Synthesis and CD structural studies of CD52 peptides and glycopeptides

Article abstract of DOI:10.1016/j.carres.2008.08.024

The syntheses of five natural and N-terminal acetylated peptides and glycopeptides of the CD52 antigen are described. Solid phase peptide synthesis was employed in the construction of the target compounds from Fmoc-protected commercial amino acids and syn

Full text of DOI:10.1016/j.carres.2008.08.024

Carbohydrate Research 343 (2008) 2894–2902
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis and CD structural studies of CD52 peptides and glycopeptides
*
Benjamin M. Swarts, Yu-Cheng Chang, Honggang Hu, Zhongwu Guo
Department of Chemistry, Wayne State University, 5101 Cass Avenue, Detroit, MI 48202, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 31 May 2008
Received in revised form 21 August 2008
Accepted 25 August 2008
Available online 30 August 2008
The syntheses of five natural and N-terminal acetylated peptides and glycopeptides of the CD52 antigen
are described. Solid phase peptide synthesis was employed in the construction of the target compounds
from Fmoc-protected commercial amino acids and synthetic glycan–asparagine conjugates. Circular
dichroism studies of the synthetic targets showed that they exist as random coils in solution, and no sig-
nificant change in secondary structure was observed when the CD52 peptide was either acetylated at the
N-terminus or glycosylated at the Asn3 residue with a disaccharide or a fucose-containing branched
trisaccharide.
Dedicated to Professor Yongzheng Hui on
the occasion of his 70th birthday
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
CD52
Peptide
Glycopeptide
N-Glycosylation
Circular dichroism (CD)
Solid phase synthesis
1. Introduction
sperm cells.14–16 Although both lymphocyte and sperm CD52 share
an identical 12 amino acid peptide, structural variations in their GPI
Asparagine glycosylation is a ubiquitous co-translational pro-
tein modification that occurs in eukaryotic and some prokaryotic
systems.1 The cellular functions of N-linked glycosylation in
eukaryotes have been extensively studied and include molecular
recognition, immune responses, modulation of protein function,
protein folding, and protein stabilization.2 The N-glycan, an oligo-
saccharide that is covalently linked to asparagine in the sequence
Asn-Xaa-Ser/Thr (where X is any amino acid except proline), has
the ability to affect protein structure and, consequently, activ-
ity.3–5 This observation has provided the impetus to design studies
that combine synthesis and spectroscopic analysis to probe the
effects of N-glycosylation on protein conformation and function.5–10
Such studies require access to pure and structurally defined sam-
ples, a task made challenging by the microheterogeneity of biosyn-
thetic glycoproteins, which generally exist as mixtures of
glycoforms. However, advances in carbohydrate and peptide syn-
thesis have enabled chemists to obtain significant quantities of
homogenous glycopeptides and small glycoproteins for compara-
tive structural analyses.11–13
anchor and N-linked glycan lead to distinct biological functions,
with the former playing a role in the human immune system and
the latter being involved in the human reproductive process.15,17
Lymphocyte CD52 antibodies have been used to treat immune-
related diseases such as leukemia,18 whereas sperm CD52-specific
antibodies have been isolated from infertile women and suggest
the potential for immunocontraceptive development.19,20
Structurally, the two forms of CD52 have the same remarkably
short peptide and exhibit one N-glycosylation site at the Asn3 res-
idue, to which complex types of glycans are linked.15,16 Interest-
ingly, the makeup of the N-linked glycans is one of the main
structural differences between the two forms. For example, bi-
and tri-antennary N-glycans, peripheral fucosylation, and the
a
(2?3)-linkage of sialic acid residues are male-specific modifica-
tions of CD52.17 The structural differences of the N-glycans
between sperm and lymphocyte CD52 seem to have a critical
influence on their bioactivity. Sperm CD52-specific antibodies bind
to the N-glycan of the antigen and may inhibit sperm-egg bind-
ing.19,20 Lymphocyte CD52 antibodies, however, recognize
a
Our interest in the effect of N-glycosylation on protein structure
lies in the human CD52 antigen, a glycosylphosphatidylinositol
(GPI)-anchored glycopeptide that is expressed on lymphocyte and
sequence of the peptide chain,18 suggesting a possible difference
in peptide structure between the two forms of CD52 and therefore
the potential for conformational change induced by N-glycosyl-
ation or GPI anchoring.
The present study aims to compare the structures of non-gly-
cosylated CD52 peptides and their glycosylated forms to determine
* Corresponding author. Tel.: +1 313 577 2557; fax: +1 313 577 8822.
E-mail address: zwguo@chem.wayne.edu (Z. Guo).
0008-6215/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2008.08.024

B. M. Swarts et al. / Carbohydrate Research 343 (2008) 2894–2902
2895
what effect a model N-glycan may have on the conformation of the
CD52 peptide backbone. For this purpose, we prepared the natural
and N-terminal acetylated CD52 peptides 1a and 1b (Fig. 1), as well
for strong base induced b-elimination or epimerization,23 care
must be taken in the selection of protecting groups and overall
synthetic strategy. Thus, partially or fully benzyl ether-protected
oligosaccharide-asparagine conjugates were used in our target
syntheses to facilitate late-stage deprotection under mild condi-
tions, and measures were taken to ensure that acid sensitive func-
tionalities would remain intact throughout each synthesis.
as glycopeptides 2 and 3a–b that contain a disaccharide b-
GlcpNAc(1?4)-b- -GlcpNAc and fucose-containing branched
trisaccharide b- -GlcpNAc(1?4)[ -Fucp(1?6)]-b- -GlcpNAc,
D-
D
a
D
a
-L
D
respectively, using 9-fluorenylmethoxycarbonyl (Fmoc)-based so-
lid phase peptide synthesis (SPPS). Next, circular dichroism (CD)
was used to examine the solution structures of the synthetic tar-
gets and determine whether Asn-N-glycosylation or N-terminal
acetylation resulted in any significant secondary structure changes.
The primary challenge of synthesizing 3a and 3b is the a-fucos-
idic linkage, which is particularly acid sensitive when it is fully
benzylated.22 Although acyl-protected fucosides are more stable
to acidic conditions,24 protection of the 2-O-position by an acyl
group, which has the neighboring group participation effect, must
be avoided in order to achieve
a-fucosylation. Our group devel-
2. Results and discussion
oped the ‘solution-phase synthesis with solid-phase workup’ strat-
egy, which employs unprotected carbohydrate and peptide
building blocks,25,26 to address the problem in an earlier synthesis
of 3b.27 Meanwhile, it was observed that an unprotected fucoside
intermediate could withstand treatment with 20% TFA, which is
strong enough to deprotect peptide side chains. Based on these re-
sults, our group developed a strategy for solid phase glycopeptide
synthesis that is compatible with all glycosidic bonds.28,29 The
strategy uses the 2-chlorotrityl resin,30 a hyper acid sensitive resin
that can release fully protected peptides upon treatment with 10%
acetic acid, as the solid phase support, and it was applied to our
synthesis of the target compounds.
