Synthesis of an N-glucoasparagine analog as a building block for a V3-loop glycopeptide from GP120 of HIV-I

Article abstract of DOI:10.1016/S0008-6215(98)00252-3

The preparative synthesis of a new N⁴-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-l-asparagine mimetic 1, starting from 2-amino-1,5-anhydro-2-deoxy-glucitol hydrochloride and Z-Asp-(OH)-OBn is described. This glycosyl-amino acid unit 1 is expected

Full text of DOI:10.1016/S0008-6215(98)00252-3

Carbohydrate Research 313 (1998) 107±116
Synthesis of an N-glucoasparagine analog as a
building block for a V3-loop glycopeptide from
GP120 of HIV-I
Andreas Schafera, Gunther Klicha, Michael Schreiberb, Hans Paulsena,
Joachim Thiema,*
a Institut fur Organische Chemie der Universitat Hamburg, Martin-Luther-King Platz 6, D-20146
Hamburg, Germany
È
È
b Bernhard-Nocht-Institut fur Tropenmedizin Abteilung medizinische Mikrobiologie, Bernhard-Nocht-
Str. 74, D-20359 Hamburg, Germany
È
Received 8 June 1998; accepted 18 September 1998
Abstract
The preparative synthesis of a new N4-(2-acetamido-2-deoxy-ꢀ-d-glucopyranosyl)-l-asparagine
mimetic 1, starting from 2-amino-1,5-anhydro-2-deoxy-glucitol hydrochloride and Z-Asp-(OH)-
OBn is described. This glycosyl-amino acid unit 1 is expected to show higher stabilities towards in
vivo conditions. Further, the use of 1 as building block for the synthesis of modi®ed glycopeptides
using solid phase support is reported. The glycopeptide Ac-SXNTRKSIHIGPGRAF-NH2 having
sugar-modi®ed Asn2 mimics parts of the V3-loop structure containing the principle neutralizing
determinant (PND) of HIV-1 and the naturally conserved glycosylation site within the V3 loop.
# 1998 Published by Elsevier Science Ltd. All rights reserved
Keywords: Glycopeptides; Mimetics; Carbohydrates; Solid phase synthesis; HIV
1. Introduction
interaction between gp120 and the two coreceptors
is mediated by the third variable region (V3 loop)
of gp120 [3]. Mutational studies with the V3 loop
suggest that coreceptor usage and HIV:cell fusion
are linked to V3 loop amino acid sequences [4].
Thus, the V3 loop of the HIV-1 gp120 envelope
glycoprotein is an important domain involved in
virus entry by making contact to target cell recep-
tors. In a second step, the V3 loop may interact
with the chemokine receptor to permit HIV:cell
fusion. This second step of HIV:cell interaction
can be inhibited by synthetic V3 loop peptides [5]
containing the highly conserved GPGRAF-motif
Binding of the HIV-1 external envelope glyco-
protein gp120 to the CD4 receptor on human cells
is the ®rst step in HIV:cell interaction and virus
entry [1]. After attachment to CD4, gp 120 inter-
acts with one of the chemokine receptors CCR5 or
CXCR4, recently identi®ed as the major cor-
eceptors for HIV-1 [2]. It was shown that the
* Corresponding author. Fax: +49-040-4123-4241;
e-mail: thiem@chemie.uni-hamburg.de
0008-6215/98/$Ðsee front matter # 1998 Published by Elsevier Science Ltd. All rights reserved
PII S0008-6215(98)00252-3

108
A. Schafer et al./Carbohydrate Research 313 (1998) 107±116
È
of the subtype B strains [6]. Thus peptides
corresponding to the V3 loop sequence GPGRAF
were able to block HIV-1 infection in vitro. One
main therapeutic aim is the extracellular blockade
of virus attachment and the fusion process which
follows. A possible strategy in blocking viral infec-
tion is the use of synthetic peptides mimicking the
gp120 V3 loop domain. Enhanced antiviral activity
was observed using multibranched peptide con-
structs [7] indicating that small V3 peptides can
interfere with gp120:coreceptor binding. Since
gp120 and especially the V3 loop is highly glyco-
sylated [8] and V3 loop glycosylation is important
for viral function and infectivity [9], we are inter-
ested in the synthesis of glycosylated V3 loop pep-
tides and the in¯uence of glycosylation on the three
dimensional structure of the V3 loop peptide.
Therefore, we report a method for the synthesis of
V3 glycopeptides containing the GPGRAF-motif
and the ¯anking glycosylation site.
Because the moderate metabolic stability of gly-
copeptides limits their potential as therapeutic
agents, modi®ed analogs such as glycopeptides
containing C-glycosylated amino acids have been
reported [10]. Herein we describe the synthesis of
the glycosyl-amino acid unit Nꢁ-(3,4,6-tri-O-acetyl-
1,5-anhydro-2-deoxyglucitol-2)-l-asparagine 1 as a
mimetic of N4-(2-acetamido-2-deoxy-ꢀ-d-gluco-
pyranosyl)-l-asparagine 2 and the incorporation of
1 as a new building block [11] in solid phase
synthesis of the potential anti-HIV active peptide 3
(Fig. 1). This glycopeptide contains a mono-
saccharide at the amino acid position which is the
naturally conserved 306 N-linked glycosylation site
of the gp120 V3 loop [9].
compound represents a new approach toward the
synthesis of glycosylamino acid mimetics which is
distinguished by the need for only a few reaction
steps starting from easily available starting materi-
als and the compatibility with most of the generally
applied protective groups.
2. Results and discussion
The modi®ed N-glucoasparagine unit 1 was
introduced into the peptide sequence as Nꢂ-Fmoc
protected derivative 9 (Scheme 1). Starting from
the known 2-acetamido-2-deoxy-glucopyranosyl
chloride 4 [12] the appropriate glucitol 5 [13] was
obtained by reduction using AIBN/tributyltin
hydride [14] in re¯uxing toluene in 84% yield.
Deacetylation of 5 was performed by acid hydroly-
sis with hydrochloric acid (2.5 N) to give the 2-
amino-1,5-anhydroglucitol hydrochloride 6 in 68%
yield.
Deacetylation procedures under alkaline reac-
tion conditions using Ba(OH)2 or NaOH [15] led to
diculties in work up resulting in low yields of 6.
