Versatile solid-phase thiolytic reduction of azido and N-Dts groups in the synthesis of haemoglobin (67-76) O-glycopeptides and photoaffinity labelled analogues to study glycan T-cell specificity

Article abstract of DOI:10.1039/a606725e

A series of O-glycosylated peptides and photoaffinity labelled glycopeptide analogues of the mouse haemoglobin-derived decapeptide Hb (67-76), VITAFNEGLK, which binds well to the MHC class II E^k molecule and is non-immunogenic in CBA/J mice, was synthesized by multiple-column peptide synthesis employing the glycosylated building blocks 1-4 and 7-21. The non-immunogenic peptide VITAFNEGLK was converted into an immunogen by introducing different tumour-associated carbohydrate moieties [β-D-GlcNAc-O-Ser/Thr, α-D-GalNAc-O-Ser/Thr (T_N-antigen) core 1 (T-antigen), core 2, core 3 and core 4] to the central position Asn-72 in the decapeptide. Previous studies suggest that T cells may be capable of recognizing epitopes which are partially defined by glycans and may be in direct contact with the T-cell receptor. In order to study the specificity of glycan interactions with the T-cell receptor a series of corresponding glycopeptides labelled with 2-azidobenzamide on the carbohydrate amino function was synthesized. The glycan structure was varied with respect to O-GlcNAc, T and T_N-antigen moieties and anomeric configuration. Throughout, efficient reduction of the N-dithiasuccinyl- and azido-functionality-containing building blocks 1, 2, 7, 8, 11, 12, 13, 16, 18 and 20 could be achieved either (i) in solution by utilizing simultaneous in situ reduction with Zn in THF-HOAc-Ac₂O or (ii) on solid-phase upon treatment with diisopropylethylamine and an excess of dithiothreitol or α-mercapto-N-methylacetamide. N-Acetylation of the resin-bound glycopeptides furnished the O-glycopeptides 24, 25 and 31-36. No further modification of the carbohydrate moiety on the solid phase was required when utilizing the N-acetylated building blocks 3, 4, 9, 10, 14, 15, 17, 19 and 21. In addition, comparative studies with solid-phase reduction were conducted for the syntheses of the O-linked glycopeptides 24, 25 and 31-36 by employing any of the building blocks 1-4 and 7-21. The photoaffinity labelled glycopeptides 39-45 were synthesized by employing building blocks 1, 2, 7, 8 and 11-13 by reduction of azido or N-Dts functionalities by thiolysis with dithiothreitol and subsequent coupling of the activated photoaffinity label 38 to the glycanamino group of the resin-bound glycopeptides. The synthesized mucin O-glycopeptides 24, 25 and 31-36 and the photoaffinity labelled analogues 39-45 were fully characterized by 1D and 2D ¹H NMR spectroscopy and by electrospray mass spectrometry.

Full text of DOI:10.1039/a606725e

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Versatile solid-phase thiolytic reduction of azido and N-Dts groups in
the synthesis of haemoglobin (67–76) O-glycopeptides and
photoaffinity labelled analogues to study glycan T-cell specificity
Ernst Meinjohanns,a Morten Meldal,a Teis Jensen,b Ole Werdelin,b
Luisa Galli-Stampino,c Søren Mouritsenc and Klaus Bock a
a
Carlsberg Laboratory, Department of Chemistry, Gamle Carlsberg Vej 10, DK-2500 Valby,
Copenhagen, Denmark
b Institute of Medical Microbiology and Immunology, University of Copenhagen, Copenhagen,
Denmark
c M & E A/S, Copenhagen, Denmark
A series of O-glycosylated peptides and photoaffinity labelled glycopeptide analogues of the mouse
haemoglobin-derived decapeptide H b (67–76), VITAF N EG LK , which binds well to the M H C class II E k
molecule and is non-immunogenic in CBA/J mice, was synthesized by multiple-column peptide synthesis
employing the glycosylated building blocks 1–4 and 7–21. The non-immunogenic peptide VITAF N EG LK
was converted into an immunogen by introducing different tumour-associated carbohydrate moieties [â-D -
G lcNAc-O-Ser/Thr, á-D -G alNAc-O-Ser/Thr (TN -antigen) core 1 (T-antigen), core 2, core 3 and core 4] to
the central position Asn-72 in the decapeptide. Previous studies suggest that T cells may be capable of
recognizing epitopes which are partially defined by glycans and may be in direct contact with the T-cell
receptor. In order to study the specificity of glycan interactions with the T-cell receptor a series of
corresponding glycopeptides labelled with 2-azidobenzamide on the carbohydrate amino function was
synthesized. The glycan structure was varied with respect to O-G lcNAc, T and TN -antigen moieties and
anomeric configuration. Throughout, efficient reduction of the N-dithiasuccinyl- and azido-functionality-
containing building blocks 1, 2, 7, 8, 11, 12, 13, 16, 18 and 20 could be achieved either (i) in solution by
utilizing simultaneous in situ reduction with Zn in TH F –H OAc–Ac2O or (ii) on solid-phase upon
treatment with diisopropylethylamine and an excess of dithiothreitol or á-mercapto-N-methylacetamide.
N -Acetylation of the resin-bound glycopeptides furnished the O-glycopeptides 24, 25 and 31–36. N o
further modification of the carbohydrate moiety on the solid phase was required when utilizing the N -
acetylated building blocks 3, 4, 9, 10, 14, 15, 17, 19 and 21. In addition, comparative studies with solid-
phase reduction were conducted for the syntheses of the O-linked glycopeptides 24, 25 and 31–36 by
employing any of the building blocks 1–4 and 7–21. The photoaffinity labelled glycopeptides 39–45 were
synthesized by employing building blocks 1, 2, 7, 8 and 11–13 by reduction of azido or N-D ts
functionalities by thiolysis with dithiothreitol and subsequent coupling of the activated photoaffinity label
38 to the glycanamino group of the resin-bound glycopeptides. The synthesized mucin O-glycopeptides
1
24, 25 and 31–36 and the photoaffinity labelled analogues 39–45 were fully characterized by 1D and 2D H
N M R spectroscopy and by electrospray mass spectrometry.
sialic acids to built the different core structures.3 Recently a new
Introduction
form of O-glycosylation has been identified in which a single
GlcNAc residue is attached via a β-O-linkage to serine or
threonine on proteins within the nuclear and cytoplasmic com-
partments of eukaryotic cells.4 This novel type of post-
translational glycosylation is both an abundant and transient
modification. The addition of O-GlcNAc appears to be highly
dynamic and responsive to extracellular stimuli in a fashion
analogous to that of protein phosphorylation.5 Aberrant glyco-
sylation on tumour cells is one of the most characteristic traits
of malignancy.6 It is therefore of significant interest to study
whether glycopeptide epitopes, mimicking post-translational
modification of proteins, can be recognized by T-cells. An
important event in the generation of an immune response is the
activation of T-cells by peptides bound to the class II proteins
of the major histocompatibility complex (MHC).7 The MHC
proteins have a unique binding motif that enables them to bind
to a variety of peptides. Antigenic peptides are derived from
exogenous proteins, which are processed into peptide fragments
of 10–25 amino acids and bound to MHC class II molecules by
antigen-presenting cells. The peptide–MHC class II complex is
then transported to the cell surface where they may be recog-
nized by CD-4 positive T-cells which then trigger the immune
Glycosylation is the most common modification of proteins.
Carbohydrates are bound to almost all the proteins on the cell
membrane and many microorganisms express glycosylated pro-
tein antigens. The presence of a glycan modulates the proper-
ties of the protein such as its solubility, protease resistance and
immunogenicity.1 Aberrant glycosylation of glycoproteins, due
to incomplete synthesis of carbohydrate chains and accumu-
lation of their precursors, is associated with oncogenesis and
has been implicated in metastasis. Among such glycoconju-
gates, TN (α-GalNAc-Ser/Thr) and sialyl-TN (α-Neu5Ac-2→6-
GalNAc-Ser/Thr) antigens, which belong to mucin-type (O-
linked) core carbohydrate antigens, are known to be cancer-
associated antigens that are expressed frequently in many
tumours but rarely in normal tissues.2 A glycoprotein is gener-
ally a heterogeneous mixture of different glycoforms: protein
molecules which differ only in the structure of the bound
carbohydrate. Heterogeneous glycosylation seems to be a
subtle mechanism of biological control. In O-linked glycosyla-
tion the oligosaccharide chains are attached to the side chain of
Ser or Thr through an α-anomeric linkage of a core α--GalNAc
residue which can be further elongated by Gal, GlcNAc, Fuc or
J. Chem. Soc., Perkin Trans. 1, 1997
871

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response. Although the majority of eukaryotic proteins are gly-
cosylated, it has not yet been demonstrated whether the glycan
part is mainly hydrolysed or remains attached during antigen
processing. It was recently demonstrated that the glycan can be
directly involved in the interaction with the T-cell receptor.8 In
the present study a peptide fragment of CBA/J mouse haemo-
globin Hb (67–76), VITAFNEGLK, has been selected to
investigate the influence of different glycosylations on T-cell
response. This peptide is non-immunogenic itself in CBA/J
mouse, but it is known to bind very well to the MHC class-II
molecule, Ek, expressed by the antigen-presenting cell (APC) of
the CBA/J mouse.9 By introducing glycan structures at the pos-
ition of the Asn residue the peptide was converted into an
immunogen.10 Preliminary studies using synthetic glycopeptide
analogues of this epitope VITAFNEGLK have shown that gly-
cosylation does not affect binding to MHC-class II and that
glycopeptide can elicit a strong T-cell response, which is glyco-
peptide specific.8d In order to investigate further the fine speci-
ficity of T-cell response against synthetic glycopeptides, a series
of glycopeptides containing O-glycan moieties such as β-
GlcNAc, α-GlcNAc, α-GalNAc and core 1–4 structures 24, 25
and 31–36 and corresponding photoaffinity labelled glycopep-
tide analogues 39–45 of immunogenic epitope structures has
been synthesized during this study. These glycopeptide ana-
logues may enable us to investigate the binding of contact res-
idues of the epitope to the T-cell receptor binding sites. A
detailed knowledge of the mechanisms by which glycopeptides
are processed in the antigen-processing pathway, presented by
the antigen-presenting cell and finally recognized by the T-cell
receptor is important for the design of peptide-based vaccines.