2.1. Synthesis of peptides 1a and 1b
The straightforward preparation of peptides 1a and 1b was car-
ried out using standard Fmoc-SPPS on an automatic peptide syn-
thesizer. Commercially available Ser-loaded resin and Fmoc-
protected amino acids were used to synthesize the target peptide
on a 0.1 mmol scale (84% yield based on weight). A portion of
the resin-bound peptide was N-acetylated, and another portion
was left unaltered at the N-terminus. Concomitant release of the
peptides from the solid support and deprotection of the amino acid
side chains was accomplished using a 95% trifluoroacetic acid (TFA)
aqueous solution containing 2.5% triethylsilane (Et3SiH). Purifica-
tion of the products by RP-HPLC gave the desired peptides 1a
and 1b. All spectroscopic data gathered for 1a were in agreement
with previously reported values,21 and 1b was positively charac-
terized using NMR and MS.
2.2.1. Synthesis of glycopeptide 2
Perbenzylated disaccharide–Asn conjugate 10 was chosen as
the glycosyl amino acid to be employed in Fmoc-SPPS en route to
glycopeptide 2 (Scheme 1). Starting from D-glucosamine, reported
methods were used to arrive at 4,31,32 which was deacetylated
and then benzylated to afford thioglycoside 5. Glycosylation of
2.2. Synthesis of glycopeptides 2, 3a, and 3b
6
33 by 5 was promoted by N-iodosuccinimide (NIS) and triflic acid
(TfOH) to give disaccharide 7 in a 75% yield. After phthalimide re-
moval and subsequent N-acetylation to afford 8, Lindlar’s catalyst
was used under a H2 atmosphere to reduce the anomeric azide to
The most widely adopted method for glycopeptide synthesis is
solid phase synthesis. However, due to the acid lability of O-glyco-
sidic bonds, particularly the fucosidic linkage,22 and the potential
H2N
O
HO
HO
O
OH
OH
OH
O
O
O
O
O
O
H
N
H
N
H
H
RHN
N
N
N
OH
N
H
N
H
N
H
N
H
N
H
N
H
O
O
O
O
O
O
O
HO
OH
O
NH2
H2N
1a
1b
R = H
R = Ac
H2N
O
O
HO
HO
O
OH
OH
OH
O
O
O
O
O
O
H
N
H
N
H
H
RHN
N
N
N
OH
N
H
N
H
N
H
N
H
N
N
H
H
O
O
O
O
O
O
O
HO
OH
NH
NHAc
H2N
O
2
R = Ac, R' = H
3a
3b
R = H, R' = α-L-fucopyranosyl
R = Ac, R' = α-L-fucopyranosyl
R'O OH
O
NHAc
O
HO
OH
OH
O
H3C
OH
OH
HO
α
( -L-fucopyranosyl)
Figure 1. CD52 peptide and glycopeptide synthetic targets.

2896
B. M. Swarts et al. / Carbohydrate Research 343 (2008) 2894–2902
give a glycosyl amine, which immediately underwent amide bond
formation using the active ester Fmoc-Asp(OBt)-OtBu to furnish 9.
Finally, treatment of the product with 20% TFA to remove the C-
terminal t-butyl group resulted in the desired disaccharide–Asn
conjugate 10.
13. Release of the glycopeptide from the solid support with 10%
acetic acid was followed by palladium-catalyzed N-glycan deben-
zylation. Finally, 20% TFA was used to deprotect the amino acid
side chains to produce the target glycopeptide 2, which was puri-
fied by RP-HPLC.
With glycosyl amino acid 10 in hand, solid phase construction
of glycopeptide 2 was commenced (Scheme 2). The preparation
of resin-linked nonapeptide 11 was carried out on an automated
peptide synthesizer using standard Fmoc-SPPS chemistry starting
from Ser-loaded 2-chlorotrityl resin. Coupling of the benzotriazole
ester of 10 with 11 was completed manually. In this case, only
1.5 equiv of 10 was used in the synthesis. The mixture of 11 and
the active ester of 10 was shaken under an argon atmosphere over-
night to give 12. The subsequent peptide chain elongation and N-
terminal acetylation followed to afford resin-bound glycopeptide
2.2.2. Synthesis of glycopeptides 3a and 3b
Partially benzylated trisaccharide–Asn conjugate 22 was chosen
as the glycosylated asparagine to be employed in Fmoc-SPPS en
route to glycopeptides 3a and 3b (Scheme 3). Our previous studies
suggested that the remaining free hydroxyl groups in 22 would
remain inert during the peptide elongation process.25 Glycosyl azide
20 was previously prepared by Shao et al.,27 while a slightly
shorter pathway leading to 20 was developed in the work presented
herein. Starting from
D-glucosamine, p-methoxybenzylidene acetal
OH
OAc
OBn
O
1. NaOMe, MeOH
ref.
O
O
2. NaH, BnBr
59%
HO
HO
AcO
AcO
BnO
BnO
OH
STol
NPhth
STol
NPhth
OBn
O
NH2
4
5
HO
N3
NPhth
NIS, TfOH
BnO
75%
N3
OBn
O
6
OBn
O
FmocHN
COOR 1. Lindlar's, H2
2. Fmoc-Asp(OBt)-OtBu
OBn
O
BnO
BnO
BnO
BnO
BnO
O
H
O
O
N
BnO
NHAc
BnO
70%
R
NHAc
O
R
R = NPhth (7)
R = NHAc (8)
Ethylenediamine;
Ac2O, pyr. (91%)
R = tBu (9)
R = H (10)
20% TFA (96%)
Scheme 1. Synthesis of disaccharide–Asn conjugate 10.