For the coupling step between 6 and Z-Asp(OH)-
OBn 7 [16], the amino acid was activated as mixed
anhydride using isobutylchloroformate [17]. This
method o€ered the advantage of using the
unblocked sugar moiety directly in aqueous solu-
tion which made the development of a protective-
group pattern obsolete. After coupling the remain-
ing hydroxy groups were acetylated to lead to 8
(75% overall). Catalytical hydrogenolysis of 8 gave
1 in quantitative yield, which was subsequently Nꢂ-
Fmoc protected (82%) leading to compound 9.
The employment of 1 as N-glucoasparagine analog
was tested by synthesizing the small peptide 10 and
subsequently the acetylated V3-loop peptide
sequence 12 using 9 (Scheme 2).
Building block 1 is characterized by a new amide
linkage between the 2-amino function of the car-
bohydrate moiety and the side chain of the amino
acid which ensures an improved stability towards
enzymes, acids and bases, in contrast to the labile
N-glycosidic linkage of 2. In addition, the
anomeric center has been reduced transforming the
cyclic acetal structure to a stable cyclic ether. This
The assembly of the O-acetylated glycopeptides
10 and 12 was done according to the peptide
sequence starting at the C-terminal end using a
fully automatic peptide synthesizer. PEGA-resin
Fig. 1. Original 2 and modi®ed 1 sugar-Asn building block and modi®ed glycopeptide structure 3.

A. Schafer et al./Carbohydrate Research 313 (1998) 107±116
È
109
[18] derivatised with Rink-linker [19] was chosen as
polymer support in order to get high amounts of
peptide±amides [20]. After cleavage of the resin
and HPLC puri®cation the O-acetylated glycopep-
tides were obtained in 21% (10) respectively 23%
(12) yield. Deacetylation was performed in aqueous
MeOH (50%) using catalytic amounts of NaOMe
(1% in MeOH). The yields after HPLC puri®ca-
tion were 60% (11) respectively 54% (3). NMR
characterisation of the peptides 10, 11 and 12 was
indicates a ꢀ-turn conformation. This interaction is
surprising, because of the small size of the peptide.
In comparison with HIV gp120 sequences as
RP342, RP142 or RP70, peptide 3 seems to gen-
erate a similar shape. Chandrasekhar et al. [22]
have shown for a similar sequence (RP) that the
evaluation of a ꢀ-turn conformation was detectable
for peptides with the GPGRAF-motif centered,
but not for peptides with this motif near the C-ter-
minus. However there is no evidence for an
induced peptide conformation by the glucitol side-
chain. Evidently, NOE interaction of the Asn-ꢃ-
NH with H-3 of the carbohydrate moiety on one
hand and with the Asn-(ꢀ2-H on the other hand
proved the linkage between the aminoglucitol and
the peptide backbone (Fig. 2, Table 1).
1
performed by H±1H-COSY NMR experiments.
For compound 3 a complete NMR assignment of
1
all protons was performed by H±1H-TOCSY and
1H±1H-NOESY spectroscopy [21]. Moreover the
assignment of several NH(i+1)!CHꢂ(i) NOEs led
to complete characterisation of 3. The NOE data
indicate that the majority of the molecule is dis-
ordered, that means no single conformation is pre-
sent in solution. However the N±N(i, i+2) NOE
between the amide protons of Gly-13 and Ala-15
This is the ®rst report on the synthesis of glyco-
sylated V3 loop peptides as a new antiviral candi-
date. In contrast to other V3 loop peptide
constructs [23] our glycosylated V3 peptide had no
Scheme 1. Synthesis of the N4-(2-acetamido-2-deoxy-ꢀ-d-glucopyranosyl)-l-asparagine analog 1 and building block 9.
Scheme 2. Synthesis of the deacetylated peptides 3 and 11.

110
A. Schafer et al./Carbohydrate Research 313 (1998) 107±116
È
inhibitory e€ects on activated T cells or PBMC
(data will be published in another context). Since
we did not observe inhibition of T cell proliferation
after incubation of T cells with the V3 loop glyco-
peptide its antiviral activity can be determined
precisely and it might be an interesting future can-
didate for safe in vivo applications. Besides the
direct antiviral e€ects observed by testing V3 loop
Fig. 2. Part of the NOESY-spectrum of compound 3. The crosspeaks (as indicated above) are shown between the 2-NH proton and
the Asn-ꢀ2-resp. Glc-H-3 which indicate the linkage between the carbohydrate moiety and the peptide unit.
Table 1
Schematic diagram showing the magnitude of various NOE connectivities observed in NOESY spectra of peptide 3

A. Schafer et al./Carbohydrate Research 313 (1998) 107±116
È
111
peptides [5,7] it is also conceivable, that glycosy-
lated V3 loop peptides compared to normal pep-
tides are more e€ective in the induction of
antibodies able to neutralize HIV-1. This might be
relevant for the development of a mixed HIV-1
vaccine based on recombinant gp120 and V3 loop
peptides.
2-Amino-1,5-anhydro-2-deoxyglucitol hydrochloride
(6).ÐA suspension of 2-acetamido-3,4,6-tri-O-
acetyl-1,5-anhydro-2-deoxy glucitol 5 [16] (2.50 g,
7.55 mmol) in 2.5 N hydrochloric acid (20 mL) was
heated under re¯ux for 2 h. The resulting clear
solution was concentrated under reduced pressure
and the oily residue was dissolved in a minimum
amount of ethanol. Diethyl ether was added drop-
wise under stirring until the product precipitated.
The crude product was ®ltered o€, recrystallized
from propanol/ethanol (1:1) and dried to a€ord
3. Experimental
ꢀ
Melting points were determined using a Mettler
1
1.02 g (68%) of 6 as a colourless solid. mp 199 C
1
FP 82 apparatus and are uncorrected. H and 13C
decomp; [ꢂ]25 +17.8 (c 1; H2O); H NMR (D2O)
d
NMR spectra were recorded on a Bruker AMX
400 or a Bruker DRX 500 spectrometer at 300 K.
