Chemical synthesis of N-acetylglucosamine derivatives and their use as glycosyl acceptors by the Mesorhizobium loti chitin oligosaccharide synthase NodC

Article abstract of DOI:10.1016/S0008-6215(99)00190-1

Rhizobial bacteria synthesize lipo-chitin oligosaccharide signal molecules (Nod factors) that are essential for the formation of symbiotic organs on the roots of host plants, a process known as nodulation. Biosynthesis of the chitin oligosaccharide moiety

Full text of DOI:10.1016/S0008-6215(99)00190-1

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Carbohydrate Research 321 (1999) 176–189
Chemical synthesis of N-acetylglucosamine derivatives
and their use as glycosyl acceptors by the
Mesorhizobium loti chitin oligosaccharide synthase NodC
Eric Kamst a, Korien Zegelaar-Jaarsveld b, Gijs A. van der Marel b,
Jacques H. van Boom b, Ben J.J. Lugtenberg a, Herman P. Spaink a,*
a Leiden Uni6ersity, Institute of Molecular Plant Sciences, Clusius Laboratory, Wassenaarseweg 64,
NL-2333 AL Leiden, The Netherlands
b Leiden Uni6ersity, Leiden Institute of Chemistry, Gorlaeus Laboratories, PO Box 9502,
NL-2300 RA Leiden, The Netherlands
Received 10 May 1999; accepted 23 July 1999
Abstract
Rhizobial bacteria synthesize lipo-chitin oligosaccharide signal molecules (Nod factors) that are essential for the
formation of symbiotic organs on the roots of host plants, a process known as nodulation. Biosynthesis of the chitin
oligosaccharide moiety in Nod factors is carried out by the rhizobial N-acetylglucosaminyltransferase NodC. The
initial acceptor or primer used for the synthesis of chitin oligosaccharides in vivo is unknown. To investigate the
acceptor specificity of NodC, we have synthesized derivatives of N-acetylglucosamine (GlcNAc) with different
aglycones and tested whether they are acceptors for NodC in vitro using a membrane preparation of an Escherichia
coli strain expressing the Mesorhizobium loti chitin oligosaccharide synthase NodC. Analysis of reaction products
using thin-layer chromatography shows that GlcNAc derivatives containing simple alkyl chains or other hydrophobic
groups linked to C-1 are acceptors for NodC. The enzyme appears to be specific for acceptors in which the aglycone
is b-linked. GlcNAc derivatives in which the methyl group of the N-acetyl moiety of GlcNAc is replaced by an
allyloxy or benzyloxy group are still used as acceptors by NodC. The original methyl group at this position therefore
does not appear to be essential for the interaction between NodC and GlcNAc. A NodC-dependent reaction product
that is more hydrophobic than GlcNAc was detected in reaction mixtures containing 5% methanol but lacking an
exogenously added acceptor. This may be due to the presence of a natural hydrophobic glycosyl acceptor for NodC
in the membranes of E. coli, but the structure of this reaction product remains to be investigated. © 1999 Elsevier
Science Ltd. All rights reserved.
Keywords: Nodulation; Polysaccharides; N-Acetylglucosamine; Glycosyl acceptors
rhizobium, Azorhizobium and Allorhizobium
(collectively called rhizobia) are able to estab-
1. Introduction
lish a symbiotic relationship with leguminous
plants such as soybean, pea, alfalfa and clover
[1–3]. This process is called nodulation and
involves the induction of a new, nodular or-
gan on the roots of the host plant in which the
bacteria convert atmospheric nitrogen into
Soil bacteria belonging to the genera Rhizo-
bium, Mesorhizobium, Sinorhizobium, Brady-
* Corresponding author. Tel.: +31-71-527-5055/5065; fax:
+31-71-527-5088.
E-mail address: spaink@rulbim.leidenuniv.nl (H.P. Spaink)
0008-6215/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(99)00190-1

E. Kamst et al. / Carbohydrate Research 321 (1999) 176–189
177
ammonia. Bacterial signal molecules, known
2. Results
as Nod factors, are essential for nodule forma-
tion [4]. Purified Nod factors induce many of
the plant root processes that normally occur
during nodulation, sometimes even leading to
the formation of complete nodules [5]. Nod
factors consist of linear oligomers of mostly
four or five b-(1“4)-linked N-acetylglu-
cosamine (GlcNAc) residues, in which the N-
acetyl group of the nonreducing-terminal
residue is replaced with a fatty acid. Biosyn-
thesis of Nod factors requires a series of en-
zymes, encoded by the rhizobial nodulation
genes [6,7].
Chemical synthesis of GlcNAc deri6ati6es.—
In order to investigate the substrate specificity
of NodC we have synthesized GlcNAc deriva-
tives with a range of aglycones (2–4, 6–13),
and in which the N-linked substituent on C-2
is also varied (14, 15). The structures of the
resulting glycosides were verified using NMR
spectroscopy and mass spectrometry. The
spectra obtained demonstrated formation of
the expected reaction products and the ab-
sence of starting materials.
The synthetic routes to compounds 2–4, 6,
and 7 are depicted in Scheme 1. b-Propyl-con-
taining monosaccharide 2 was prepared by
hydrolysis of the phthaloyl group of allyl 3,4,6
The nodulation protein NodC is a rhizobial
N-acetylglucosaminyltransferase [8], which
synthesizes chitin oligosaccharides [9,10]. We
have recently shown that the synthesis of
chitin oligosaccharides by NodC proceeds by
the addition of GlcNAc residues from the
donor UDP-GlcNAc to O-4 of the nonreduc-
ing-terminal residue of the growing chain [11].
The enzyme is able to use free GlcNAc and
the artificial glycoside GlcNAc-1-O-p-nitro-
phenyl as acceptors. The use of hydrophobic
sugar derivatives as artifical glycosyl acceptors
greatly simplifies quantification of enzyme ac-
tivity, since the reaction products can be read-
ily purified using reversed-phase cartridges
[12]. Artificial acceptors are not only used to
assay glycosyltransferase activities but also to
characterize substrate specificity. This ap-
proach has been used for many glycosyltrans-
ferases involved in protein glycosylation.
Mammalian b-(1“4)-galactosyltransferases,
for instance, were shown to have a broad
acceptor specificity [13,14], whereas a related
N-acetylglucosaminyltransferase from the
snail Lymnea stagnalis shows a clear prefer-
ence for acceptors containing a terminal Glc-
NAc residue in b-(1“6) linkage to galactose
or N-acetylgalactosamine [15]. These enzymes
are so-called nonprocessive glycosyltrans-
ferases; they transfer only a single monosac-
charide to an acceptor. NodC, however,
belongs to the processive glycosyltransferases,
which produce linear oligosaccharides or
-tri-O-acetyl-2-deoxy-2-phthalimido-b- -glu-
D
copyranoside (a gift of R. Lagas, Leiden Uni-
versity, Leiden, The Netherlands) followed by
acetylation and selective deacetylation. Glyco-
sylation of allyl alcohol with oxazoline [16]
under the influence of Me3SiOTf resulted, af-
ter deacetylation, in the b-allyl-containing
derivative 3. Hydrogenolysis of hexyl 2-
acetamido-2-deoxy-4,6-di-O-benzyl-b- -glu-
D
copyranoside (a gift of R. Lagas, Leiden Uni-
versity, Leiden, The Netherlands) under
standard conditions gave 4. Hexyl 4,6-O-ben-
zylidene-2-deoxy-3-O-p-methoxybenzyl-2-ph-
thalimodo-b- -glucopyranoside was sub-
D
mitted to DDQ (2,3-dichloro-5,6-dicyano-1,4-
benzoquinone) and acid hydrolysis to give a
triol, the phthaloyl of which was hydrolyzed.
The resulting compound was completely
acetylated and subsequently O-deacetylated to
give the desired hexyl derivative 6. The synthe-
sis of compound 7 was achieved by N-iodo-
succinomide (NIS)-assisted glycosylation of
benzyl alcohol [17] followed by hydrolysis of
the phthaloyl group, acetylation, and selective
deacetylation. The synthesis of compound 8
(Scheme 2) started with the condensation of
2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-b-
D-glucopyranoside with a spacer molecule to
give, after deacetylation, compound 11. Hy-
drogenolysis of triol 11 resulted in the free
amine-containing derivative 8, which was N-
substituted with a photo-affinity label yielding
compound 10. Derivative 9 was acquired fol-
lowing the same sequence of reactions starting
with an N-protected tyramine derivative.
polysaccharides
by
repetitively
adding
monosaccharide residues to a growing chain.
We herein describe the synthesis of several
derivatives of GlcNAc and their ability to act
as acceptors for M. loti NodC.

