acetyl-a-D-galacto-
[12]
pyranosyl)trichloroacetimidate (8)
in the presence of catalytic amounts of
.
BF3 OEt2 (!9). Acidic hydrolysis of the benzylidene protecting group (!10)
and removal of the carbobenzoxy protecting group by hydrogenolysis yielded
the free primary amine, which was directly dansylated to afford the fluor-
escent-labeled compound 11. Finally, the target molecule 3 was obtained by
´
Zemplen deacetylation. Since attempts to perform a selective enzymatic acety-
lation on the 6-OH of the GlcNAc moiety using a lipase failed,[3] the starting
material 12 for the chemical fucosylation was obtained by regioselective acety-
lation of the 6-position at low temperature. The lactose derivative 4 (Sch. 1) was
synthesized starting from the commercially available lactose octaacetate 13 by
direct glycosylation of 3-benzyloxycarbonylamino-1-propanol, promoted by
.
BF3 OEt2 as Lewis acid. 14 was obtained stereoselectively, although in
moderate yield. Deprotection of the primary amine followed by dansylation
´
(!15) and Zemplen deacetylation provided the lactose derivative 4.
In the next step, type 1 and type 2 disaccharides 3 and 4 were analyzed for
their ability to act as acceptors for FucT III using 14C-labeled GDP-fucose.[13]
For this purpose, we expressed a new construct of FucT III [Other constructs

S. Rabbani et al.
792
Scheme 1: Synthesis of fluorescent-labeled lacto-N-biose 3 and lactose 4: a)
HO(CH2)3NHCbz, TMSOTf, Cl(CH2)2Cl, reflux, 88% [10]; b) MeONa, MeOH, rt, then
benzaldehyde-dimethylacetal, cat. pTsOH, DMF, 81%; c) 8 [11], BF3†OEt2, CH2Cl2, 08C to rt,
78%; d) TFA/H2O 7 : 3, CH2Cl2, 08C, 83%; e) Pd/C, H2, MeOH, rt, then dansyl chloride, TEA,
CH2Cl2, rt, 72%; f) MeONa, MeOH, 57%; g) AcCl, pyridine, CH2Cl2, 2 788C, 74%; h)
HO(CH2)3NHCbz, BF3†OEt2, CH2Cl2, rt, 41%; i) Pd/C, H2, MeOH, rt, then dansyl chloride, TEA,
CH2Cl2, rt, 58%; k) MeONa, MeOH, rt, 77%.
of FucT III have already been expressed in various expression systems][14] in a
secretable form. FucT III cDNA[9] lacking the first 35 amino acids of the coding
region was fused to a secretory sequence and a protein A binding domain. This
construct was used for the transfection of Sf9 cells leading to the secreted
enzyme, which could easily be purified by affinity chromatography.
The enzyme shows predominantly a(1–4)-activity (see lacto-N-biose, Table 1).
With the nonnatural substrate 3, an improved synthetic productivity

Fluorescent-labeled Milk Oligosaccharides
793
compared to the natural substrate lacto-N-biose was observed. The
productivity with LacNAc and the type 2 derivative 4 is comparable. The
maximum rate possible, when both donor and acceptor are saturated (kcat), is
often affected by rate-limiting substrate binding or product release steps.
The specificity constant (kcat/Km) combines the effects of both rate and
binding and therefore gives a better indication of the synthetic productivity
of the enzyme. Since the major factor governing specificity is the stability of
the enzyme-bound transition state, the specificity constant reflects how well
the transition states, which are formed during the conversion of the substrates,
are stabilized by the protein.[15] As expected for substrate 3, the specificity
constant is in the same order as the one for the natural acceptor substrate
lacto-N-biose. The specificity constant for substrate 4, however, is only 5%
compared to the natural substrate (see Table 1), but is expected to still be
Scheme 2: Chemical and enzymatic fucosylation of fluorescent-labeled lacto-N-biose 3
˚
and lactose 4: a) CuBr2, Bu4NBr, MS 3 A, DMF/CH2Cl2 1:1, rt, 96%; b) MeONa, MeOH, rt, quant.;
c) Pd(OH)2, H2, MeOH, then dansyl chloride, TEA, MeOH, 52%; d) FucT III, GDPfucose, MnCl2,
Na-cacodylate buffer, BSA, CIAP, 378C, overnight [3 ! 1 (88%); 4 ! 2 (54%)].

S. Rabbani et al.
794
sufficient for a preparative application.[16] These data therefore prompted us to
apply the enzymatic fucosylation reaction for the preparative synthesis of the
fluorescent-labeled target molecules 1 and 2 (Sch. 2).
In addition, the Lewisa derivative 1 was also synthesized by a chemical
glycosydation (Sch. 2) using thioglycoside 16[17] as donor. In the first
attempt, the electrophile was generated by the conditions described by
Ogawa et al.[18] using cupric bromide as thiophilic promoter. Although the
presence of an acylamino function is known to be potentially problematic, the
fucosylation worked successfully, giving the Lewisa derivative 17 in almost
quantitative yield. Unfortunately, the product retained traces of the copper
salt, which could not be removed by washing with EDTA or concentrated
ammonia solutions or by repeated filtrations through silica gel. Because of
the paramagnetic properties of copper, the 1H NMR spectrum contained
broad lines and was impossible to interpret, whereas the 13C NMR spectrum
was in good agreement with the expected structure. For the final confirmation
of the structure of the trisaccharide 17, the glycosylation was repeated using
NIS-TfOH as promoter. This change of the conditions resulted in a much
lower yield (26%), but allowed the full characterization of 17. After deacetyla-
tion leading to 18, the removal of the copper contamination was readily
achieved. Therefore, the cupric bromide-promoted glycosylation turned out to
be the method of choice, despite the inconvenience of the contamination with
copper salts. Final deprotection (i.e., removal of the benzyl and the carboben-
zoxy protecting groups by hydrogenolysis) followed by dansylation afforded flu-
orescent-labeled Lewisa trisaccharide 1.
CONCLUSION
With FucT III, an excellent specificity constant for type 1 substrate 3 (137%
with respect to the natural substrate lacto-N-biose) was obtained, leading to
1 in a high yield (see Sch. 1 Table 1). The enzyme, however, exhibits only a
weak affinity for the lactose derivative 4 (5% compared to the natural substrate
lacto-N-biose). However, the kinetic parameters proved to be sufficient for the
preparative synthesis of the Lewisx derivative 2. The specificity constant for 4
Table 1: Kinetic parameters of FucT III for the natural substrates lacto-N-
biose and NAc-lactosamine and the non-natural derivatives 3 and 4
kcat
Km
[mM]
kcat/Km
Substrate
[min21
]
[mM21†min21
]
Lacto-N-biose (type 1)
LacNAc (type 2)
3
4
6.09
0.82
8.92
7.06
182
691
194
33.46
1.19
45.97
1.58
4470