In 2D experiments 512 FIDs with 4096 complex
data points and 32 scans each were acquired. The
NOESY spectra were acquired with a 300 ms mix-
ing time. The water resonance was suppressed by
low-power presaturation during the relaxation
delay (1.5 s). Data were processed on a PC using
2D WIN±NMR (950901.6) software (Bruker
Franzen Analytik GmbH). The data were zero-®l-
led twice in the t1 dimension, and multiplied with a
squared sinebell function (SSB 2) in both dimen-
sions. Chemical shifts are given in ppm relative to
internal acetone (ꢃ 2.225 ppm) for solutions in
D2O/H2O and TMS (ꢃ 0.000 ppm) for other solu-
tions. FAB mass spectra were measured on a
double-focused VG-Analytical 70±250 S mass
spectrometer with 3-nitrobenzyl alcohol as matrix.
Optical rotations were recorded on a Perkin±Elmer
Polarimeter 241, [a]-values are given in units of
ꢃ 3.15 (ddd, 1 H, J1a,1e 11.2, J1a,2 11.2, J1e,2 5.1 Hz,
H-2), 3.23±3.32 (m, 2 H, H-4, H-5), 3.41 ( dd, 1 H,
J1a,1e 11.2, J1a,2 11.2 Hz; H-1a), 3.50 (dd, 1 H, J2,3
10.2, J3,4 8.1 Hz; H-3), 3.58 (dd, 1 H, J5,6 5.6, J6,6*
12.2 Hz; H-6), 3.75 (dd, 1 H, J5,6* 2.0, J6,6* 12.2 Hz;
H-6*), 4.05 (dd, 1 H, J1a,1e 11.2, J1e,2 5.1 Hz; H-1e);
13C NMR (D2O) ꢃ 51.87 (C-5), 61.10 (C-6), 66.44
(C-1), 70.23 (C-4), 74.56 (C-3), 80.98 (C-2). Anal.
Calcd. for C6H14NO4Cl: C, 36.17; H, 7.09; N, 7.03.
Found: C, 36.33; H, 7.07; N 7.02.
Na-Benzyloxycarbonyl-Ng-[3,4,6-tri-O-acetyl-
1,5-anhydro-2-deoxyglucitol-2]-l-asparagine benzyl
ester (8).ÐN-Methylmorpholine (570 ꢄL, 5.14 mmol)
was added to a stirred and cooled solution ( 10 ꢀC)
of Nꢂ-benzyloxycarbonyl-l-asparagine benzyl ester
7 (1.93 g, 5.41 mmol) in dry THF (30 mL). Iso-
butylchloroformate (740 ꢄL, 5.68 mmol) was
added slowly and stirring was continued for 30 min
at 10 ꢀC. To a stirred suspension of 2-amino-1,5-
anhydro-2-deoxy-glucitol hydrochloride 6 (972 mg,
4.87 mmol) in THF (15 mL) and triethylamine
(0.68 mL, 4.87 mmol) water (2 mL) was added
dropwise.
10 deg cm2 g . MPLC was performed on silica
gel Silitech 12±26, 6 ꢄm (ICN) at 300±500 kPa.
HPLC was performed on a Merck/Hitachi HPLC
system with LiChrospher RP-18 columns
1
1
The resulting solution was added slowly to the
ꢀ
1
(250Â25 mm; 7 ꢄm; ¯ow rate 10 cm3 min ) for
cooled ( 10 C) solution of 6 under vigorous stir-
ꢀ
preparative separation and LiChrospher RP-18
1
ring. The mixture was stirred for 10 min at 10 C
column (250Â4 mm; 7 ꢄm; ¯ow rate 10 cm3 min )
and then allowed to warm to room temperature
over 2 h. After evaporation the oily residue was
dissolved in dry pyridine (25 mL) and treated with
acetic anhydride (8 mL, 84.6 mmol). The mixture
was stirred at room temperature for 16 h and then
the solvent was removed under reduced pressure
(codestillation with toluene). Puri®cation of the
crude product by MPLC chromatography on silica
gel (EtOAc/light petroleum 1:2) gave 8 (2.3 g,
for analytical use with bu€er A (0.1% TFA in
water) and bu€er B (0.1% TFA in acetonitrile).
Peak detection was performed with a photodiode
array detector at 215 nm. Peptide 9 and 11 were
synthesized on a Pioneer Perseptive Applied Bio-
systems continues ¯ow peptide synthesizer. DMF
used for peptide synthesis was analysed for free
amines by addition of Dhbt-OH prior to use.
Reagents for peptide synthesis were purchased as
follows: Dhbt-OH and TBTU from Fluka; HATU
and Fmoc amino acids from Perseptive Applied
Biosystems.
ꢀ
75%) as a colourless solid. M.p. 157 C; [ꢂ]25
d
+38.2 (c 1, CH2Cl2); 1H NMR (CDCl3) ꢃ 1.91 (s, 3
H; Ac±H3) 1.96 (s, 3 H; Ac±H3), 2.01 (s, 3 H; Ac±
H3), 2.57 (dd,1 H, Jꢂ,ꢀ 4.6, Jꢀ,ꢀ* 15.8 Hz; Asn-ꢀ-H),

112
A. Schafer et al./Carbohydrate Research 313 (1998) 107±116
È
2.76 (dd, 1 H, Jꢂ,ꢀ* 5.1, Jꢀ,ꢀ* 15.8 Hz; Asn-ꢀ*-H),
2.95 (dd, 1 H, J1a,1e 11.2 Hz, J1a,2 11.2 Hz; H-1a),
3.42 (ddd, 1 H, J4,5 9.7, J5,6 2.5, J5,6* 5.1 Hz; H-5),
3.95 (dd, 1 H, J1a,1e 11.2, J1e,2 5.1 Hz; H-1e), 4.01
(m, 1 H, H-2), 4.02±4.18 (m, 2H, J5,6 2.5, J5,6*
5.1 Hz; H-6, H-6*), 4.52 (ddd, 1 H, Jꢂ,ꢀ 4.6, Jꢂ,ꢀ*
5.1, Ja,Asn±NH 8.1 Hz; Asn-ꢂ-H), 4.82 (dd, 1 H, J2,3
10.7, J3,4 10.2 Hz; H-3), 4.95 (dd, 1 H, J3,4 10.7, J4,5
9.7 Hz; H-4), 5.03 (s, 2 H; CH2±Bn), 5.10 (s, 2 H;
CH2±Bn), 5.75 (d, 1 H, JNH-2,2 7.6 Hz; NH-2), 5.88
(d, 1 H, Jꢂ,Asn-NH 8.1 Hz; Asn±NH), 7.23±7.31 (m,
10 H, Ar); 13C NMR (CDCl3) ꢃ 19.58, 19.65, 19.72
(3ÂAc±CH3), 36.88 (Asn-ꢀ-C), 49.44 (C-2), 49.83
(Asn-ꢂ-C), 61.32 (C-6), 66.08 (Bn±CH2), 66.47
(Bn±CH2), 66.90 (C-1), 67.10 (C-4), 73.02 (C-3),
75.51 (C-5), 127.06 , 127.19, 127.42, 127.52, 127.60,
134.31, 135.13 (2ÂAr), 168.27 (Z±CO), 168.84
(Ac±CO), 169.0 (NH±CO), 169.5 (CO) 169.8, 171.2
(2ÂAc±CO); MS (FAB): 629.2 (M+H)+. Anal.