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E. Kamst et al. / Carbohydrate Research 321 (1999) 176–189
lowed by acetylation, purification, and selec-
tive deacetylation.
Synthesis of GlcNAc derivatives containing
a-linked aglycones corresponding to com-
Use of chemically synthesized GlcNAc
deri6ati6es as acceptors by NodC.—GlcNAc
derivatives were added to reaction mixtures
containing membranes from an E. coli strain
expressing the M. loti chitin oligosaccharide
synthase NodC [18]. After incubation of these
mixtures with UDP-[14C]GlcNAc, reaction
products were analyzed using thin-layer chro-
matography (TLC). Two factors indicate
whether NodC uses the GlcNAc derivative as
an acceptor: (i) the appearance of NodC-de-
pendent spots that are more hydrophobic than
free chitin oligosaccharides, and (ii) the ab-
sence of these reaction products when Glc-
NAc derivatives are not added to the reaction
mixture, or when control preparations lacking
NodC are used. A reduction in the formation
of chitinpentaose, the natural major reaction
pounds 2 and 3 is depicted in Scheme 3.
Propyl 2-acetamido-2-deoxy-a- -glucopyra-
D
noside (12) was prepared by standard Fischer-
glycosylation of N-benzyloxycarbonylamino-
D
-glucopyranoside (15) with allyl alcohol, fol-
lowed by hydrogenolysis and N-acetylation.
The a-allyl derivative 13 was obtained by Fis-
cher-glycosylation of GlcNAc with allyl alco-
hol followed by acetylation, column
chromatography, and subsequent selective
deacetylation.
The influence of the substituent at the
amino group of GlcNAc for recognition by
NodC was investigated with compounds 14
and 15 (Scheme 3) having an allyloxy and a
benzyloxy function, respectively. To obtain
these compounds, glucosamine was treated
with either allyl- or benzylchloroformate, fol-
Scheme 1. Chemical synthesis of GlcNAc derivatives containing b-glycosidically linked aglycones. Reagents used were: (a) EtOH,
,
H2NNH2·H2O D; (b) pyridine, Ac2O; (c) MeOH, KOt-Bu; (d) DCE, Me3SiOTf, 50 °C; (e) DCE, AllOH, MS 4 A, Me3SiOTf; (f)
5:2:0.5 iPrOH–H2O–AcOH, N2, Pd/C, H2; (g) 8:1 CH2Cl2–H2O, DDQ; (h) AcCl, NEt3; (i) 1:1 MeOH–THF, KOt-Bu; (j) DCE,
,
THF, BnOH, MS 4 A, NIS, Me3SiOTf.

E. Kamst et al. / Carbohydrate Research 321 (1999) 176–189
179
Scheme 2. Chemical synthesis of GlcNAc derivatives containing b-glycosidically linked nitrogen-containing aglycones. Reagents
,
used were: (a) CH3CN, MS 4 A, SnCl4 D; (b) MeOH, KOt-Bu; (c) 1:1 iPrOH–H2O, N2, Pd/C, H2; (d) DMF, DIPEA.
C-1 of GlcNAc. Compounds 8 and 9 both
bear an aglycone terminating with a free
amino group and do not seem to be used as
primers by NodC. Judging from spot intensi-
ties (Fig. 1, lanes 8, 9, and H2O) and the
quantification of the radioactive counts using
the PhosphorImager (data not shown), the
level of chitinpentaose synthesized by NodC is
not substantially altered by the presence of
these GlcNAc derivatives, which indicates that
they are probably not even bound by NodC.
The other compounds in panel A all function
as acceptors. Differences in hydrophobicity
between these acceptors correlate with the dif-
ferences in migration of their respective reac-
tion products (Fig. 1). Due to the intense spot
of GlcNAc it not always possible to see all
expected reaction products (e.g. lanes 6, 7, and
10). The intense spots migrating between those
for chitinpentaose and GlcNAc observed with
compounds 1, 2, and 3 (Fig. 1) are likely to
represent oligosaccharides synthesized using
these GlcNAc derivatives as starter units,
since these spots do not migrate with exactly
product of M. loti NodC [18], relative to the
amount of chitinpentaose formed in a corre-
sponding control reaction lacking an exoge-
nously added acceptor indicates that the
GlcNAc derivative is bound by NodC, regard-
less of whether or not it acts as an acceptor.
In control reactions, either water, methanol or
dimethylsulfoxide (DMSO) was added since
these are the solvents used for dissolving the
GlcNAc derivatives. The results of this analy-
sis are shown in Fig. 1 and summarized in
Table 1. In the absence of exogenously added
acceptors, the major product of M. loti NodC
is chitinpentaose (Fig. 1, lanes labelled H2O,
MeOH, and DMSO). Surprisingly, the pres-
ence of methanol resulted in the appearance of
a NodC-dependent spot (Fig. 1, lane MeOH)
that is more hydrophobic and therefore mi-
grates faster than GlcNAc and that was not
detected in a standard reaction mixture lack-
ing methanol (Fig. 1, lane H2O).
Table 1 (panel A) shows the series of com-
pounds bearing acyl chains of various lengths
and hydrophobicity b-glycosidically linked to

180
E. Kamst et al. / Carbohydrate Research 321 (1999) 176–189
Table 1
Acceptor activity of NodC a
Scheme 3. Chemical synthesis of GlcNAc derivatives contain-
ing a-glycosides, or amide-bound substituents. Reagents used
were: (a) H2O, NaHCO3, Z-Cl; (b) AllOH, AcCl; (c) 1:1
iPrOH–H2O, Pd/C, H2; (d) MeOH, Ac2O; (e) pyridine,
Ac2O; (f) MeOH, KOt-Bu; (g) H2O, NaHCO3, AllocCl.
the same Rf values as free chitin oligosacchar-
ides, and are absent when the GlcNAc deriva-
tives are omitted from the reaction mixtures.
The largest amount of reaction product was
obtained with compounds 4, 5, 6, 7, 10, and
11. In all cases, the formation of free chitin-
pentaose was reduced in comparison with con-
trol reactions to which only solvent was
added, particularly strongly with compounds
4, 5, 10, and 11. This indicates that these
GlcNAc derivatives, which are the most hy-
drophobic, are more efficiently bound by
NodC than the other compounds tested.
Next, we investigated whether NodC spe-
cifically uses acceptors with b-linked aglycones
since the GlcNAc residues in chitin oligosac-
charides are b-(1“4) glycosidically linked.
The two hydrophobic aglycones that are b-
glycosidically linked in glycosides 2 and 3
(Table 1, panel A) were therefore coupled in
a-linkage to C1 of GlcNAc, resulting in com-
pounds 12 and 13 (panel B). Elongation of the
a-linked derivatives by NodC was not de-
tectable (Fig. 1). The only apparent NodC-de-
pendent spot in these samples is the
methanol-dependent spot discussed above.
a The scores are based on the TLC analysis shown in Fig. 1.
The compounds are divided into categories A, B and C on the
basis of the difference in conformational or positional linkage
of the indicated substitions. With compounds that scored
negatively (−) no detectable incorporation could be detected.
The compounds scoring positively (+) as acceptor lead to the
production of new metabolites. +/− indicates a marginal
incorporation. Asterisks indicate compounds that are used as
acceptors by NodC and in addition lead to a reduction in
chitinpentaose formation by more than 90%, compared with
control reactions to which only solvents were added. Com-
pounds were tested at a concentration of 1 mM in the
presence of 10 mM UDP-GlcNAc.
However, the intensity of the spot represent-
ing chitinpentaose in these cases is less than
that in the control reaction (Fig. 1, lanes 12,
13, and MeOH), suggesting that compounds
12 and 13 may be bound by NodC even
though they do not apparently function as
primers. In contrast, the b-linked analogues 2
and 3 are clearly used as substrates by NodC