Fluorescent-labeled Milk Oligosaccharides
795
is comparable to the one for LacNAc, indicating that the N-acetyl group is not
contributing to the stabilization of the transition state formed during the con-
version of the enzyme-bound substrate to the product.
In conclusion, a comparison of the two approaches applied to the synthesis
of trisaccharide 1 indicates that overall the purely chemical and the chemo-
enzymatic approaches are equally efficient. However, the main differences
are as follows: in the chemical approach the fluorescent label is introduced in
the last step, whereas in the chemo-enzymatic approach the fluorescently
labeled acceptor is enzymatically fucosylated. The latter furnishes us with
the possibility of using a variety of GDP-fucose analogs[19] to form a library
of trisaccharides in one final step. Moreover, FucT III shows a remarkable sub-
strate tolerance for structurally modified type 1 disaccharides. Although the
reaction rates with type 2 or lactose derivatives are much lower, they are
still sufficient for preparative applications.
EXPERIMENTAL
Dry solvents and liquid reagents were distilled prior to use: dichloromethane and
pyridine were distilled from calcium hydride; 1,2-dichloroethane, DMF, and
˚
methanol were dried on 4A molecular sieves; all reaction vessels, after being
dried, were kept under argon. Organic solutions were dried over
anhydrous sodium sulfate, and the solvent was evaporated at reduced pressure
below 408C. All reactions were monitored by TLC on silica gel 60 F–254 plates
(Merck), spots being developed with 5% sulfuric acid in methanol/water (1: 1)
or with phosphomolybdate-based reagent. Silica gel Merck 60 (230–400 mesh)
was used for flash chromatography. Optical rotations were measured at rt with
a Perkin-Elmer 241 polarimeter. 1H NMR and 13C NMR spectra were recorded
on Bruker AC 300 and Varian Gemini 200 spectrometers in solution. Chemical
shifts are given in ppm (d), relative to SiMe4 as internal standard; J values are
given in Hz. IR spectra were measured by a Perkin-Elmer 1420 spectropho-
tometer (NaCl crystal windows). Mass spectrometry was performed on a
Thermo Quest Finningan LCQTM DECA spectrometer. Mass spectra were
recorded using negative and positive electrospray (ES) as indicated. Elemental
analyses were performed using the Carlo Erba elemental analyzer 1108.
3-(Benzyloxycarbonylamino)propyl 2-acetylamino-4,6-
O-benzylidene-2-deoxy-b-D-glucopyranoside (7)
Compound 6 (2.42 g, 4.50 mmol) was dissolved in anhydrous methanol
(10 mL) and 0.5M sodium methoxide (2.7 mL) was added. After 2 h the
reaction mixture was neutralized with IR 120 resin, Hþ form filtered, and con-
centrated. The crude material was dissolved in DMF (20 mL), benzaldehyde

S. Rabbani et al.
796
dimethylacetal (0.65 g, 4.28 mmol) and p-toluenesulfonic acid (0.01 g) were
added, the solution was distilled under vacuum at 608C until TLC (ethyl
acetate) revealed the disappearence of the starting material, and it was concen-
trated. The crude material was diluted with dichloromethane and then washed
with 5% NaHCO3 solution. The organic layer was dried and concentrated to
afford the title compound 7 (1.74 g, 81%) as a foam. [a]D 238.1 (C 0.9,
1
CHCl3); H NMR (CDCl3) d 1.40–1.95 (m, 2H, CH2), 2.10 (s, 3H, CH3CONH),
2.92–3.12 (m, 1H, CHaHbNH), 3.16–3.40 (m, 2H, H-2, CHaHbNH), 3.55–3.86
(m, 5H, H-4, H-5, H-6a, CH2O), 3.90–4.04 (dt, 1H, J2,3 ¼ J3,4 9.2 Hz, J3,OH
3.4 Hz, H-3), 4.16 (d, 1H, J1,2 8.0 Hz, H-1), 4.30 (dd, 1H, J6a,6b 10.3 Hz, J6b,5
4.9 Hz, H-6b), 4.90–5.20 (m, 3H, CH2Ph, NH), 5.57 (s, 1H, benzylidene),
7.25–7.60 (m, 10H, Ph), 7.70 (d, 1H, J 7.7 Hz, NHCOCH3); 13C NMR (CDCl3)
d 23.3 (s, CH3CONH), 30.9 (t, CH2), 37.0 (t, CH2NH), 59.6 (d, C-2), 66.6, 66.8,
67.8 (3t, C-6, CH2O, CH2Ph), 69.2, 74.8, 82.5 (3d, C-3, C-4, C-5,), 102.1,
102.63 (C-1, CHPh), 127.1–129.8 (10d, CHAr), 137.2, 137.9 (2s, CqAr), 158.0
(s, NHCO), 174.9 (s, CH3CO); IR (nujol): 3300, 1690 cm21; ESI/MS (positive
ion mode), m/e 501 [M þ 1]þ; Anal. Calcd for C26H32N2O8: C, 62.39; H, 6.44;
N, 5.60. Found: C, 62.61; H, 6.12; N, 5.33.
3-(Benzyloxycarbonylamino)propyl 2,3,4,6-tetra-O-acetyl-
b-D-galactopyranosyl-(1!3)-2-acetamido-4,6-
O-benzylidene-2-deoxy-b-D-glucopyranoside (9)
Compound 7 (1.00 g, 2.0 mmol) and donor 8 (1.48 g, 3.0 mmol) were dis-
solved in dichloromethane (50 mL) under inert atmosphere, then at 08C
.
BF3 OEt2 (1 mL, 0.1M solution in dichloromethane) was added. After
stirring for 3 h at rt, the solution was neutralized by addition of Et3N and con-
centrated. Flash chromatography of the residue (ethyl acetate) gave 9 (1.30 g,
78%) as a foam. [a]D 2 16.2 (C 0.5, CHCl3); 1H NMR (CDCl3) d 1.65–1.90
(m, 2H, CH2), 1.95–2.10 (5s, 15H, COCH3), 3.15–3.40 (m, 3H, CH2NH, H-2),
3.45–3.80 (m, 5H, H-4, H-5, H-6a, H-50, CHaHbO), 3.85–4.00 (m, 2H, H-60a,
CHaHbO), 4.10 (m, 1H, H-60b), 4.30 (dd, 1H, J6a,6b 10.3 Hz, J6b,5 4.6 Hz,
H-6b), 4.45 (t, 1H, J 9.0 Hz, H-3), 4.75 (d, 1H, J 8.0 Hz, H- 10), 4.90 (dd, 1H,
J3 ,4 10.3 Hz, J3 ,2 3.3 Hz, H-30), 5.05–5.25 (m, 5H, CH2Ph, NH, H-10, H-20),
5.30 (d, 1H, H-40), 5.50 (s, 1H, benzylidene), 6.10 (d, 1H, J 7.1 Hz,
NHCOCH3), 7.25–7.60 (m, 10H, Ph); 13C NMR (CDCl3) d 20.5, 20.5, 20.5,
20.5, 23.4 (5q, CH3CO), 29.5 (t, CH2), 37.9 (t, CH2NH), 57.6 (d, C-2), 60.9 (t),
66.0 (d), 66.6 (t), 66.8 (d), 67.4 (t), 68.6 (t), 69.4 (d), 70.5 (d), 71.0 (d), 77.2 (d),
80.5 (d), 100.0, 100.4, 101.4 (3d, C-1, C-10, CHPh), 126.0–129.1 (10d, CHAr),
136.6, 137.1 (2s, CqAr), 156.5 (s, NHCO), 169.4, 170.0, 170.0, 170.0, 170.8
(5s, CH3CO); IR (nujol): 3400, 1740, 1700, 1650; ESI/MS (positive ion mode),
m/e 831 [M þ 1]þ; Anal. Calcd for C40H50N2O17: C, 57.83; H, 6.07; N, 3.37.
Found C, 57.59; H, 5.80; N, 3.58.
0
0
0
0