Calcd. for C31H36N2O12: C, 59.21; H, 5.78; N,
4.46. Found: C, 59.52; H, 5.73; N, 4.34.
solution of 1 (380 mg, 0.97 mmol) in 5 mL sat.
ꢀ
NaHCO3-solution at 0 C. The reaction mixture
was allowed to warm to room temperature and
stirred for 30 min, water (25 mL) was added and
the solution was washed with diethyl ether (25 mL)
and EtOAc (2Â25 mL). Then the aqueous phase
ꢀ
was cooled to 0 C and acidi®ed with hydrochloric
acid (5 N) to pH 2. The precipitated product was
extracted with EtOAc (4Â25 mL) and the combined
organic layers were washed with water (3Â50 mL)
and saturated NaCl solution (3Â50 mL). The
organic layer was dried over MgSO4 and con-
centrated under reduced pressure to give 520 mg of
9 (86%) as a colourless solid.
M.p. 218 ꢀC; [ꢂ]25 +8.6 (c 1, DMSO); 1H
d
NMR ([D6]DMSO) ꢃ 1.90 (s, 3 H; Ac±H3), 1.96 (s,
3 H; Ac±H3), 1.99 (s, 3 H; Ac±H3), 2.39 (dd, 1H,
Jꢂ,ꢀ 8.1, Jꢀ,ꢀ* 15.2 Hz; Asn-ꢀ-H), 2.55 (dd, 1 H,
Jꢂ,ꢀ* 5.6, Jꢀ,ꢀ* 15.2 Hz; Asn-ꢀ*-H), 3.23 (dd, 1H,
J1a,1e 11.2, J1a,2 11.2 Hz; H-1a), 3.67 (ddd, 1 H, J4,5
10.5, J5,6 2.2, J5,6* 5.1 Hz; H-5), 3.73 (dd, 1 H, J1a,1e
11.2, J1e,2 5.6 Hz; H-1e), 3.97±4.00 (m, 1 H, Ja,ꢀ
8.1, Jꢂ,b* 5.6, Asn-ꢂ-H), 4.00 (dd, 1 H, J5,6 2.2, J6,6*
12.2 Hz; H-6), 4.09 (dd, 1 H, J5,6* 5.1, J6,6* 12.2 Hz;
H-6*), 4.18±4.31 (m, 4 H, J1a,2 11.2, J1e,2 5.6, J2,3
9.66 Hz; H-2, Fmoc±CH2, Fmoc±CH), 4.78 (dd, 1
H, J2,3 9.7 J3,4 9.7 Hz; H-3), 4.97 (dd, 1 H, J3,4 9.7,
J4,5 10.5 Hz; H-4), 7.29±7.93 (m, 8 H; Fmoc±Ar);
13C NMR ([D6]DMSO) ꢃ 21.22, 21.33 (3ÂAc±
CH3), 36.71 (Asn-ꢀ-C), 47.40 (Fmoc±H), 49.46 (C-
2), 51.25 (Asn-ꢂ-C), 63.01 (C-6), 66.58 (Fmoc±
CH2), 67.84 (C-1), 69.67 (C-4), 74.35 (C-3), 76.16
(C-5), 120.96, 126.41, 127.99, 128.57, 141.53,
144.52, 144.59 (Fmoc±Ar), 156.64 (Fmoc±CO),
163.42 (NH±CO), 170.37, 170.92, 171.12 (3ÂAc±
CO), 173.54 (CO). MS (FAB): 627.2 (M+H)+.
Anal. Calcd. for C31H34N2O12: C, 59.42; H, 5.47;
N, 4.47. Found: C, 59.17; H, 5.51; N, 4.44.
General procedure for solid-phase synthesis of
peptides (3) and (10±12).ÐThe standard synthesis
protocol from Perseptive Applied Biosystems syn-
thesizer was performed using 4 equivalents of N-
Fmoc-protected amino acid derivatives and piper-
idine (20% in DMF) as Fmoc removal reagent.
Fmoc-Rink-PEGA resin (0.180 mmol/g substitu-
tion) was used in a 278 mg scale (50 ꢄmol) for each
peptide. DMF was used as solvent for all reactions.