The most efficient and reliable approach to the synthesis of a
wide range of O-glycopeptides is the use of suitable protected
O-glycosylated serine and threonine amino acids as building
blocks in the stepwise assembly of glycopeptides.11 Direct gly-
cosylation of active esters derivatives, Nα-Fmoc-Ser-OPfp and
Nα-Fmoc-Thr-OPfp has been developed which alleviates the
manipulation of protecting groups in the glycosyl amino acids
and allows direct use in the multiple-column solid-phase syn-
thesis.12 Solid-phase methods utilizing Nα-Fmoc-protected gly-
cosylated amino acid building blocks are the most flexible and
reliable way to prepare a series of glycopeptides.13 In the ‘active
ester’ approach the preactivated Fmoc-AA-OPfp can be glyco-
sylated directly to provide building blocks for multiple-column
peptide synthesis (MCPS).11
This report describes the efficient multiple-column glycopep-
tide synthesis of a series of O-glycopetide analogues 24, 25 and
31–36 employing either the use of the acetamido building
blocks 3, 4, 9, 10, 14, 15, 17, 19 and 21 or alternatively the use
of the N-dithiasuccinyl (N-Dts)- and azido (N3)-containing
building blocks 1, 2, 7, 8, 11–13, 16, 18 and 20. The syntheses of
the N-Dts- and N3-containing building blocks 1, 2, 7, 8, 11–13,
16, 18 and 20 and the N-acetylated building blocks 9, 10, 14, 15,
17, 19 and 21 has been reported previously.14,15 The reduction of
the amino group precursors N3 and N-Dts groups are import-
ant transformations in the solid-phase synthesis of glycopep-
tides to introduce the amino functions. The N-Dts functionality
can be cleaved efficiently by thiolytic reduction on a solid phase
by employing dithiothreitol (DTT) or β-mercaptoethanol
(BME) in the presence of diisopropylethylamine (DIPEA) as a
catalyst.15,16 The parent amino functions can be successfully
modified on the solid phase to the corresponding N-acetyl
glycopeptide.15,16 Azides, on the other hand, are, in general,
readily reduced in solution to their corresponding amines by a
range of reducing agents [e.g. LiAlH4, H2–Lindar catalyst, Zn–
HCl, PPh3–water, H2S–pyridine, Bu3SnH–azoisobutyronitrile
(AIBN), NaBH4–phase-transfer catalyst, thioacetic acid]. Most
of these methods unfortunately suffer from disadvantages in
relation to general applicability, selectivity and particularly
convenience for reductions on a solid phase where homo-
geneous conditions are required. It has been shown that azido
sugars, on the other hand, can be reduced by dithiols under
weakly basic conditions on a solid phase.17 As for the mechan-
ism, initial attack by a thiolate anion on the azide with sub-
sequent cyclization to a cyclic disulfide and the amine, respect-
ively, is proposed. Reduction of azides with thioacetic acid and
simultaneous N-acetylation is cumbersome and inconvenient
due to the long reaction times required and the inherent forma-
tion of N-thioacetates as impurities. In order to circumvent
these difficulties different thiols were investigated for the reduc-
tion of azide- and N-Dts-containing glycopeptides on a solid
phase in the present report. A different strategy for the in situ
reduction and N-acetylation of azido and N-Dts groups by
means of Zn-reduction in tetrahydrofuran (THF)–Ac2O–
HOAc to yield the N-acetylated building blocks 9, 10, 14, 15,
17, 19 and 21 for the MCPS of mucin core 1, 2, 3 and 4 O-
glycopeptides has previously been described.14 The 2-azido and
the 2-N-Dts group have been employed as amino group pre-
cursors to obtain, stereoselectively, the α- (azido-approach) or
β-linked (N-Dts approach) amino sugars, respectively. The syn-
thetic utility of DTT or N-methyl-α-mercaptoacetamide (MCA)
for the rapid and mild reduction of the azide- and the N-Dts
functionalities both in solution and on solid phase in the MCPS
of the glycopeptides 24, 25 and 31–36 is described. Further-
more, MCPS and characterization of the seven photoaffinity
labelled glycopeptide analogues 39–45 is reported. The MCPS
was successfully achieved via the incorporation of the pre-
viously described N-Dts and azido building blocks 1, 2, 7, 8 and
11–13 into standard MCPS, followed by DTT-reduction on the
solid phase and coupling of the photoaffinity label 38 to the
free amino group of the glycan during the MCPS procedure.
The general application of glycosylated building blocks with
thiol reduction of azido and N-Dts groups in the synthesis of a
range of O-glycopeptides is described. In addition we report the
1H NMR and electrospray mass spectrometry (ESMS) char-
acterization of all the O-glycopeptides 24, 25 and 31–36 and
39–45.
Results and discussion
The present paper describes the synthesis of a large variety of
O-glycopeptides and their photoaffinity-labelled analogues of
the Hb (67–76) peptide. One of the most efficient methods for
the synthesis of such a series of glycopeptides is MCPS per-
formed in a Teflon block with several synthesis columns
employing glycosylated amino acids as building blocks. The
protecting group patterns of the building blocks has previously
been shown to be suitable for the solid-phase synthesis of O-
glycopeptides. The O-glycopeptides were assembled by Fmoc-
based solid-phase synthesis. The use of Fmoc allows mild
deprotection with piperidine in N,N-dimethylformamide
(DMF) without β-elimination of the carbohydrate. O-Acetyl
groups were employed for protection of carbohydrate hydroxy
groups, allowing mild deprotection with sodium methoxide in
methanol. The carboxy group is activated as its pentafluoro-
phenyl (Pfp) ester for aminolysis and formation of the peptide
bonds. The reactivity of the Pfp esters was further enhanced
by addition of 3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine
(DHBT-OH). The use of DHBT-OH as an auxiliary nucleo-
phile allows the progress of formation of the amide bond to be
followed visually. The peptide syntheses were performed on
Macrosorb resin, derivatized by the acid-labile hydroxymethyl-
phenoxyacetic acid (HMPA) linker, using a two-step reaction
cycle (coupling and Fmoc deprotection). The linker was
coupled to the resin by activation with O-(benzotriazol-1-yl)-
N,N,NЈ,NЈ-tetramethyluronium tetrafluoroborate (TBTU) and
N-ethylmorpholine (NEM). The first amino acid, lysine, was
coupled as Fmoc-Lys(Boc)-OH by activation with 1-
mesitylsulfonyl-3-nitro-1,2,4-triazole (MSNT) and N-methyl-
imidazole. Unchanged amino groups were capped by 20%
acetic anhydride in DMF. The resin was washed thoroughly
872
J. Chem. Soc., Perkin Trans. 1, 1997

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after each Fmoc-removal and acylation step. Fmoc cleavages
were effected by treatment with 20% piperidine in DMF. The
successive amino acids were coupled as Pfp esters. The glyco-
sylated Fmoc-amino acid-OPfp esters were coupled as usual
amino acids in MCPS by using extended reaction times and
only a small excess of the building block. Acidolytic cleavage
and deprotection of the peptide moiety of the completed gly-
copeptides with 95% aq. trifluoroacetic acid (TFA) and final
deacetylation with sodium methoxide in methanol gave the
desired glycopeptides. The crude glycopeptides were then puri-
fied by semipreparative C-18 HPLC and analysed by ESMS and
1H NMR spectroscopy.