Fluorescent-labeled Milk Oligosaccharides
797
3-(Benzyloxycarbonylamino)propyl (2,3,4,6-tetra-O-acetyl-
b-D-galactopyranosyl)-(1 ! 3)-2-acetamido-2-deoxy-b-
D-glucopyranoside (10)
To a stirred solution of compound 9 (0.090 g, 0.11 mmol) in dichloromethane
(1 mL) at 08C trifluoroacetic acid (1 mL, 70% aqueous solution) was added.
After stirring for 10 min at 08C, the solution was neutralized by addition of
satd. aqueous Na2CO3, extracted with dichloromethane (3 ꢀ 10 mL), and con-
centrated. Benzaldehyde was coevaporated with toluene. Flash chromato-
graphy (ethyl acetate) of the crude material afforded 10 (0.070 g, 83%) as a
1
foam. [a]D 20.2 (C 1, CHCl3); H NMR (CDCl3) d 1.68–1.75 (m, 2H, CH2),
1.97, 1.97, 2.05, 2.07, 2.14 (5s, 15H, COCH3), 3.15–3.60 (m, 6H, CH2NH,
H-3, H-30, H-5, CHaHbO), 3.65–3.75 (m, 2H, CHaHbO, OH), 3.80–3.95
(m, 3H, 2H-6, OH), 3.95–4.05 (t, 1H, H-50), 4.10–4.20 (m, 3H, 2H-60, H-2),
4.55 (d, 1H, J 7.9 Hz, H-10), 4.72 (d, 1H, J 8.3 Hz, H-1), 5.00 (dd, 1H, J2 ,3
0
0
10.4 Hz, J4 ,3 3.3 Hz, H-30), 5.05–5.20 (m, 3H, CH2Ph, NH), 5.23 (dd, 1H,
H-20), 5.35 (d, 1H, H-40), 6.25 (d, 1H, J2,NH7.7 Hz, NHCOCH3), 7.30–7.45
(m, 5H, Ph); 13C NMR (CDCl3) d 20.5, 20.5, 20.5, 20.7, 23.4 (5q, CH3CO),
29.7 (t, CH2), 37.6 (t, CH2NH), 56.5 (d, C-2), 61.5, 62.7 (2t, C-6, C-60), 66.8
(2t, CH2O, CH2Ph), 66.9 (d), 68.9 (d), 70.1 (d), 70.7 (d), 71.1 (d), 75.3 (d), 83.8
(d), 99.7, 101.4 (2d, C-1, C-10), 128.0, 128.2, 128.2, 128.6, 128.6 (5d, CHAr),
137.5 (s, CqAr), 156.5 (s, NHCO), 169.4, 170.0, 170.1, 170.5, 170.8 (5s,
CH3CO); IR (nujol): 3300, 1740, 1700 cm21; ESI/MS (positive ion mode), m/e
743 [M þ 1]þ; Anal. Calcd for C33H46N2O17: C, 53.37; H, 6.24; N, 3.77. Found:
C, 53.09; H, 6.51; N, 3.51.
0
0
3-(5-Dimethylamino-naphthalene-1-sulfonylamino)propyl
(2,3,4,6-tetra-O-acetyl-b-D-galactopyranosyl)-(1!3)-2-
acetamido-2-deoxy-b-D-glucopyranoside (11)
Compound 10 (0.106 g, 0.14 mmol) was dissolved in methanol (4 mL), and
10% Pd-C (0.100 g) and glacial acetic acid (five drops) were added. The
mixture was kept in a hydrogen atmosphere with vigorously stirring for 1 h.
The mixture was filtered over a Celite pad and concentrated. The crude
material was dissolved in 5 mL of dichloromethane/methanol (7 : 3), and
dansyl chloride (0.039 g, 0.14 mmol) and Et3 N (0.024 mL, 0.172 mmol) were
added. After stirring for 1 h at rt, the reaction was diluted with dichloro-
methane (20 mL) and washed with water (15 mL). The aqueous layer was sep-
arated and extracted with dichloromethane (3 ꢀ 15 mL), the combined organic
layers were dried and concentrated. The residue was purified by flash chrom-
atography (ethyl acetate) affording the title compound 11 (0.087 g, 72%) as a
1
yellow, amorphous solid. [a]D 21.75 (C 1, CH3OH); H NMR (CDCl3) d 1.60–
1.70 (m, 2H, CH2), 1.95, 1.98, 2.03, 2.06, 2.13 (5s, 15H, CH3CO), 2.87 (s, 6H,

S. Rabbani et al.
798
N(CH3)2), 2.98–3.05 (m, 2H, CH2NH), 3.30 (m, 1H, H-2), 3.40–3.46 (m, 2H, 2H-
6), 3.56–3.64 (m, 2H, CHaHbO), 3.71–3.77 (m, 1H, H-50), 3.83–3.95 (m, 4H, H-4,
CHaHbO, 2 H-60), 3.98–4.03 (m, 1H, H-50), 4.10–4.12 (m, 2H, 2H-60), 4.18
(m, 1H, H-3), 4.57 (d, 1H, J1,2 8.0 Hz, H-1), 4.78 (d, 1H, J1 ,2 8.1 Hz H-10), 5.00
0
0
(dd, 1H, J2 ,3 10.5 Hz, J4 ,3 3.5 Hz, H-30), 5.20 (dd, 1H, H-20), 5.35 (d, 1H,
H-40), 5.86 (t, 1H, J 5.9 Hz, NHSO2), 6.46 (d, 1H, J 7.0 Hz, NH), 7.17 (d, 1H, J
7.6 Hz, HAr), 7.48–7.57 (m, 2H, HAr), 8.17–8.29 (m, 2H, 2HAr), 8.53 (d, 1H, J
8.5 Hz, HAr); 13C NMR (CDCl3) d 20.5, 20.5, 20.5, 20.7, 23.6 (5q, CH3CO), 29.0
(t, CH2), 40.5 (t, CH2NH), 45.4 (2q, (CH3)2N), 56.6 (d, C-2), 61.4 (t), 62.7 (t),
67.0 (t), 67.2 (t), 68.8 (t), 70.0 (d), 70.8 (d), 71.0 (d), 75.6 (d), 83.4 (d), 99.8,
101.4 (2d, C-1, C-10), 115.3 (d, CH Dans), 118.7 (d, CH Dans), 123.2 (d, CH
Dans), 128.4 (d, CH Dans), 129.2 (d, CH Dans), 129.6 (s, Dans), 129.9
(s, Dans), 130.5 (d, CH Dans), 134.8 (s, CSO2), 152.0 (s, CN(CH3)2), 169.5,
170.0, 170.1, 170.5, 171.2 (5s, CH3CO); IR (nujol): 3250, 1750 cm21; ESI/MS
(positive ion mode), m/e 842 [M þ 1]þ; Anal. Calcd for C37H51N3O17S: C,
52.79; H, 6.11; N, 4.99. Found: C, 52.53; H, 6.35; N, 4.72.
0
0
0
0
3-(5-Dimethylamino-naphthalene-1-sulfonylamino)propyl
b-D-galactopyranosyl-(1!3)-2-acetamido-2-deoxy-
b-D-glucopyranoside (3)
To a stirred solution of compound 11 (0.087 g, 0.10 mmol) in methanol
(1 mL), 1M sodium methoxide in anhydrous methanol (0.155 mL) was added.
After stirring for 1 h at rt, the solution was neutralized by addition of IRC-86
resin, Hþ form; the resin was filtered off and washed with methanol and the
filtrate was concentrated. Flash chromatography of the residue (ethyl
acetate/methanol/water, 7 : 2 : 1) afforded 3 (0.040 g, 57 %) as a yellow solid.
[a]D 22.4 (C 0.25, CD3OD); 1H NMR (CD3OD) d 1.59–1.66 (m, 2H, CH2),
1.89 (s, 3H, NHCOCH3), 2.90 (s, 6H, N(CH3)2), 2.92–2.96 (m, 2H, CH2NH),
3.18–3.90 (m, 14H, CH2O, H-2, H-3, H-4, H-5, 2 H-6, H-20, H-30, H-40, H-50,
2H-60), 4.27 (d, 1H, J1,2 7.5 Hz, H-1), 4.39 (d, 1H, J1 ,2 8.0 Hz, H-10), 7.29
(d, 1H, J 7.0 Hz, HAr), 7.58–7.63 (m, 2H, HAr), 8.20 (dd, 1H, J 7.0 Hz, J
1.0 Hz, HAr), 8.37 (d, 1H, J 8.7 Hz, HAr), 8.58 (d, 1H, J 8.5 Hz, HAr); 13C NMR
(CD3OD) d 23.5 (q, CH3CONH), 31.2 (t, CH2), 41.4 (t, CH2NH), 46.1 (2q,
(CH3)2N)), 56.6 (d, C-2), 62.8, 63.1, 68.0 (3t, C-6, C-60, CH2O), 70.6 (d), 71.0
(d), 72.7 (d), 75.0 (d), 77.4 (d), 77.9 (d), 85.5 (d), 102.6, 105.9 (2d, C-1, C-10),
116.8 (d, CH Dans), 120.9 (d, CH Dans), 124.6 (d, CH Dans), 129.4 (d, CH
Dans), 130.3 (d, CH Dans), 131.3 (s, Dans), 131.4 (d, CH Dans), 131.5
(s, Dans), 131.4 (s, CSO2), 153.6 (s, CN(CH3)2), 174.5 (s, CH3CONH); IR
(nujol): 3300, 1630, 1570, 1320 cm21; ESI/MS (negative ion mode), m/e 672
[M-1]2; Anal. Calcd for C29H43N3O13S: C, 51.70; H, 6.43; N, 6.24. Found: C,
51.48; H, 6.62; N, 6.09.
0
0