The building block 9 was coupled manually outside
the synthesizer in equimolar value. The peptide
synthesis was ®nished by acetylation with acetic
anhydride (50% in DMF) and cleavage of the gly-
copeptides from the polymer with concurrent
Ng -[3,4,6-tri-O-acetyl-1,5-anhydro-2-deoxy-
glucitol-2]-l-asparagine (1).ÐTo a solution of 8
(500 mg, 0.8 mmol) in dry methanol (25 mL) Pd/C
(50 mg) was added and the mixture was stirred
under hydrogen atmosphere (1 bar) for 8 h. The
catalyst was ®ltered o€ and the solution was con-
centrated under reduced pressure. Yieldꢀ: 323.5 mg
(100%), colourless foam. M.p. 115 C; [ꢂ]25
+14.3 (c 1, H2O); H NMR (D2O) ꢃ 2.10 (s, 3 H;
d
1
Ac±H3), 2.12 (s, 3 H; Ac±H3), 2.14 (s, 3 H; Ac±H3),
2.94 (d, 2 H, Jꢂ,ꢀ 5.6 Hz; Asn-ꢀ-H), 3.53 (dd, 1 H,
J1a,1e 11.7, J1a,2 11.2 Hz; H-1a), 3.92 (ddd, 1 H, J4,5
10.2, J5,6 4.1, J5,6* 2.0 Hz; H-5), 4.04 (dd, 1 H, J1a,1e
11.7, J1e,2 5.6 Hz; H-1e), 4.21 (dd, 1 H, J5,6 4.1,
J6,6* 12.7 Hz; H-6), 4.28 (ddd, 1 H, J1a,2 11.2, J1e,2
5.6, J2,3 9.2 Hz; H-2), 4.30 (t, 1 H, Jꢂ,ꢀ 5.6 Hz; Asn-
ꢂ-H), 4.37 (dd, 1 H, J5,6* 2.0, J6,6* 12.7 Hz; H-6*),
5.05 (dd, 1 H, J2,3 9.2, J3,4 9.7 Hz; H-3), 5.22 (dd, 1
H, J3,4 9.7, J4,5 10.2 Hz; H-4); 13C NMR (D2O) ꢃ
20.45, 20.47, 20.52 (3ÂAc±CH3), 35.36 (Asn±ꢀ-C),
49.36 (C-2), 51.56 (Asn±ꢂ-C), 62.64 (C-6), 67.29
(C-1), 69.22 (C-4), 74.57 (C-3), 75.89 (C-5), 172.25
(Ac±CO), 173.29 (CO), 173.83, 174.08 (2ÂAc±CO);
MS (FAB): 405.2 (M+H)+. Anal. Calcd. for
C16H24N2O10: C, 47.52; H, 5.98; N, 6.93. Found:
C, 47.48; H, 5.95; N, 6.85.
Na-(90 -Fluorenylmethyloxycarbonyl)-Ng-[3,4,6-
tri-O-acetyl-1,5-anhydro-2-deoxy-glucitol-2]-l-
asparagine (9).ÐA solution of 9-¯uorenylmethyl-
succinimidyl carbonate (327 mg, 0.97 mmol) in
DMF (4 mL) was added to a vigorous stirred

A. Schafer et al./Carbohydrate Research 313 (1998) 107±116
È
113
removal of the Boc, tBu, Pbf and trityl side-chain
protecting groups by treating the resin with aq
95% CF3CO2H (2 mL) two times for each 1 h. The
resin was washed with aq 95% CF3CO2H
(5Â2 mL, 2 min each). The combined ®ltrates were
evaporated and the residue was coevaporated sev-
eral times with toluene and 3:1 toluene±MeOH.
Deacetylated compounds 11 and 3 were available
by dissolving glycopeptides 10 resp. 12 in aq 50%
MeOH (2 mL) and addition of a 1% solution of
NaOMe in MeOH (&50 ꢄL) until pH 9.0. The
reaction was stopped by the addition of HOAc
(10 ꢄL).The deprotection was followed by analy-
tical HPLC.
(60% overall yield: 13%). 1H NMR (D2O, pD 3.5)
ꢃ 1.15 (d, 3 H, Jꢀ,ꢁ 6.4 Hz, Thr-ꢁ-H3), 1.16 (d, 3 H,
Jꢀ,ꢁ 6.1 Hz, Thr-ꢁ-H3), 2.05, (s, 3 H, Ac±H3), 2.71
(dd, 1 H, Jꢂ,ꢀ 6.2 Hz, Jꢀ,ꢀ* 15.5 Hz, Asn-ꢀ-H), 2.84
(dd, 1 H, Jꢂ,ꢀ* 6.1 Hz, Jꢀ,ꢀ* 15.5 Hz, Asn-ꢀ*-H),
2.84 (dd, 1H, Jꢂ,ꢀ 7.6 Hz, Jꢀ,ꢀ* 16.5 Hz, Asn±ꢀ±
H), 2.94 (dd, 1H Jꢂ,ꢀ* 5.3 Hz, Jꢀ,ꢀ* 16.5 Hz, Asn-
ꢀ*-H), 3.12 (dd, 1 H, J1a,1e 10.4 Hz, J1a,2 10.7 Hz,
H-1a), 3.25 (ddd, 1 H, J4,5 9.5 Hz, J5,6 2.2 Hz, J5,6*
5.7 Hz, H-5), 3.32 (dd, 1 H, J3,4 9.0 Hz, J4,5 9.5 Hz,
H-4), 3.43 (dd, 1 H, J2,3 9.4 Hz, J3,4 9.0 Hz, H-3),
3.61 (dd, 1 H, J5,6 2.2 Hz, J6,6* 12.3 Hz, H-6) 3.73
(ddd, 1 H, J1a,2 10.7 Hz, J1e,2 5.1 Hz, J2,3 9.4 Hz, H-
2), 3.78 (dd, 1 H, J5,6* 5.7 Hz, J6,6* 12.3 Hz, H-6*),
3.80 (dd, 1 H, J1a,1e 10.4 Hz, J1e,2 5.1Hz, H-1e),
4.14 (dd, 1 H, Jꢂ,ꢀ 4.6 Hz, Jꢀ,ꢁ 6.4 Hz, Thr-ꢀ-H),
4.26 (d, 1 H, Jꢂ,ꢀ 4.6 Hz, Thr-ꢂ-H), 4.26 (dd, 1 H,
Jꢂ,ꢀ 6.1 Hz, Jꢀ,ꢁ 6.1 Hz, Thr-ꢀ-H), 4.26 (d, 1 H, Ja,ꢀ
6.1 Hz, Thr-ꢂ-H), 4.73 (dd, 1 H, Jꢂ,ꢀ 6.2 Hz, Jꢂ,ꢀ*
6.1 Hz, Asn-ꢂ-H), 4.79 (dd, 1 H, Jꢂ,ꢀ 7.6 Hz, Jꢂ,ꢀ*
5.3 Hz, Asn-ꢂ-H). MS (MALDI±TOF) Calcd for
C24H40N6O14 (M)+: 636.26. Found: (M+Na)+
658.3.