E. Kamst et al. / Carbohydrate Research 321 (1999) 176–189
181
Fig. 1. NH2-TLC analysis of NodC reaction products synthesized in the presence of hydrophobic GlcNAc derivatives as
acceptors. Samples were taken from reaction mixtures with membranes containing NodC protein (+ lanes) or control membranes
lacking NodC (− lanes). Control reactions were performed with NodC reaction mixtures to which the following solvents had
been added: H2O (solvent for compounds 8 and 9), DMSO (solvent for compound 15), or methanol (MeOH, solvent for the
remaining GlcNAc derivatives). The numbers beneath the lanes correspond to the numbers of the GlcNAc derivatives listed in
Table 1. Reactions were performed in the presence of 10 mM UDP-[14C]GlcNAc. GlcNAc derivatives were added to a final
concentration of 1 mM and the final solvent concentration was 5%. Lane R contains a standard mixture of purified, radiolabelled
chitin-oligosaccharides prepared as described [18]. Roman numerals indicate the migration positions of GlcNAc (I) and chitin
oligosaccharides ranging from chitinbiose (II) to chitinpentaose (V). The top of each chromatogram shown corresponds to the
front of the mobile phase. *: The appearance of a spot that migrates slower than chitinpentaose in the initial experiment shown
in the second panel from the left probably represents an artefact since it was not observed in later experiments, of which an
example is shown in the right-hand panel.
scribed in our present report is therefore an
unexpected finding but can potentially be ex-
plained in three ways. First, methanol could
release membrane components, which may
then be used as glycosyl acceptor by NodC. A
second possibility is that this spot corresponds
to a compound that is also produced in the
standard reaction mixture lacking methanol,
but is now rendered soluble in the presence of
methanol. The third possible explanation is
that the presence of methanol somehow stimu-
lates or reveals a modification of the reaction
products of NodC by an endogenous E. coli
enzyme. Structural analysis of this reaction
product is required in order to distinguish
between these possibilities.
(Fig. 1). NodC therefore appears to be specific
for b-glycosides.
The chemical synthesis of two GlcNAc
analogs in which the N-acetyl is replaced is
shown in Scheme 3. In these derivatives 14
and 15 (Table 1, panel C) an allyloxy or
benzyloxy moiety replaces the acetyl-CH3
group. Both compounds are used as acceptors
by NodC (Fig. 1), showing that the CH3 of
the N-acetyl group of GlcNAc is not essential
for interaction with NodC. Glycoside 15 is
clearly less efficiently bound by NodC than is
compound 14. This suggests that in order to
be bound to NodC, the N-linked group on
C-2 should not be very large.
The data reported here show that NodC
does not display a strict specificity towards the
structure of the aglycone in the GlcNAc
derivatives that were used. Only those com-
pounds that contain a free amino group in the
aglycone are not acceptors. Their inability to
act as acceptors is probably due to a low
affinity of NodC for these compounds, rather
than a reduction in the reaction velocity, since
these GlcNAc derivatives do not inhibit the
synthesis of free chitin oligosaccharides by
NodC. The most hydrophobic GlcNAc
derivatives were found to be used most effi-
ciently as acceptors. This could be due to a
higher affinity of NodC for these acceptors
but could also be caused by an increased
3. Discussion
NodC is a bacterial chitin oligosaccharide
synthase that is capable of using free GlcNAc
and GlcNAc-1-O-p-nitrophenyl as acceptors
[11]. It is, however, not known how chitin
oligosaccharide synthesis is initiated in vivo.
Previous results have indicated that prenyl-py-
rophosphate carriers are not required [10,11].
In addition, NodC-dependent hydrophobic re-
action products were not detected in vivo or in
vitro in reaction mixtures following extraction
with organic solvents [11]. The detection of a
hydrophobic NodC-dependent product in re-
action mixtures containing 5% methanol de-

182
E. Kamst et al. / Carbohydrate Research 321 (1999) 176–189
accumulation of these compounds in mem-
branes, leading to higher local concentrations
in the vicinity of NodC.
sium methoxide, and subsequently distilled.
Pyridine was refluxed for 18 h in the presence
of calcium hydride and then distilled. Diethyl
ether was distilled from LiAlH4. Acetonitrile
There is a remarkable difference in the ratios
of the oligosaccharides of different chain length
that are produced in the presence of various
GlcNAc derivatives. The most obvious expla-
nation for this phenomenon is that the nature
of the modifications in the GlcNAc analogue
influence the binding affinity of NodC for the
oligosaccharide intermediates and therefore
leads to a different ratio between the kinetics of
release of the end products and elongation with
additional GlcNAc units. The fact that in the
case of the incorporation of pNP-GlcNAc [11]
there is no effect of varying concentrations of
this compound on the relative quantities of the
products with different chain lengths shows
that this phenomenon cannot be explained by
assuming a mechanism in which the GlcNAc
analogs are incorporated as chain terminators.
The simple rationale behind this is that, in such
an alternative mechanism, an increasing con-
centration of chain terminators should lead to
a relative increase in the production of shorter
chain length products, which was not observed
[11].
Compound 10 contains an azido group on
the phenyl moiety linked to the reducing termi-
nus of GlcNAc. Upon exposure to UV radia-
tion, this azido group is known to become
highly reactive towards compounds bearing the
RꢀNHꢀR moiety, such as is found in the
protein backbone, and results in the formation
of a covalent bond. Since compound 10 is
clearly used as an acceptor by NodC, it is
expected to be a valuable tool for identifying
the acceptor-binding site in this enzyme.
Until recently, synthetic glycosides have only
been used to study non-processive glycosyl-
transferases. The results described here, how-
ever, clearly show that synthetic glycosides are
also useful for assaying enzyme activity and
investigating the substrate specificity of glyco-
syltransferases that act in a processive manner.
,
(p.a., Rathburne) was dried over 4 A molecular
sieves (Aldrich). Tetrahydrofuran (THF, p.a.,
,
E. Merck) was stored over 4 A molecular sieves
before use. Toluene and diethyl ether were
stored over sodium wire. Methanol was stored
,
over 3 A molecular sieves. Dichloromethane
,
and 1,2-dichloroethane were stored over 4 A
molecular sieves. Solvents used for column
chromatography were of technical grade and
distilled before use. Reactions were performed
under anhydrous conditions at room tempera-
ture (rt), unless stated otherwise. Solvents were
evaporated under reduced pressure at 40 °C.
TLC analyses were conducted on Schleier &
Schu¨ll DC Fertigfolien (F 1500 LS 254). Com-
pounds were visualized using UV light and
charring with 1:4 H2SO4 –ethanol. Those com-
pounds that contain free amines were stained
using a ninhydrin solution (0.3 g dissolved in a
mixture of 3 mL AcOH and 100 mL EtOH).
Column chromatography was performed on
columns of silica gel (Baker, 0.063–0.200 nm).
Petroleum ether used for elution of the columns
was low-boiling (40–60 °C). Gel-filtration was
performed on Sephadex LH20 (Pharmacia).
Structural analysis of synthetic GlcNAc
1
deri6ati6es.—13C and H NMR spectra were
recorded using a Bruker DPX-300 spectrometer
at 75 and 300 MHz, respectively. Chemical
shifts are given in ppm using Me4Si in CDCl3
as internal reference. In addition, final reaction
products were analyzed using mass spectrome-
try. Spectra were recorded on a Finnigan MAT
TSQ-70 equipped with a custom-made electro-
spray interface (ESI).
Chemical synthesis of GlcNAc deri6ati6es.—
Compounds 1 and 5 were kind gifts from S.H.
van Leeuwen (Leiden University, Leiden, The
Netherlands) and M. Palcic (University of Al-
berta, Edmonton, Canada), respectively.
Propyl 2-acetamido-2-deoxy-i-
noside (2).—Starting material was allyl 3,4,6-
-gluco-
D
-glucopyra-
4. Experimental
tri-O-acetyl-2-deoxy-2-phthalimido-b-
D
pyranoside, a gift from R. Lagas (Leiden Uni-
versity, Leiden, The Netherlands). 13C NMR
(CDCl3): l 20.0, 20.2, 20.3 (CH3 Ac), 54.2 (C-2),
61.6 (C-6), 68.6, 70.4, 71.4, (C-3, C-4, C-5), 69.8
General methods.—Toluene, 1,2-dichloroe-
thane, and CH2Cl2 were distilled from P2O5.
Methanol was dried by refluxing with magne-

