Chemical synthesis of analogs of the glycopeptide contulakin-G, an analgetically active conopeptide from Conus geographus

Article abstract of DOI:10.1016/j.carres.2005.11.010

Cone snails are marine predators that use immobilizing venoms for catching prey. Chemical analysis of the venoms has revealed a variety of biologically active small and intermediate size peptides rich in post-translational modifications (modified amino ac

Full text of DOI:10.1016/j.carres.2005.11.010

Carbohydrate
RESEARCH
Carbohydrate Research 341 (2006) 9–18
Chemical synthesis of analogs of the glycopeptide contulakin-G,
an analgetically active conopeptide from Conus geographus
Ulrika Westerlind and Thomas Norberg*
Department of Chemistry, Swedish University of Agricultural Sciences, SE-750 07 Uppsala, Sweden
Received 17 August 2005; received in revised form 31 October 2005; accepted 11 November 2005
Available online 1 December 2005
Abstract—Cone snails are marine predators that use immobilizing venoms for catching prey. Chemical analysis of the venoms has
revealed a variety of biologically active small and intermediate size peptides rich in post-translational modifications (modified amino
acids, glycosylation). The glycopeptide contulakin-G (pGlu-Ser-Glu-Glu-Gly-Gly-Ser-Asn-Ala-[b-D-Galp-(1!3)-a-D-GalpNAc-
(1!]Thr-Lys-Lys-Pro-Tyr-Ile-Leu-OH) is a potent analgesic from Conus geographus venom. The in vivo activity of synthetic
contulakin-G was previously found to be significantly higher compared to that of a peptide lacking the glycan. In order to further
investigate the importance of the glycan, we have now synthesized analogs of contulakin-G where the glycan chain O-linked to threo-
nine has been altered either to b-D-Galp-(1!3)-b-D-GalpNAc-, a-D-Galp-(1!3)-a-D-GalpNAc-, or b-D-Galp-(1!6)-a-D-Galp-
NAc-. The glycopeptides were assembled on a Wang resin using commercially available Fmoc amino acids and synthetically
prepared Fmoc-protected threonine derivatives carrying O-acetyl protected sugar chains. The final products were thoroughly char-
acterized by NMR and mass spectroscopy.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Keywords: Contulakin-G; Toxin; Peptide; Synthesis; Glycosylated; Glycopeptide; N-Acetyl galactosamine; Galactose
1. Introduction
getic activity of synthetic contulakin-G was found to be
significantly higher compared to that of its synthetic
unglycosylated counterpart. Similar observations4–8 of
differences in biological activity between glycosylated
and non-glycosylated peptides or proteins have been
made also in other fields. These observations all touch
on the general question of the function of glycans of gly-
copeptides and glycoproteins in Nature. We believe that
the Conus glycopeptides are among the best model com-
pounds available to address this question, since they
have distinct biological activities and are comparatively
small molecules which can be readily prepared and mod-
ified by chemical synthesis. In addition, the synthetic
products can, as has been shown9 for contulakin-G, be
subjected to conformational studies by NMR spectro-
scopy, which opens up possibilities to understand struc-
ture–activity relationships on a more fundamental
molecular level. As part of a program directed at synthe-
sis of glycosylated conopeptides and analogs, we have
previously10 synthesized contulakin-G analogs where the
sugar chain was either shortened (to a-D-GalpNAc-)
Cone snails are a group of interesting marine predators
who catch their prey with the aid of immobilizing ven-
oms. Chemical analysis of the venoms has revealed a
great variety of small and intermediate size peptides,
each with a distinct and very interesting neurobiological
activity.1 The peptides are rich in post-translational
modifications, such as sulfation, hydroxylation (of pro-
line), bromination (of tryptophan), c-carboxylation (of
glutamic acid) and O-glycosylation (of serine and threo-
nine). We have recently reported2 studies of contulakin-
G, a glycopeptide from Conus geographus venom.
Analysis and chemical synthesis confirmed the structure
of contulakin-G as pGlu-Ser-Glu-Glu-Gly-Gly-Ser-
Asn-Ala-[b-D-Galp-(1!3)-a-D-GalpNAc-(1!]Thr-Lys-
Lys-Pro-Tyr-Ile-Leu-OH (Scheme 1). Contulakin-G is a
potent analgesic in a number of pain models3. The anal-
*
Corresponding author. Tel.: +46 18 671578; fax: +46 18 673477;
e-mail: thomas.norberg@kemi.slu.se
0008-6215/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2005.11.010

10
U. Westerlind, T. Norberg / Carbohydrate Research 341 (2006) 9–18
Scheme 1.
or shifted in position (to Ser 7). Both these analogs, as
well as a synthetic unglycosylated analog, showed signif-
icantly lower analgetic activity in a mouse tail flick
latency assay.10 In an NMR study,9 subtle differences in
conformational preferences between these analogs and
native contulakin-G were found. We have now synthe-
sized three new analogs (21, 23, and 25) of contulakin-
G where either an anomeric linkage or the intersugar
glycosylation position is altered (see Scheme 1). In com-
pound 21, the disaccharide linkage to Thr is changed
from the native a configuration to b; in compound 23
the interglycosidic linkage is changed from the native
b configuration to a; and in compound 25, the original
b-(1!3) intersugar glycosylation position is changed
to b-(1!6). This communication reports the chemical
synthesis and spectroscopic characterization of 21, 23,
and 25. Biological studies and conformational analysis
of the synthesized glycopeptides will be reported else-
where in the near future.
sponding derivatives 8 and 18 were novel compounds,
and were therefore synthesized as described below.
Derivative 8 (Scheme 2): Brief treatment of phenyl
2,3,4,6-tetra-O-benzyl-1-thio-b-D-galactopyranoside12
(1) with bromine in dichloromethane produced the cor-
responding glycosyl bromide, which was reacted in situ
with
4-methylphenyl
2-azido-4,6-O-benzylidene-2-
deoxy-1-thio-b-D-galactopyranoside11 (2) and tetraethyl-
ammonium bromide in the presence of molecular
sieves to give the a-linked disaccharide derivative 3
(63%). Glycosylation (DMTST) of Boc-L-threonine
t-butyl ester13 with 3 gave, as a major product, the
a-linked disaccharide–threonine derivative 4 (31%). For
reasons unclear this yield could not be improved, de-
spite several attempts. Catalytic hydrogenation (Pd/C)
followed by acetylation with acetic anhydride in pyri-
dine gave 6 (64%), which was treated with TFA in
dichloromethane to give 7, which was reacted in situ
with Fmoc-OSu to give the disaccharide–threonine
derivative 8 (77%), suitable for the use in peptide
synthesis.