H2N-Ser-2ClTrt
tBu
H2N-Asp-Thr-Ser-Gln-Thr-Ser-Ser-Pro-Ser-2ClTrt
Fmoc-
SPPS
tBu tBu tBu Trt tBu tBu tBu
tBu
11
10
, DCC, HOBt, NMP
O
C
FmocHN
BnO
BnO
BnO
BnO
Asp-Thr-Ser-Gln-Thr-Ser-Ser-Pro-Ser-2ClTrt
O
O
H
N
O
tBu tBu tBu Trt tBu tBu tBu
tBu
BnO
NHAc
12
NHAc
O
Peptide elongation
AcHN
Gly
Gln
N-terminal acetylation
O
Trt
HN
C
BnO
BnO
BnO
BnO
Asp-Thr-Ser-Gln-Thr-Ser-Ser-Pro-Ser-2ClTrt
O
O
H
N
O
tBu tBu tBu Trt tBu tBu tBu
tBu
BnO
13
NHAc
NHAc
O
41% (est.) CH2Cl2/AcOH/TFE (8:1:1)
78%
42%
Pd-C, H2, CH2Cl2/MeOH
20% TFA, Et3SiH, CH2Cl2
O
2ClTrt = Peptide
O
resin
2
Cl
Scheme 2. Solid phase synthesis of glycopeptide 2.

B. M. Swarts et al. / Carbohydrate Research 343 (2008) 2894–2902
2897
1434 was synthesized in four steps. 3-O-Benzylation of 14 followed
by regioselective acetal ring opening gave glycosyl acceptor 15 di-
rectly. The resultant p-methoxybenzyl (PMB) group on the 6-O-po-
sition can be easily and selectively removed under mild conditions.
Alcohol 15 underwent glycosylation with glycosyl bromide 1635
in the presence of silver triflate (AgOTf). The disaccharide product
contained a small quantity of an inseparable impurity, so full char-
acterization of the products was completed after acidic removal of
OH
OPMB
1. NaH, BnBr
pMPh
O
ref.
ref.
2. 1.0 M HCl, NaBH3CN
O
O
O
HO
HO
O
HO
OAc
OH
N3
14 NPhth
N3
HO
BnO
71%
O
NH2
D-Glucosamine
15 NPhth
AcO
Br
16 NPhth
1. AgOTf
AcO
69%
OAc
HO
OH
BnO
OH
O
OBn
O
2. 5% TFA
AcO
AcO
OH
OH
OBn
SEt
O
O
O
N3
BnO
L-Fucose
18
NPhth
17
NPhth
85%
CuBr2, Bu4NBr
BnO
O
OBn
BnO
OBn
OBn
O
O
OBn
HO
FmocHN
COOR
1. Lindlar's, H2
O
HO
2. Fmoc-Asp(OBt)-OAll
R'O
O
H
O
O
HO
O
R
N
R'O
BnO
NHAc
O
R
72%
O
R'O
N3
NHAc
BnO
O
21
R = All (
)
Pd(PPh3)4,
N-methylaniline (78%)
22
R = H (
)
19
R = NPhth, R' = Ac (
R = NHAc, R' = H (
)
Ethylenediamine;
20
)
Ac2O, pyr; NaOMe (86%)
Scheme 3. Synthesis of trisaccharide–Asn conjugate 22.
11
22, DCC, HOBt, NMP
BnO
O
OBn
O
OBn
O
C
FmocHN
HO
O
Asp-Thr-Ser-Gln-Thr-Ser-Ser-Pro-Ser-2ClTrt
tBu tBu tBu Trt tBu tBu tBu
HO
O
H
O
tBu
HO
N
BnO
NHAc
23
NHAc
O
Peptide elongation
20% Piperidine
NH2
BnO
O
OBn
Gly
Gln
HN
OBn
O
C
Trt
O
HO
HO
HO
O
Asp-Thr-Ser-Gln-Thr-Ser-Ser-Pro-Ser-2ClTrt
tBu tBu tBu Trt tBu tBu tBu
O
H
O
tBu
N
BnO
NHAc
24
NHAc
O
Ac2O, MeOH
43%
CH2Cl2/AcOH/TFE (8:1:1)
CH2Cl2/AcOH/TFE (8:1:1)
40%
87%
31%
Pd-C, H2, CH2Cl2/MeOH
20% TFA, Et3SiH, CH2Cl2
85% Pd-C, H2, CH2Cl2/MeOH
28%
20% TFA, Et3SiH, CH2Cl2
3a
3b
Scheme 4. Solid phase synthesis of glycopeptides 3a and 3b.

2898
B. M. Swarts et al. / Carbohydrate Research 343 (2008) 2894–2902
the PMB group. The resulting alcohol 17 was fucosylated by 1836
stereoselectively according to the conditions (CuBr2–n-Bu4NBr)
set forth by Lemieux et al.37 The transformation from 19 to 20
was achieved in a 3-step sequence, including removal of the N-
phthalyl and O-acetyl groups, acetylation, and then O-deacetyla-
tion. O-Deacetylation at this stage was preferential due to potential
complications in late-stage basic deprotection. Next, Lindlar’s cat-
alyst was employed under a H2 atmosphere to reduce the anomeric
azide and produce a glycosyl amine, which underwent amide bond
formation with the active ester Fmoc-Asp(OBt)-OAll to furnish 21.
In contrast to the previous trisaccharide–Asn conjugate,27 in which
the carboxylic group was protected by a t-butyl group, an allyl
group was used to protect the carboxylic group in 21.38 The allyl
ester could be deprotected without acid, thus allowing fucose to
remain benzylated. Indeed, treatment of 21 with Pd(PPh3)4 and
N-methylaniline resulted in the desired trisaccharide–Asn conju-
gate 22, which was subsequently used in Fmoc-SPPS.39
Fmoc-SPPS construction of glycopeptides 3a and 3b (Scheme 4)
began with the manual coupling of the benzotriazole active ester of
glycosyl-Asn conjugate 22 to the 2-chlorotrityl-resin anchored
nonapeptide 11. The resulting glycodecapeptide was then elon-
gated at the N-terminus by manual Fmoc-SPPS to give fully
protected, resin-bound glycopeptide 24. Both the natural and N-
terminal acetylated trisaccharide glycopeptides, 3a and 3b, were
accessible from 24. Glycopeptide 3a was obtained via cleavage of
24 from the solid support followed by palladium-catalyzed N-gly-
can debenzylation and TFA-mediated side chain deprotection as
described above. Care was taken during the debenzylation of 3a
to use aldehyde-free solvents in order to prevent N-terminal meth-
ylation via reductive amination. After the benzyl protecting groups
were removed, the fucosidic linkage became stable to 20% TFA
treatment, which was used to achieve amino acid side chain depro-
tection. Glycopeptide 3b was afforded by first carrying out solid
phase N-terminal acetylation, then completing the same protocols
of glycopeptide release and global deprotection described for 3a.