E. Kamst et al. / Carbohydrate Research 321 (1999) 176–189
183
3.22–3.91 (m, 8 H, H-2, H-3, H-4, H-5, H-6,
CH2 Propyl), 4.39 (d, 1 H, H-1, J1,2 8.04 Hz).
MS: [M+Na]+ for C11H21NO6: m/z 286.0.
(CH2 Allyl), 96.7 (C-1), 117.5 (CH2 Allyl),
123.2 (CH Phth), 130.9 (qC Phth), 132.9,
134.1 (CH Phth, CH Allyl), 169.2, 169.8,
170.4 (CꢁO Ac). Hydrazine monohydrate (0.5
mL, 10.3 mmol) was added to a solution of
the glucopyranoside derivative (0.50 g, 1.1
mmol) in EtOH (96%, 30 mL). The reaction
mixture was stirred under reflux for 17 h and
subsequently cooled. The solids were filtered
and the filtrate was concentrated. The crude
amine-containing derivative (propyl 2-amino-
Allyl 2-acetamido-2-deoxy-i- -glucopyra-
D
noside (3).—The fresh oxazoline, prepared as
described [16] (937 mg, 3.0 mmol) was dried
by evaporation with toluene and subsequently
dissolved in dichloroethane (15 mL). Accord-
ing to Refs. [19,20], allyl alcohol (0.61 mL, 9.0
,
mmol) and 4 A molecular sieves were added
and the resulting mixture was stirred at rt for
30 min. Trimethylsilyl trifluoromethanesul-
fonate (0.115 mL, 0.59 mmol) was added and
the reaction mixture was stirred for 19 h. The
reaction mixture was neutralized with Et3N.
The solids were filtered and the filtrate was
concentrated. The reaction product was a 2:1
mixture of oxazoline and product. The pure
product allyl 2-acetamido-3,4,6-tri-O-acetyl-2-
2-deoxy-b- -glucopyranoside) was dried by
D
evaporation with toluene and dissolved in
pyridine (5 mL). Acetic anhydride (2.0 mL,
21.3 mmol) was added to the solution and the
reaction mixture was stirred for 20 h at rt. The
reaction mixture was concentrated and the
residue was taken up in EtOAc (15 mL). The
resulting solution was washed with HCl (3%,
10 mL), water (10 mL), and NaHCO3 (10%,
10 mL). The organic phase was dried
(MgSO4), filtered, and concentrated. The
crude compound was purified by column
deoxy-b- -glucopyranoside was obtained in
D
21% yield after column chromatography (50–
100% EtOAc in petroleum ether). 13C NMR
(CDCl3): l 20.2 (CH3 Ac), 22.7 (CH3 NHAc),
53.9 (C-2), 62.0 (C-6), 68.7, 71.1, 72.2, (C-3,
C-4, C-5), 69.6 (CH2 allyl), 99.4 (C-1), 117.0
(CH2 allyl), 133.4 (CH allyl), 169.1, 170.2,
170.3, 170.6 (CꢁO Ac). The acetylated sugar
(245 mg, 0.63 mmol) was dissolved in MeOH
(6.0 mL) and potassium tert-butanolate (7 mg,
0.06 mmol) was added. The solution was
stirred for 5 h. The reaction mixture was
neutralized with Dowex 50W×4 and the
solids were filtered. The filtrate was concen-
trated and the residue was purified by column
chromatography (0–30% MeOH in EtOAc) to
give the pure product 3 in 74% yield.
chromatography
(20–100%
EtOAc
in
petroleum ether) to give the pure compound
propyl
oxy-b-
2-acetamido-3,4,6-tri-O-acetyl-2-de-
D
-glucopyranoside (87%). 13C NMR
characterisation showed that the allyl group
was converted to the propyl function. 13C
NMR (CDCl3): l 9.4 (CH3 Propyl), 19.7 (CH3
Ac), 21.8 (CH3 NHAc), 22.0 (CH2 Propyl),
53.7 (C-2), 61.7 (C-6), 68.5, 70.8, 72.2 (C-3,
C-4, C-5), 70.9 (CH2 Propyl), 100.1 (C-1),
169.2, 170.0, 170.4, 171.0 (CꢁO Ac). Potas-
sium tert-butanolate (4 mg, 0.04 mmol) was
added to a solution of this saccharide (354
mg, 0.91 mmol) in MeOH (4 mL). After stir-
ring for 5 h, the reaction mixture was neutral-
ized with Dowex 50W×4 and subsequently
filtered. The filtrate was concentrated and the
pure product 2 was obtained in 86% yield
after column chromatography (80% petroleum
ether to 30% MeOH in EtOAc).
13C NMR (CD3OD): l 23.0 (CH3 NHAc),
56.8 (C-2), 62.4 (C-6), 70.5 (CH2 allyl), 71.7,
75.6, 77.3, (C-3, C-4, C-5), 101.4 (C-1), 117.1
(CH2 allyl), 135.0 (CH allyl), 173.5 (CꢁO
1
NHAc). H NMR (CD3OD): l 1.97 (s, 3 H,
CH3 NHAc), 3.24–4.39 (m, 8 H, H-2, H-3,
H-4, H-5, H-6, CH2 Allyl), 4.43 (d, 1 H, H-1,
J1,2 8.0 Hz), 5.04–5.31 (m, 2 H, CH2 Allyl),
5.79–5.98 (m, 1 H, CH Allyl). MS: [M+Na]+
for C11H19NO6: m/z 284.1
13C NMR (CD3OD): l 10.7 (CH3 Propyl),
23.0 (CH3 NHAc), 23.4 (CH2 Propyl), 56.9
(C-2), 62.3 (C-6), 71.6, 75.5, 77.2 (C-3, C-4,
C-5), 71.6 (CH2 Propyl), 102.2 (C-1), 173.7
(CꢁO NHAc). 1H NMR (CD3OD): l 0.91 (t, 3
H, CH3 Propyl, JH,H 7.3 Hz), 1.51–1.61 (m, 2
H, CH2 Propyl), 1.97 (s, 3 H, CH3 NHAc),
Hexyl 2-acetamido-2-deoxy-i-
anoside (4).—Starting material was hexyl 2-
-glu-
D
-glucopyr-
acetamido-2-deoxy-4,6-di-O-benzyl-b-
D
copyranoside, a gift from R. Lagas (Leiden
University, Leiden, The Netherlands). 13C