Fluorescent-labeled Milk Oligosaccharides
799
3-(Benzyloxycarbonylamino)propyl (2,3,4,6-tetra-O-acetyl-
b-D-galactopyranosyl)-(1!3)-6-O-acetyl-2-acetamido-
2-deoxy-b-D-glucopyranoside (12)
To a stirred solution of compound 10 (0.460 g, 0.62 mmol) in dichloro-
methane (7.5 mL) at 2788C pyridine (0.250 mL, 3.10 mmol) and acetyl
chloride (0.140 mL, 1.86 mmol) were added. The solution was stirred at
2 788C for 10 min, then quenched by addition of methanol and concentrated.
The residue was purified by flash chromatography (ethyl acetate/methanol,
9 : 1), affording the title compound 12 (0.360 g, 74%) as a white, amorphous
1
solid. [a]D 21.3 (C 1, CHCl3); H NMR (CDCl3) d 1.62–1.82 (m, 2H, CH2),
1.95–2.14 (6s, 18H, CH3CO), 3.14–3.24 (m, 2H, CH2NH), 3.34–3.52
(m, 4H, H-3, H-4, H-5, CHaHbO), 3.82–3.92 (m, 2H, CHaHbO, OH), 4.00
(m, 1H, H-50), 4.09–4.18 (m, 3H, H-2, 2 H-60), 4.23 (d, 1H, J6a,6b 12.0 Hz,
J6a,5 5.0 Hz, H-6a), 4.40 (d, 1H, H-6b), 4.54 (d, 1H, J1 ,2 8.0 Hz, H-10), 4.69
0
0
(d, 1H, J1,2 8.5 Hz, H-1), 4.99 (dd, 1H, J3 ,2 11.0 Hz, J3 ,4 3.5 Hz, H-30),
5.02–5.12 (m, 3H, CH2Ph, NH), 5.21 (dd, 1H, H-20), 5.36 (d, 1H, H-40),
6.10 (d, 1H, NHCOCH3), 7.30–7.45 (m, 5H, Ph); 13C NMR (CDCl3) d 20.5–
23.4 (6q, CH3CO), 30.4 (t, CH2), 38.4 (t, CH2NH), 57.6 (d, C-2), 62.2, 63.9
(2t, C-6, C-60), 67.4, 67.6 (2t, CH2O, CH2Ph), 69.5 (2d), 69.9 (d), 71.5 (d),
71.9 (d), 74.2 (d), 84.4 (d), 100.0, 102.4 (2d, C-1, C-10), 128.7–129.3 (5d,
CHAr), 137.6 (s, CqAr), 157.6 (s, NHCO), 169.4–170.8 (6s, CH3CO); IR
(nujol): 3300, 1750, 1660 cm21; ESI/MS (positive ion mode), m/e 785
[M þ 1]þ; Anal. Calcd for C35H48N2O18: C, 53.57; H, 6.17; N, 3.57. Found:
C, 53.78; H, 6.29; N, 3.34.
0
0
0
0
3-(Benzyloxycarbonylamino)propyl (2,3,4,6-tetra-O-acetyl-
b-D-galactopyranosyl)-(1!4)-2,3,6-tri-O-acetyl-
b-D-glucopyranoside (14)
To a stirred solution of octa-O-acetyl-b-lactose 13 (0.548 g, 0.81 mmol)
and 3-benzyloxycarbonylamino-1-propanol (0.203 g, 0.97 mmol) in dichloro-
.
methane (11.5 mL) at 08C BF3 OEt2 (0.200 mL, 1.61 mmol) was added. The
solution was stirred at rt for 17 h, then Et3N was added until neutralization.
The solvent was removed under reduced pressure. Flash chromatography
(hexane/ethyl acetate, 4:6) afforded 14 (0.274 g, 41%) as a foam. [a]D –6.6
(C 1.2, CHCl3); 1H NMR ( CDCl3) d 1.75–1.77 (m, 2H, CH2), 1.95, 2.00,
2.03, 2.03, 2.04, 2.07, 2.13 (7s, 21H, CH3CO), 3.15–3.18 (m, 2H, CH2NH),
3.52–3.60 (m, 2H, CHaHbO, H-5), 3.76 (t, 1H, J3,4 ¼ J4,5 9.4 Hz, H-4),
3.80–3.90 (m, 2H, H-50, CHaHbO, 4.03–4.20 (m, 3H, H-6a, 2H-60), 4.40–
4.55 (m, 3H, H-1,0 H-10, H-6b), 0 4.87 (dd, 1H, J1,2 9.5 Hz, J2,3 8.0 Hz, H-2),
0
4.95 (dd, 1H, J2 ,3 10. 5 Hz, J3 ,4 3.5 Hz, H-30), 5.00–5.21 (m, 4H, CH2Ph,
0