Purification by RP-HPLC gave the desired glycopeptides 3a and
3b. Characterization of 3a was carried out using NMR and MS,
while all spectroscopic data gathered for 3b were in agreement
with previously reported values.27
2.3. CD structural studies
The aqueous solution conformations of the synthetic CD52 pep-
tides and glycopeptides 1–3 were studied using CD spectroscopy,
which has previously been used to probe the effects of glycosyl-
ation on peptide structure.40–42 The CD spectra of 1a, 1b, 2, 3a,
and 3b are very similar with peak minima at 196, 201, 201, 198,
and 200 nm, respectively (Fig. 2). These features are suggestive of
random coil conformations for all derivatives as clearly described
in the literature.40,43 It was thus concluded that, in this case, nei-
ther N-glycosylation nor N-terminal acetylation led to any ordered
secondary conformation of the CD52 peptide backbone.
2.4. Conclusion
The free peptide of the human CD52 antigen and four N-terminal
acetylated and/or Asn-glycosylated derivatives were synthesized
by SPPS. Benzylated glycosyl asparagine conjugates were employed
as building blocks in the solid phase synthesis of glycopeptides. To
avoid strong acid treatment that may affect the
a-fucosidic bond,
2-chlorotrityl resin, which is particularly acid-labile, was used as
the solid phase support so that the glycopeptides could be released
under mildly acidic conditions compatible with all glycosidic link-
ages. CD structural studies of the synthetic peptides and glycopep-
tides led us to conclude that neither N-glycosylation nor N-terminal
acetylation had a significant influence on the random coil confor-
mation of the CD52 peptide, even though the fucose-containing
branched trisaccharide is relatively sterically hindered. We believe
that this is probably a result of the unique structure and properties
of the CD52 peptide, as it is extremely short and hydrophilic. How-
ever, the conformation of the CD52 antigen when it is GPI-anchored
onto the cell surface is still unknown.
3. Experimental
3.1. General methods
1H NMR spectra were recorded at 400 or 500 MHz with chemi-
cal shifts reported in ppm (d) downfield from tetramethylsilane
(TMS) or relative to CHCl3 (7.26 ppm) unless otherwise noted. Cou-
pling constants (J) are reported in hertz (Hz). 13C NMR spectra were
recorded at 125 MHz. Thin layer chromatography (TLC) was per-
formed on Silica Gel GF254 plates with detection by UV or charring
with a 5% H2SO4 in EtOH solution. Molecular sieves 4 Å (4 Å MS)
were dried in high vacuum at 170–180 °C for 6–10 h immediately
before use. Anhydrous solvents were obtained from a solvent puri-
fication system, while commercial anhydrous reagents were used
without further purification. Automated peptide synthesis was car-
ried out on an Applied Biosystems 433A peptide synthesizer using
fresh reagents and solvents purchased from Applied Biosystems.
2,5-Dihydroxybenzoic acid (DHB) was used as the MALDI TOFMS
matrix, and samples were dissolved in either H2O–CH3CN (1:1)
containing 1% TFA or CH2Cl2–MeOH (1:1) for unprotected and fully
protected compounds, respectively.
3.2. CD52 peptide 1b
Resin-bound, fully protected CD52 peptide 1a21 was subjected
to acetylation conditions (pyridine–Ac2O 2:1) for 4 h. After the resin
was washed thoroughly and filtered, the desired N-terminal acety-
lated peptide 1b was released by treatment with 95% aqueous TFA
containing 2.5% Et3SiH as the cation scavenger. After precipitation
by cold ether and lyophilization, 1b was purified by RP-HPLC (Supe-
lco Discovery C18, 250 Â 10 mm, eluent 5% CH3CN in H2O, 2 mL/
min, tR = 14.9 min). 1H NMR (500 MHz, D2O, DHO at d 4.79 as
Figure 2. CD spectra of 1a, 1b, 2, 3a, and 3b in H2O at room temperature. The molar
ellipticities of 1a, 1b, 2, 3a, and 3b were normalized from concentrations of
6.6 Â 10À2
,
3.2 Â 10À2
,
8.0 Â 10À2
,
6.2 Â 10À2
,
and 9.2 Â 10À2 mM, respectively.
Each curve represents the average of five scans.

B. M. Swarts et al. / Carbohydrate Research 343 (2008) 2894–2902
2899
reference): d 4.72 (t, J = 7.0 Hz, 1H), 4.54–4.43 (m, 4H), 4.41–4.39
(m, 3H), 4.35–4.31 (m, 2H), 4.25 (dd, J = 5.0, 6.5 Hz, 1H), 3.94–
3.73 (m, 11H), 2.95 (dd, J = 6.5, 17.0 Hz, 1H), 2.87 (dd, J = 6.0,
15.5 Hz, 1H), 2.85 (dd, J = 7.0, 17.0 Hz, 1H), 2.77 (dd, J = 8.0,
15.5 Hz, 1H), 2.40–2.29 (m, 5H), 2.21–2.08 (m, 2H), 2.07 (s, 3H),
2.05–1.96 (m, 5H), 1.22 (d, J = 2.0 Hz, 3H), 1.20 (d, J = 1.5 Hz, 3H);
13C NMR (D2O, 125 MHz): d 178.04, 177.99, 175.16, 174.97,
174.71, 174.54, 174.00, 173.61, 173.34, 173.18, 172.38, 172.27,
172.09, 172.00, 171.91, 171.53, 170.03, 67.19, 67.08, 61.74, 61.28,
61.03, 60.93, 60.74, 59.46, 59.18, 56.13, 56.05, 55.58, 53.85, 53.41,
53.37, 50.69, 48.26, 42.70, 36.17, 36.02, 31.23, 31.15, 29.52, 26.87,
26.72, 24.70, 21.90, 18.92, 18.87; HR ESIMS: [M+Na]+ calcd for
C47H75N15O25Na, 1272.4956; found: m/z 1272.4932.