184
E. Kamst et al. / Carbohydrate Research 321 (1999) 176–189
NMR (CDCl3): l 13.9 (CH3 Hexyl), 23.4
(CH3 NHAc), 22.5, 25.6, 29.4, 31.5 (4×CH2
Hexyl), 58.1 (C-2), 69.0, 69.5 (C-6, CH2
Hexyl), 74.9, 75.6, 78.3 (C-3, C-4, C-5), 100.2
(C-1), 127.5, 127.7, 128.0, 128.2 (CH Bn),
138.1, 138.3 (qC Bn), 172.2 (CꢁO NHAc). The
benzylated glycoside (200 mg, 0.41 mmol) was
dissolved in a 5:2:0.5 mixture of isopropanol–
water–AcOH (5 mL). After stirring for 30
min under bubbling nitrogen, palladium on
charcoal was added and hydrogen was bub-
bled through for 3 h. The hydrogen was re-
moved by bubbling nitrogen and sub-
sequently, the palladium was filtered over a
bed of hyflo. The filtrate was concentrated
and the residue was purified by silica gel chro-
matography (0–40% MeOH in EtOAc). The
final product 4 was obtained in 76% yield.
13C NMR (CDCl3): l 14.3 (C-6 hexyl), 23.0
(CH3 NHAc), 23.4, 26.4, 30.3, 32.4 (4×CH2
hexyl), 56.9 (C-2), 62.4 (C-6), 70.5 (C-1 hexyl),
71.7, 75.6, 77.3 (C-3, C-4, C-5), 102.3 (C-1),
173.5 (CꢁO NHAc).
23.2, 23.5, 25.1, 31.2, 33.0 (5×CH2 cyclo-
hexyl), 56.9 (C-2), 68.6 (C-6), 66.0, 68.4, 77.1,
82.0 (C-3, C-4, C-5, CH cyclohexyl), 97.1,
101.6 (C-1, CH CHPh), 123.2 (CH Phth),
126.2, 128.1, 129.0 (CH Ar), 131.4 (qC Phth),
133.9 (CH Phth), 137.0 (qC CHPh). Accord-
ing to [22], acetyl chloride (0.11 mL, 1.5
mmol) was added to a solution of this crude
monosaccharide in a 1:1 mixture of MeOH–
CH2Cl2 (14 mL) at 0 °C. The ice-bath was
removed and the reaction was stirred for 1.5 h
at rt. The reaction mixture was neutralized
with Et3N and the solvents were evaporated.
The residue was purified by column chro-
matography (30–100% EtOAc in petroleum
ether) to give the pure cyclohexyl 2-deoxy-2-
phthalimido-b- -glucopyranoside in 71% yield
D
over two steps. 13C NMR (CD3OD): l 24.1,
24.4, 26.4, 32.2, 34.0 (5×CH2 cyclohexyl),
58.6 (C-2), 62.6 (C-6), 72.5, (d.i.), 77.3, 78.0
(C-3, C-4, C-5, CH cyclohexyl), 97.6 (C-1),
124.1 (CH Phth), 132.8 (qC Phth), 135.5 (CH
Phth). This triol (370 mg, 0.98 mmol) was
dissolved in EtOH (96%, 30 mL) and heated
at reflux temperature for 18 h in the presence
of hydrazine monohydrate (0.5 mL, 10.3
mmol) as described [23,24]. The solution was
cooled to rt and a white precipitate was
formed. The phthaloyl salts were removed by
filtration and washed with EtOH. The filtrate
was concentrated to give the crude amine,
which was dried by evaporation with toluene.
The dry residue was dissolved in pyridine (5
mL) and Ac2O (1.0 mL, 10.6 mmol) was
added. After stirring for 18 h, the solution was
concentrated and once rotory-evaporated with
toluene. The residue was redissolved in EtOAc
(10 mL) and washed with HCl (3%, 10 mL),
water (10 mL), and NaHCO3 (10%, 10 mL).
The organic solution was dried (MgSO4),
filtered, and concentrated. The crude product
was purified by column chromatography (10–
80% EtOAc in petroleum ether). Concentra-
tion of the appropriate fractions gave the
desired derivative in 81% yield. 13C NMR
(CDCl3): l 20.3 (CH3 Ac), 22.7 (CH3 NHAc),
23.4, 23.4, 25.1, 31.2, 32.8 (5×CH2 cyclo-
hexyl), 54.6 (C-2), 62.1 (C-6), 69.0, 71.0, 72.2,
77.2 (C-3, C-4, C-5, CH cyclohexyl), 98.7 (C-
1), 169.1, 170.1, 170.3 (CꢁO Ac). To a solu-
tion of the fully acetylated sugar (328 mg, 0.79
Cyclohexyl 2-acetamido-2-deoxy-i-
copyranoside (6).—Starting material was cy-
clohexyl 4,6-O-benzylidene-2-deoxy-3-O-p-
methoxybenzyl-2-phthalimido-b- -glucopyra-
D-glu-
D
noside, a gift from R. Lagas (Leiden Univer-
sity, Leiden, The Netherlands). 13C NMR
(CDCl3): l 23.3, 23.6, 25.2, 31.3, 33.0 (5×
CH2 cyclohexyl), 54.7, 56.0 (C-2, CH3 PMB),
68.7 (C-6), 65.9, 74.1, 77.1, 82.9 (C-3, C-4,
C-5, CH cyclohexyl), 97.1, 100.6 (C-1, CH
CHPh), 113.2 (CH PMB), 122.9 (CH Phth),
125.9, 128.1, 128.5, 129.6 (CH Ar), 130.0 (qC
PMB), 131.4 (qC Phth), 133.6 (CH Phth),
137.3 (qC Ar), 158.7 (qC PMB). The pro-
tected saccharide (0.83 g, 1.38 mmol) was
dissolved in an 8:1 mixture of CH2Cl2–water
(7.0 mL) and 2,3-dichloro-5,6-dicyano-1,4-
benzoquinone (0.47 g, 2.1 mmol) was added at
rt [21]. The reaction mixture was stirred for 1
h, then the solids were filtered over a path of
hyflo. The filtrate was diluted with CH2Cl2 (10
mL). The organic solution was washed with
water (10 mL) and subsequently with
NaHCO3 (10%, 10 mL, twice), dried (MgSO4),
filtered, and concentrated. The crude product
cyclohexyl 4,6-O-benzylidene-2-deoxy-2-ph-
thalimido-b-
D
-glucopyranoside was used with-
13
out further purification. C NMR (CDCl3): l