S. Rabbani et al.
800
H-20, NH), 5.17 (t, 1H, J2,3 ¼ J3,4 9.5 Hz, H-3), 5.34 (d, 1H, H-40), 7.15–7.40
(m, 5H, Ph); 13C NMR (CDCl3) d 20.3–20.6 (7q, CH3CO), 29.5 (t, CH2),
38.1 (t, CH2NH), 60.8, 61.9 (2t, C-6, C-60), 66.4 (t), 66.7 (d), 67.3 (t), 69.1
(d), 69.7 (d), 70.6 (d), 70.9 (d), 71.6 (d), 72.7 (d), 76.1 (d), 100.3, 100.9 (2d,
C-1, C-10), 127.9, 127.9, 127.9 (3d, CHAr), 128.3, 128.3 (2d, CHAr), 136.7
(s, CqAr), 156.4 (s, NHCO), 168.9, 169.4, 169.6, 169.8, 169.9, 170.1, 170.2
(7s, CH3CO); IR (nujol): 3391, 1751, 1725 cm21; ESI/MS (positive ion
mode), m/e 845 [M þ 18]þ; Anal. Calcd for C37H49NO20: C, 53.69; H, 5.97;
N, 1.69. Found: C, 53.42; H, 5.81; N, 1.57.
3-(5-Dimethylamino-naphthalene-1-sulfonylamino)propyl
(2,3,4,6-tetra-O-acetyl-b-Dgalactopyranosyl)-(1!4)-
2,3,6-tri-O-acetyl-b-D-glucopyranoside (15)
Compound 14 (0.340 g, 0.41 mmol) was dissolved in methanol (4 mL), and
10% Pd-C (0.400 g) and glacial acetic acid (two drops) were added. The
mixture was kept in a hydrogen atmosphere with vigorous stirring for 1 h.
The mixture was filtered over a Celite pad and concentrated. The crude
material was dissolved in dichloromethane (5 mL), and dansyl chloride
(0.110 g, 0.41 mmol) and Et3 N (0.07 mL, 0.49 mmol) were added. After
stirring for 2 h at rt the reaction was quenched by addition of methanol, then
diluted with dichloromethane (20 mL) and washed with water (15 mL). After
phase separation the aqueous layer was extracted with dichloromethane
(3 ꢀ 15 mL), and the combined organic layers were dried and concentrated.
The residue was purified by flash chromatography (hexane/ethyl acetate,
1:1) affording 15 (0.220 g, 58%) as an amorphous solid. [a]D –15.8 (c 0.9,
CHCl3); 1H NMR (CDCl3) d 1.60–1.70 (m, 2H, CH2), 1.90–2.20 (7s, 21H,
CH3CO), 2.90 (s, 6H, 2 N(CH3)2), 2.92–3.05 (m, 2H, CH2NH), 3.45–3.60
(m, 2H, CHaHbO, H-5), 3.72–3.91 (m, 3H, CHaHbO, H-4, H-50), 4.04–4.15
(m, 3H, H-6a, 2H-60), 4.37 (d, 1H, J1,2 7.8 Hz, H-1), 4.50–4.57 (m, 2H, H-6b,
H-10), 4.80 (t, 1H, J1,2 ¼ J2,3 8.8 Hz, H-2), 4.97 (dd, 1H, J2 ,3 10.0 Hz, J3 ,4
0
0
0
0
3.7 Hz, H-30), 5.07–5.22 (m, 3H, H-20, H-3, NH), 5.35 (d, 1H, H-40), 7.19
(d, 1H, J 7.5 Hz, HAr), 7.48–7.62 (m, 2H, HAr), 8.23 (d, 1H, J 7.1 Hz, HAr),
8.31 (d, 1H, J 8.6 Hz, HAr), 8.54 (d, 1H, J 8.5 Hz, HAr); 13C NMR (CDCl3) d
20.4–20.7 (72, CH3CO), 29.4 (t, CH2), 40.4 (t, CH2NH), 45.4, 45.4 (2q,
(CH3)2N), 60.8, 61.8 (2t, C-6, C-60), 66.6 (d), 67.1 (t, CH2O), 69.1 (d), 70.7 (d),
71.0 (d), 71.6 (d), 72.8 (d), 76.1 (d), 77.0 (d), 100.3, 101.0 (2d, C-1, C-10), 115.1
(d, CH Dans), 118.9 (d, CH Dans), 123.2 (d, CH Dans), 128.2 (d, CH Dans),
129.4 (d, CH Dans), 129.9 (2s, Dans), 130.3 (d, CH Dans), 135.0 (s, CSO2),
152.0 (s, CN(CH3)2), 169.0–170.5 (7s, CH3CO); IR (nujol): 3370, 1750, 1650,
1375 cm21; ESI/MS (positive ion mode), m/e 927 [M þ 1]þ; Anal. Calcd for
C41H54N2O20S: C, 53.13; H, 5.87; N, 3.02. Found: C, 52.88; H, 6.13; N, 3.21.

Fluorescent-labeled Milk Oligosaccharides
801
3-(5-Dimethylamino-naphthalene-1-sulfonylamino)propyl
b-D-galactopyranosyl-(1!4)- b-D-glucopyranoside (4)
Compound 15 (0.086 g, 0.09 mmol) was dissolved in methanol (2 mL), and
1M sodium methoxide in anhydrous methanol (0.220 mL) was added. After
stirring for 2 h at rt, the solution was neutralized by addition of IRC-86 resin,
Hþ form; the resin was filtered off and the filtrate was concentrated. The
residue was purified by flash chromatography (ethyl acetate/methanol/
water, 7 : 2 : 1), affording 4 (0.045 g, 77%) as a yellow foam. [a]D –2.0 (C 1.4,
1
CH3OH); H NMR (CD3OD) d 1.65 (m, 2H, CH2), 2.87 (s, 6H, N(CH3)2), 2.99
(t, 2H, J 6.6 Hz, CH2NH), 3.14–3.83 (m, 14H, CH2O, H-2, H-3, H-4, H-5, 2
H-6, H-20, H-30, H-40, H-50, 2H-60), 4.14 (d, 1H, J1,2 7.8 Hz, H-1), 4.35 (d, 1H,
0
J1 ,2 7.0 Hz, H-10), 7.25 (d, 1H, J 7.5 Hz, HAr), 7.51–7.62 (m, 2H, HAr), 8.18
0
(d, 1H, J 6.8 Hz, HAr), 8.35 (d, 1H, J 8.6 Hz, HAr), 8.54 (d, 1H, J 8.5 Hz, HAr);
13C NMR (CD3OD) d 30.9 (t, CH2), 41.1 (t, CH2NH), 45.9, 45.9 (2q, (CH3)2 N),
62.0, 62.5 (2t, C-6, C-60), 68.0 (t, CH2O), 70.3 (d), 72.6 (d), 74.7 (d), 74.9 (d),
76.4 (d), 76.4 (d), 77.1 (d), 80.8 (d), 104.1, 105.2 (2d, C-1, C-10), 116.5 (d, CH
Dans), 120.6 (d, CH Dans), 124.3 (d, CH Dans), 129.1 (d, CH Dans), 130.2
(d, CH Dans), 131.2 (d, CH Dans), 131.3 (2s, Dans), 137.1 (s, CSO2), 153.3
(s, CN(CH3)2); IR (nujol): 3320, 1310; ESI/MS (positive ion mode), m/e 633
[M þ 1]þ; Anal. Calcd for C27H40N2O13S: C, 51.26; H, 6.37; N, 4.43. Found: C,
51.49; H, 6.51; N, 4.18.
3-(Benzyloxycarbonylamino)propyl (2,3,4,6-tetra-O-acetyl-
b-D-galactopyranosyl)- (1!3)-[2,3,4-tri-O-benzyl-a-L-
fucopyranosyl-(1!4)]-6-O-acetyl-2-acetamido-2-deoxy-
b-D-glucopyranoside (17)
Compound 12 (0.176 g, 0.22 mmol) and fucosyl donor 16 (0.312 g,
0.67 mmol) were dissolved in dichloromethane (2 mL) and DMF (2 mL) under
inert atmosphere; this solution was transferred into a flask containing
˚
powdered molecular sieves 3 A (2.00 g), Bu4NBr (0.361 mg, 1.12 mmol), and
CuBr2 (0.223 mg, 1.00 mmol). The mixture was stirred at rt for 2 h, then
filtered over a Celite pad and washed with NaHCO3 saturated aqueous
solution and brine; after phase separation the aqueous layer was extracted
with dichloromethane and the combined organic layers were dried and concen-
trated. The residue was purified by flash chromatography (dichloromethane/
methanol, 20 : 1) affording the title compound 17 in almost quantitative yield
1
(960%0 )00 as a foam. [a]D –10.0 (C 1, CHCl3); H NMR (CDCl3) d 1.23 (d, 3H,
J
,
6.8 Hz, H-600), 1.60–1.80 (m, 2H, CH2), 1.91–2.05 (6s, 18H, CH3CO),
5
6
3.10–3.45 (m, 3H, CH2NH, CHaHbO), 3.60–4.20 (m, 12H, H-2, H-3, H-4,
H-5, CHaHbO, 2 H-60, H-50, H-200, H-300, H-400, H-500), 4.35–4.52 (m, 3H, 2 H-6,
H-100), 4.65–5.20 (m, 13H, H-1, H-10, 4 CH2Ph, H-30, H-20, NH), 5.33 (d, 1H,