Na2SO4, filtered, and concentrated under vacuum. The residue was
purified by silica gel chromatography to give 8 as white foam
(156 mg, 91%). NMR data of 8 were consistent with literature-
reported values.44 MALDI TOFMS: [MÀN2+Na]+ calcd for
C51H57N3O10Na, 894.4; found: m/z 895.0; [M+Na]+ calcd for
C51H57N5O10Na, 922.4; found: m/z 922.8.
a
c
3.6. N -(9-Fluorenylmethoxycarbonyl)-N -[2-acetamido-3,4,6-
tri-O-benzyl-2-deoxy-b- -glucopyranosyl)-(1?4)-2-acetamido-
D
3,6-di-O-benzyl-2-deoxy-b-D-glucopyranosyl]-L-asparagine tert-
butyl ester (9)
To a solution of Fmoc-Asp-OtBu in CH2Cl2 (10 mL) and NMP
(1 mL) were subsequently added HOBt and DCC. The mixture was
stirred at rt for 1 h to give the active ester. Meanwhile, a mixture
of 8 and Lindlar’s catalyst in CH2Cl2–MeOH was stirred at rt for
2.5 h under an H2 atmosphere. After filtration through Celite and
concentration in vacuum, the resulting residue was dissolved in
CH2Cl2 (2 mL), and then the freshly prepared active ester solution
was added (DCU was filtered off before addition). The mixture
was stirred at rt overnight, concentrated, and purified directly by
silica gel chromatography to give 9 as white foam (70%). 1H NMR
(500 MHz, CD3OD–CDCl3 1:5): d 7.71 (d, 2H, J = 8.0 Hz), 7.56 (d,
2H, J = 7.5 Hz), 7.40–7.06 (m, 32H), 4.79–4.67 (m, 4H), 4.61–4.53
(m, 3H), 4.51 (d, 1H, J = 10.5 Hz), 4.42 (t, 1H, J = 5 Hz), 4.40–4.31
(m, 5H), 4.23 (t, 1H, J = 7.5 Hz), 4.16 (t, 1H, J = 7.0 Hz), 4.04 (t, 1H,
J = 7.0 Hz), 3.96 (t, 1H, J = 8.5 Hz), 3.84 (t, 1H, J = 10.0 Hz), 3.64–
3.49 (m, 7H), 3.49–3.41 (m, 2H), 3.28–3.23 (m, 1H), 2.77 (ddd,
2H, J = 16 Hz, 3.5 Hz), 1.78 (s, 3H), 1.73 (s, 3H), 1.37 (s, 9H); MALDI
TOFMS: [M+Na]+ calcd for C74H82N4O15Na, 1289.6; found: m/z
1289.9.
3.3. p-Methylphenyl 3,4,6-tri-O-benzyl-2-deoxy-2-phthalimido-
1-thio-b-D-glucopyranoside (5)
After 4 (5.00 g, 9.23 mmol) was treated with NaOMe in metha-
nol (0.05 M, 40 mL) at rt for 2 h, the solution was neutralized to
pH 6–7 by Amberlyst H+ resin. After filtration of the resin, the fil-
trate was concentrated to afford a white powder that was directly
used for the next step. To a solution of the triol intermediate
(3.00 g, 7.22 mmol) in DMF (60 mL) were added tetrabutylam-
monium iodide (TBAI, 300 mg, 0.76 mmol) and benzyl bromide
(5.71 mL, 45.9 mmol). The solution was cooled to 0 °C and then
NaH (60% dispersion in mineral oil, 1.6 g, 40.0 mmol) was added
slowly. The reaction mixture was stirred at 0 °C for 1 h and then
stirred at rt overnight. The reaction mixture was quenched with
aqueous NH4Cl and extracted with CH2Cl2. The organic phase was
washed with aqueous NH4Cl and brine, then dried over Na2SO4
and filtered. The solvent was evaporated, and the crude product
was purified by silica gel chromatography to give 5 as white foam
(2.9 g, 59%). 1H NMR (400 MHz, CDCl3): d 7.90–7.60 (m, 4 H),
7.42–7.26 (m, 10H), 7.04–6.82 (m, 5H), 5.51 (d, 1H), 4.76–4.90 (m,
2H), 4.74–4.56 (m, 3H), 4.50–4.36 (m, 2H), 4.26 (t, 1H), 3.90–3.66
(m, 4H), 2.29 (s, 3H); MALDI TOFMS: [M+Na]+ calcd for C42H39NO6-
NaS, 708.2; found: m/z 708.4.
a
c
3.7. N -(9-Fluorenylmethoxycarbonyl)-N -[2-acetamido-3,4,6-
tri-O-benzyl-2-deoxy-b- -glucopyranosyl)-(1?4)-2-acetamido-
3,6-di-O-benzyl-2-deoxy-b- -glucopyranosyl]- -asparagine (10)
D
D
L
To a solution of 9 (55 mg, 43 lmol) in CH2Cl2 (4 mL) were added
triethylsilane (0.25 mL) and TFA (1 mL). After the mixture was stir-
red for 2.5 h at rt, Et3N was added to quench the reaction, which
was then concentrated and subjected to silica gel chromatography
to give 10 as white foam (50 mg, 96%). 1H NMR (400 MHz, CD3OD–
CDCl3 1:5): d 7.76 (d, J = 7.2 Hz, 2H), 7.63 (d, J = 7.2 Hz, 2H), 7.37–
7.16 (m, 32H), 5.71 (d, J = 2.4 Hz, 1H), 4.78–4.52 (m, 9H), 4.49–
4.44 (4H), 4.39–4.31 (m, 2H), 4.26–4.16 (m, 3H), 3.93–3.73 (m,
6H), 3.66–3.53 (m, 5H), 3.37 (s, br, 1H), 2.86 (dd, J = 5.6, 16.4 Hz,
1H), 2.76 (dd, J = 7.2, 15.2 Hz, 1H), 2.04 (s, 3H), 1.86 (s, 3H); MALDI
TOFMS: [M+Na]+ calcd for C70H74N4O15Na, 1233.5; found: m/z
1233.4.
3.4. O-(-3,4,6-Tri-O-benzyl-2-deoxy-2-phthalimido-b-
glucopyranosyl)-(1?4)-3,6-di-O-benzyl-2-deoxy-2-
phthalimido-b-D-glucopyranosyl azide (7)
D-
To a solution of 5 (1.0 g, 1.46 mmol) and 6 (514 mg, 1.00 mmol)
in anhydrous CH2Cl2 (50 mL) was added 4 Å MS under an Ar atmo-
sphere. The reaction mixture was cooled to À30 °C, and NIS
(520 mg, 2.31 mmol) was added. TfOH (41 lL, 0.46 mmol) was
then added dropwise over a 5-min period. The reaction mixture
was stirred for 20 min, and was then filtered through Celite. The fil-
trate was washed with aqueous NaHCO3, Na2S2O3, and brine solu-
tion. The organic layer was dried over Na2SO4, filtered, and
concentrated to give yellowish oil. Silica gel chromatography
yielded 7 (800 mg, 75%) as clear syrup. NMR data were consistent
with literature-reported values.44 MALDI TOFMS: [MÀN2+Na]+
calcd for C63H57N3O12Na, 1070.4; found: m/z 1070.2; [M+Na]+ calcd
for C63H57N5O12Na, 1098.4; found: m/z 1098.2.