E. Kamst et al. / Carbohydrate Research 321 (1999) 176–189
185
mmol) in a 1:1 mixture of MeOH–THF (8
mL) was added potassium tert-butanolate (18
mg, 0.16 mmol). The mixture was stirred for
18 h, neutralized with Dowex 50W×4,
filtered, and concentrated. Purification of the
residue was achieved by silica gel column
chromatography. The column was eluted with
a gradient of 0–30% MeOH in EtOAc to yield
the product 6 in 59% yield.
CH2 Bn), 5.19 (t, 1 H, H-3/H-4, JH,H 9.5 Hz),
5.38 (d, 1 H, H-1, J1,2 8.8 Hz), 5.79 (dd, 1 H,
H-3/H-4, JH,H 9.1 Hz, JH,H 10.6 Hz), 7.06–
7.13 (m, 5 H, CH Ar), 7.70–7.77 (m, 4 H, CH
Ar). The acetylated saccharide (788 mg, 1.5
mmol) was dissolved in EtOH (96%, 45 mL)
and hydrazine hydrate (0.73 mL, 15.0 mmol)
was added. The resulting solution was heated
under reflux for 18 h and subsequently cooled
to rt. The solids were filtered and the filtrate
was concentrated to dryness. The residue was
roto-evaporated with toluene and dissolved in
pyridine (8.0 mL) and acetic anhydride (4.0
mL, 42.3 mmol). The reaction was stirred at rt
for 21 h and then concentrated. The residue
was redissolved in EtOAc (20 mL) and
washed with HCl (3%, 15 mL), water (15 mL),
and NaHCO3 (10%, 15 mL). The organic
phase was dried (MgSO4), filtered, and con-
centrated. The crude product was purified by
column chromatography (20–100% EtOAc in
petroleum ether) to give the pure product
13C NMR (CD3OD): l 23.1 (CH3 NHAc),
24.4, 24.5, 26.5, 32.3, 34.2 (5×CH2 cyclo-
hexyl), 57.5 (C-2), 62.6 (C-6), 71.9, 75.6, 77.4,
77.7 (C-3, C-4, C-5, CH cyclohexyl), 100.6
(C-1), 173.4 (CꢁO NHAc). MS: [M+Na]+
for C14H25NO5: m/z 326.1.
Benzyl 2-acetamido-2-deoxy-i- -glucopy-
D
ranoside (7).—The thio-ethyl donor (480 mg,
1.0 mmol) [17] was dried by evaporation with
toluene and subsequently dissolved in 1,2-
dichloroethane (16 mL). Benzyl alcohol (0.21
mL, 2.0 mmol) was added and the mixture
,
was stirred in the presence of 4 A molecular
sieves under a blanket of argon for 30 min.
The mixture was cooled with an ice-bath and
a solution of N-iodosuccinimide (270 mg, 1.2
mmol) and trimethylsilyl trifluoromethanesul-
fonate (33 mL, 0.17 mmol) in THF (8 mL)
was added. The ice-bath was removed and the
reaction mixture was stirred for 6 h at rt. The
reaction mixture was neutralized with Et3N
(0.3 mL) and filtered over a path of hyflo. The
filtrate was diluted with EtOAc (20 mL) and
washed with Na2S2O3 (1 M, 15 mL). The
organic layer was dried (MgSO4), filtered, and
concentrated. The pure product benzyl 2-ph-
benzyl
2-acetamido-3,4,6-tri-O-acetyl-2-de-
oxy-b-
D
-glucopyranoside in 81% yield. 13C
NMR (CDCl3): l 20.3 (CH3 Ac), 22.7 (CH3
NHAc), 54.0 (C-2), 62.0 (C-6), 68.6, 71.4, 72.3
(C-3, C-4, C-5), 70.4 (CH2 Bn), 99.4 (C-1),
127.6, 128.1 (CH Bn), 136.8 (qC Bn), 169.3,
170.5 (CꢁO Ac). To a solution of the acety-
lated derivative (262 mg, 0.5 mmol) dissolved
in 1:1 THF–MeOH (5 mL) was added potas-
sium tert-butanolate (6 mg, 0.05 mmol). After
stirring for 4 h, the reaction mixture was
neutralized with Dowex 50W×4, filtered and
the solvents were evaporated. The residue was
purified by column chromatography (0–20%
MeOH in EtOAc) to give the pure compound
7 (72%).
thalimido-3,4,6-tri-O-acetyl-2-deoxy-b- -glu-
D
copyranoside was obtained in 90% yield after
column chromatography (0–40% EtOAc in
petroleum ether).
13C NMR (CD3OD): l 23.0 (CH3 NHAc),
57.3 (C-2), 62.9 (C-6), 71.5 (CH2 Bn), 72.2,
77.9, 78.0 (C-3, C-4, C-5), 101.8 (C-1), 128.8,
129.3 (CH Bn). MS: [M+H]+ for
C15H21NO6: m/z 334.1.
13C NMR (CDCl3): l 19.8, 20.0, 20.2 (CH3
Ac), 54.1 (C-2), 61.5 (C-6), 68.5, 70.1, 71.4
(C-3, C-4, C-5), 71.3 (CH2 Bn), 96.7 (C-1),
123.0 (CH Phth), 127.3, 127.7 (CH Bn), 130.8
(qC Phth), 133.8 (CH Phth), 136.2 (qC Bn),
166.9 (CꢁO Phth), 169.0, 169.5, 170.1 (CꢁO
Ac).
3-Aminopropyl 2-acetamido-2-deoxy-i-
glucopyranoside (8).—The monosaccharide
-glu-
D
-
donor 1,3,4,6-tetra-O-acetyl-2-deoxy-b-
D
1H NMR (CDCl3): l 1.86, 2.03, 2.14 (3×s,
3 H, CH3 Ac), 3.40–3.55 (m, 1 H, H-2), 3.87
(ddd, 1 H, H-5, J5,4 10.2 Hz, J5,6 2.2 Hz, J5,6
copyranoside [25] (778 mg, 2.0 mmol) and
3-(benzyloxycarbonylamino)propanol [26] (1.0
g, 5.0 mmol) were dried by evaporation of
toluene and dissolved in MeCN (20 mL) ac-
2
4.4 Hz), 4.19 (dd, 1 H, H-6, J6,6 12.4 Hz, J6,5
2.2 Hz), 4.31–4.39 (m, 1 H, H-6), 4.69 (AB,
,
cording to Ref. [27]. 4 A molecular sieves were

186
E. Kamst et al. / Carbohydrate Research 321 (1999) 176–189
Then, the reaction mixture was placed under
nitrogen for 20 min and subsequently the
solids were filtered over a path of hyflo. The
filtrate was concentrated and purified by
column chromatography (0–100% MeOH in
EtOAc in the presence of 1% Et3N) to give the
unprotected product 8 (78%).
added and the mixture was stirred for 30 min
at rt under a blanket of argon. Tin tetrachlo-
ride (0.28 mL, 2.4 mmol) was added and the
reaction mixture was heated at reflux tempera-
ture for 18 h. The reaction mixture was
cooled, diluted with EtOAc (20 mL), and the
solids were filtered over a path of hyflo. The
filtrate was washed with KF (1 M, 15 mL) and
water (15 mL). The organic phase was dried
(MgSO4), filtered, and concentrated. The
residue was purified by column chromatogra-
phy (30% petroleum ether“10% MeOH in
EtOAc) to yield the pure product 3-(benzy-
loxycarbonylamino)propyl 2-acetamido-3,4,6-
4-(Aminoethyl)phenyl 2-acetamido-2-deoxy-
i-
D
-glucopyranoside (9).—The monosaccha-
-gluco-
ride 1,3,4,6-tetra-O-acetyl-2-deoxy-b-
D
pyranoside (389 mg, 1.0 mmol) and the Z-pro-
tected tyramine [28] (542 mg, 2.0 mmol) were
coupled according to Ref. [27]. Starting mate-
rials were dried by evaporation with toluene.
The dry components were dissolved in MeCN
tri-O-acetyl-2-deoxy-b- -glucopyranoside in
D
78% yield. 13C NMR (CDCl3): l 20.3, 20.6
(CH3 Ac), 22.7 (CH3 NHAc), 29.3, 36.7 (2×
CH2 Spacer), 53.7 (C-2), 61.8 (C-6), 66.2, 66.4
(CH2 Spacer, Z), 68.4, 71.4, 72.9 (C-3, C-4,
(10 mL) and stirred in the presence of 4 A
,
molecular sieves under a blanket of argon for
30 min. Tin tetrachloride (0.14 mL, 1.2 mmol)
was added and the resulting mixture was
heated at reflux temperature for 18 h. The
reaction mixture was cooled down and subse-
quently filtered over a path of hyflo. The
filtrate was diluted with EtOAc (10 mL) and
subsequently washed with KF (1 M, 10 mL)
and water (10 mL). The organic layer was
dried (MgSO4), filtered, and concentrated. The
pure product 4-[2-(benzyloxycarbonylamino)-
ethyl]phenyl 2-acetamido-3,4,6-tri-O-acetyl-2-
1
C-5), 100.9 (C-1, JC,H 161.8 Hz), 127.3, 127.7,
128.2 (CH Z), 136.6 (qC Z), 156.6 (CꢁO Z),
169.0, 170.3, 170.6 (CꢁO Ac, NHAc). To a
solution of this acetylated monosaccharide
(839 mg, 1.6 mmol) in MeOH (16 mL), potas-
sium tert-butanolate (18 mg, 0.16 mmol) was
added. After stirring for 4 h, TLC analysis
showed the reaction to be complete. The reac-
tion mixture was neutralized with Dowex
50W×4 and the solids were filtered. The
filtrate was concentrated and the residue was
purified by column chromatography (0–30%
MeOH in EtOAc) to yield the desired com-
pound 3-(benzyloxycarbonylamino)propyl 2-
deoxy-b- -glucopyranoside was obtained in
D
51% yield after column chromatography (50–
100% EtOAc in petroleum ether). 13C NMR
(CHCl3): l 20.3 (CH3 Ac), 22.5 (CH3 NHAc),
34.9, 42.1 (2×CH2 Tyramine), 54.0 (C-2),
61.9 (C-6), 66.3 (CH2 Z), 68.5, 71.5, 72.1 (C-3,
C-4, C-5), 98.7 (C-1), 116.8, 127.8, 128.2,
128.3, 129.5 (CH Ar), 133.2 (qC Ar), 155.5
(CꢁO Z), 169.4, 170.5, 171.2 (CꢁO Ac,
acetamido-2-deoxy-b- -glucopyranoside (11,
D
13
Scheme 2) (76%). C NMR (CD3OD): l 23.1
(CH3 NHAc), 30.5, 38.5 (2×CH2 Spacer),
57.0 (C-2), 62.5 (C-6), 66.9, 67.6 (CH2 Spacer,
Z), 71.7, 75.9, 77.4 (C-3, C-4, C-5), 102.3
(C-1), 128.5, 128.8, 129.3 (CH Z), 138.0 (qC
1
NHAc). H NMR (CHCl3): l 2.00, 2.03, 2.04,
2.05 (4×CH3 Ac, NHAc), 2.73 (t, 2 H, CH2
Tyramine, JH,H 6.9 Hz), 3.38 (q, 2 H, CH2
Tyramine, JH,H −JH,NH 6.9 Hz), 3.77–3.85 (m,
1 H, H-5), 4.06–4.20 (m, 2 H, H-2, H-6), 4.27
1
Z), 158.5 (CꢁO Z), 173.8 (CꢁO NHAc). H
NMR (CD3OD): l 1.68–1.78 (m, 2 H, CH2
spacer), 1.98 (s, 3 H, CH3 NHAc), 3.15–3.95
(m, 10 H, H-2, H-3, H-4, H-5, H-6, 2×CH2
spacer), 4.33 (d, 1 H, J1,2 8.8 Hz), 5.06 (s, 2 H,
CH2 Z), 7.31–7.33 (m, 5 H, CH Z). The
spacer-containing monosaccharide (507 mg,
1.23 mmol) was dissolved in a 1:1 mixture of
isopropanol–water (10 mL). The solution was
de-aired by nitrogen and subsequently palla-
dium on charcoal was added. Hydrogen was
bubbled through the reaction mixture for 2 h.
2
(dd, 1 H, H-6, J6,6 12.4 Hz, J6,5 5.1 Hz), 5.07
(s, 2 H, CH2 Z), 5.12 (t, 1 H, H-3/H-4, JH,H
9.9 Hz), 5.24 (d, 1 H, H-1, J1,2 8.8 Hz), 5.40 (t,
1 H, H-3/H-4, JH,H 9.9 Hz), 6.79–7.36 (m, 9
H, CH Ar). To a solution of this monosaccha-
ride (306 mg, 0.51 mmol) in MeOH (5 mL)
was added potassium tert-butanolate (6 mg,
0.05 mmol). After stirring for 4 h, TLC analy-
sis showed the reaction to be complete. The