2. Results and discussion
Derivative 18 (Scheme 3): Treatment of 911 with 2,2-
dimethoxypropane and p-toluenesulfonic acid in aceto-
nitrile gave 3,4-isopropylidene acetal 10 as the major
product (39%). Glycosylation of 10 with 1114 and silver
triflate at ꢀ30 ꢁC gave the b-linked disaccharide deriva-
2.1. Synthesis of glycosylated Fmoc amino acids
The disaccharide–Fmoc threonine derivative 19 was
available from previous work11, whereas the corre-

U. Westerlind, T. Norberg / Carbohydrate Research 341 (2006) 9–18
11
˚
Scheme 2. Reagents and conditions: (i) Br2; (ii) Et4NBr, 4 A MS, CH2Cl2/DMF, 63%; (iii) DMTST, N-Boc-threonine t-butylester, 31%; (iv) Pd(C),
H2, EtOH; (v) Ac2O, pyridine, 64%; (vi) TFA/CH2Cl2; (vii) Fmoc-OSu/Na2CO3/H2O/dioxane, 77%.
Scheme 3. Reagents and conditions: (i) Me2C(OMe)2, CH3CN, TsOH, 39%; (ii) AgOTf, toluene/CH2Cl2, 86%; (iii) DMTST, N-Fmoc-threonine
phenacylester, 56%; (iv) HOAc, H2O, 80 ꢁC; (v) Ac2O, pyridine; (vi) AcSH, rt, 67%; (vii) Zn, HOAc, 89%.
tive 12 (86%). Glycosylation of Fmoc-L-threonine
phenacyl ester11 with 12 promoted by DMTST15 gave,
as a major product, the a-linked disaccharide–threonine
derivative 14 (56%). A compound tentatively assigned as
the b-linked isomer was also isolated (20%). Treatment
of 14 with successively, aq acetic acid (to remove 3,4-iso-
propylidene acetal), pyridine/acetic anhydride, and thio-
acetic acid (to convert the azido function to acetamido)
gave compound 17 (67%). Finally, phenacyl ester was
removed by treatment with zinc in acetic acid to give
the disaccharide–threonine derivative 18 (89%), suitable
for use in peptide synthesis.
2.2. Glycopeptide synthesis
Solid-phase synthesis of 21, 23, and 25 was carried out
manually using Fmoc chemistry, with t-butyl ether side
chain protection for tyrosine and serine, N-t-butoxycar-
bonyl side chain protection for lysine, and t-butyl ester
side chain protection for glutamic acid. A small glass ves-
sel with an upper glass stopper and a bottom sintered
glass frit was used for the reactions. Agitation was by
rotation of the vessel during synthesis of 21, and by nitro-
gen bubbling up through the bottom frit during synthesis
of 23 and 25. It was noted that the latter agitation mode

12
U. Westerlind, T. Norberg / Carbohydrate Research 341 (2006) 9–18
gave considerably better total yields, mainly because of
less resin leakage from the vessel. Starting with Leu-
Wang resin, the next amino acid (Ileu, 5 equiv), activated
with PyBOP/HOBt (5 equiv) and DIPEA (10 equiv) in
DMF, was added. The Fmoc group was then removed
using 20% piperidine in DMF. The Kaiser ninhydrin
test16 was performed after each coupling and after depro-
tection of the Fmoc group. The coupling/deprotection
cycles were repeated using the appropriate amino acids
until the entire peptide sequence had been assembled.
Coupling of the glycosylated amino acid was performed
with only 2 equiv of activated amino acid. The Kaiser
tests indicated that the reactions had gone virtually to
completion after 2 h except for the Asn and pGlu cou-
plings, where longer reaction times and repetition of the
couplings twice with fresh reagents had to be carried
out. After the last coupling cycle, the resin was subjected
to cleavage conditions (TFA–H2O–thioanisole) and the
released O-acetylated glycopeptide was preliminary puri-
fied. After O-deacetylation with sodium methoxide in
methanol the material was finally purified by RP-HPLC.
The total yields of pure 21, 23, and 25 were 9%, 37%, and
56%, respectively, calculated from the amounts of resin
used. The identity of the glycopeptides was verified by
mass spectroscopy as well as with 1H NMR spectroscopy.
recorded at 303 K with a Bruker DRX 400 for CDCl3
solutions (internal CHCl3, dH 7.26 ppm and dC
77.00 ppm at 303 K), unless otherwise stated. Only
selected NMR data are reported. Assignments were
corroborated by appropriate 2-D experiments. The
HRMS and MALDI-MS spectra were recorded with
Agilent MSD-TOF and Bruker Reflex 3 instruments,
respectively. In the latter case, dihydroxybenzoic acid
(DHB) was used as matrix. Optical rotations [a]D were
measured at room temperature (22–24 ꢁC) with a
Perkin–Elmer 241 polarimeter. TLC was performed
on Silica Gel F254 (Merck, Darmstadt, Germany) with
detection by UV-light and/or by staining with 5% sulfu-
ric acid in ethanol. Column chromatography was per-
˚
formed on Matrex silica gel 60 A (35–70 lm, Amicon)
unless otherwise stated. Semipreparative HPLC were
carried out using a Waters HPLC system consisting
of a 600S controller, a 616 gradient pump, a 481 UV
detector set at 254 nm, and a Nucleosil C-18 column
(10 · 250 mm, 5 lm particle size). Acetonitrile and
0.1% aq TFA was used as solvents. Isolute cartridges
(C-18 EC) were from International Sorbent Technology,
Mid Glamorgan, UK. Molecular sieves (powdered
˚
4 A) were dried at 270 ꢁC/0.5 Torr overnight. Dry
dichloromethane was prepared by distillation from
P2O5. Dimethyl(thiomethyl)sulfonium triflate (DMTST)
was prepared essentially as previously described,15 and
was stored under dry nitrogen at ꢀ20 ꢁC. DMF
(amine-free, for peptide synthesis), N-hydroxybenzo-
triazole (HOBt), and N,N-diisopropylethylamine (DI-
PEA) was from Sigma–Aldrich (St. Louis, USA).
Fmoc-protected amino acids were from Bachem AG
(Bubendorf, Switzerland). Benzotriazole-1-yl-oxy-tris-
pyrrolidinophosphonium hexafluorophosphate (Py-
BOP) and Leu-Wang resin were from Novabiochem/
Merck Biosciences AG (Switzerland). Other reagents
and solvents were purchased with high commercial
quality and were used without further purification
unless otherwise stated.