3.8. Resin-bound glycopeptide 11
Ser-loaded 2-chlorotrityl resin (417 mg, 0.25 mmol) was sub-
jected to eight cycles of Fmoc-SPPS on an automated peptide syn-
thesizer to prepare resin-bound nonapeptide 11 (661 mg, 93% by
weight) using HBTU-DIEA as the condensation reagent and 20%
piperidine as the deprotection reagent. The product was then used
in the SPPS construction of glycopeptides 2, 3a, and 3b.
3.5. O-(2-Acetamido-3,4,6-tri-O-benzyl-2-deoxy-b-
glucopyranosyl)-(1?4)-2-acetamido-3,6-di-O-benzyl-2-
deoxy-b- -glucopyranosyl azide (8)
D-
D
3.9. CD52 glycopeptide 2
After a mixture of 7 (200 mg, 0.19 mmol), ethylenediamine
(1 mL), and 1-butanol (5 mL) was stirred at 90 °C overnight, it
was concentrated to dryness under vacuum. The resulting residue
was dissolved in Ac2O–pyridine (1:2, 3 mL) and stirred overnight at
rt. After the reaction mixture was diluted with EtOAc (50 mL), the
solution was washed with saturated NaHCO3 and brine, dried over
A solution of glycosyl amino acid 10 (37 mg, 30
(8 mg, 60 mol), and DCC (12 mg, 60 mol) in NMP (1 mL) was
stirred at rt for 1 h and then transferred to a SPPS vessel containing
resin-bound nonapeptide 11 (56 mg, 20 mol). The mixture was
shaken at rt overnight under an Ar atmosphere, after which the
resin was filtered off and washed thoroughly with NMP to give
lmol), HOBt
l
l
l

2900
B. M. Swarts et al. / Carbohydrate Research 343 (2008) 2894–2902
solid supported glycopeptide 12. After Fmoc removal with 20%
piperidine in NMP, the resin was subjected to two manual Fmoc-
SPPS cycles using the benzotriazole active esters of Fmoc-
Gln(Trt)-OH and Fmoc-Gly-OH to afford the desired peptide se-
quence. After final Fmoc deprotection and N-terminal acetylation
of half of the sample with Ac2O to give 13, the resin was treated
with CH2Cl2–AcOH–TFE (8:1:1) at rt for 2 h to release the glyco-
peptide. The resin was filtered off and the cleavage mixture was di-
luted with hexanes and concentrated under vacuum to give a
white solid (13 mg, 41% based on 11). To the resulting fully pro-
33% HBr in HOAc (100 lL) at 0 °C. After being stirred at rt for 5 h,
the reaction mixture was washed with saturated aqueous NaHCO3
and brine, dried over Na2SO4, and concentrated under vacuum to
give glycosyl bromide 16 as syrup that was directly used in the gly-
cosylation reaction. After a mixture of 16, 15 (40 mg, 0.07 mmol),
and MS 4 Å (50 mg) in dry CH2Cl2 (2 mL) was stirred at rt for 1 h,
it was cooled to À50 °C. To this mixture was added AgOTf
(38 mg, 0.15 mmol). The reaction mixture was then warmed to rt
and stirred for 3 h, at which point the MS were filtered off, and
the resulting filtrate was washed with NaHCO3 and brine, then
dried over Na2SO4 and concentrated under vacuum. The residue
was not fully separable by silica gel chromatography, as another
compound co-eluted with the desired disaccharide. Thus, to a solu-
tion of the intermediate in CH2Cl2 (4 mL) were added TFA (0.2 mL)
and Et3SiH (0.5 mL). After the reaction mixture was stirred for
2.5 h, it was quenched with Et3N, concentrated, and purified by sil-
ica gel chromatography to give pure 17 (43 mg, 69% yield over two
steps). Spectroscopic data of 17 were identical to literature-
reported values.27
tected glycopeptide (13 mg, 4 lmol) were added solvents CH2Cl2–
MeOH (1:1, 1 mL) and 10% Pd–C (13 mg) under a H2 atmosphere.
After MALDI MS showed complete reaction, the catalyst was
filtered off and the solvent was evaporated to get the debenzylated
product (8 mg, 78% based on previous step), which was treated
with 20% TFA in CH2Cl2 (1 mL) at rt for 2.5 h. After co-evaporation
with toluene and thorough washing of the residue with diethyl
ether, the product 2 was lyophilized and then purified by RP-HPLC
(Supelco Discovery C18, 250 Â 10 mm, eluent 2.0% i-PrOH in H2O,
2 mL/min, tR = 16.1 min, 2.2 mg, 42% based on the previously given
yield). 1H NMR (500 MHz, D2O, DHO at d 4.79 as reference): d 5.05
(d, J = 10.0 Hz, 1H), 4.69 (t, J = 7.0 Hz, 1H), 4.61 (d, J = 8.0 Hz, 1H),
4.55 (t, J = 5.0 Hz, 1H), 4.51 (t, J = 5.0 Hz, 1H), 4.47–4.40 (m, 4H),
4.37–4.32 (m, 2H), 4.28–4.26 (m, 2H), 4.01–3.81 (m, 15H), 3.78–
3.74 (m, 4H), 3.69–3.64 (m, 2H), 3.60–3.55 (m, 2H), 3.52–3.49
(m, 2H), 2.89 (dd, J = 6.0, 16.0 Hz, 1H), 2.77 (dd, J = 7.5, 15.0 Hz,
2H), 2.69 (dd, J = 6.5, 16.0 Hz, 1H), 2.42–2.30 (m, 5H), 2.22–2.11
(m, 2H), 2.08 (s, 6H), 2.06–2.04 (m, 2H), 2.02 (s, 3H), 2.01–1.99
(m, 2H), 1.24 (s, 3H), 1.23 (s, 3H); 13C DEPT NMR CH carbons
(D2O, 125 MHz): 101.53, 78.91, 78.38, 76.35, 76.08, 73.62, 73.12,
69.87, 67.20, 66.85, 61.08, 59.44, 59.19, 57.35, 56.45, 55.76,
55.56, 53.83, 53.46, 53.29, 51.80, 50.22; HR ESIMS: [M+2H]2+ calcd
for C63H103N17O35, 828.8401; found: m/z 828.8363.