Na-Acetyl-l-threonyl-Ng-[3,4,6-tri-O-acetyl-1,5-
anhydro-2-deoxy-glucitol-2]-l-asparagyl-l-aspartyl-
l-threonylamide (10).ÐAcylation times for all
amino acids were 30 min according to the standard
protocol, but 24 h in case of building block 9. The
cleavage of 10 from the polymer was followed by
puri®cation using HPLC [bu€er A-bu€er B, 100/
0!50/50 (10 min)]. Yield: 8 mg (21%) (calculated
1
on the substitution of the resin). H NMR (D2O,
pD 3.5) ꢃ 1.14 (d, 3 H, Jꢀ,ꢁ 6.4 Hz, Thr-ꢁ-H3), 1.18
(d, 3 H, Jꢀ,ꢁ 6.4 Hz, Thr-ꢁ-H3), 2.01 (s, 3 H, Ac±
H3), 2.04 (s, 3 H, Ac±H3), 2.04 (s, 3 H, Ac±H3),
2.06 (s, 3 H, Ac±H3), 2.63 (dd, 1 H, Ja,ꢀ 7.1 Hz,
Jꢀ,ꢀ* 15.4 Hz, Asn-ꢀ-H), 2.77 (dd, 1 H, Jꢂ,ꢀ* 6.2 Hz,
Jꢀ,ꢀ* 15.4 Hz, Asn-ꢀ*-H), 2.83 (dd, 1 H, Ja,ꢀ 7.3 Hz,
Jꢀ,ꢀ* 17.1 Hz, Asn-ꢀ*-H), 2.92 (dd, 1 H, Jꢂ,ꢀ*
5.6 Hz, Jꢀ,ꢀ* 17.1 Hz, Asn-ꢀ*-H), 3.41 (dd, 1 H,
J1a,1e 11.5 Hz, J1a,2 11.4 Hz, H-1a), 3.82 (ddd, 1 H,
J4,5 9.9 Hz, J5,6 2.0 Hz, J5,6* 4.6 Hz, H-5), 3.92 (dd,
1 H, J1a,1e 11.5 Hz, J1e,2 5.5 Hz, H-1e), 4.02 (ddd, 1
H, J1a,2 11.4 Hz, J1e,2 5.5 Hz, J2,3 10.4 Hz, H-2),
4.02 (dd, 1 H, J5,6 2.0 Hz, J6,6* 13.0 Hz, H-6) 4.12
(dd, 1 H, Jꢂ,ꢀ 4.6 Hz, Jꢀ,ꢁ 6.4 Hz, Thr-ꢀ-H), 4.24 (d,
1 H, Ja,ꢀ 4.6 Hz, Thr-ꢂ-H), 4.25 (dd, 1 H, Ja,ꢀ
5.1 Hz, Jꢀ,ꢁ 6.4 Hz, Thr-ꢀ-H), 4.25 (d, 1 H, Jꢂ,ꢀ
5.1 Hz, Thr-ꢂ-H), 4.29 (dd, 1 H, J5,6* 4.6 Hz, J6,6*
13.0 Hz, H-6*), 4.69 (dd, 1 H, Jꢂ,ꢀ 7.1 Hz, Jꢂ,ꢀ*
6.2 Hz, Asn-ꢂ-H), 4.77 (dd, 1 H, Jꢂ,ꢀ 7.3 Hz, Ja,ꢀ*
5.6 Hz, Asn-ꢂ-H), 4.96 (dd, 1 H, J3,4 9.2 Hz, J4,5
9.9 Hz, H-4), 5.12 (dd, 1 H, J2,3 10.4 Hz, J3,4
9.2 Hz, H-3). MS (FAB) Calcd for C30H46N6O17
(M)+: 762.74. Found: (M+Na)+ 785.8 (81%),
(M+H)+ 763.8 (100%), (M±NH2)+ 746.8 (37%).
Na-Acetyl-l-threonyl-Ng-[1,5-anhydro-2-deoxy-
glucitol-2]-l-asparagyl-l-aspartyl-l-threonylamide
(11).ÐThe reaction time was 3 h. The ®nal product
was puri®ed by HPLC [bu€er A-bu€er B, 100/
0!85/15 (5 min)!60/40 (10 min)]. Yield: 4.0 mg
Na-Acetyl-l-seryl-Ng-[3,4,6-tri-O-acetyl-1,5-
anhydro - 2 - deoxy - glucitol - 2] - l - asparagyl - l -
asparagyl-l-threonyl-l-arginyl-l-lysyl-l-seryl-l-
isoleucyl-l-histidyl-l-isoleucyl-l-glycyl-l-prolyl-l-
glycyl - l - arginyl - l - alanyl - l - phenylalanylamide
(12).ÐAcylation times for all amino acids were
90 min, but in case of building block 9 24 h.
The cleavage of one third aliquot of the product
12 was followed by puri®cation using HPLC
[bu€er A±bu€er B, 100/0!80/20 (5 min)!70/30
(30 min)!50/50 (5 min)]. Yield: 7.6 mg (23%) (cal-
1
culated on the substitution of the resin). H NMR
(D2O, pD 3.5) ꢃ 0.83 (dd, 3 H, Jꢁ*a,ꢃ 7.6 Hz, Jꢁ*b,ꢃ
7.9 Hz, Ile-ꢃ-H3), 0.83 (dd, 3 H, Jꢁ*a,ꢃ 7.4, Jꢁ*b,ꢃ
7.6 Hz, Ile-ꢃ-H3), 0.85 (d, 3H, Jꢀ,ꢁ 6.9 Hz, Ile-ꢁ-
H3), 0.90 (d, 3 H, Jꢀ,ꢁ 6.9 Hz, Ile-ꢁ-H3), 1.13 (m, 1
H, Jꢁ*a,ꢁ*b 20.3 Hz, Jꢁ*b,ꢃ 7.9 Hz, Ile-ꢁ*b-H), 1.15
(m, 1 H, Jꢁ*a,ꢁ*b 20.3 Hz, Jꢁ*b,ꢃ 7.6 Hz, Ile-ꢁ*b-H),
1.22 (d, 3 H, Jꢀ,ꢁ 6.4 Hz, Thr-ꢁ-H), 1.28 (d, 1H,
Jꢂ,ꢀ 7.1 Hz, Ala-ꢀ-H), 1.38 (m, 1H, Jꢁ*a,ꢁ*b 20.3 Hz,
Jꢁ*a,ꢃ 7.6 Hz, Ile-ꢁ*a-H), 1.44 (m, 1 H, Jꢁ*a,ꢁ*b
20.3 Hz, Jꢁ*a,ꢃ 7.4 Hz, Ile-ꢁ*a-H), 1.57 (ddt, 2 H,
Jꢀ,ꢁ 8.6 Hz, Jꢀ*,ꢁ 5.7 Hz, Jꢁ,ꢃ 6.9 Hz, Arg-ꢁ-H2),
1.57 (m, 2H, Jꢁ,ꢃ 8.5 Hz, Lys-ꢁ-H2), 1.64 (ddt, 2 H,
Jꢀ,ꢁ 9.1 Hz, Jꢀ*ꢁ 6.0 Hz, Jꢁ,ꢃ 6.9 Hz, Arg-ꢁ-H2), 1.68
(ddt, 2 H, Jꢁ,ꢃ 8.5 Hz, Jꢃ," 7.6 Hz, Jꢃ,"* 8.1 Hz, Lys-
ꢃ-H2), 1.75 (m, 1 H, Jꢀ,ꢁ 8.6 Hz, Arg-ꢀ-H), 1.79 (m,
1 H, Lys-ꢀ-H), 1.81 (m, 1 H, Jꢂ,ꢀ 7.4 Hz, Jꢀ,ꢁ
6.9 Hz, Ile-ꢀ-H), 1.81 (m, 1 H, Jꢀ,ꢁ 9.1 Hz, Arg-ꢀ-

Table 2
1H NMR data (H2O/D2O 9:1, pH 3.5, 500 MHz) for glycopeptide 3 coupling constants (Hz) in parentheses
ꢃ(ppm)
NH Hꢂ1 Hꢂ2 Hꢀ1 Hꢀ2 Hꢁ1a Hꢁ1b Hꢁ2 Hꢃ1 Hꢃ2 H"1 H"2 Ac/NH H-1a H-1b H-2 H-3 H-4 H-5 H-6a H-6b
Arom.