1
The H NMR chemical shifts measured for the amino
acid residues in the glycosylated peptides 21, 23, and 25
are similar to those9 in the natural peptide contulakin-
G, except for signals from NH and a-H protons near or
at the glycosylation site. The amino acid sequence of
the peptide was confirmed by sequential assignment
using the daN(i, i + 1) NOE connectivities. The site of gly-
cosylation of compounds 21, 23, and 25 was confirmed by
the existence of a NOE between H-1 of GalpNAc and b-
H of Thr-10. The b-glycosidic bond between the glycan
and Thr-10 of 21 was confirmed by an upfield chemical
shift of H-1 of GalpNAc as compared with native contu-
lakin-G, as well as by the observed large (8–10 Hz) cou-
pling constant. The 1!3 linkage of 21 was confirmed
by a NOE interaction between the b-D-Galp anomeric
proton and H-3 of b-D-GalpNAc. The glycosidic bond
between H-1 of the a-D-Galp and H3 of a-D-GalpNAc
of 23 was also verified by a NOE interaction. The down-
field chemical shift as well as the small (3.7 Hz) coupling
constant of H-1 of a-D-Galp of 23 confirmed the presence
of an a-glycosidic bond instead of the natural b. The 1!6
interglycosidic linkage in 25 was confirmed by a NOE
interaction between the b-D-Galp anomeric proton and
H-6 of a-D-GalpNAc.
3.2. 4-Methylphenyl 2-azido-4,6-O-benzylidene-2-deoxy-
1-thio-3-O-(2,3,4,6-tetra-O-benzyl-a-D-galacto-
pyranosyl)-b-D-galactopyranoside (3)
Bromine (1.29 mL, 25.38 mmol) was added to a stirred
and cooled (ice) mixture of 112 (11.87 g, 18.75 mmol)
and dichloromethane (60 mL). After 40 min at 0 ꢁC
cyclohexene (a few drops) was added until color indi-
cated no more change and then 211 (3.0 g, 7.50 mmol),
molecular sieves (13 g), tetraethylammonium bromide
(5.43 g), and DMF (1.50 mL) were added. The mixture
was stirred at rt for 48 h. Pyridine (5 mL) was added
and the mixture was filtered. The filtrate was washed
with aq 1 M sulfuric acid, aq 1 M sodium hydrogen car-
bonate and water, dried, and concentrated. The residue
was purified by column chromatography to give 3
3. Experimental
3.1. General methods
Compounds were concentrated under reduced pres-
sure (bath temperature <40 ꢁC). NMR spectra were

U. Westerlind, T. Norberg / Carbohydrate Research 341 (2006) 9–18
13
(4.17 g, 4.69 mmol, 63%), [a]D +9 (c 0.1, CHCl3); 1H
NMR data: d 5.48 (1H, s, CH benzylidene), 5.17 (1H,
d, J = 3.4 Hz, H-10), 4.94 (1H, d, J = 11.5 Hz, OBn),
4.81 (1H, d, J = 11.5 Hz, OBn), 4.68 (1H, d,
J = 11.5 Hz, OBn), 4.57 (1H, d, J = 11.5 Hz, OBn),
4.53 (1H, d, J = 11.7 Hz, OBn), 4.49 (3H, m, OBn),
4.35 (1H, dd, J = 12.5, 1.5 Hz, H-6a), 4.26 (2H, m, H-
1, H-4), 4.10 (1H, dd, J = 6.6, 5.6 Hz, H-50), 4.04 (2H,
m, H-20, H-30), 3.98 (1H, dd, J = 12.5, 1.5 Hz, H-6b),
3.94 (1H, br d, J < 1 Hz, H-40), 3.88 (1H, dd,
J = 10.0 Hz, H-2), 3.70 (1H, dd, J = 10.0, 3.2 Hz, H-
3), 3.60 (1H, dd, J = 9.8, 6.6 Hz, H-6a0), 3.47 (1H, dd,
J = 9.8, 5.6 Hz, H-6b0), 3.26 (1H, m, H-5). HRMS:
Calcd for C54H59N4O9S: 939.39973. Found: 939.39953
(M+NH4+).
1.119 mmol) in ethanol (50 mL). The mixture was
flushed first with nitrogen and then with hydrogen,
and stirred for 48 h at rt under a hydrogen atmosphere.
After nitrogen flushing, the solution was filtered and
concentrated to give 5. This material was dissolved in
pyridine–acetic acid 2:1 (48 mL) and was stirred for
24 h. The residue was then concentrated and co-concen-
trated with toluene. Column chromatography (toluene–
ethyl acetate 1:1) of the residue gave 6 (635 mg,
1
0.711 mmol, 64%). [a]D +81 (c 0.3, CHCl3); H NMR
data: d 6.54 (1H, d, J = 10.0 Hz, NHThr), 6.14 (1H, d,
J = 10.3 Hz, NHAc), 5.47 (1H, br d, J = 2.9 Hz, H-40),
5.34 (1H, br d, J = 2.7 Hz, H-4), 5.30 (2H, m, H-10,
H-20), 5.10 (1H, dd, J = 10.5, 2.9 Hz, H-30), 4.86 (1H,
br d, J = 3.4 Hz, H-1), 4.67 (1H, ddd, J = 11.0, 10.3,
3.4 Hz, H-2), 4.36 (1H, m, H-6a0), 4.35 (1H, m, H-50),
4.28 (1H, m, CHThrb), 4.25 (1H, m, CHThra), 4.18
(1H, m, H-5), 4.10 (2H, m, H-6ab), 3.99 (1H, dd,
J = 11.0, 2.7 Hz, H-3), 3.94 (1H, dd, J = 9.1Hz, H-
6b0), 2.25 (3H, s, OAc), 2.19 (3H, s, OAc), 2.14 (3H, s,
OAc), 2.06 (3H, s, OAc), 2.05 (3H, s, OAc), 2.00 (3H,
s, NHAc), 1.94 (3H, s, OAc), 1.46 (9H, s, t-Bu), 1.44
(9H, s, t-Bu), 1.33 (3H, d, J = 6.4 Hz, Thr-CH3).
HRMS: Calcd for C39H61N2O21: 893.37613. Found:
893.37643 (M+H+).