a
c
3.12. N -(9-Fluorenylmethoxycarbonyl)-N -[2-acetamido-
3,4,6-tri-O-benzyl-2-deoxy-b- -glucopyranosyl)-(1?4)-[O-2,3,4-
tri-O-benzyl- -fucopyranosyl)-(1?6)]-2-acetamido-3-O-
D
a
benzyl-2-deoxy-b-D-glucopyranosyl]-L-asparagine allyl ester
(21)
Glycosyl azide 20 (127 mg, 0.133 mmol) was dissolved in a mix-
ture of CH2Cl2 (1 mL) and MeOH (3 mL). Lindlar’s catalyst (127 mg)
was then added and the reaction mixture was stirred under a H2
atmosphere at rt for 3 h. After filtration of the catalyst by silica
gel, the reaction mixture was concentrated under vacuum to give
the glycosyl amine intermediate. Meanwhile, to a solution of
Fmoc-Asp(OH)-OAll (79 mg, 0.20 mmol) in CH2Cl2 (3 mL) and
NMP (0.1 mL) were added DCC (49 mg, 0.24 mmol) and HOBt
(32 mg, 0.24 mmol). After stirring for 1 h, DCU was filtered off
and the active ester solution was directly transferred to a solution
of the glycosyl amine in CH2Cl2 (1 mL). The reaction mixture was
stirred at rt under an Ar atmosphere overnight, then concentrated
and purified by silica gel chromatography to give the glycosyl-Asn
conjugate 21 (122 mg, 72%). 1H NMR (400 MHz, CDCl3–CD3OD
3:1): d 7.56 (d, J = 8.0 Hz, 2H), 7.40 (d, J = 7.2 Hz, 2H), 7.21–7.05
(m, 24H), 5.72–5.65 (m, 1H), 5.11 (d, J = 16.8 Hz, 1H), 5.02 (d,
J = 10.4 Hz, 1H), 4.77 (d, J = 11.6 Hz, 1H), 4.72 (d, J = 11.2 Hz, 1H),
4.68–4.55 (m, 4H), 4.44–4.40 (m, 6H), 4.23 (dd, J = 7.6, 10.8 Hz,
1H), 4.04–3.99 (m, 2H), 3.87–3.84 (m, 2H), 3.74 (dd, J = 3.2,
9.6 Hz, 1H), 3.71–3.66 (m, 4H), 3.62–3.56 (m, 5H), 3.35–3.27 (m,
4H), 3.13–3.07 (m, 4H), 2.62 (dd, J = 6.0, 16.4 Hz, 1H) 2.49 (dd,
J = 4.0, 16.4 Hz, 1H), 1.77 (s, 3H), 1.67 (s, 3H), 0.95 (d, J = 6.4 Hz,
3H); HR ESIMS: [M+Na]+ calcd for C72H82N4O19Na, 1329.5471;
found: m/z 1329.5414.
3.10. 3-O-Benzyl-2-deoxy-6-O-(p-methoxybenzyl)-2-
phthalimido-b-D-glucopyranosyl azide (15)
To a solution of 14 (2.20 g, 4.86 mmol) in DMF (15 mL) at 0 °C
was added NaH (60% dispersion in mineral oil, 1.24 g, 7.27 mmol).
After stirring for 30 min, benzyl bromide (0.86 mL, 7.27 mmol) was
added to the reaction mixture, which was stirred at rt overnight.
After quenching excess NaH with MeOH, the reaction mixture
was concentrated under vacuum to remove most of the DMF, then
diluted with EtOAc and washed with saturated NaHCO3, brine,
dried over Na2SO4, and concentrated under vacuum. Purification
by silica gel chromatography gave the benzylated intermediate
(2.11 g, 3.9 mmol, 80%), which was then dissolved in THF and
cooled to 0 °C. To this solution were added MS 4 Å (1.0 g), NaBH3CN
(2.4 g, 39 mmol), and 1.0 M HCl in dry Et2O (to pH 1–2). After 1 h,
MS were filtered off, and the filtrate was diluted with EtOAc. The
organic layer was washed with cold NaHCO3 and brine, then dried
over Na2SO4, filtered, and concentrated under vacuum. Silica gel
chromatography gave 15 as colorless syrup (1.9 g, 89%). 1H NMR
(500 MHz, CDCl3): d 7.83–7.71 (m, 4H), 7.29 (d, J = 8.5 Hz, 2H),
6.89–7.04 (m, 7H), 5.36 (d, J = 9.5 Hz, 1H), 4.75 (d, J = 12.0 Hz, 1
H), 4.59 (d, J = 12.0 Hz, 1H), 4.53 (d, J = 11.5 Hz, 2H), 4.26 (dd,
J = 8.5, 10.5 Hz, 1H), 4.08 (dd, J = 9.5, 10.5 Hz, 1H), 3.81–3.85 (m,
2H), 3.82 (s, 3H) 3.70–3.79 (m, 2H). HR ESIMS: [M+Na]+ calcd for
C29H28N4O7Na, 567.1856; found: m/z 567.1832.
a
c
3.13. N -(9-Fluorenylmethoxycarbonyl)-N -[2-acetamido-
3,4,6-tri-O-benzyl-2-deoxy-b- -glucopyranosyl)-(1?4)-[O-2,3,4-
tri-O-benzyl- -fucopyranosyl)-(1?6)]-2-acetamido-3-O-
benzyl-2-deoxy-b- -glucopyranosyl]- -asparagine (22)
D
a
D
L
To a solution of 21 (77 mg, 0.058 mmol) in CH2Cl2–MeOH (1:1,
2 mL) were added N-methylaniline (100 L) and Pd(PPh3)4 (12 mg,
l
0.01 mmol). The reaction mixture was stirred overnight at rt, then
concentrated under vacuum and purified by silica gel chromato-
graphy to give 22 (57 mg, 78%). 1H NMR (500 MHz, CD3OD, d 4.78
as reference): d 7.69 (d, J = 7.2 Hz, 2H), 7.55–7.52 (m, 2H), 7.29–
7.15 (m, 24H), 4.98 (d, J = 11.6 Hz, 1H), 4.90 (d, J = 10.0 Hz, 1H),
4.84 (d, J = 2.4 Hz, 1H), 4.71–4.66 (m, 4H), 4.61 (d, J = 8.8 Hz, 1H),
4.54 (d, J = 10.4 Hz, 1H), 4.47 (d, J = 11.2 Hz, 1H), 4.24–4.23 (m,
3H), 4.11 (t, J = 6.4 Hz, 1H), 4.01 (d, J = 5.6 Hz, 1H), 3.90–3.81 (m,
3.11. O-(2-Acetamido-2-deoxy-b-
2,3,4-tri-O-benzyl- -fucopyranosyl)-(1?6)]-2-acetamido-3-O-
benzyl-2-deoxy-b- -glucopyranosyl azide (17)
D-glucopyranosyl)-(1?4)-[O-
a
D
To a solution of 1,2,3,4-tetra-O-acetyl-2-deoxy-2-phthalimido-
D-glucopyranose (75 mg, 0.15 mmol) in CH2Cl2 (2 mL) was added
b-

B. M. Swarts et al. / Carbohydrate Research 343 (2008) 2894–2902
2901
4H), 3.78–3.68 (m, 5H), 3.53 (t, J = 9.6 Hz, 1H), 3.47–3.42 (m, 2H),
3.38 (t, J = 9.8 Hz, 1H), 3.31–3.28 (m, 3H), 3.19–1.17 (m, 1H), 2.65
(s, br, 2H), 1.91 (s, 3H), 1.84 (s, 3H), 1.05 (d, J = 5.6 Hz, 3H); HR
ESIMS: [M+Na]+ calcd for C69H78N4O19Na, 1289.5158; found: m/z
1289.5116.