E. Kamst et al. / Carbohydrate Research 321 (1999) 176–189
187
CD3OD–DMSO): l 57.6, 60.1 (C-2), 62.6 (C-
6), 72.1, 72.3, 73.2, 75.7, 77.9 (C-3, C-4, C-5),
92.5, 97.0 (C-1), 129.1, 129.6 (CH Z), 138.5
(qC Z), 158.0 (CꢁO Z). A solution of this
monosaccharide (1.57 g, 5.0 mmol) in allyl
alcohol (40 mL) containing acetyl chloride
(2%, 0.8 mL) was heated at reflux temperature
for 43 h. The reaction mixture was neutralized
with Et3N and subsequently concentrated. The
crude product allyl 2-benzyloxycarbony-
reaction mixture was neutralized with Dowex
50W×4 and the solids were filtered. The
filtrate was concentrated and the residue was
redissolved in 1:1 isopropanol–water (10 mL).
The solution was de-aired by nitrogen and
palladium on charcoal was added. Hydrogen
was bubbled through the reaction mixture for
2 h. The reaction mixture was placed under
nitrogen for 20 min and the solids were subse-
quently filtered over a path of hyflo. The
filtrate was concentrated and purified by
column chromatography (0–100% MeOH and
1% Et3N in EtOAc) to yield the tyramine-con-
taining monosaccharide 9 in 65% yield.
lamino-2-deoxy- -glucopyranose was purified
D
by column chromatography (80–100% EtOAc
in petroleum ether) to give first the a anomer
in 55% yield. Further elution of the column
with 10% MeOH in EtOAc gave the b anomer
13C NMR (CD3OD): l 23.0 (CH3 NHAc),
36.4, 43.1 (2×CH2 Tyramine), 57.2 (C-2),
62.4 (C-6), 71.7, 75.5, 78.1 (C-3, C-4, C-5),
101.0 (C-1), 117.9, 130.8 (CH Tyramine),
133.5 (qC Tyramine), 157.8 (qC Tyramine),
173.9 (CꢁO NHAc).
in 6% yield. Allyl 2-acetamido-2-deoxy-a- -
D
glucopyranoside 13C NMR (CD3OD): l 58.2
(C-2), 63.7 (C-6), 68.5 (CH2 Z), 70.1 (CH2
Allyl), 73.2, 73.9, 74.8 (C-3, C-4, C-5), 98.9
1
(C-1, JC,H 169.4 Hz), 118.5 (CH2 Allyl),
1H NMR (CD3OD): l 1.98 (s, 3 H, CH3
NHAc), 2.69–3.94 (m, 10 H, H-2, H-3, H-4,
129.8, 130.4 (CH Z), 136.4 (CH Allyl), 142.5
(qC Z), 159.8 (CꢁO Z). Allyl 2-benzyloxycar-
H-5, H-6, 2 CH2 Tyramine), 5.01 (d, 1 H,
bonylamino-2-deoxy-b-
-glucopyranoside 13C
D
–
H-1, J1,2 8.4 Hz), 6.97 (d, 2 H, CH Tyramine,
JH,H 8.8 Hz), 7.16 (d, 2 H, CH Tyramine, JH,H
8.8 Hz). MS: [M+H]+ for C16H24N2O6: m/z
341.1.
NMR (CD3OD): l 58.9 (C-2), 62.7 (C-6), 67.3
(CH2 Z), 70.8 (CH2 Allyl), 72.1, 75.8, 77.8
1
(C-3, C-4, C-5), 102.2 (C-1, JC,H 160.2), 117.0
(CH Allyl), 128.8, 129.4 (CH Z), 135.5 (CH2
Allyl).
1-O-[N-(4-Azidobenzoyl)-3-amino]propyl-2-
acetamido-2-deoxy-i-
D
-glucopyranoside (10).
The a-linked monosaccharide (200 mg, 0.57
mmol) was dissolved in a 1:1 mixture of iso-
propanol–water (10 mL). The solution was
de-aired with nitrogen and subsequently palla-
dium on charcoal was added. Hydrogen was
bubbled trough the mixture for 30 min. The
solution was satured with nitrogen and subse-
quently the solids were filtered through a bed
of hyflo. The filtrate was concentrated and the
residue was dried by evaporation with toluene.
The crude product was redissolved in MeOH
(29 mL) and the solution was cooled with an
ice-bath. Acetic anhydride (0.91 mL, 9.6
mmol) was added, and the solution was stirred
for 45 min at 0 °C. The reaction mixture was
concentrated and purified by column chro-
matography. Elution of the column with 20%
petroleum ether to 50% MeOH in EtOAc gave
the pure propyl-containing product 12 in 84%
yield.
—Compound 8 (5 mg, 18 mmol) was dis-
solved in DMF (0.5 mL) and subsequently the
photoaffinity label succinimidyl 4-azidoben-
zoic acid (5 mg, 19 mmol) and di-iso-propy-
lethylamine (6.3 mL, 36 mmol) were added.
The reaction mixture was stirred for 55 h
under exclusion of light. Then the reaction
mixture was concentrated and the residue was
rotory-evaporated with toluene and tested
without further purification.
Propyl 2-acetamido-2-deoxy-h- -gluco-
D
pyranoside (12).—Glucosamine-hydrochloride
(13.6 g, 63.1 mmol) was dissolved in water
(100 mL) and NaHCO3 (13.0 g, 155.0 mmol)
was added. The solution was cooled in an
ice-bath and benzyl chloroformate (15.0 mL,
106.0 mmol) was added in portions. The solu-
tion was stirred for 17 h at 4 °C until it
became a suspension. The product (2-benzy-
loxycarbonylamino-2-deoxy-
D
-glucopyrano-
13C NMR (CD3OD): l 11.0 (CH3 Propyl),
22.6 (CH3 NHAc), 23.6 (CH2 Propyl), 55.4
(C-2), 62.6 (C-6), 70.5 (CH2 Propyl), 72.3,
side, 15, Scheme 3) was filtered, washed with
EtOAc and dried (88%).13C NMR (1:1