3.3. N-tert-Butoxycarbonyl O-[2-azido-4,6-O-benzyl-
idene-2-deoxy-3-O-(2,3,4,6-tetra-O-benzyl-a-D-galacto-
pyranosyl)-a-D-galactopyranosyl]-L-threonine
tert-butylester (4)
A mixture of 3 (3.65 g, 4.103 mmol), N-tert-butoxycar-
bonyl-L-threonine tert-butylester13 (1.69 g, 6.157 mmol)
˚
and powdered 4 A molecular sieves (13 g) in dry dichlo-
romethane–diethyl ether–toluene (27–20–20 mL) was
stirred at rt for 5 min and then DMTST (2.50 g,
9.69 mmol) was added. After 30 min, pyridine
(2.69 mL) was added to quench the reaction. The mix-
ture was diluted with diethyl ether and the solids were
filtered off. The filtrate was washed with aq 2 M sulfuric
acid, aq 1 M sodium hydrogen carbonate, dried (magne-
sium sulfate), and concentrated. Column chromatogra-
phy (toluene–ethyl acetate 10:1) of the residue gave 4
3.5. N-9-Fluoroenylmethoxycarbonyl O-[2-acetamido-
4,6-di-O-acetyl-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl-a-D-
galactopyranosyl)-a-D-galactopyranosyl]-L-threonine (8)
A solution of 6 (550 mg, 0.616 mmol) in dry dichloro-
methane (14 mL) was mixed with TFA (14 mL) and
the mixture was stirred at 35 ꢁC for 24 h, then evapo-
rated and coevaporated with toluene to give crude 7,
which was dissolved in a mixture of dioxane (14 mL)
and H2O (14 mL). Solid Na2CO3 (160 mg, 1.50 mmol)
was added, and the mixture was ultrasonicated for
15 min and was then cooled to 0 ꢁC and Fmoc-OSu
(252 mg, 0.747 mmol) was added and stirring was con-
tinued at rt for 6 h, after which the material was acidi-
fied to pH 1.5 with aq HCl, diluted with water and
extracted three times with chloroform. Column chroma-
tography (gradient from 100% chloroform to 20% meth-
anol in chloroform) of the residue gave 8 (456 mg,
1
(1.37 g, 1.28 mmol, 31%), [a]D +114 (c 0.3, CHCl3); H
NMR data: d 5.41 (2H, m, CH benzylidene, NHThr),
5.28 (1H, d, J = 2.9 Hz, H-10), 5.11 (1H, d, J = 2.9 Hz,
H-1), 4.95 (1H, d, J = 11.5 Hz, OBn), 4.82 (1H, d,
J = 11.7 Hz, OBn), 4.70 (1H, d, J = 11.7 Hz, OBn),
4.58 (3H, m, OBn), 4.51 (2H, abq, J = 12.0 Hz, OBn),
4.39 (2H, m, H-4, CHThrb), 4.22 (1H, dd, J = 12.7,
J < 1, H-6a), 4.17–4.06 (5H, m, H-3, H-20, H-30, H-50,
CHThra), 4.01 (1H, m, H-40), 3.99 (1H, m, H-6b),
3.93 (1H, dd, J = 10.5, 2.9 Hz, H-2), 3.62 (3H, m, H-5,
H-6ab0), 1.51 (9H, s, t-Bu), 1.45 (9H, s, t-Bu), 1.24
(3H, d, J = 6.6 Hz, Thr-CH3). HRMS: Calcd for
C60H76N5O14: 1090.53833. Found: 1090.53684 (M+
NH4+).
1
0.476 mmol, 77%), [a]D +80 (c 0.4, CHCl3); H NMR
data: d 7.77 (2H, d, J = 7.3 Hz, Fmoc), 7.58 (2H, dd,
J = 7.3, 4.9 Hz, Fmoc), 7.41 (2H, dd, J = 7.3 Hz,
Fmoc), 7.30 (2H, ddd, J = 7.3, 9.5, 3.2 Hz, Fmoc),
6.88 (1H, d, J = 9.5 Hz, NHThr), 6.23 (1H, d,
J = 10.3 Hz, NHAc), 5.41 (1H, br d, J = 2.7 Hz, H-40),
5.36 (1H, br d, J = 2.5 Hz, H-4), 5.30 (2H, m, H-10,
H-20), 5.07 (1H, dd, J = 10.8, 2.7 Hz, H-30), 5.00 (1H,
d, J = 3.9 Hz, H-1), 4.61 (1H, dd, J = 10.8, 6.6 Hz,
FmocCH2), 4.55 (1H, dd, J = 11.0, 3.9 Hz, H-2), 4.49
(1H, m, CHThrb), 4.48 (1H, m, CHThra), 4.42 (1H,
3.4. N-tert-Butoxycarbonyl O-[2-acetamido-4,6-di-O-
acetyl-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl-a-D-galacto-
pyranosyl)-a-D-galactopyranosyl]-L-threonine tert-butyl-
ester (6)
A suspension of palladium on carbon (10%, 1.50 g) in
ethanol (50 mL) was added to a solution of 2 (1.20 g,

14
U. Westerlind, T. Norberg / Carbohydrate Research 341 (2006) 9–18
dd, J = 10.8, 6.6 Hz, FmocCH2), 4.33 (1H, dd, J = 11.0,
4.2 Hz, H-6a0), 4.20 (3H, m, H-5, H-50, FmocCH), 4.12
(2H, m, H-6ab), 3.97 (1H, dd, J = 11.0, 2.5 Hz, H-3),
3.89 (1H, dd, J = 11.0, 9.3 Hz, H-6b0), 3.71 (1H, s,
COOH), 2.22 (3H, s, OAc), 2.17 (3H, s, OAc), 2.12
(3H, s, NHAc), 2.07 (6H, s, OAc), 2.01 (3H, s, OAc),
1.94 (3H, s, OAc), 1.29 (3H, d, J = 6.4 Hz, Thr-CH3),
13C NMR data: d 174.79 (OAc), 172.96 (NHAc),
172.04 (COOH), 170.72 (OAc), 170.49 (OAc), 170.34
(OAc), 170.18 (OAc), 170.03 (OAc), 157.24 (NHCOO),
144.25 (FmocAr), 144.20 (FmocAr), 141.58 (FmocAr),
141.58 (FmocAr), 127.97 (FmocAr), 127.91 (FmocAr),
127.29 (FmocAr), 127.18 (FmocAr), 125.06 (FmocAr),
125.06 (FmocAr), 120.19 (FmocAr), 120.19 (FmocAr),
99.54 (H-1), 92.12 (H-10), 76.92 (CHThrb), 68.67
(H-3), 67.69 (H-30), 67.64 (H-40), 67.35 (H-50), 66.61
(H-5), 66.46 (FmocCH2), 65.59 (H-20), 64.94 (H-4),
62.53 (H-6), 62.07 (H-60), 58.64 (CHThra), 47.90
(H-2), 47.61 (FmocCH), 22.44 (NHAc), 21.06 (OAc),
20.92 (OAc), 20.87 (OAc), 20.79 (OAc), 20.78 (OAc),
20.68 (OAc), 18.93 (Thr-CH3). HRMS: Calcd
for C45H55N2O21: 959.32918. Found: 959.32913 (M+
H+).