lem in the selective removal of C-2 amino-protecting groups in
the presence of adjacent active esters. The N-Dts and acet-
amido building blocks 1, 2, 3 and 4, respectively, were then used
in comparative studies for the MCPS of the β-O-GlcNAc gly-
copeptides 24 and 31. Comparison of the thiolytic solid-phase
reduction conditions employing different thiols (see Table 1)
showed that rapid and quantitative cleavage of the N-Dts
amino protecting group could be achieved by thiolysis with any
of the thiols such as DTT, MCA, BME or propane-1,3-dithiol
(PDT). Concerning yields, the use of DTT and MCA is
superior to the less expensive but less efficient use of BME and
PDT (see Table 1). Employing the N-acetylated β-O-GlcNAc
building blocks 3 and 4 served the same purpose to synthesize
the corresponding β-O-GlcNAc glycopeptides 24 and 31. The
comparative results (see Table 2) showed that, due to the omis-
sion of further deprotection and N-acetylation steps on the
solid phase, generally higher yields were obtained by use of the
β-O-GlcNAc building blocks 3 (80% compared with 78%) and 4
(80% compared with 68%). However, these yields do not take
into account the loss of compound during reduction of 1 and 2
Table 2 Comparative results obtained by employing the O-linked
building blocks 1–4 and 7–19 in the synthesis of O-glycopeptides 24, 25
and 31–36. Expected and observed molecular masses for the individual
glycopeptides 24, 25 and 31–36 analysed by ESMS
Relative
Molecular
Compound formula
molecular
mass
Building
ESMSa block
Yield
(%)b
Scheme 1 Synthesis of the β--O-GlcNAc building blocks 3 and 4;
reagents and conditions: i, Zn in THF–Ac2O–HOAc (3:2:1)
24
24
31
31
25
25
32
32
33
33
34
34
35
35
36
36
C57H94N12O20 1267.46
C58H96N12O20 1281.49
C57H94N12O20 1267.46
C58H96N12O20 1281.49
C64H106N12O25 1443.76
C72H119N13O30 1646.83
C66H109N13O25 1484.68
C74H122N14O30 1687.88
1267.63
1267.65
1281.72
1281.78
1267.79
1267.64
1267.63
1267.72 10
1444.43 13
1444.43 15
1647.35 16
1647.38 17
1485.22 18
1485.28 19
1688.35 20
1688.36 21
1
3
2
4
7
9
8
78c
80
68c
80
Synthesis of the â-O-GlcNAc glycopeptides 24 and 31
The N-Dts-concept afforded the stereocontrolled synthesis of
the β-O-GlcNDts-containing building blocks 1, 2, 16, 18 and 20
and the N-Dts group could be removed either (i) by zinc reduc-
tion in THF–acetic anhydride–acetic acid with simultaneous
N-acetylation in solution or (ii) by thiolysis with DTT or BME
on the solid phase. By treatment of the N-Dts-protected deriva-
tives 1 and 2 with zinc in THF–acetic anhydride–acetic acid
(3:2:1) the N-acetylated β--GlcNAc building blocks 3 and
4 were obtained after silica gel chromatography and recrystal-
lization in 70 and 76% yield, respectively. Formation of the
bicyclic lactams 5 and 6 as minor by-products (less than 5%)
was observed during the conversion (see Scheme 1). The
bicyclic lactams 5 and 6 are formed by intramolecular dis-
placement of the Pfp ester by the amino group on C-2. This side
reaction, forming a seven-membered ring, is a common prob-
82c
85
63c
75
52c
76
45c
64
45c
62
35c
55
Glycopeptides were detected as [glycopeptide ϩ H]ϩ ions. b Obtained
after preparative reversed-phase HPLC. c Thiolysis conditions: DTT
(0.2 mol dmϪ3), DIPEA (0.1 mmol dmϪ3) in dichloromethane and sub-
sequent N-acetylation with 20% Ac2O in DMF.
a
Table 1 Reduction of N-Dts- and azido group-containing glycopeptides 22 and 23 with subsequent N-acetylation to obtain the glycopeptides 24
and 25 by employing different thiolytic reagents and DIPEA as a catalyst (see Scheme 2)
Building
block
Thiol
(0.2 )
Reaction time
on solid phase
Yields
(%)
Entry
Glycopeptide
Base
1
2
3
4
5
6
7
8
N-Dts 1
N-Dts 1
N-Dts 1
N-Dts 1
Azide 7
Azide 7
Azide 7
Azide 7
24
24
24
24
25
25
25
25
DTT
McA
BME
PDT
DTT
McA
BME
PDT
DIPEA
(0.1 m)
DIPEA
(0.3 )
DIPEA
(0.3 )
DIPEA
(0.3 )
DIPEA
(0.1 m)
DIPEA
(0.3 )
DIPEA
(0.3 )
DIPEA
(0.3 )
2 × 10 min
2 × 10 min
2 × 10 min
2 × 10 min
2 × 2 h
93,a
78b
92,a
74b
89,a
67b
87,a
62b
99,a
82b
98,a
78b
65,a
48b
15,a
7b
2 × 2 h
2 × 2 h
2 × 2 h
a
Yields were determined from chromatographic data (analytical HPLC and percentage of product determined by absorption at 220 nm). b Yields
after preparative HPLC (based on 50 mg resin with the loading 0.2 mmol gϪ1 specified by the supplier).
J. Chem. Soc., Perkin Trans. 1, 1997
873

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to 3 and 4, respectively. This loss becomes more important with
increasing complexity of the building blocks, e.g. 16 and 20.
and glycopeptide analogue synthesis, the selectivity between the
reduction of N-Dts and azido functionalities in solution and on
the solid-phase was investigated. Therefore, different conditions
for deprotection of the Dts group in the presence of azides were
studied. The previously described compound 2618 was therefore
treated with different thiols. It could be shown that it is possible
to reduce both the N-Dts and azido group simultaneously, util-
izing DTT or MCA as reducing reagent and DIPEA as catalyst.
After N-acetylation the corresponding 1,2-bisacetamido com-
pound 28 could be obtained in almost quantitative yield
(Scheme 3). By use of the less reactive PDT in the presence of
DIPEA it was possible to reduce selectively the N-Dts group of
substrate 26 without affecting the azido group. After N-
acetylation the β-azide 27 was obtained in 94% yield. Further-
more, this selective reduction was used for selective removal of
N-Dts in the presence of azido groups on the solid phase. To
study this reaction the resin-bound glycopeptide 29 was syn-
thesized on the solid phase by employing the previously
described building block 16 containing both an N-Dts and an
azido group. Our results concerning reduction of azides on the
solid phase (see Table 1) showed that utilizing PDT as reducing
agent, even at prolonged reaction times, only reduces the azido
group slightly (15% after 4 h) while the N-Dts group was com-
pletely cleaved after only about 10 min. By employing PDT as
reducing reagent for the glycopeptide 29, thiolytic removal of N-
Dts could be obtained selectively in the presence of the azido
group. After acetylation, cleavage and HPLC separation the
glycopeptide 30 was obtained in 76% yield (Scheme 4). No
reduction of the azido group was observed. The use of the less
reactive PDT as thiolytic reagent allowed the selective reduc-
tion of N-Dts in the presence of the less reactive azido group.
The successful thiolysis of the azide can be easily detected by 1H
NMR spectroscopy. Upon transformation of azide to acet-
Reduction of the azido group on the solid phase
The azido approach, on the other hand, offers the possibility of
synthesizing predominantly the α-linked O-GalNAc-serine and
-threonine building blocks. Glycosylation of Fmoc-Ser-OPfp or
Fmoc-Thr-OPfp esters with 2-azido-2-deoxygalactosyl donors
affords, with high stereoselectivity and good yields, the corre-
sponding α-linked building blocks. The azido group can be
reduced and N-acetylated in the same way as the N-Dts group
either (i) prior to incorporation of the building blocks to MCPS
by zinc reduction in THF–acetic anhydride–acetic acid or (ii)
after incorporation on the solid phase by employing DTT or
MCA as reducing reagents. In Table 1 are listed both the ana-
lytical and the isolated yields for the reduction of the resin-
bound azido group-containing glycopeptide by means of the
different thiolytic reagents. The results demonstrate the ease
and applicability of the thiolytic azido-reduction procedure on
the solid phase. The rate of reduction varies considerably with
the employed thiol. High reduction yields were obtained by
using DTT and MCA (99 and 98%). In contrast, employment
of the less reactive thiolytic reagents BME and PDT gave yields
which were low(er) (65 and 15%). Due to the higher yields of
the obtained glycopeptide 25 (see Table 1) the azido-reduction
procedures employing the DTT–DIPEA and MCA–DIPEA
thiolysis procedure were preferred and consequently used in the
following glycopeptide syntheses.