Ser-1
8.302 4.392
(6.3) (5.7)
(5.3)
3.847 3.804
(11.9)
2.044
Asn-2
Asn-3
8.544 4.731
(7.8) (5.4)
(7.6)
8.364 4.740
(7.3) (6.0)
(7.5)
2.850 2.741
(15.9)
8.160 3.870 3.199 3.799 3.505 3.375 3.314 3.855 3.374
(8.5) (11.0) (10.8) (10.1) (8.7) (9.7) (2.2) (12.3)
(5.4)
(5.8)
2.842 2.746
(15.9)
7.493
Thr-4
Arg-5
8.109 4.278
(7.5) (4.5)
8.265 4.310
(7.3) (6.3)
(8.5)
4.234
(6.4)
1.843 1.755 1.612
(7.5) (6.6)
1.188
1.612 3.173 3.173
(6.9) (6.0)
7.136
Lys-6
Ser-7
Ile-8
8.250 4.303
(7.8) (6.2)
(8.0)
8.252 4.431
(7.3) (5.0)
(5.3)
1.806 1.719 1.411
(15.9)
1.411 1.654 1.654 2.967 2.967
(5.8) (7.4) (7.4)
(8.6)
3.822 3.780
(11.5)
8.055 4.153
(7.7) (7.5)
1.796
(7.1)
(7.3)
3.192 3.098
(15.3)
1.314 1.106 0.815 0.798
(19.8) (7.5)
(7.3)
His-9
Ile-10
8.559 4.718
(7.6) (6.4)
(8.9)
8.210 4.191
(7.9) (8.2)
8.556
(1.4)
7.232
1.793
(7.0)
(6.7)
1.416 1.127 0.871 0.808
(20.2) (7.8)
(7.3)
Gly-11 8.300 4.114 4.033
(5.9) (17.3)
(5.5)
Pro-12
4.350
(8.5)
(4.6)
2.252 1.961 2.000
(13.5) (7.0) (6.6)
(7.7)
2.000 3.645 3.594
(7.2) (10.0)
Gly-13 8.469 3.910 3.910
(5.9)
Arg-14 8.089 4.259
(6.7) (6.4)
(8.1)
1.750 1.663 1.532
(5.7) (3.6) (6.1)
1.532 3.131 3.131
(6.1)
7.106
Ala-15 8.262 4.240
(6.6) (7.2)
1.258
Phe-16 8.075 4.533
(7.3) (6.0)
(8.6)
3.123 2.990
(13.9)
7.463
7.041
7.36±7.22

A. Schafer et al./Carbohydrate Research 313 (1998) 107±116
È
115
H), 1.82 (m, 1 H, Jꢀ*ꢁ 5.7 Hz, Arg-ꢀ*-H), 1.84 (m,
1 H, Jꢂ,ꢀ 7.9 Hz, Jꢀ,ꢁ 6.9 Hz, Ile-ꢀ-H), 1.85 (m, 1 H,
Jꢀ*,ꢁ 6.0 Hz, Arg-ꢀ*-H), 1.86 (m, 1 H, Lys-ꢀ*-H),
1.99 (ddt, 1 H, Jꢂ,ꢀ 6.4 Hz, Jꢀ,ꢀ* 11.9 Hz, Jꢀ,ꢁ
6.9 Hz, Pro-ꢀ-H), 2.04 (dddt, 2 H, Jꢀ,ꢁ 6.9 Hz, Jꢀ*,ꢁ
7.2 Hz, Jꢁ,ꢃ 6.4 Hz, Jꢁ,ꢃ* 9.4 Hz, Pro-ꢁ-H2), 2.05 (s,
3 H, Ac±H3), 2.07 (s, 3 H, Ac±H3), 2.08 (s, 3 H,
Ac±H3), 2.10 (s, 3 H, Ac±H3), 2.28 (ddt, 1 H, Jꢂ,ꢀ*
9.2 Hz, Jꢀ,ꢀ* 11.9 Hz, Jꢀ*,ꢁ 7.2 Hz, Pro-ꢀ*-H), 2.68
(dd, 1 H, Jꢂ,ꢀ 7.1 Hz, Jꢀ,ꢀ* 15.6 Hz, Asn-ꢀ-H), 2.76
(dd, 1 H, Jꢂ,ꢀ 7.6 Hz, Jꢀ,ꢀ* 16.0 Hz, Asn-ꢀ-H), 2.80
(dd, 1 H, Jꢂ,ꢀ* 6.4 Hz, Jꢀ,ꢀ* 15.6 Hz, Asn-ꢀ*-H),
2.86 (dd, 1 H, Jꢂ,ꢀ* 6.0 Hz, Jꢀ,ꢀ* 16.0 Hz, Asn-ꢀ*-
H), 2.99 (dd, 2 H, Jꢁ,ꢃ 7.6 Hz, Jꢁ*,ꢃ 8.1 Hz, Lys-"-
H2), 3.02 (dd, 1 H, Jꢂ,ꢀ 8.5 Hz, Jꢀ,ꢀ* 14.2 Hz, Phe-
ꢀ-H), 3.10 (dd, 1 H, Jꢂ,ꢀ 7.8 Hz, Jꢀ,ꢀ* 15.8 Hz, His-
ꢀ-H), 3.15 (dd, 1 H, Jꢂ,ꢀ 6.0 Hz, Jꢀ,ꢀ* 14.2 Hz, Phe-