S. Rabbani et al.
802
J 40,30 3.5 Hz, H-40), 6.20 (d, 1H, NHCOCH3), 7.20–7.50 (m, 20H, Ph); 13C NMR
(CDCl3) d 14.0 (d, C-600), 20.5–24.6 (6q, CH3CO), 29.7 (t, CH2), 37.7 (t, CH2NH),
53.5 (d, C-2), 59.7, 60.5, 62.7 (3t, C-6, C-60, CH2O), 66.1 (t, CH2Ph), 66.5
(t, CH2Ph), 66.8 (d), 67.0 (d), 68.4 (d), 70.6 (d), 71.2 (2d), 71.9 (d), 72.6
(t, CH2Ph), 74.4 (t, CH2Ph), 75.5 (d), 76.8 (d), 77.0 (d), 80.3 (d), 96.1 (d, C-100),
100.3 (2d, C-1, C-10), 127.1–128.7 (20d, CHAr), 136.7, 137.9, 138.5, 138.7 (4s,
CqAr), 156.8 (s, NHCO), 169.9–170.6 (6s, CH3CO); IR (nujol): 3450,
1650 cm21. ESI/MS (positive ion mode), m/e 1218 [M þ 18]þ; Anal. Calcd for
C62H76N2O22: C, 61.99; H, 6.38; N, 2.33. Found: C, 62.21; H, 6.11; N, 2.12.
3-(Benzyloxycarbonylamino)propyl b-D-galactopyranosyl-
(1!3)-[2,3,4-tri-O-benzyl-a-L-fucopyranosyl-(1!4)]-2-
acetamido-2-deoxy-b-D-glucopyranoside (18)
Compound 17 (0.096 g, 0.08 mmol) was dissolved in methanol (1 mL), and
1M sodium methoxide in anhydrous methanol (0.01 mL) was added. After
stirring for 1 h at rt, the solution was neutralized by addition of IRC-86 resin,
Hþ form; the resin was filtered off and the filtrate was concentrated. The
residue was purified by flash chromatography (ethyl acetate/methanol, 8:2)
affording 18 in quantitative yield as a foam. [a]D –94.0 (C 1, CH3OH); 1H
NMR (CD3OD) d 1.20 (d, 3H, J6 ,5 6.8 Hz, C-600), 1.50–1.66 (m, 2H, CH2), 1.83
00 00
(s, 3H, CH3CONH), 2.90–3.05 (m, 2H, CH2NH), 3.10–3.90 (m, 18H, H-2, H-3,
H-4, H-5, 2 H-6, H-20, H-30, H-40, H-50, 2 H-60, H-200, H-300, H-400, H-500, CH2O),
4.12 (d, 1H, J1,2 7.5 Hz, H-1), 4.17 (d, 1H, J1 ,2 7.0 Hz, H-10), 4.34–4.67
0
0
(m, 6H, 3 CH2Ph), 4.76–4.82 (m, 2H, CH2Ph), 4.88 (d, 1H, J1 ,2 3.5 Hz, H-100),
00 00
6.80–7.15 (m, 20H, Ph). 13C NMR (CDCl3)
d
14.0 (q, C-600), 24.8
(q, CH3CONH), 30.9 (t, CH2), 38.7 (t, CH2NH), 57.6 (d, C-2), 59.6, 61.3, 63.3,
67.4, 68.0, 68.2, 68.5 (7t, 4 CH2Ph, C-6, C-60, CH2O), 72.4 (d), 73.4 (d), 73.9
(d), 74.8 (d), 75.8 (d), 76.4 (d), 77.0 (d), 77.4 (d), 78.3 (d), 79.8 (d), 80.9 (d), 98.6
(d, C-100), 102.4, 104.9 (2d, C-1, C-1’), 128.5–130.0 (20d, CHAr), 139.5–140.4
(4s, CqAr), 158.9 (s, NHCO), 174.8 (s, CH3CONH); IR (nujol): 3300, 1750,
1660 cm21; ESI/MS (positive ion mode), m/e 991 [M þ 1]þ; Anal. Calcd for
C52H66N2O18: C, 63.02; H, 6.71; N, 2.83. Found: C, 63.28; H, 6.49; N, 3.04.
3-(5-Dimethylamino-naphthalene-1-sulfonylamino)propyl
b-D-galactopyranosyl-(1!3)- [a-L-fucopyranosyl-
(1 ! 4)]-2-acetamido-2-deoxy-b-D-glucopyranoside (1)
Compound 18 (0.080 g, 0.08 mmol) was dissolved in methanol (0.8 mL), and
10% Pd(OH)2-C (0.080 g) and glacial acetic acid (two drops) were added. The
mixture was kept in a hydrogen atmosphere with vigorous stirring for 96 h.
The mixture was filtered over a Celite pad and concentrated. The residue
was dissolved in methanol (2 mL), and dansyl chloride (0.024 g, 0.09 mmol)

Fluorescent-labeled Milk Oligosaccharides
803
and Et3 N (0.013 mL, 0.097 mmol) were added. After stirring for 56 h at rt, the
solvent was removed. The crude product was purified by flash chromatography
(ethyl acetate/methanol/water, 8 : 2.5 : 1) affording the title compound 1
1
(0.036 g, 52%) as an amorphous yellow solid. [a]D –57.7 (C 0.5, CH3OH); H
NMR (CD3OD) d 1.19 (d, 3H, J6 ,5 6.5 Hz, C-600), 1.58–1.65 (m, 2H, CH2),
00 00
1.85 (s, 3H, CH3CONH), 2.89 (s, 6H, N(CH3)2), 2.92 (t, 2H, J 7.0 Hz,
CH2NH), 3.32–3.97 (m, 18H, CH2O, H-2, H-3, H-4, H-5, 2 H-6, H-20, H-30,
H-40, H-50, 2H-60, H-200, H-300, H-400, H-500), 4.36–4.37 (2d, 2H, J 7.8 Hz and J
8.0 Hz, H-1 and H-10), 5.02 (d, 1H, J 4.0 Hz, H-100), 7.28 (d, 1H, J 7.5 Hz, HAr),
7.56–7.63 (m, 2H, HAr), 8.19 (d, 1H, J 7.5 Hz, HAr), 8.35 (d, 1H, J 8.5 Hz,
13
H
Ar); 8.55 (d, 1H, J 8.5 Hz, HAr); C NMR (CD3OD) d 16.6 (q, C-600), 23.2
(q, CH3CONH), 30.9 (t, CH2), 41.1 (t, CH2NH), 45.8 (q, 2C, (CH3)2 N), 57.2
(d, C-2), 61.3, 62.8, 67.6 (3t, C-6, C-60, CH2O), 69.8 (d), 70.0 (d), 71.1 (d), 72.3
(d), 73.6 (d),73.7 (d), 74.8 (d), 76.7 (d), 76.7 (d), 77.4 (d), 78.5 (d), 99.6 (d, C-
100), 102.3 and 104.9 (2d, C-1, C-10), 116.4 (d, CH Dans), 120.8 (d, CH Dans),
124.4 (d, CH Dans), 129.2 (d, CH Dans), 130.1 (d, CH Dans), 130.9 (d, CH
Dans), 131.1 (s, Dans), 131.2 (s, Dans), 137.0 (s, CSO2), 153.2 (s, CN(CH3)2),
174.1 (s, CH3CONH); IR (nujol): 3400, 1690 cm21; ESI/MS (positive ion
mode), m/e 820 [M þ 1]þ; Anal. Calcd for C35H53N3O17S: C, 51.27; H, 6.52; N,
5.13. Found: C, 51.02; H, 6.79; N, 4. 79.
Cloning, Expression, and Purification of Fucosyltransferase
III (EC 2.4.1.65)
FucT III (EMBL accession no. X53578[14]) was expressed in Sf9 insect cells
using the baculovirus expression system. FucT III cDNA lacking in the first 35
amino acids of the coding region was fused to the untranslated
g-interferon secreting sequence and to the IgG binding domain of protein A.
The construct was then cloned into baculovirus transfer vector pVL1392 (Phar-
Mingen). After transfection of Sf9 cells in monolayer, recombinant virus was
produced, and the titer was amplified to 1.109 pfu/mL. A confluent monolayer
culture of Sf9 insect cells (1 ꢀ 106 cells/mL) in SF900II medium supplemented
with 5% fetal calf serum, 100 U/mL penicillin, and 0.1 mg/mL streptomycin
was infected with recombinant baculovirus at the m.o.i of 5 and incubated at
278C. The supernatant containing the secreted enzyme was collected 48 h post-
infection, centrifuged, and filtered through a Millipore membrane (0.2 mm).
The filtrate was adjusted to pH 7.6 with 2.5 M Tris-HCl solution and loaded
onto an IgG-Sepharose-6 column (Amersham Biosciences) attached to an
FPLC system (BioRad). Two washing steps with 10 bed volumes of buffer con-
taining 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, and 0.05% Tween 20 followed
by two bed volumes of 5 mM NH4OAc (pH 5.0) were performed. The enzyme
was eluted with 10 mL of 500 mM NH4OAc (pH 3.4). The collected fractions
were immediately neutralized with 2.5 M Tris-HCl solution and concentrated