Selective reduction of N-Dts in the presence of azido groups in
solution and on the solid phase
In the course of studies concerning the development of ortho-
gonal protecting-groups schemes for solid-phase glycopeptide
Building blocks 1–4 and 7–15 employed in the MCPS of α- and β-O-GlcNAc, T-antigen and TN -antigen glycopeptides
874
J. Chem. Soc., Perkin Trans. 1, 1997

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Building blocks 16–21 employed in the MCPS of core 2, core 3 and core 4 glycopeptides
Scheme 2 Reduction of the N-Dts glycopeptide 22 and the azido glycopeptide 23 in the MCPS of the glycopeptides 24 and 25, employing different
thiols (see Table 1); reagents: i, thiol, DIPEA; ii, Ac2O, DMF; iii, SPPS; iv, piperidine, DMF; v, 95% aq. TFA; vi, NaOMe
amide the H-1 proton characteristically shifts upfield while H-2
shifts downfield from δ ~3.84 (30) to δ ~4.29 (34), respectively
(see Table 3). Another indication for the conversion of azide
into acetamide is the resonance for C-2 in the 13C NMR spec-
trum shifting characteristically from δC 59.2 (30) to δC 47.1 (34).
Syntheses of the mucin O-glycopeptides 25 and 32–36
Due to the high yields obtained in stereospecific α-gly-
cosylations using 2-azido-2-deoxyglycosyl donors the T- and
TN -antigen building blocks 7–10 and 12–15 were conveniently
used to synthesize the T- and TN -glycopeptides 25, 32 and 33.
J. Chem. Soc., Perkin Trans. 1, 1997
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Table 3 1H NMR data for the O-linked glycopeptides 24, 25 and 30–36 measured in CD3CO2D–D2O (1:1) at 600 MHz at 300 K
30
24
31
25
32
33
34
35
36
Val
Ile
NH
α-H
β-H
γ-H3
γ-H3
3.93
2.23
1.01
1.00
3.94
2.23
1.01
0.99
3.94
2.23
0.99
1.01
3.95
2.23
1.01
1.00
3.92
2.17
1.01
0.99
3.91
2.20
1.00
0.99
3.95
2.23
1.00
1.01
3.95
2.22
1.01
1.00
3.95
2.21
1.00
1.00
NH
8.36
8.37
8.37
8.39
8.40
8.41
8.36
8.37
8.37
α-H
4.32
4.34
4.32
4.33
4.28
4.30
4.33
4.33
4.33
β-H
1.83
1.83
1.82
1.82
1.83
1.81
1.83
1.82
1.80
γ-H2
γ-H3
δ-H3
1.51/1.15
0.87
0.85
1.52/1.15
0.89
0.86
1.51/1.15
0.87
0.85
1.52/1.17
0.85
0.87
1.51/1.15
0.86
0.84
1.50/1.15
0.87
0.84
1.51/1.15
0.88
0.85
1.51/1.14
0.89
0.85
1.51/1.14
0.88
0.84
Thr
NH
α-H
β-H
γ-H3
8.14
4.42
4.17
1.12
8.12
4.43
4.20
1.14
8.13
4.42
4.17
1.11
8.11
4.39
4.19
1.14
8.14
4.35
4.17
1.12
8.14
4.37
4.15
1.12
8.14
4.43
4.17
1.12
8.13
4.41
4.16
1.19
8.12
4.44
4.15
1.20
Ala
NH
α-H
β-H3
8.00
4.39
1.29
8.01
4.43
1.27
7.98
4.40
1.29
8.01
4.30
1.25
8.20
4.39
1.23
8.03
4.33
1.29
8.00
4.38
1.28
7.99
4.36
1.27
7.99
4.38
1.26
Phe
NH
α-H
β-H2
1-H
2-H
3-H
8.00
4.81
3.17/2.98
7.31
7.26
7.98
4.65
3.15/2.91
7.25
7.22
8.04
4.72
3.15/3.01
7.26
7.22
7.90
4.60
3.06/2.91
7.28
7.23
8.08
4.75
3.16/3.01
7.31
7.25
8.04
4.79
3.18/3.00
7.31
7.25
8.00
4.82
3.17/2.99
7.30
7.25
7.99
4.81
3.18/2.99
7.30
7.25
7.98
4.82
3.17/2.98
7.29
7.25
7.23
7.10
7.16
7.15
7.24
7.23
7.23
7.23
7.23
Ser or Thr
NH
α-H
β-H/β-HЈ
γ-H3
8.26
4.59
4.23
1.20
8.06
4.56
4.07/3.93
7.90
4.49
4.26
1.10
8.03
4.56
3.98/3.84
7.99
4.39
4.29
1.29
8.26
4.55
4.28
1.23
8.27
4.59
4.24
1.20
8.16
4.56
4.26
1.20
8.23
4.57
4.26
1.20
Glu
NH
8.20
8.07
7.95
8.11
8.23
8.19
8.20
8.07
8.19
α-H
4.45
4.41
4.41
4.38
4.36
4.42
4.46
4.54
4.48
β-H2
γ-H2
2.12/1.99
2.49
2.17/1.99
2.48
2.16/1.99
2.49
2.05/1.89
2.41
2.12/1.97
2.49
2.12/1.96
2.49
2.13/1.98
2.50
2.12/1.95
2.46
2.13/1.96
2.47
Gly
Leu
NH
α-H2
8.24
4.06/3.88
8.06
3.98
8.08
3.98
8.12
3.91/3.86
8.27
3.96/3.84
8.27
4.01/3.87
8.23
4.06/3.88
8.13
4.39
8.21
4.04/3.98
NH
7.99
7.84
7.85
7.87
7.98
8.01
7.98
8.13
8.02
α-H
β-H2
γ-H
4.40
1.60
1.61
4.40
1.60
1.61
4.40
1.60
1.62
4.39
1.59
1.62
4.36
1.60
1.60
4.38
1.60
1.62
4.39
1.60
1.61
4.43
1.61
1.61
4.42
1.60
1.61
δ-H3
0.88/0.91
0.90/0.85
0.88/0.84
0.90/0.85
0.90/0.87
0.86/0.90
0.88/0.91
0.87/0.92
0.88/0.91
Lys
NH
8.15
8.10
8.11
8.14
8.20
8.21
8.14
8.15
8.15
α-H
4.43
4.43
4.43
4.40
4.54
4.40
4.43
4.42
4.43
β-H2
γ-H2
δ-H2
ε-H2
NH2
1.91/1.75
1.45
1.70
3.03
7.46
1.90/1.74
1.44
1.70
3.02
7.46
1.90/1.75
1.44
1.69
3.02
7.46
1.90/1.75
1.45
1.70
3.03
7.48
1.90/1.75
1.44
1.68
3.01
7.46
1.90/1.76
1.43
1.69
3.01
7.49
1.91/1.74
1.45
1.70
3.03
7.47
1.91/1.72
1.44
1.70
3.03
7.47
1.91/1.73
1.44
1.70
3.03
7.46
GalNAc
1-H
5.01
5.08
(2.14)
4.47
4.02
4.04
3.85
4.03
3.92
5.13
(1.98)
4.37
4.38
4.05
3.88
4.02
3.86
4.92
(2.41)
4.27
3.96
4.24
3.73
4.01
4.01
4.91
(2.35)
4.29
3.96
4.02
4.31
4.35
4.35
4.86
(2.27)
4.23
3.95
4.06
4.26
4.38
4.38
4.90
(2.41)
4.27
3.95
4.06
4.32
4.42
4.38
2-H
3-H
4-H
5-H
6-H
6-HЈ
3.84
3.95
4.02
4.35
4.36
4.35
876
J. Chem. Soc., Perkin Trans. 1, 1997

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Table 3 (contd.)
30
24
31
25
32
33
34
35
36
GlcNAc
1-H
4.56
4.57
(7.95)
4.56
(8.00)
4.52
(7.98)
4.66
(8.05)
4.53
(7.88)
4.67
(8.03)
3.72,
3.69
3.47,
3.56
3.53,
3.44
3.48,
3.45
3.90,
3.94
3.88,
3.92
2-H
3-H
4-H
5-H
6-H
6-HЈ
3.76
3.45
3.58
3.59
3.89
3.90
3.76
3.61
3.47
3.47
3.96
3.76
3.73
3.62
3.45
3.45
3.93
3.93
3.74
3.45
3.57
3.57
3.94
3.94
3.69
3.64
3.52
3.41
3.89
3.78
Gal
1-H
2-H
3-H
4-H
5-H
6-H
6-HЈ
4.43
3.58
3.64
3.95
3.64
3.82
3.80
4.44
3.55
3.61
3.92
3.60
3.79
3.75
4.45
3.57
3.62
3.93
3.64
3.81
3.78
NH
NHAc
2.03
2.03
2.03
2.02
2.02
2.02
2.03,
2.02
2.02
2.03,
2.03,
2.01
Scheme 3 Reduction of compound 26 containing N-Dts and azido
functionalities; reagents and conditions: i, 0.2 mol PDT, 0.3 mol DIPEA
in CH2Cl2; ii, 0.2 mol DTT, 0.1 mmol DIPEA in CH2Cl2; iii, Ac2O–
pyridine (1:2)
Employing the azido building blocks 7, 8, 12 and 13 the azido
groups could be efficiently reduced on the solid phase by treat-
ment with DTT–DIPEA in dichloromethane on the solid
phase. After N-acetylation with Ac2O in DMF the T- and TN -
glycopeptides 25, 32 and 33 were obtained in fairly high yields
[25 (68%), 32 (63%) and 33 (52%), respectively]. Utilizing the
alternative route of reduction and simultaneous in situ N-
acetylation of the azido building blocks prior to incorporation
onto solid-phase, gave in general higher yields [25 (85%), 32
(75%) and 33 (76%), respectively]. However, taking into
account the loss of compounds during the additional synthetic
step in the synthesis of the building blocks 9, 10 and 12 the use
of azide-containing building blocks is a valuable alternative
because of the effectiveness of the mild reduction procedure
carried out on the solid phase. Even though azides can be
reduced and transformed into the N-acetamido function with
thioacetic acid, the DTT reduction method offers other advan-
tages. The reaction times are generally shorter and no inherent
formation of impurities like N-thioacetates was observed. The
DTT method is extremely mild and generally applicable for
azide reduction on the solid phase.