ꢀ*-H), 3.16 (t, 2 H, Jꢁ,ꢃ 6.9 Hz, Arg-ꢃ-H2), 3.20 (t,
2 H, Jꢁ,ꢃ 6.9 Hz, Arg-ꢃ-H2), 3.22 (dd, 1 H, Jꢂ,ꢀ*
7.1 Hz, Jꢀ,ꢀ* 15.8 Hz, His-ꢀ*-H), 3.47 (dd, 1 H,
J1a,1e 11.4 Hz, J1a,2 11.4 Hz, H-1a), 3.62 (dt, 1 H,
Jꢁ,ꢃ 6.4 Hz, Jꢃ,ꢃ* 13.5 Hz, Pro-ꢃ-H), 3.70 (dt, 1 H,
Jꢁ,ꢃ* 9.4 Hz, Jꢃ2,ꢃ* 13.5 Hz, Pro-ꢃ*-H), 3.82 (t, 2 H,
Jꢂ,ꢀ 5.0 Hz, Ser-ꢀ-H2), 3.83 (dd, 1 H, Jꢂ,ꢀ 5.3 Hz,
Jꢀ,ꢀ* 11.7 Hz, Ser-ꢀ-H), 3.85 (dd, 1 H, Jꢂ,ꢀ* 5.3 Hz,
Jꢀ,ꢀ* 11.7 Hz, Ser-ꢀ*-H), 3.87 (ddd, 1 H, J4,5
9.7 Hz, J5,6 1.9 Hz, J5,6* 6.0 Hz, H-5), 3.93 (s, 2 H,
Gly-ꢂ-H2), 3.98 (dd, 1 H, J1a,1e 11.4 Hz, J1e,2
7.0 Hz, H-1e), 4.05 (d, 1 H, Jꢂ2,ꢂ* 16.4 Hz, Gly-ꢂ-
H), 4.14 (d, 1 H, Jꢂ2,ꢂ* 16.4 Hz, Gly-ꢂ*-H), 4.17
(ddd, 1 H, J1a,2 11.4 Hz, J1e,2 7.0 Hz, J2,3 9.9 Hz, H-
2), 4.18 (d, 1 H, Jꢂ,ꢀ 7.4 Hz, Ile-ꢂ-H), 4.19 (dd, 1 H,
J5,6 1.9 Hz, J6,6* 13.0 Hz, H-6), 4.22 (d, 1 H, Jꢂ,ꢀ
7.9 Hz, Ile-ꢂ-H), 4.26 (m, 2 H, Jꢂ2,ꢀ 6.4 Hz, Thr-ꢂ-
H, Thr-ꢀ-H), 4.26 (d, 1 H, Jꢂ2,ꢀ 7.1 Hz, Ala-ꢂ-H),
4.28 (m, 1 H, Lys-ꢂ-H), 4.32 (dd, 1 H, J5,6* 6.0 Hz,
J6,6* 13.0 Hz, H-6*), 4.32 (m, 1 H, Arg-ꢂ-H), 4.34
(m, 1 H, Arg-ꢂ-H), 4.40 (t, 1 H, Jꢂ,ꢀ 5.3 Hz, Ser-ꢂ-
H), 4.46 (t, 1 H, Jꢂ,ꢀ 5.0 Hz, Ser-ꢂ-H), 4.47 (dd, 1
H, Jꢂ,ꢀ 6.4 Hz, Jꢂ,ꢀ* 9.2 Hz, Pro-ꢂ-H), 4.54 (dd, 1
H, Jꢂ,ꢀ 8.5 Hz, Jꢂ,ꢀ* 6.0 Hz, Phe-ꢂ-H), 4.72 (dd, 1
H, Jꢂ,ꢀ 7.1 Hz, Jꢂ,ꢀ* 6.4 Hz, Asn-ꢂ-H), 4.75 (dd, 1
H, Jꢂ,ꢀ 7.8 Hz, Jꢂ,ꢀ* 7.1 Hz, His-ꢂ-H), 4.76 (dd, 1
H, Jꢂ,ꢀ2 7.6 Hz, Jꢂ2,ꢀ2* 6.0 Hz, Asn-ꢂ-H), 4.99 (dd,
1 H, J3,4 9.4 Hz, J4,5 9.7 Hz, H-4), 5.17 (dd, 1 H,
J2,3 9.9 Hz, J3,4 9.4 Hz, H-3), 7.2±7.4 (m, 5 H, Phe±
Ar±H5), 7.26 (d, 1 H, J2,4 1.4 Hz, His-4-H), 8.60 (d,
1 H, J2,4 1.4 Hz, His-2-H). MS (MALDI±TOF):
Calcd for C89H142N28O29 (M)+: 2067.05. Found:
(M+H)+ 2068.1.
arginyl-l-lysyl-l-seryl-l-isoleucyl-l-histidyl-l-iso-
leucyl-l-glycyl-l-prolyl-l-glycyl-l-arginyl-l-alanyl-
l-phenylalanylamide (3).ÐThe reaction time was
4 h. The ®nal product was puri®ed by HPLC [buf-
fer A±bu€er B, 100/0!85/15 (5 min)!75/25
(40 min)!60/40 (5 min)]. Yield: 3.9 mg (55%,
overall yield: 12%). MS (MALDI±TOF) Calcd for
C83H136N28O26 (M)+: 1941.02. Found: (M+H)+
1942.2
Acknowledgements
Support of this work by the Fonds der Che-
mischen Industrie and the Deutsche For-
schungsgemeinschaft (SFB 470) is gratefully
acknowledged.
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