ture was stirred for another 50 min at ꢀ30 to ꢀ40 ꢁC,
after which TLC (toluene–ethyl acetate 6:4) indicated
no more change. Pyridine (1 mL) was added to neutral-
ize the mixture, and the mixture was poured into diethyl
ether (100 mL). The solid was filtered off and washed
with diethyl ether and dichloromethane. The filtrate
was washed with aq sodium thiosulfate (1 M), aq sulfu-
ric acid (1 M), and aq sodium hydrogen carbonate
(0.5 M), dried and evaporated. The residue was purified
by column chromatography (toluene–ethyl acetate 7:3)
to give 12 (1.35 g, 1.98 mmol, 86%), [a]D +19 (c 0.3,
CHCl3); 1H NMR data: d 7.27 (2H, d, J = 8.4 Hz,
Ph), 7.15 (2H, d, J = 8.4 Hz, Ph), 5.40 (1H, dd,
J = 3.4, 1.0 Hz, H-40), 5.22 (1H, dd, J = 10.4, 8.0 Hz,
H-20), 5.01 (1H, dd, J = 10.4, 3.4 Hz, H-30), 4.58 (1H,
d, J = 8.0 Hz, H-10), 4.30 (1H, d, J = 10.6 Hz, H-1),
4.07 (2H, m, H-4, H-3), 3.36 (1H, dd, J = 10.6, 6.9 Hz,
H-2), 2.35 (3H, s, S–Ph–Me), 2.15, 2.04, 1.98, 1.98 (each
3H, s, OAc). HRMS: Calcd for C30H43N4O13S:
699.25418. Found: 699.25534 (M+NH4+).
3.8. N-(9-Fluorenylmethyloxycarbonyl)-O-[2-azido-3,4-
O-isopropylidene-2-deoxy-6-O-(2,3,4,6-tetra-O-acetyl-b-
D-galactopyranosyl)-a-D-galactopyranosyl]-L-threonine
phenacylester (14)
3.6. 4-Methylphenyl 2-azido-3,4-O-isopropylidene-2-
deoxy-1-thio-b-D-galactopyranoside (10)
A mixture of 12 (0.673 g, 0.987 mmol), N-(9-fluorenyl-
methyloxycarbonyl)-L-threonine phenacylester11 13
p-Toluenesulfonic acid (50–100 mg, enough to make the
mixture acidic to indicator paper) was added to a solu-
tion of 911 (2.63 g, 8.43 mmol) and 2,2-dimethoxypro-
pane (1.45 g, 13.9 mmol) in dry acetonitrile (40 mL).
After 2 h or when TLC (dichloromethane–EtOAc 9:1)
indicated complete reaction, the mixture was neutralized
with pyridine (0.6 mL) and concentrated to a syrup. The
residue was purified by column chromatography
(dichloromethane–ethyl acetate 9:1) to give 10 (1.15 g,
3.27 mmol, 39%), [a]D +18 (c 0.1, CHCl3); 1H NMR
data (DMSO-d6, d for (CD3)2SO = 2.50): d 7.37 (2H,
d, J = 8.4 Hz, Ph), 7.15 (2H, d, J = 8.4 Hz, Ph), 4.85
(1H, br t, J = 5.4 Hz, H-6-OH), 4.67 (1H, d,
J = 10.6 Hz, H-1), 4.15 (2H, m, H-3, H-4), 3.86 (1H,
dd, J = 6.2 Hz, H-5), 3.54 (2H, m, H-6), 3.33 (1H, m,
H-2), 2.27 (s, 3H, S–Ph–Me), 1.31, 1.24 (two s, each
3H, isopropylidene Me).
˚
(0.645 g, 1,40 mmol), powdered 4 A molecular sieves
(3.19 g), dry dichloromethane (6.9 mL), dry diethyl ether
(5.0 mL), and dry toluene (5.0 mL) was stirred at room
temperature for 5 min, then DMTST (0.753 g,
2.92 mmol) was added in portions over 5 h (until TLC
indicated no more change). Pyridine (0.67 mL) was
added to quench the reaction and the mixture was
diluted with diethyl ether. The solid was filtered off and
washed with ether and dichloromethane. The filtrate
was washed with aq sulfuric acid (2 M), aq sodium
hydrogen carbonate (1 M), dried, and evaporated. The
residue was purified by column chromatography (tolu-
ene–ethyl acetate 7:3) to give 14 (0.561 g, 0.552 mmol,
56%), [a]D +6 (c 0.3, CHCl3); 1H NMR data: 7.74–
7.93 (Fmoc aromatics), 7.36–7.65 (phenacyl aromatics),
7.11–7.34 (m, Fmoc aromatics), 5.82 (1H, d, J = 9.4 Hz,
NH-Thr) 5.55 (1H, d, J = 16.7 Hz, phenacyl CH2), 5.38
(1H, d, J = 3.5 Hz, H-40), 5.32 (1H, d, J = 16.7 Hz,
phenacyl CH2), 5.30 (1H, d, J = 3.5 Hz, H-1), 5.23
(1H, dd, J = 10.2, 7.9 Hz, H-20), 5.01 (1H, dd, J =
10.2, 3.5 Hz, H-30), 4.58 (1H, d, J = 7.9 Hz, H-10),
4.53 (1H, br d, a-CH-Thr), 3.52 (1H, dd, J = 7.9,
3.5 Hz, H-2), 2.15, 2.04, 2.04, 1.98 (each 3H, s, OAc),
1.44 (3H, d, J = 6.4 Hz, CH3–Thr). HRMS: Calcd for
C50H57N4O19: 1017.36115. Found: 1017.35995 (M+H+).
A compound assigned (data not shown) as the corre-
sponding b isomer of 14 was also isolated (0.204 g,
0.201 mmol, 20%).