Scheme 4 Selective reduction of N-Dts in the presence of an azido
group on the solid phase; reagents and conditions: i, 0.2 mol PDT, 0.3
mol DIPEA in CH2Cl2, 2 × 20 min, room temp.; ii, 15% Ac2O in DMF,
2 × 10 min; iii, SPPS; iv, 20% piperidine in DMF; v, 95% aq. TFA; vi,
1% NaOMe in MeOH (pH 8.5)
sponding building blocks12–21 for solid-phase peptide synthesis
(SPPS) of O-glycopeptides was reported.14 Simultaneous in situ
reduction of N-Dts and azido functionalities in precursors 12,
13, 16, 18 and 20 with zinc dust in THF–acetic acid in the
presence of acetic anhydride offered a method for the synthesis
of the N-acetylated building blocks 14, 15, 17, 19 and 21. These
Efficient and reliable procedures for the synthesis of mucin
core 1, 2, 3 and 4 O-glycopeptides, the synthesis of the corre-
J. Chem. Soc., Perkin Trans. 1, 1997
877

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building blocks were used directly in MCPS of mucin core 1, 2,
3 and 4 O-glycopeptides, omitting the need for further reduc-
tion steps on the solid phase. For comparative studies the build-
ing blocks 12–21 were then used in MCPS of the MHC class II
binding glycopeptides 33–36. For the synthesis of the glycopep-
tides 33–36 the N-Dts- and azide-containing building blocks 12,
13, 16, 18 and 20 and the corresponding N-acetamido building
blocks 14, 15, 17, 19 and 21 were used. As the key step the
simultaneous reduction of N-Dts and azido functionalities was
achieved by thiolysis with DTT (0.1 )–DIPEA (0.1 m) in
dichloromethane. After subsequent N-acetylation with acetic
anhydride in DMF the peptide synthesis was continued. Due to
instability of the N-Dts group towards secondary amines to
form urea derivatives, it is necessary to remove the N-Dts group
before resuming the peptide synthesis using piperidine or mor-
pholine for removal of Fmoc. The N-Dts group has to be
removed before continuation of the peptide synthesis! After coup-
ling of the last amino acid, Fmoc deprotection, cleavage from
the resin with 95% TFA, O-deacetylation and preparative
HPLC purification, the pure O-glycopeptides were obtained in
yields of 35–64% based on the loading of the resin (Table 2).
Depending on the method of synthesis, reduction on the
building-block stage or reduction during peptide assembly, the
mucin core 1, 2, 3 and 4 O-glycopeptides 33–36 could be
obtained in 52 or 76% (33); 45 or 64% (34); 45 or 62% (35) and
35 or 55% (36) yield, respectively. The purity of the glycopep-
tides 33–36 was excellent as indicated by analytical HPLC. In
the case of the azide- and N-Dts-containing building blocks the
corresponding glycopeptides were obtained without any prob-
lems during assembly or reduction when utilizing building
block 20 with two N-Dts and one azido functionality. This
shows that the azido and N-Dts building blocks 12, 13, 16, 18
and 20 and the acetamido building blocks 14, 15, 17, 19 and 21
can be used alternatively and independently in the assembly of
O-glycopeptides on a solid phase. Compounds 33–36 were fully
affinity tag 38 which can be activated to produce a reactive
nitrene. The photoaffinity labelling technique requires the syn-
thesis of photolabile reagents to produce reactive intermediates,
such as carbenes or nitrenes, which insert rapidly into the
amino acid residues of the biomolecule and tag the target mol-
ecule for isolation and structural determination. Several photo-
labile probes such as diazo ketones, diazoacetates and aromatic
azides have been used. The most frequently employed are aro-
matic azides. Thus, aromatic azides produce long lived nitrene
intermediates, have remarkable chemical stability, a long-
wavelength absorption upon photolysis and they are readily
synthesized. The DHBT-activated 2-azidobenzoic acid 38
was selected for the present investigation. The synthesis of the
photoaffinity tag 38 is depicted in Scheme 5. Treatment of
the commercially available compound 37 with 0.5 mol equiv.
of dicyclohexylcarbodiimide (DCCI) in DMF at room tem-
perature for 12 h afforded the DHBT-active ester 38, in a
Scheme 5 Synthesis of the photoaffinity label 38; reagents and con-
ditions: i, DCCI, DMF, 24 h, room temp.
high yield of 93% after silica gel chromatography, as yellowish
crystals. MCPS, performed in a Teflon block with several syn-
thesis columns, was found to be efficient in the synthesis of the
photoaffinity labelled glycopeptides 39–45. The synthesis was
carried out on Macrosorb resin modified with the acid-labile
HMPA linker using acylation reactions utilizing stock solutions
of preactivated Fmoc-amino acid-OPfp esters with DHBT-OH
catalysis and subsequent Fmoc deprotection with 20% piperi-
dine in DMF. The glycosylated Fmoc-amino acid-OPfp
esters 1, 2, 7, 8, 11–13 were coupled as usual amino acids in
MCPS using extended reaction times and only a small excess of
the building block (1.6 mol equiv.). The use of DHBT-OH
allows the acylation reaction to be monitored visually due to
the yellow ion-pair formed with residual amine. The key step in
the present strategy is the DTT reduction of the resin bound N-
Dts and azido glycopeptides and subsequent coupling of the
photoaffinity label 38 to the corresponding amino groups on
the solid phase (see Schemes 6 and 7). For the stereoselective
introduction of amino glycans into the glycopeptides the azido
and the N-Dts groups were employed to obtain stereoselectively
α- and β-linked amino sugar glycopeptides, respectively. The
previously described N-Dts 1 and 2 and the azido building
blocks 7, 8, 11–13 were used. For the reduction of the amino
group precursors on the solid phase, DTT was used. The Mac-
1
characterized by ESMS and 1D and 2D H NMR spectroscopy
(see Table 3). The chemical shifts of the anomeric carbohydrate
protons vary significantly in comparison with the other carbo-
hydrate protons. Connectivities of the α, β, γ, δ and ε protons
were accessed by 1H–1H 2D homonuclear chemical shift correl-
ation (COSY) and nuclear Overhauser enhancement in rotat-
ing frame (ROESY) experiments.
Synthesis of the photoaffinity labelled glycopeptides 39–45
It has been demonstrated that the glycans in the Hb (67–76)
glycopeptide analogues are in direct contact with the T-cell
receptor and specific recognition of the glycan on the glycopep-
tide was observed. In order to identify more specifically the
binding site of the T-cell receptor, corresponding photoaffinity
labelled glycopeptides have been synthesized. These analogues
are modified on the carbohydrate amino group by the photo-
O-Glycopeptides 29–36 synthesized by multiple-column solid-phase synthesis. The asterisk * indicates site of glycosylation.
878
J. Chem. Soc., Perkin Trans. 1, 1997

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Scheme 7 Principle of SPPS of photoaffinity labelled glycopeptides
41–45 utilizing compounds 7, 8, 11–13; reagents and conditions: i, DTT
(0.2 mol), DIPEA (0.1 mmol) in CH2Cl2; ii, 38 in DMF, 6 h; iii, 20%
piperidine in DMF; iv, Fmoc-AA-OPfp (3 mol equiv.), DHBT-OH (1
mol equiv.); v, 95% aq. TFA; vi, 1% NaOMe in MeOH (pH 9)
Scheme 6 Principle of SPPS of photoaffinity labelled glycopeptides 39
and 40 utilizing compounds 1 and 2; reagents and conditions: i, DTT
(0.2 mol), DIPEA (0.1 mmol) in CH2Cl2; ii, 38 in DMF, 6 h; iii, 20%
piperidine in DMF; iv, Fmoc-AA-OPfp (3 mol equiv.), DHBT-OH (1
mol equiv.); v, 95% aq. TFA; vi, 1% NaOMe in MeOH (pH 9)
TFA–water (95:5). Removal of the acetyl groups from the
carbohydrate moiety was achieved with sodium methoxide (1%)
in methanol at pH 8.5 (pH paper). The labelled glycopeptides
39–45 were then subjected to semipreparative HPLC purifi-
cation before being characterized by ESMS and 1H NMR spec-
troscopy. After lyophilization the pure labelled glycopeptides
were obtained in yields from 61 to 78% based on the loading of
the resin (see Table 4). The synthesis was achieved without any
problems concerning the reduction of N-Dts and azide groups
and subsequent acylation with the photoaffinity label 38. This
reaction procedure can be generally applied in the synthesis of
modifications of the amino function of amino sugars in
glycopeptides.