white solid (12 mg, 40% based on 11). The remaining deprotection
steps were carried out according to the protocol described for 3a to
arrive at 3b, which was purified by RP-HPLC (Supelco Discovery
C18, 250 Â 10 mm, eluent 1.5% i-PrOH in H2O, 2 mL/min,
tR = 17.9 min, 1.9 mg, 28% based on the previously given yield).
Spectroscopic data of 3b were consistent with literature-reported
values.27
3.14. CD52 glycopeptide 3a
A solution of glycosyl amino acid 10 (51 mg, 40
(12 mg, 90 mol), and DCC (18 mg, 90 mol) in NMP (1 mL) was
stirred at rt for 1 h and then transferred to a SPPS vessel contain-
ing resin-bound nonapeptide 11 (56 mg, 20 mol). The mixture
l
mol), HOBt
3.16. Circular dichroism spectroscopy
l
l
CD spectra of all derivatives were recorded on a Chirascan circu-
lar dichroism spectrometer equipped with a water bath to control
the temperature at 25 °C. The solutions of 1a, 1b, 2, 3a, and 3b
were prepared from dry samples in ddH2O to give concentrations
of 6.6 Â 10À2, 3.2 Â 10À2, 1.6 Â 10À2, 4.2 Â 10À2, and 1.8 Â 10
À2 mM, respectively. The molar ellipticity was normalized using
l
was shaken at rt overnight under an Ar atmosphere, after which
the resin was filtered off and washed thoroughly with NMP to
give resin supported glycopeptide 12. After Fmoc removal with
20% piperidine in NMP, the resin was subjected to two manual
Fmoc-SPPS cycles using the benzotriazole active esters of
Fmoc-Gln(Trt)-OH and Fmoc-Gly-OH to give the desired peptide
sequence. After final Fmoc deprotection to give 24, half of the re-
sin was treated with CH2Cl2–AcOH–TFE (8:1:1) at rt for 2 h to
release the glycopeptide. The resin was filtered off and the
cleavage mixture was diluted with hexanes and concentrated
under vacuum to give a white solid (13 mg, 43% based on 11).
the equation
of each peptide analog.
D
e
= h/(32.98 Â C), in which h is the CD absorbance
Acknowledgments
This work was supported by NSF (CHE-0407144 and 0715275).
We thank Dr. B. Shay and Dr. L. Hryhorczuk for the MS measure-
ments, and Dr. B. Ksebati for his help with some NMR
measurements.
To the resulting fully protected glycopeptide (13 mg, 4.3 lmol)
were added CH2Cl2–MeOH (1:1, 1 mL) and 10% Pd–C (13 mg) un-
der a H2 atmosphere (MeOH used in the debenzylation was re-
fluxed with NaBH4 for several hours and then distilled to
reduce any formaldehyde present and thus prevent N-methyla-
tion via reductive amination). After MALDI MS showed complete
reaction, the catalyst was filtered off and the solvent was evap-
orated to get the debenzylated product (10 mg, 87% based on
previous step), which was treated with 20% TFA in CH2Cl2
(1 mL) at rt for 2.5 h. After co-evaporation with toluene and
thorough washing of the residue with diethyl ether, the product
3a was lyophilized and then purified by RP-HPLC (Supelco Dis-
covery C18, 250 Â 10 mm, eluent 1.5% i-PrOH in H2O, 2 mL/
min, tR = 17.9 min, 2.1 mg, 31% based on the previously given
yield). 1H NMR (500 MHz, D2O, DHO at d 4.79 as reference): d
5.05 (d, J = 10.0 Hz, 1H), 4.90 (d, J = 4.0 Hz, 1H), 4.67 (d,
J = 8.5 Hz, 1H), 4.54 (t, J = 5.5 Hz, 1H), 4.53–4.51 (m, 1H), 4.49–
4.44 (m, 2H), 4.43–4.40 (m, 2H), 4.37–4.32 (m, 2H), 4.27 (t,
J = 6.0 Hz, 1H), 4.14 (dd, J = 6.5, 13.0 Hz, 1H), 3.95–3.86 (m,
11H), 3.85–3.74 (m, 8H), 3.70–3.68 (m, 2H), 3.57 (t, J = 8.5 Hz,
1H), 3.53–3.46 (m, 2H), 2.90 (dd, J = 5.0, 16.5 Hz, 1H), 2.86–
2.77 (m, 3H), 2.42–2.37 (m, 4H), 2.35–2.32 (m, 1H), 2.21–2.17
(m, 1H), 2.12–2.10 (m, 1H), 2.09 (s, 3H), 2.08–2.04 (m, 2H),
2.03 (s, 3H), 2.02–2.00 (m, 2H), 1.24–1.20 (m, 9H); 13C NMR
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.carres.2008.08.024.
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(D2O, 125 MHz):
d 177.99, 177.78, 174.93, 174.74, 173.84,
173.58, 173.16, 172.59, 172.23, 172.13, 171.99, 171.89, 171.52,
170.05, 167.70, 101.29, 99.48, 78.66, 78.43, 76.10, 75.34, 73.67,
72.99, 72.02, 69.90, 69.70, 68.35, 67.19, 66.98, 66.69, 61.98,
61.28, 61.07, 61.00, 60.75, 59.41, 59.16, 56.07, 55.78, 55.58,
53.90, 53.84, 53.78, 53.33, 51.26, 50.24, 48.25, 40.54, 36.32,
31.22, 31.10, 29.51, 27.01, 26.87, 24.68, 22.41, 22.31, 18.94,
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