3.7. 4-Methylphenyl-2-azido-3,4-O-isopropylidene-2-
deoxy-6-O-(2,3,4,6-tetra-O-acetyl-b-D-galacto-
pyranosyl)-1-thio-b-D-galactopyranoside (12)
A mixture of 10 (0.81 g, 2.30 mmol), 1114 (0.97 g,
˚
2.36 mmol), powdered 4 A molecular sieves (4 g), dry
toluene (7 mL), and dry dichloromethane (17 mL) was
stirred at rt for 10 min, then cooled to ꢀ30 to ꢀ40 ꢁC
while a solution of silver triflate (1.3 g, 5.06 mmol) in
dry toluene (26 mL) was added (during 5 min). The mix-

U. Westerlind, T. Norberg / Carbohydrate Research 341 (2006) 9–18
15
3.9. N-(9-Fluorenylmethyloxycarbonyl)-O-[2-acetamido-
3,4-di-O-acetyl-2-deoxy-6-O-(2,3,4,6-tetra-O-acetyl-b-D-
galactopyranosyl)-a-D-galactopyranosyl]-L-threonine
phenacylester (17)
J = 7.3 Hz, Fmoc), 7.33 (2H, dd, J = 7.8, 7.3 Hz,
Fmoc), 6.13 (1H, d, J = 9.1 Hz, NHAc), 5.82 (1H, d,
J = 9.78 Hz, NHThr), 5.37 (1H, d, J = 3.2 Hz, H-40),
5.34 (1H, m, H-4), 5.17 (1H, dd, J = 10.5, 7.8 Hz, H-
20), 5.13 (1H, dd, J = 10.5, 2.9 Hz, H-3), 5.03 (1H, d,
J = 3.9 Hz, H-1), 5.00 (1H, d, J = 10.5, 3.2 Hz, H-30),
4.54 (1H, dd, J = 10.8, 6.6 Hz, FmocCH2), 4.48 (1H,
d, J = 7.8 Hz, H-10), 4.46–4.34 (4H, m, H-2, CHThra,
CHThrb, FmocCH2), 4.33 (1H, dd, J = 11.0, 4.2 Hz,
H-6a0), 4.28–4.15 (3H, m, FmocCH, H-5, H-6a0), 4.05
(1H, dd, J = 11.3, 6.6 Hz, H-6b0), 3.89 (1H, dd,
J = 6.6 Hz, H-50), 3.83 (1H, dd, J = 10.7, 4.4 Hz, H-
6a), 3.61 (1H, dd, J = 8.6, 10.8 Hz, H-6b), 2.16 (3H, s,
OAc), 2.13 (3H, s, OAc), 2.05 (6H, s, OAc), 2.03 (3H,
s, NHAc), 2.00 (3H, s, OAc), 1.99 (3H, s, OAc), 1.31
(3H, d, J = 6.1 Hz, Thr-CH3); 13C NMR data: d
172.32 (NHAc), 170.85 (COOH), 170.42 (OAc), 170.19
(OAc), 170.19 (OAc), 170.13 (OAc), 170.09 (OAc),
169.19 (OAc), 156.67 (NHCOO), 143.77 (FmocAr),
143.68 (FmocAr), 141.33 (FmocAr), 141.33 (FmocAr),
127.83 (FmocAr), 127.80 (FmocAr), 127.17 (FmocAr),
127.15 (FmocAr), 125.11 (FmocAr), 124.98 (FmocAr),
120.11 (FmocAr), 120.03 (FmocAr), 101.17 (H-10),
99.37 (H-1), 76.91 (CHThrb), 70.90 (H-50), 70.82 (H-
30), 68.57 (H-20), 68.23 (H-3), 68.19 (H-6), 67.89 (H-4),
67.74 (H-5), 67.15 (FmocCH2), 66.98 (H-40), 61.14 (H-
60), 58.57 (CHThra), 48.09 (H-2), 47.27 (FmocCH),
22.99 (NHAc), 20.75 (OAc), 20.73 (OAc), 20.73 (OAc),
20.64 (OAc), 20.62 (OAc), 20.57 (OAc), 18.50 (Thr-
CH3). HRMS: Calcd for C45H55N2O21: 959.32918.
Found: 959.32925 (M+H+).
A mixture of 14 (0.544 g, 0.535 mmol) and 80% acetic
acid (2.5 mL) was stirred at 90 ꢁC for 2 h, after which
TLC (toluene–ethyl acetate 1:2) detected complete con-
version into a slower-moving compound. The solution
was concentrated and co-concentrated several times
with toluene and once with pyridine. The residue (con-
taining crude 15) was taken up in pyridine–acetic anhy-
dride 2:1 (2.5 mL). After allowing to stand overnight at
rt, the mixture was concentrated and co-concentrated
with toluene several times to give crude 16, which was
dissolved in thioacetic acid (2.5 mL) and the solution
was left at rt for 48 h, after which TLC (toluene–ethyl
acetate 1:2) indicated complete conversion. The mixture
was purified by column chromatography (stepwise gra-
dient: toluene, toluene–ethyl acetate 1:1, and toluene–
ethyl acetate 1:2) to give 17 (0.387 g, 0.359 mmol,
1
67%), [a]D +4 (c 0.2, CHCl3); H NMR data: d 7.74–
7.93 (Fmoc aromatics), 7.36–7.65 (phenacyl aromatics),
7.11–7.34 (m, Fmoc aromatics), 6.00 (1H, d,
J = 10.0 Hz, NHAc), 5.85 (1H, d, J = 9.1 Hz, NH–
Thr), 5.62 (1H, d, J = 16.7 Hz, phenacyl CH2), 5.36
(3H, m, H-1, H-4, H-40), 5.25 (1H, d, J = 16,4 Hz,
phenacyl CH2), 5.16 (2H, m, H-3, H-20), 4.98 (1H, dd,
J = 10.5, 3.5 Hz, H-30), 4.64 (1H, m, H-2), 4.48 (5H,
m, a,b-Thr, FmocCH2, H-10), 1.64, 1.97, 1.98, 2.04,
2.05, 2.12, 2.16, (each 3H, s, Ac), 1.46 (3H, d, J =
6.1 Hz, CH3–Thr). HRMS: Calcd for C53H61N2O22:
1077.37105. Found: 1077.37041 (M+H+).
3.11. General procedures for glycopeptide synthesis
3.10. N-(9-Fluorenylmethyloxycarbonyl)-O-[2-acetamido-
3,4-di-O-acetyl-2-deoxy-6-O-(2,3,4,6-tetra-O-acetyl-b-D-
galactopyranosyl)-a-D-galactopyranosyl]-L-threonine (18)
The solid-phase synthesis of 21, 23, and 25 was carried
out manually using Fmoc chemistry, with t-butyl ether
side chain protection for tyrosine and serine, N-t-butoxy-
carbonyl side chain protection for lysine, and t-butyl
ester side chain protection for glutamic acid. Starting
with Leu-Wang resin (loading level 0.75 mmol/g), the
next amino acid (Ileu, 5 equiv), activated with PyBOP/
HOBt (5 equiv) and DIPEA (10 equiv) in DMF, was
added. The Fmoc group was then removed using
20% piperidine in DMF. The Kaiser ninhydrin test16
was performed after each coupling and after deprotec-
tion of the Fmoc group. The coupling/deprotection cy-
cles were repeated using the appropriate amino acids
until the entire peptide sequence had been assembled.