rosorb resin (100 mg) was, after incorporation of the building
blocks 1, 2, 7, 8, 11–13, thoroughly washed with dichlorometh-
ane. The azido and N-Dts reduction was then performed by
treatment of the resin with a solution of DTT (0.2 )–DIPEA
(0.1 m) in dichloromethane for 6 h (1 × 2 min, 2 × 3 h). The
transformation of the azido group into the corresponding
amino function was followed by IR spectroscopy by observ-
ation of complete disappearance of the azide absorption band
at νmax 2117 cmϪ1. In addition the Kaiser test was used to follow
the formation of amino groups on the solid phase. After com-
plete conversion into the amino function, the resin was washed
successively with dichloromethane and DMF. Further modifi-
cation of the amino group with the photoaffinity label was
achieved by adding a solution of 3 mol equiv. of the active-
DHBT ester 38 dissolved in DMF to the resin. Coupling of ester
38 was complete after 6 h. After Fmoc-deprotection, peptide
synthesis was continued by employing Fmoc-amino acid-OPfp
esters and DHBT-OH. After the addition of the last amino acid
residue, the Fmoc group was removed and the glycopeptide
was deprotected and cleaved from the resin by treatment with
Conclusions
In the present work an efficient MCPS protocol which allows
the simultaneous and parallel synthesis of a large variety of
mucin O-glycopeptides and their photoaffinity labelled ana-
logues has been described. Comparative studies demonstrated
that any of the building blocks 1–4 and 7–21 are well suited
for the MCPS of the O-glycopeptides 24, 25 and 31–36. The
use of the acetamido building blocks 3, 4, 9, 10, 14, 15, 17, 19
and 21 gave overall a similar yield when compared with the
J. Chem. Soc., Perkin Trans. 1, 1997
879

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The mild conditions used in the conversion of N-Dts and azide
into the corresponding amino function by means of thiols
shows that both groups are useful precursors of amines and
that conversion can be employed selectively during synthetic
procedures. Application of this new concept in other areas of
peptide synthesis on a solid phase are currently under investi-
gation. The corresponding immunological studies of all the
glycopeptides 24, 25, 31–36 and 39–45 will be published
elsewhere.
Experimental
General procedures
All solvents were purchased from Labscan Ltd. (Dublin, Ire-
land). Vacuum liquid chromatography (VLC) was performed
on pre-dried Merck silica gel 60 H (0.040–0.060 mm, heating at
120 ЊC for 24 h) with solvents dried over molecular sieves. TLC
was performed on Merck Silica Gel 60 F254 aluminium sheets
with detection by charring with sulfuric acid and by UV light,
when applicable. DMF was freshly distilled by fractional distil-
lation at reduced pressure and kept over molecular sieves (3 Å).
Dichloromethane was distilled from phosphorus pentaoxide
and kept over molecular sieves (3 Å). Pyridine was distilled and
kept over molecular sieves (3 Å). Light petroleum was the 60–
80 ЊC fraction. Concentrations were performed under reduced
pressure at temperatures below 40 ЊC. MgSO4 was used to dry
organic extracts. Suitably protected Nα-Fmoc-amino acid-OPfp
esters were purchased from Bachem (Bubendorf, Switzerland)
and NovaBiochem (Switzerland). Macrosorb SPR 250 resin
from Sterling Organics (UK), NEM and DHBT-OH from
Fluka, threo-1,4-dimercaptobutane-2,3-diol (DTT) from
Aldrich, DIPEA, MCA and PDT from Sigma. Electrospray
mass spectrometry was performed in the positive mode on a
Fisons VG Quattro Instrument. MALDI-TOF MS was per-
formed on a Finnigan MAT 2000 using a matrix of α-cyano-4-
hydroxycinnamic acid. 1H and 13C NMR spectra were recorded
on a Bruker AM 600 MHz or DRX 250 MHz spectrometer.
Chemical shifts are given in ppm and referenced to internal
SiMe4 (δH 0.00) or CDCl3 (δC 77.00) at 300 K. J-Values are given
in Hz. For spectra recorded in CD3CO2D–water the HOAc sig-
nal at δH 2.03 was used as internal reference. For the assignment
of signals 1H–1H COSY, 1H–1H double-quantum-filtered
phase-sensitive COSY and NOE in rotating frame (ROESY)
experiments were used. Analytical and semipreparative
reversed-phase HPLC separations were performed on a Waters
HPLC system using analytical RCM (8 × 100 mm) and Delta
PAK (15 µm; 300 Å; 25 × 200 mm) C-18 columns with a flow rate
of 1 cm3 minϪ1 and 10 cm3 minϪ1, respectively. Detection was at
215 and 280 nm with a photodiode array detector (Waters M
991). Solvent System A: 0.1% TFA in water; B: 0.1% TFA in
90% acetonitrile–10% water.
Photoaffinity labelled glycopeptides 39–45 synthesized by MCPS. The
asterisk * indicates site of glycosylation.
Table 4 Results of the SPPS of the photoaffinity labelled glycopep-
tides 39–45 by use of the building blocks 1, 2, 7, 8, 11–13. N-Dts and
azido reduction a and coupling of the photoaffinity label 38 performed
on the solid phase. Comparison of expected and observed molecular
masses for the individual glycopeptides 39–45 analysed by ESMS
Molecular
formula
Relative
molecular mass
Yield
(%)c
Compound
ESMSb
39
40
41
42
43
44
45
C62H96N16O19
C63H98N16O19
C62H96N16O19
C63H98N16O19
C63H98N16O19
C68H106N16O24
C69H108N16O24
1369.56
1383.58
1369.56
1383.58
1383.58
1531.69
1545.72
1370.76
1384.75
1370.82
1384.77
1384.53
1532.70
1546.61
64
63
78
73
71
63
61
a
Reduction conditions: dithiothreitol (DTT) (0.2 mol dmϪ3), diiso-
propylethylamine (DIPEA) (0.1 mmol dmϪ3) in dichloromethane.
b Glycopeptides were detected as [glycopeptide ϩ H]ϩ ions. c Yields were
determined based on the loading of the resin (0.2 mmol gϪ1) and
obtained after semipreparative reversed-phase HPLC.
use of their precursors 1, 2, 7, 8, 12, 13, 16, 18 and 20 with
solid-phase reduction, in particular for the larger building
blocks. The N-Dts- and azido group-containing building
blocks possess, on the other hand, some favourable proper-
ties. Mild reduction conditions for the conversion of N-Dts
and azido to the corresponding amino function by treatment
with thiols (DTT and MCA) does not induce any decom-
position of the glycopeptide and it offers the possibility of
further modification of the sugar amino function into the
acetamido or photoaffinity labelled glycopeptides 24, 25, 31–
36 and 39–45. Selective reduction of N-Dts in the presence of
the more stable azido group can be achieved by treatment with
the less reactive PDT both in solution and on the solid phase.
For the introduction of the photoaffinity label onto the glyco-
peptides the readily available DHBT-activated 2-azidobenzoic
acid 38 was selected. Incorporation of the building blocks 1,
2, 7, 8 and 11–13, subsequent reduction of the N-Dts or azido
functionalities by treatment with DTT, and coupling of the
photoaffinity label 38 onto the corresponding amino group
affords excellent yields of the labelled glycopeptides 39–45.
The outlined protocol for affinity labelling proved to be
advantageous in many respects. A difficult synthesis of the
corresponding labelled building blocks prior to incorporation
into the peptide was avoided because acylation of the glycosyl
amino group with the photoaffinity label 38 could be effected
efficiently on the solid phase. Similarly, a large variety of other
labels can be attached to the amino function of amino sugars
on the solid phase. Applying these MCPS protocols the
O-GlcNAc and mucin O-glycopeptides 24, 25 and 31–36 and
the photoaffinity labelled glycopeptide analogous 39–45 could
be obtained in excellent yields and purity (see Tables 2 and 4).
O-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-â-D-gluco-
pyranosyl)-N-(fluoren-9-ylmethoxycarbonyl)-L-serine penta-
fluorophenyl ester 3
Compound 1 (1 g, 1.11 mmol) was dissolved in THF–acetic
anhydride–acetic acid [50 cm3 (3:2:1)] and zinc (~750 mg,
activated with 2% aq. CuSO4) was added. The mixture was
stirred for 6 h at room temperature, diluted with THF (100
cm3), filtered through Celite, rinsed several times with THF
and concentrated. Purification by VLC-chromatography on
dried silica gel [ethyl acetate–light petroleum (2:1)] and sub-
sequent crystallization afforded the title compound 3 (633 mg,
70%) as crystals; NMR data, mp and optical rotation were in
agreement with those previously reported.19 A small fraction of
the lactam 5 (less than 5%) was isolated from the purification
by silica gel chromatography. [α]2D7 ϩ17.5 (c 1.7, CDCl3);
C31H34N2O11 [MALDI-MS (M ϩ H)ϩ Calc. m/z, 596.53. Obs.
m/z, 597.57]; δH (250 MHz, CDCl3) 6.35 (1 H, NH), 5.86 (1 H,
J 5.2, NH-Fmoc), 5.15 (1 H, 4-H), 5.06 (1 H, 3-H), 4.55 (1 H,
880
J. Chem. Soc., Perkin Trans. 1, 1997

View Online
J1,2 7.73, 1-H), 4.43 and 4.37 (2 H, FmocCH2), 4.25 and 4.15 (2
H, 6-H2), 4.14 (1 H, FmocCH), 3.75 (1H, 5-H) and 3.68 (1 H,
2-H).