S. Rabbani et al.
804
by ultrafiltration (Amicon, YM 30) to a 50 mL volume. The protein concen-
tration (325.5 mm/mL) was determined by Bradford assay using bovin serum
albumin as standard. The enzyme activity (10.75 U/mg) was measured by
the FucT III assay.
FucT III Assay
The assay format published by Palcic et al.[20] was slightly modified. The assay,
run in duplicates, was conducted in a total volume of 30 mL containing 50 mM
cacodylate (pH 6.5), 20 mM MnCl2, 0.150 mM GDP-fucose, 55 000 cpm GDP-
[14C]fucose (287 mCi/mmol, Amersham Pharmacia), 3 mg BSA (Sigma), 0.01 to
4 mM acceptor substrate, and 3 mU of FucT III. After 30 min of incubation at
378C, the reaction was stopped by addition of 1 mL water and loaded onto Sep-
Pak-C18 cartridges (Waters). The cartridges were washed twice with 5 mL bi
distilled water and eluted with 5 mL methanol. The transfer rate of [14C]fucose
onto the acceptor substrate was determined by liquid scintillation counting.
Determination of Kinetic Parameters for the Substrates
Lacto-N-biose, LacNAc, 3 and 4
The kinetic parameters were determined using 0.150 mM of GDP-fucose
and concentrations of the acceptor substrates ranging from 0.01 to 4 mM.
The reaction mixtures were prepared as described above. Under the above
conditions the formation of products was shown to be linear in time. Kinetic
constants were obtained from double-reciprocal plots by linear regression
analysis of Lineweaver-Burk plots.
Enzymatic Synthesis of 3-(5-Dimethylamino-naphthalene-1-
sulfonylamino)propyl b-D-Galactopyranosyl-(1!3)-[a-L-
fucopyranosyl-(1!4)]-2-acetamido-2-deoxy-b-D-
glucopyranoside (1)
The reaction was conducted in 500 mL total volume of bidistilled water con-
taining Na-cacodylate buffer (150 mL, 250 mM, pH 6.5), MnCl2-solution (50 mL,
250 mM), disaccharide 3 (2.3 mg, 3.4 mmol), GDP-fucose (4.1 mg, 6.9 mmol), and
bovine serum albumin (0.7 mg, Fluka). The mixture was vortexed and incu-
bated overnight at 378C with fucosyltransferase III (150 mL, 500 mU) and
calf intestine alkaline phosphatase (2 ml, 2U, Roche). After a TLC (CH2Cl2/
methanol/water, 10 : 4 : 0.8) control indicated the total consumption of 3, the
turbid solution was centrifuged and the supernatant passed over RP-18
column (Sep-Pack-C18 cartridge, Waters). The column was washed with
water and eluted with methanol. After evaporation of the solvent, the
residue was chromatographed on RP-18 (H2O/MeOH gradient 1 : 0 to 1 : 1) to

Fluorescent-labeled Milk Oligosaccharides
805
yield, after a final lyophilization from water, trisaccharide 1 (1.5 mg, 88%) as
colorless powder with identical spectroscopic properties as 1 obtained by
chemical means.
Enzymatic Synthesis of 3-(5-Dimethylamino-naphthalene-1-
sulfonylamino)propyl b-D-Galactopyranosyl-(1!3)-[a-L-
fucopyranosyl-(1!4)]-b-D-glucopyranoside (2)
Compound 2 was synthesized according to the procedure described for 1,
except that the reaction time was increased to 48 h. Starting from disaccharide
4 (2.3 mg, 3.6 mmol) trisaccharide 2 (2.5 mg, 54%) was obtained. [a]D –46.61 (C
0.14, CH3OH); 1H NMR (CD3OD) d 1.18 (d, 3H, J 6
,
00 00
6.5 Hz, C-600), 1.63–1.66
5
(m, 2H, CH2), 2.89 (s, 6H, N(CH3)2), 2.94–3.00 (m, 2H, CH2NH), 3.38–3.92
(m, 18H, CH2O, H-2, H-3, H-4, H-5, 2 H-6, H-20, H-30, H-40, H-50, 2H-60;, H-200,
H-300, H-400, H-500), 4.12 (d, 1H, J 7.8 Hz, H-10), 4.39 (d, 1H, J 7.2 Hz, H-1), 5.43
(d, 1H, J 3.9 Hz, H-100), 7.28 (d, 1H, J 7.5 Hz, HAr), 7.56–7.61 (m, 2H, HAr),
8.19 (d, 1H, J 7.3 Hz, HAr), 8.35 (d, 1H, J 8.7 Hz, HAr), 8.56 (d, 1H, J 8.5 Hz,
13
H
Ar); C NMR (CD3OD) d 17.0 (q, C-600), 31.2 (t, CH2), 41.4 (t, CH2NH), 46.2
(2q, (CH3)2 N), 61.3, 63.2, 67.8 (3t, C-6, C-60, CH2O), 68.3 (d), 70.4 (d), 70.8
(d), 71.6 (d), 73.3 (d), 74.2 (d), 74.9 (d), 75.3 (d), 76.8 (d), 77.1 (d), 77.6 (d),
79.5 (d), 100.9, 104.2, 104.6 (3C, C-1, C-10, C-100), 116.8 (d, CH Dans), 121.0
(d, CH Dans), 124.8 (d, CH Dans), 129.5 (d, CH Dans), 130.0 (d, CH Dans),
130.6 (d, CH Dans), 131.4 (s, Dans), 131.5 (s, Dans), 137.6 (s, CSO2), 153.6
(s, CN(CH3)2); ESI/MS (positive ion mode), m/e 779 [M þ 1]þ.
ACKNOWLEDGMENT
Financial support was generously provided by the Swiss National Science
Foundation. In addition, we thank YAMASA Corp., Tokyo, Japan, for the
generous gift of GDP-fucose.
REFERENCES
[1] (a) Kurz, C.; Rudloff, S.; Baier, W.; Klein, N.; Strobel, S. Oligosaccharides in human
milk: structural, functional, and metabolic aspects. Annu. Rev. Nutr. 2000, 20,
699–722; (b) Newburg, D.S.; Neubaur, S.H. Handbook of Milk Composition.
Academic: San Diego, CA, 1995, 273–349; (c) Stahl, B.; Thurl, S.; Zeng, J.;
Karas, M.; Hillenkamp, F.; Steup, M.; Sawatzski, G. Oligosaccharides from
human milk as revealed by matrix-assisted laser desorption/ionization mass
spectrometry. Anal. Biochem. 1994, 223, 218–226.
[2] (a) Newburg, D.S. Human milk glycoconjugates that inhibit pathogens. Curr. Med.
Chem. 1999, 6, 117–127; (b) Ofek, I.; Beachey, E.H. Bacterial adherence. Adv.
Intern. Med. 1980, 25, 503–523; (c) Boedecker, E.C. Enterocyte adherence of
Escherichia coli: its relation to diarrheal disease. Gastroentereology 1982, 83,
489–492; (d) Gothefors, L.; Olling, S.; Winberg, J. Breast feeding and biological