A small glass vessel with an upper glass stopper and a
bottom sintered glass frit was used for the solid-phase
reactions. Agitation was by rotation of the vessel during
synthesis of 21, and by nitrogen bubbling up through
the bottom frit during synthesis of 23 and 25. After
the last coupling cycle, the resin was subjected to
cleavage conditions (95% TFA, 2.5% thioanisole, and
2.5% H2O) and the released O-acetylated glycopeptide
A mixture of 17 (0.295 g, 0.274 mmol) and 90% acetic
acid (8.6 mL) was stirred and heated to 35 ꢁC, while acti-
vated zinc powder (1.443 g) was added in portions dur-
ing 2 h, after which TLC (ethyl acetate–acetic acid 6:1)
indicated complete reaction. The mixture was filtered
(minding not to let the solids getting too dry on the fil-
ter), washed carefully with acetic acid and ethanol–ethyl
acetate 1:1, and the filtrate was evaporated. The residue
was triturated with ethyl acetate–dichloromethane 1:1
(6 mL), the mixture was filtered, the filtrate was concen-
trated and the residue was purified by column chroma-
tography, eluting first with 1,2-dichloroethane and
then with increasing concentrations of methanol (nine
steps, from 2% to 10% and five steps, from 12% to
20%). The residue was freeze-dried from benzene to give
18 (0.234 g, 0.244 mmol, 89%), [a]D +57 (c 0.1, CHCl3);
1H NMR data: d 7.78 (2H, d, J = 7.8 Hz, Fmoc), 7.63
(2H, dd, J = 7.3, 2.5 Hz, Fmoc), 7.41 (2H, dd,

16
U. Westerlind, T. Norberg / Carbohydrate Research 341 (2006) 9–18
Table 1. 1H NMR chemical shifts (ppm) of compounds 21, 23, and 25 at 295 K in H2O–D2O (95–5%)
Residue
pGlu1
Compound
NH
aH
bH
Others
21
23
25
4.43
4.43
4.42
2.12
2.12
2.10
cCH2 2.58, 2.44
cCH2 2.58, 2.44
cCH2 2.57, 2.45
Ser2
21
23
25
8.49
8.49
8.49
4.50
4.48
4.49
3.89
3.90
3.91
Glu3
Glu4
Gly5
Gly6
Ser7
21
23
25
8.60
8.59
8.59
4.45
4.44
4.45
2.01
2.01
2.01
cCH2 2.49, 2.17
cCH2 2.50, 2.18
cCH2 2.51, 2.17
21
23
25
8.44
8.47
8.46
4.42
4.42
4.41
2.01
2.01
2.01
cCH2 2.49, 2.17
cCH2 2.50, 2.18
cCH2 2.51, 2.17
21
23
25
8.50
8.47
8.50
4.01
4.00
4.01
21
23
25
8.32
8.31
8.31
4.03
4.03
4.03
21
23
25
8.35
8.35
8.36
4.49
4.47
4.45
3.89
3.90
3.91
Asn8
Ala9
Thr10
Lys11
Lys12
Pro13
Tyr14
Ile15
21
23
25
8.52
8.49
8.51
4.77
4.76
4.75
2.86, 2.78
2.87, 2.79
2.87, 2.80
21
23
25
8.11
8.10
8.12
4.36
4.50
4.47
1.41
1.41
1.44
21
23
25
8.08
8.66
8.34
4.48
4.55
4.54
4.27
4.35
4.33
cCH3 1.33
cCH3 1.33
cCH3 1.33
21
23
25
8.09
8.56
8.40
4.35
4.36
4.37
1.77
1.83
1.71
NH2 7.54, cCH2 1.47, dCH2 1.72, eCH2 3.03
NH2 7.55, cCH2 1.47, dCH2 1.68, eCH2 3.02
NH2 7.53, cCH2 1.46, dCH2 1.69, eCH2 3.01
21
23
25
8.38
8.40
8.38
4.58
4.41
4.49
1.80
1.85
1.74
NH2 7.54, cCH2 1.47, dCH2 1.72, eCH2 3.03
NH2 7.59, cCH2 1.48, dCH2 1.74, eCH2 3.05
NH2 7.57, cCH2 1.48, dCH2 1.74, eCH2 3.03
21
23
25
4.41
4.42
4.41
2.25, 1.83
2.28, 1.84
2.27, 1.85
cCH2 2.10, dCH2 3.84, 3.66
cCH2 2.04, dCH2 3.87, 3.67
cCH2 2.03, dCH2 3.86, 3.64
21
23
25
8.14
8.24
8.25
4.59
4.59
4.58
3.02
3.02
3.00
Ar 7.13, 6.84
Ar 7.13, 6.84
Ar 7.13, 6.84
21
23
25
7.96
7.95
7.95
4.15
4.14
4.14
1.81
1.80
1.80
cCH2 1.42, 1.12, cCH3 0.89, dCH3 0.84
cCH2 1.41, 1.12, cCH3 0.88, dCH3 0.84
cCH2 1.41, 1.13, cCH3 0.89, dCH3 0.84
Leu16
21
23
25
8.19
8.25
8.28
4.29
4.30
4.30
1.65
1.66
1.67
cCH2 1.64, dCH3 0.95, 0.90
cCH2 1.65, dCH3 0.96, 0.91
cCH2 1.64, dCH3 0.96, 0.90
was preliminary purified. After O-deacetylation with so-
dium methoxide in methanol, the material was finally
purified by RP-HPLC (for purification details, see be-
low). The synthesized peptides 21, 23, and 25 were char-
acterized by NMR spectroscopy using a Bruker DRX
600 MHz spectrometer equipped with a 2.5 mm micro-
probe. A 5 mg sample of each glycopeptide was dis-
1
solved in 0.10 mL D2O or H2O–D2O (95–5%). 1-D H

U. Westerlind, T. Norberg / Carbohydrate Research 341 (2006) 9–18
17
Table 2. 1H NMR chemical shifts (ppm) for the glycan part of compounds 21, 23, and 25 at 295 K in D2O
Compound
H-1
H-2
H-3
H-4
H-5
H-6
NH
NAc
Gal
21
23
25
4.47
5.14
4.40
3.53
3.83
3.63
3.64
3.72
3.91
3.93
4.00
3.66
3.67
ND
3.66
3.79
ND
3.77
GalNAc
21
23
25
4.57
4.97
4.93
4.02
4.23
4.12
3.88
4.03
3.94
4.20
3.99
4.05
3.71
4.05
4.22
3.65
3.79
4.09, 3.85
8.32
7.57
7.62
2.06
2.07
2.04
NMR and 2-D DQF-COSY, TOCSY, NOESY, and
HSQC-DEPT were recorded at 295 K. For experiments
in D2O, the residual HDO peak was suppressed by pre-
saturation and in H2O–D2O (95–5%) the WATER-
GATE pulse sequence was used. The 2-D spectra were
recorded in phase-sensitive mode using the TIPPI
method. The DQF-COSY, TOCSY, and NOESY spec-
tra were acquired with 4K data points in t2 and 512
increments in t1, and a D1 relaxation time of 1.5 s. In
the TOCSY experiments mixing times of 60 and 80 ms
were used and the NOESY spectra were recorded at a
filtrate was concentrated and triturated with cold diethyl
ether. The solvent was decanted and the precipitate was
dissolved in water and lyophilized. The residue was dis-
solved in water (5 mL) and applied to a C-18 Isolute car-
tridge (4 g, pre-washed with first 20 mL MeOH, then
100 mL of water). The cartridge was washed with
20 mL of water and then the material was eluted with
10–40% acetonitrile and lyophilized to give 20 (39 mg,
0.017 mmol). The material was treated with 0.5 M so-
dium methoxide (0.10 mL) in methanol (1 mL) for 1 h,
neutralized with 0.05 mL TFA and was then lyophilized.