1-H), 5.15 (1 H, dd, 3-H), 6.11 (1 H, d, NH) and 7.02 (1 H, d,
NH); δC(62.9 MHz; CDCl3) (inter alia) 20.6, 20.7, 20.8, 23.1
and 23.4 (COCH3), 53.4 (C-2), 61.7 (C-6), 67.63 (C-3), 72.9
(C-4), 73.5 (C-5) and 80.34 (C-1).
O-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-â-D-glucopyrano-
syl)-N-(fluoren-9-ylmethoxycarbonyl)-L-threonine pentafluoro-
phenyl ester 4
3-(2-Azidobenzoyloxy)-4-oxo-3,4-dihydro-1,2,3-benzotriazine 38
DHBT-OH (3.26 g, 20 mmol) was dissolved in DMF (20 cm3)
and DCCI (2.07 g, 10 mmol) was added. The solution was
stirred for 24 h at room temperature [TLC: light petroleum–
ethyl acetate (1:1)], filtered through Celite and concentrated.
The residue was washed with diethyl ether and purified by
chromatography on silica gel [light petroleum–ethyl acetate
(2:1)] to yield compound 38 (2.86 g, 93%) as yellowish crystals,
mp 131 ЊC [Found: ESMS: (M ϩ H)ϩ, 308.57. C14H8N6O3
requires M, 308.26]; δH (250 MHz; CDCl3) 7.25–7.43 (2 H, m),
7.66–7.77 (1 H, ddd), 7.82–7.94 (1 H, ddd), 8.02–8.11 (1 H,
ddd), 8.20–8.34 (2 H, m) and 8.38–8.48 (1 H, dd); δC(62.9 MHz;
CDCl3) 120.2, 125.1, 126.2, 129.5, 133.2 and 135.9 (arom. C).
Compound 2 (1 g, 1.09 mmol) was dissolved in THF–acetic
anhydride–acetic acid [50 cm3 (3:2:1)] and zinc (~750 mg, acti-
vated with 2% aq. CuSO4) was added. The mixture was stirred
for 6 h, diluted with freshly distilled THF (100 cm3), filtered
through Celite, rinsed several times with THF and concen-
trated. Purification on pre-dried silica gel [ethyl acetate–light
petroleum (2:1)] and subsequent crystallization afforded the
title compound 4 (693 mg, 76%) as crystals; NMR data, mp
and optical rotation were in agreement with those previously
reported.19 A small fraction of the cyclic lactam 6 (less than
5%) was isolated from the silica gel purification, [α]2D7 ϩ17.5 (c
1.7, CDCl3); C31H34N2O11 [MALDI-MS (M ϩ H)ϩ Calc. m/z,
610.62. Obs. m/z, 611.57]; δH (250 MHz; CDCl3) 6.25 (1 H,
NH), 5.86 (1 H, JN H ,H α 5.2, NH-Fmoc), 5.14 (1 H, 4-H), 4.99
(1 H, 3-H), 4.74 (1 H, Hα-Thr), 4.69 (1 H, J 7.73, 1-H), 4.43 and
4.37 (2 H, Fmoc-CH2), 4.35 (1 H, Hβ-Thr), 4.21 and 4.11 (2 H,
6-H2), 4.14 (1 H, Fmoc-CH), 3.67 (1 H, 5-H), 3.62 (1 H, 2-H)
and 1.31 (3 H, J 6.02, Hγ-Thr); δC(62.9 MHz; CDCl3) 96.05
(C-1), 72.97 (C-5), 72.89 (C-3), 71.92 (Fmoc-CH2), 67.59 (Cβ-
Thr), 67.51 (C-4), 62.05 (C-6), 59.15 (C-2), 58.41 (Cα-Thr),
47.55 (Fmoc-CH), 21.14, 20.94, 20.93 (3 × OAc) and 12.83
(Cγ-Thr).
General procedure for the simultaneous reduction and N-acetyl-
ation of the N-Dts and azido groups of the building blocks 1, 2, 7,
8, 11–13, 16, 18 and 20
The appropriate building block (50 mg) was dissolved in THF–
acetic acid–acetic anhydride [5 cm3 (3:1:2)]. Zinc dust (acti-
vated for 2 min in 2% aq. copper sulfate and washed twice with
water) was added and the mixture was stirred for 2 h. The solu-
tion was filtered through Celite, washed with THF–acetic acid,
concentrated and dried in vacuo. The residue was dissolved in
dichloromethane (10 cm3), washed three times with water, dried
over MgSO4 and concentrated to give the crude product. The
1
2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-â-D-glucopyranosyl
azide 27
acetamido building blocks were essentially pure by H NMR
spectroscopy and could be employed directly in SPPS without
further purification. Alternatively the acetamido building
blocks 3, 4, 9, 10, 14, 15, 17, 19 and 21 were purified by chroma-
tography on pre-dried silica gel for characterization.
The glycosyl azide 26 (500 mg, 1.1 mmol) was dissolved in dry
dichloromethane (20 cm3) and a solution of PDT (0.2 ) and
DIPEA (0.3 ) in dichloromethane was added. The solution
was stirred at room temperature until TLC [toluene–ethyl
acetate (2:1)] showed complete disappearance of the starting
material. The solution was concentrated and the residue was dis-
solved in pyridine (10 cm3)–acetic anhydride (5 cm3). The solu-
tion was kept at room temperature for 2 h, concentrated and
co-concentrated with toluene. The product was purified by
VLC on silica gel [toluene–ethyl acetate (2:1)] to yield the title
acetamide 27 (408 mg, 94%) [Found: ESMS: (M ϩ H)ϩ,
373.56. C14H20N4O8 requires M, 372.34]; δH (250 MHz; CDCl3)
1.99 (3 H, s, NHAc), 2.04 (6 H, s, Ac), 2.06 (3 H, s, Ac), 3.83
(1 H, ddd, J4,5 9.2, J5,6a 2.0, J5,6b 5.0, 5-H), 3.95 (1 H, ddd, J1,2
9.4, J2,N H 9.0, J2,3 10.0, 2-H), 4.21 (1 H, dd, J6a,6b 12.4, 6-Ha),
4.32 (1 H, dd, 6-Hb), 4.8 (1 H, d, 1-H), 5.14 (1 H, dd, J3,4 9.2,
4-H), 5.28 (1 H, dd, 3-H) and 5.65 (1 H, d, NH); δC(62.9
MHz; CDCl3) (inter alia) 20.9, 21.0, 21.1 and 23.6 (COCH3),
53.8 (C-2), 61.9 (C-6), 68.2 (C-4), 72.0 (C-3), 73.6 (C-5) and
88.2 (C-1).
Preparation of the Macrosorb SPR 250 resin
Syntheses of the O-glycopeptides were performed in DMF using
the Macrosorb SPR 250 resin. The resin (5 g, loading 0.2 mmol
g
Ϫ1) was packed into a 50 cm3 disposable syringe (Discardit II,
Beckton Dickinson) fitted with a Teflon filter. The syringe was
connected to a suction flask through a Teflon tube with a manual
2-way Teflon valve and the resin was swelled in DMF (20 cm3)
for 30 min; excess of reagents, DMF etc. was removed by apply-
ing vacuum. The resin was derivatized with the hydroxymethyl-
phenoxy acetic acid HMPA-linker: The HMPA-linker (455 mg,
2.5 mmol), TBTU (750 mg, 2.37 mmol) and NEM (315 mm3, 2.5
mmol) were dissolved in DMF (20 cm3) and after 5 min added to
the resin. After 2 h the resin was washed carefully with DMF
(5 × 20 cm3) and dichloromethane (5 × 10 cm3). Nα-Fmoc-
Lys(Boc)-OH (1.75 mg, 3.75 mmol) and N-methylimidazole
(231 mg, 223 mm3, 2.81 mmol) were dissolved in dichlorometh-
ane (20 cm3) and MSNT (1.11 g, 3.71 mmol) was added. After 5
min the solution was added to the resin and the mixture was kept
for 3 h. The resin was then washed thoroughly with dichloro-
methane (5 × 10 cm3) and DMF (5 × 10 cm3), and unreactive
amino groups of the resin were acetylated with a solution of 20%
acetic anhydride in DMF (10 cm3). After 20 min the resin was
washed successively with DMF (5 × 10 cm3) and diethyl ether
(5 × 10 cm3) and dried in vacuo.