S. Rabbani et al.
properties of faecal. E coli strains. Acta Pediatr. Scand. 1975, 64, 807–812;
806
(e) Andersson, B.; Porras, O.; Hansan, L.A.; Lagergard, T.; Svanborg-Eden, C. Inhi-
bition of attachment of Streptococcus pneumoniae and Haemophilus influenzae by
human milk and receptor oligosaccharides. J. Infect. Dis. 1986, 153, 232–237;
(f) Holmgren, J.; Svennerholm, A.M.; Lindblad, M. Receptor-like glycocompounds
in human milk that inhibit classical and El Tor Vibrio cholerae cell adherence
(hemagglutination). Infect Immun. 1983, 39, 147–154; (g) Crane, J.K.;
Azar, S.S.; Stam, A.; Newburg, D.S. Oligosaccharides from human milk block
binding and activity of the Escherichia coli heat-stable enterotoxin (STa) in the
T84 intestinal cells. J. Nutr. 1994, 124, 2358–2364.
[3] La Ferla, B.; Prosperi, D.; Lay, L.; Russo, G.; Panza, L. Synthesis of building blocks
of human milk oligosaccharides. Fucosylated derivatives of the lacto- and neolacto-
series. Carbohydr. Res. 2002, 337, 1333–1342, and references cited therein.
[4] (a) Ernst, B.; Wagner, B.; Baisch, G.; Katopodis, A.; Winkler, T.; Oehrlein, R.
Substrate specificity of fucosyltransferase III: an efficient synthesis of sialyl
Lewisv-, sialyl Lewisa-derivatives and mimetics thereof. Can. J. Chem. 2000, 78,
892–904; (b) Ernst, B.; Oehrlein, R. Substrate and donor specificity of glycosyl-
tansferases. Glycoconj. J. 1999, 16, 161–170, and references cited therein;
(c) Hindsgaul, O.; Palcic, M.M. Glycosyltransferases in the synthesis of oligosac-
charide analogs. Trends Glycosci. Glycotech. 1996, 8, 37–49; (d) Ichikawa, Y.;
Look, G.C.; Wong, C-H. Enzyme-catalyzed oligosaccharide synthesis. Anal.
´
Biochem. 1992, 202, 215–221; (e) David, S.; Auge, C.; Gautheron, C. Enzymatic
methods in preparative carbohydrate chemistry. Adv. Carbohydr. Chem.
Biochem. 1991, 49, 175–189; (f) Drueckhammer, D.G.; Hennen, W.J.;
Pederson, R.L.; Barbas, C.F.; Gautheron, C.M.; Krach, T.; Wong, C-H. Enzyme
catalysis in synthetic carbohydrate chemistry. Synthesis 1991, 7, 499–525;
(g) Toone, E.J.; Simon, E.S.; Bednarski, M.D.; Whitesides, G.M. Enzyme-catalyzed
synthesis of carbohydrates. Tetrahedron 1989, 45, 5365–5422.
[5] Mechref, Y.; Novotny, M.V. Structural investigations of glycoconjugates at high
sensitivity. Chem. Rev. 2002, 102, 321–369.
[6] Shibaev, V.N.; Veselovsky, V.V.; Lozanova, A.V.; Maltsev, S.D.; Danilov, L.L.;
Forsee, W.T.; Xing, J.; Cheung, H.C.; Jedrzejas, M.J. Synthesis of dolichyl phos-
phate derivatives with fluorescent label at the omega-end of the chain, new tools
to study protein glycosylation. Bioorg. Med. Chem. Lett. 2000, 10, 189–192.
[7] Gege, C.; Oscarson, S.; Schmidt, R.R. Synthesis of fluorescent-labeled sialyl Lewisx
glycosphingolipids. Tetrahedron Lett. 2001, 42, 377–380.
[8] Fujimoto, K.; Miyata, T.; Aoyama, Y. Saccharide-directed cell recognition and
molecular delivery using macrocyclic saccharide clusters: masking of hydrophobi-
city to enhance the saccharide specificity. J. Am. Chem. Soc. 2000, 122, 3558–3559.
[9] Sasaki, K.; Kurata, K.; Funayama, K.; Nagata, M.; Watanabe, E.; Ohta, S.;
Hanai, N.; Nishi, T. Expression cloning of a novel alpha 1,3- fucosyltransferase
that is involved in biosynthesis of the sialyl Lewisx carbohydrate determinants in
leukocytes. J. Biol. Chem. 1994, 269, 14730–14737.
[10] Kamst, E.; Zegelaar-Jaarsveld, K.; van der Marel, G.A.; van Boom, J.H.;
Lugtenberg, B.J.J.; Spaink, H.P. Chemical synthesis of N-acetylglucosamine
derivatives and their use as glycosylacceptors by the Mesorhizobium loti chitin
oligosaccharide synthase Nod. Carbohydr. Res. 1999, 321, 176–189.
[11] Evans, M.E. Cyclic acetals. In Methods in Carbohydrate Chemistry; Whistler, R.L.,
BeMiller, J.N., Eds.; Academic Press: New York, 1980; Vol. 8, 313–315.

Fluorescent-labeled Milk Oligosaccharides
807
[12] Schmidt, R.R.; Stumpp, M. Synthese von 1-Thioglycosiden. Liebigs Ann. Chem.
1983, 1249–1256.
[13] Palcic, M.M.; Sujino, K. Assays for glycosyltransferases. Trends Glycosci.
Glycotech. 2001, 13, 361–370.
[14] (a) Kukowska-Latallo, J.F.; Larsen, R.D.; Nair, R.P.; Lowe, J.B. A cloned human
cDNA determines expression of a mouse stage-specific embryonic antigen and
the Lewis blood group alpha (1,3/4)-fucosyltransferase. Genes & Dev. 1990, 4,
1288–1303; (b) De Vries, T.; Srnka, C.A.; Palcic, M.M.; Swiedler, S.J.; van den
Eijnden, D.H.; Macher, B.A. Acceptor specificity of different length constructs of
human recombinant a(1,3/4)-fucosyltransferases. J. Biol. Chem. 1995, 270,
8712–8722; (c) Costa, J.; Nimtz, M.; Grabenhorst, E.; Conradt, H.S. Stable
expression of the Golgi form and secretory variants of human fucosyltransferase
III from BHK-21 cells. Purification and characterization of an engineered trun-
cated form from the culture medium. J. Biol. Chem. 1997, 272, 11613–11621;
(d) Morais, V.A.; Serpa, J.; Palma, A.S.; Costa, T.; Maranga, L.; Costa, J. Expression
and characterization of recombinant human alpha (1,3/4)-fucosyltransferase III
from Spodoptera frugiperda (Sf9) and Trichoplusia ni (Tn) cells using the baculo-
virus expression system. Biochem J. 2001, 353, 719–725.
[15] Seto, N.O.L.; Palcic, M.M.; Compston, C.A.; Li, H.; Bundle, D.R.; Naranga, S.A.
Sequential interchance of four amino acids from blood group B to blood group A
glycosyltransferase boosts catalytic activity and progressively modifies sub-
strate recognition in human recombinant enzymes. J. Biol. Chem. 1997, 272,
14133–14138.
[16] Palcic, M.M.; Hindsgaul, O. Flexibility in the donor substrate specificity of
b1,4-galactosyltransferase: application in the synthesis of complex carbohydrates.
Glycobiology 1991, 1, 205–209.
[17] Sato, S.; Ito, T.; Nukuda, Y.; Ogawa, T. Synthetic studies on cell-surface glycans. 49.
Total synthesis of X hapten, III3 Fuca-nLc4 Cer. Carbohydr. Res. 1987, 167,
197–210.
[18] Sato, S.; Mori, M.; Ito, Y.; Ogawa, T. An efficient approach to O-glycosides through
CuBr2-Bu4NBr mediated activation of glycosides. Carbohydr. Res. 1986, 155,
C6–C10.
[19] Baisch, G.; Oehrlein, R. Convenient chemoenzymatic synthesis of b-purine-
diphosphate sugars (GDP-fucose-analogues). Bioorg. Med. Chem. 1997, 5,
383–391.
[20] Palcic, M.M.; Heerze, I.D.; Pierce, M.; Hindsgaul, O. The use of hydrophobic
synthetic glycosides as acceptors in glycosyltransferase assays. Glycoconj.
J. 1988, 5, 49–63.