The deacetylated sample was purified by semi-prepara-
tive RP-HPLC using 10–50% acetonitrile in 0.1% aq
TFA to give 21 (19 mg, 0.0092 mmol, 9%). MALDI
MS: Calcd for C88H141N20O37: 2069.98. Found:
1
mixing time of 400 ms. The H NMR chemical shifts
were measured using acetone (d = 2.225) as internal
reference.
1
3.12. b-b-Contulakin (21)
2070.12 (M+H+). H NMR assignments are given in
Tables 1 and 2.
The manual solid-phase synthesis of b-contulakin (21)
was performed starting from Fmoc-Leu-Wang resin
(133 mg, 0.10 mmol). The resin was swelled with dichlo-
romethane for 30 min and washed five times with DMF.
The Fmoc group was removed using 20% piperidine in
DMF (3 · 10 min) and washed five times with DMF.
A mixture of Fmoc protected Ile (5 equiv, 177 mg,
0.5 mmol), PyBOP (260 mg, 0.5 mmol), HOBt (77 mg,
0.5 mmol), and DIPEA (10 equiv, 0.166 mL, 1.0 mmol)
in DMF (2 mL) were then added to the resin and the
mixture was agitated for 2 h. After complete coupling,
the resin was washed five times with DMF. The Fmoc
deprotections and amino acid couplings were then re-
peated according to the above procedure until the entire
peptide sequence had been finished. Coupling of the gly-
cosylated amino acid 1911 was performed with agitation
for 24 h using only 2 equiv of the amino acid and cou-
pling reagent and 4 equiv of DIPEA. The aspargine
and pyroglutamate residues were coupled overnight
and the couplings had to be repeated twice with fresh re-
agents due to sluggish reactions (according to the Kaiser
test).16 After coupling of the pyroglutamate residue, the
resin was washed five times each with DMF, isopropa-
nol, and diethyl ether and then dried overnight. The
material was cleaved from the resin by addition of
3 mL cleavage cocktail (95% TFA, 2.5% thioanisole,
and 2.5% H2O) and agitation for 3 h. The resin was then
filtered off and washed twice with cleavage cocktail. The
3.13. a,a-Contulakin (23)
This compound was synthesized similarly to 21 starting
with Fmoc-Leu-Wang resin (67 mg, 0.050 mmol), using
glycosylated amino acid 8 (96 mg, 0.10 mmol). The gly-
copeptide was cleaved from resin and purified on a C-18
cartridge to give 22 (81 mg, 0.035 mmol). The material
was de-O-acetylated and purified by semi-preparative
RP-HPLC to give 23 (38 mg, 0.0184 mmol, 37%). MAL-
DI MS: Calcd for C88H141N20O37: 2069.98. Found:
2070.36 (M+H+); calcd for C88H140N20O37Na:
2091.96. Found: 2092.40 (M+Na+) 1H NMR assign-
ments are given in Tables 1 and 2.
3.14. 1,6-Contulakin (25)
This compound was synthesized similarly to 21 starting
with Fmoc-Leu-Wang resin (133 mg, 0.10 mmol), using
glycosylated amino acid 18 (192 mg, 0.2 mmol). The gly-
copeptide was cleaved from resin and purified on a C-18
cartridge to give 24 (132 mg, 0.057 mmol). The material
was de-O-acetylated and purified by semi-preparative
RP-HPLC to give 25 (116 mg, 0.056 mmol, 56%). MAL-
DI MS: Calcd for C88H141N20O37: 2069.98. Found:
2070.51 (M+H+); calcd for C88H140N20O37Na:
2091.96. Found: 2092.52 (M+Na+). H NMR assign-
ments are given in Tables 1 and 2.
1

18
U. Westerlind, T. Norberg / Carbohydrate Research 341 (2006) 9–18
5. Andersson, L. K.; Dolphin, G. T.; Kihlberg, J.; Baltzer, L.
Acknowledgements
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6. Delorme, E.; Lorenzini, T.; Giffin, J.; Martin, F.; Jacob-
sen, F.; Boone, T.; Elliott, S. Biochemistry 1992, 31, 9871–
9876.
7. Higuchi, M.; Oheda, M.; Kuboniwa, H.; Tomonoh, K.;
Shimonaka, Y.; Ochi, N. J. Biol. Chem. 1992, 267, 7703–
7709.
8. Corthay, A.; Backlund, J.; Broddefalk, J.; Michaelsson,
E.; Goldschmidt, T. J.; Kihlberg, J.; Holmdahl, R. Eur. J.
Immunol. 1998, 28, 2580–2590.
9. Kindahl, L.; Sandstro¨m, C.; Craig, A. G.; Norberg, T.;
Kenne, L. Can. J. Chem. 2002, 80, 1022–1031.
10. Wagstaff, J.; Kang, J.; Layer, R.; Imperial, J.; Schmidt, K.;
Olivera, B. M.; Craig, A. G.; Sandstro¨m, C.; Norberg, T.,
in preparation.
We thank Dr. Anthony Craig (Salk institute, La Jolla,
CA, USA), for stimulating conversations and helpful
advice, Per Persson (Analytical Chemistry Department,
BioVitrum AB, Stockholm, Sweden) for generous help
with HRMS data, Dr. Frank Lindh (IsoSep AB, Upp-
sala), for advice and help with HPLC purifications,
and Dr. Corine Sandstro¨m, for expert advice and help
with NMR spectroscopy.
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