188
E. Kamst et al. / Carbohydrate Research 321 (1999) 176–189
6.01 (m, 1 H, CH Allyl). MS: [M+Na]+ for
C11H19NO6: m/z 284.1.
1
72.7, 73.6 (C-3, C-4, C-5), 98.3 (C-1, JC,H
1
169.4 Hz), 173.6 (CꢁO NHAc). H NMR
2 - Allyloxycarbonylamino - 2 - deoxy -
D
- glu-
(CD3OD): l 0.96 (t, 3 H, 7.3 Hz, CH3 Propyl),
1.57–1.71 (m, 2 H, CH2 Propyl), 1.98 (s, 3 H,
CH3 NHAc), 3.30–3.84 (m, 8 H, H-2, H-3,
H-4, H-5, H-6, CH2 Propyl), 4.78 (d, 1 H,
H-1, J1,2 3.7 Hz, H-1). MS: [M+Na]+ for
C11H21NO6: m/z 286.0.
copyranose (14).—Allyl chloroformate (0.9
mL, 8.4 mmol) was added in portions to a
cooled (0 °C) solution of glucosamine-hy-
drochloride (1.1 g, 5 mmol) and NaHCO3
(420 mg, 12.3 mmol) in water (100 mL). The
solution was stirred for 17 h at 4 °C. Then, the
solution was concentrated and the residue was
dried by evaporation with toluene. The crude
product was dissolved in pyridine (15 mL) and
Ac2O (3.8 mL, 40 mmol) was added. After
stirring for 19 h, the reaction mixture was
concentrated and co-evaporated with toluene.
The residue was taken up in EtOAc (50 mL)
and washed with HCl (3%, 40 mL), water (40
mL), and NaHCO3 (10%, 40 mL). The or-
ganic phase was dried (MgSO4), filtered, and
concentrated. The crude product was purified
by column chromatography (30–80% EtOAc
in petroleum ether) to yield the acetylated
product 1,3,4,6-tetra-O-acetyl-2-allyloxycar-
Allyl 2-acetamido-2-deoxy-h- -glucopyra-
D
noside (13).—GlcNAc (664 mg, 3.0 mmol)
was dissolved in allyl alcohol (24 mL). Acetyl
chloride (2%, 0.48 mL) was added and the
resulting solution was stirred for 20 h at reflux
temperature. Then, the reaction mixture was
neutralized with Et3N and concentrated. The
crude product was dried by evaporation with
toluene and dissolved in pyridine (10 mL) and
Ac2O (5 mL, 53.2 mmol). After stirring for 18
h the mixture was concentrated and evapo-
rated with toluene. The residue was taken up
in EtOAc (30 mL) and subsequently washed
with HCl (3%, 25 mL), water (25 mL) and
NaHCO3 (10%, 25 mL). The resulting organic
layer was dried (MgSO4), filtered and concen-
trated. The pure product allyl 2-acetamido-
bonylamino-2-deoxy- -glucopyranose in 72%
D
yield. 13C NMR (CDCl3): l 19.9, 20.1 (CH3
Ac), 52.1, 54.0 (C-2), 61.0 (C-6), 65.1 (CH2
Alloc), 67.4, 69.0, 70.0, 67.7, 71.8 (C-3, C-4,
C-5), 90.1, 91.8 (C-1), 117.0 (CH Alloc), 132.1
(CH2 Alloc), 155.3 (CꢁO Alloc), 168.3, 168.7,
169.9, 170.3 (CꢁO Ac).
3,4,6-tri-O-acetyl-2-deoxy-a- -glucopyrano-
D
side was obtained by column chromatography
(10–50% EtOAc in petroleum ether) in 67%
yield. 13C NMR (CDCl3): l 20.0 (CH3 Ac),
22.1 (CH3 NHAc), 51.1 (C-2), 61.5 (C-6), 67.2,
67.8, 70.5, (C-3, C-4, C-5), 68.1 (CH2 Allyl),
1H NMR (CDCl3): l showed that the major
compound in this anomeric mixture was the a
anomer. Potassium tert-butanolate (22 mg,
0.20 mmol) was added to a solution of this
sugar (779 mg, 2.0 mmol) in MeOH (6 mL).
After stirring for 6 h, the reaction mixture was
neutralized with Dowex 50W×4 and subse-
quently filtered. The filtrate was concentrated
and the residue was purified by silica gel chro-
matography. The column was eluted with 20%
petroleum ether to 30% MeOH in EtOAc. The
pure compound 14 was obtained in 78% yield.
13C NMR (CD3OD): l 57.3 (C-2), 62.6 (C-
6), 66.5 (CH2 Alloc), 72.1, 72.7 (d.i.), 75.8,
77.7 (C-3, C-4, C-5), 92.8, 97.0 (C-1), 117.6
(CH Alloc), 134.1 (CH2 Alloc).
1
95.9 (C-1, JC,H 170.9 Hz), 117.6 (CH2 Allyl),
132.8 (CH Allyl), 168.8, 170.3, 170.4, (CꢁO
Ac), 173.3 (CꢁO NHAc). Potassium tert-bu-
tanolate (17 mg, 0.15 mmol) was added to a
solution of the acetylated compound (580 mg,
1.5 mmol) in MeOH (15 mL). After stirring
for 5 h, the reaction mixture was neutralized
with Dowex 50W×4 and the solids were
filtered. The filtrate was concentrated and the
crude product was purified by silica gel chro-
matography (0–40% MeOH in EtOAc) to give
the pure a-glycoside 13 in 73% yield.
13C NMR (CD3OD): l 22.6 (CH3 NHAc),
55.2 (C-2), 62.5 (C-6), 69.0 (CH2 Allyl), 70.6,
72.1, 72.6, (C-3, C-4, C-5), 97.5 (C-1), 117.5
(CH2 Allyl), 135.3 (CH Allyl), 173.5 (CꢁO
NodC enzyme preparation.—The E. coli
strain BL21(DE3) containing a plasmid carry-
ing the cloned nodC gene from M. loti strain
E1R [18] was used as the source of NodC
protein. Expression of nodC was induced in
1
NHAc). H NMR (CD3OD): l 1.98 (s, 3 H,
CH3 NHAc), 3.31–4.29 (m, 8 H, H-2, H-3,
H-4, H-5, CH2 Allyl), 4.83 (d, 1 H, H-1, J1,2
3.4 Hz), 5.01–5.36 (m, 2 H, CH2 Allyl), 5.84–

E. Kamst et al. / Carbohydrate Research 321 (1999) 176–189
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189
exponentially growing cells by the addition of
isopropylthiogalactopyranoside (IPTG), fol-
lowed by disruption of the bacteria using son-
ication and isolation of membranes by
differential centrifugation as described previ-
ously [18]. Membrane pellets were resus-
pended in buffer (100 mM Tris–HCl, 20 mM
MgCl2, pH 7.5) in 1/100 of the original culture
volume and stored at −80 °C until use.
Enzyme reactions.—Reaction mixtures con-
tained 1 mM acceptor, 10 mM UDP-
[14C]GlcNAc (0.05 mCi, 230 mCi/mmol;
Amersham International, Amersham, UK),
and 10 mL of membranes in a final reaction
volume of 20 mL. After an incubation time of
30 min at 20 °C, reactions were stopped by
boiling for 2 min, followed by centrifugation
in a micro centrifuge for 5 min. Reaction
products were then analyzed by loading 2 mL
of the supernatant onto an NH2-TLC plate
which was developed using 60% MeCN. Spots
were visualized using a PhosphorImager in
combination with the ImageQuant software
(Molecular Dynamics, Sunnyvale, CA).
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The authors wish to thank Drs S.H. van
Leeuwen and M. Palcic for supplying us with
compounds 1 and 5, respectively. In addition
we wish to acknowledge Dr P.P.G. van der
Holst for his help in part of the experiments
and Dr R. Lagas for his gift of allyl 3,4,6-tri-
O-acetyl-2-deoxy-2-phthalimido-b-
ranoside and hexyl 2-acetamido-2-deoxy-
-glucopyranoside. This
D
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4,6-di-O-benzyl-b-
D
work was funded by the Netherlands Organi-
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