1,2-Bisacetamido-3,4,6-tri-O-acetyl-1,2-dideoxy-â-D-gluco-
pyranose 28
A solution of DTT (0.2 ) and DIPEA (0.1 m) in dichloro-
methane was added to the β-azide 26 (500 mg, 1.1 mmol),
dissolved in dry dichloromethane (20 cm3). The mixture was
stirred at ambient temperature for 30 min [TLC, chloroform–
methanol (5:1)], concentrated and redissolved in a mixture of
pyridine (10 cm3) and acetic anhydride (5 cm3). The mixture
was kept for 2 h at room temperature, concentrated, co-
concentrated with toluene and then purified by VLC on silica
gel [toluene–ethyl acetate (2:1)] to yield title compound 28 (418
mg, 96%) [Found: ESMS: (M ϩ H)ϩ, 389.57. C16H24N2O9
requires M, 388.38]; δH (250 MHz; CDCl3) 1.99, 2.02, 2.10, 2.12
and 2.14 [15 H, 5 s, NHAc (2×) and Ac (3×)], 3.80 (1 H, ddd,
J4,5 10.2, J5,6a 4.3, J5,6b 2.1, 5-H), 4.16 (1 H, ddd, J1,2 8.3, J2,3
10.2, J2,N H 8.0, 2-H), 4.12 (1 H, J6a,6b 12.5, 6-Ha), 4.33 (1 H, dd,
6-Hb), 5.08 (1 H, dd, J3,4 9.2, 4-H), 5.09 (1 H, dd, J1,N H 8.3,
General procedures for SPPS of the O-glycopeptides
The derivatized resin was transferred into a custom-made 20
column Teflon synthesis block (100 mg resin per column). Nα-
Fmoc deprotection was effected by successive 2 min and 20 min
treatments of the resin with 20% piperidine in DMF (1 cm3).
The washing procedure (10 times with DMF) was repeated after
each coupling/Fmoc deprotection step. Each Nα-Fmoc-amino
acid-OPfp ester (3 mol equiv.) and DHBT-OH (1 mol equiv.)
was dissolved in DMF (0.6 cm3). In the case of the glycosylated
J. Chem. Soc., Perkin Trans. 1, 1997
881

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building blocks only 1.6 mol equiv. were employed. The solu-
tions were added to the resins and then left for 4 h. After each
coupling step the resin was washed, the Nα-Fmoc group was
removed and the resin was washed as described above.
undesired side reaction was detected after the completion of the
glycopeptide synthesis.
Multiple-column peptide syntheses of the O-GlcNAc and mucin
O-glycopeptides 24, 25 and 31–36
Comparative studies for the reduction of N-Dts and azido groups
with DTT, MCA, BME and PDT on a solid phase (see Table 1)
Comparative studies for the reduction of N-Dts and azido
groups on a solid phase were performed in a custom-made
Teflon block with 20 wells. The derivatized Macrosorb resin was
transferred into the Teflon block (50 mg resin per column),
Fmoc deprotection was effected by treatment with 20% piperi-
dine in DMF, and amino acids were coupled as OPfp-esters (3
mol equiv.) with DHBT-OH (1 mol equiv.) added as auxiliary
nucleophile. Incorporation of the glycosylated N-Dts and azido
building blocks into the glycopeptides was accomplished by
utilizing Nα-Fmoc-Ser(Ac3-β--GlcNDts)-OPfp 1 and Nα-
Fmoc-Ser(Ac3-α--GalN3)-OPfp 7 (1.6 mol equiv.) in the pres-
ence of DHBT-OH (1 mol equiv.). After coupling of the N-Dts-
and azide-containing building blocks, reduction was performed
on the solid phase by thiolysis, alternatively with DTT (0.2 )–
DIPEA (0.1 m), MCA (0.2 )–DIPEA (0.3 ), BME (0.2
)–DIPEA (0.3 ) or with PDT (0.2 )–DIPEA (0.3 ) in
dichloromethane in three portions. The first portion was sucked
quickly through the resin, followed by two additional portions,
which were removed after 10 min (N-Dts reduction) and 2 h
(azide reduction), respectively. After a thorough wash with
dichloromethane (5 vol.) and DMF (10 vol.) the amino group
was acetylated with acetic anhydride in DMF (20%). The
residual amino acids were then coupled in the usual way. After
completion of the syntheses the resin was washed with dichlo-
romethane and dried. Cleavage of the glycopeptide was
achieved with 95% TFA for 2 h, followed by filtration and con-
centration and precipitation by addition of diethyl ether. The
glycopeptides were then O-deacetylated by treatment with 2%
sodium methoxide in methanol and analysed by analytical and
semipreparative HPLC, using a linear gradient 0 to 50% B for
30 min and then 50 to 100% for 10 min (analytical HPLC) and 0
to 50% B for 50 min and then 50 to 100% B for 10 min (semi-
preparative HPLC), respectively. Yields and comparative data
are presented in Table 1. 1H NMR data are presented in Table 3.
The derivatized resin was transferred into a 20 column Teflon
block (100 mg/column, loading 0.2 mmol gϪ1). Throughout the
syntheses the cleavage of the Nα-Fmoc moiety was achieved by
treatment with 20% piperidine in DMF. All amino acids were
then coupled to the peptide as their Pfp esters (3 mol equiv.
each) with DHBT-OH (1 mol equiv.) added as an auxiliary
nucleophile. Acylation times were 2–4 h throughout the syn-
theses. Incorporation of the glycosylated serine and threonine
amino acids into the glycopeptide was achieved by utilizing
either the building blocks 1, 2, 7, 8, 12, 13, 16, 18 and 20 or the
reduced and N-acetylated building blocks 3, 4, 9, 10, 14, 15, 17,
19 and 21. The appropriate building blocks (1.6 mol equiv.) were
coupled in the presence of DHBT-OH (1 mol equiv.). The reac-
tion time for the glycosylated building blocks was 8 h. After
thorough washing of the resin with DMF (5 vol.) and di-
chloromethane (10 vol.) the reduction of the N-Dts and azido
groups on the solid phase was achieved by adding DTT (0.2 )
and DIPEA (0.1 m) in dichloromethane (1 × 2 min and 2 × 3
h in the case of azido reduction and 1 × 2 min and 2 × 10 min in
the case of N-Dts reduction). The amino groups were then N-
acetylated with 20% acetic anhydride in DMF after washing of
the resin with DMF. After incorporation of the last amino acid,
valine, the terminal Nα-Fmoc group was cleaved, and the resin
was washed successively with DMF and diethyl ether and
dried. The cleavage of the O-glycopeptides from the HMPA-
linker and simultaneous amino acid deprotection were per-
formed by treatment with 95% aq. TFA for 2 h. After cleavage
the solution was filtered from the resin and the resin was
washed three times with 95% aq. TFA. The combined filtrates
were concentrated, co-concentrated with toluene and the gly-
copeptides were then precipitated by several triturations with
diethyl ether. Residual solvent was removed under reduced
pressure and the acetylated glycopeptides were dissolved in dry
methanol (2 mg cmϪ3) and O-deacetylated by addition of a
catalytic amount of 2% sodium methoxide in methanol until
pH paper indicated pH 8.5. The mixture was then stirred for
4 h at room temperature, neutralized with solid CO2 and con-
centrated. The crude glycopeptides were dissolved in water and
purified by semipreparative HPLC, using a linear gradient
0–50% B in 50 min and then 50–100% in 10 min. The purified
glycopeptides 24, 25 and 31–36 were then lyophilized from
water and fully characterized by ESMS and 1H NMR spec-
troscopy. Yields and ESMS data are presented and compared in
Table 2. 1H NMR data are presented in Table 3.
Selective reduction of N-Dts, in the presence of azide, on the solid
phase
The N-Dts- and azido group-containing core 2 building block
16 (1.6 mol equiv.) was coupled to the peptide on the HMPA-
derivatized Macrosorb resin. Selective N-Dts reduction was
accomplished by two 10 min treatments of the resin with PDT
(0.2 ) and DIPEA (0.3 ) in dichloromethane. After a
thorough wash with dichloromethane (5 vol.) and DMF (10 vol.)
the amino group was acetylated with acetic anhydride in DMF
(20%). The synthesis of the glycopeptide 30 was then continued
by the same procedure as described above. Purification of com-
pound 30 was achieved by semipreparative HPLC to afford a
Multiple-column peptide syntheses of the photoaffinity labelled
glycopeptides 39–45
The MCPS of the photoaffinity labelled glycopeptides was per-
formed analogously to the synthesis of the O-glycopeptides.
Incorporation of the glycosylated N-Dts and azido building
blocks 1, 2, 7, 8, 11, 12 and 13 (1.6 mol equiv.) in the presence of
DHBT-OH (1 mol equiv.) and the subsequent thiolytic reduc-
tion were performed as previously described. Coupling of the
photoaffinity label 38 to the free amino group was achieved,
after thorough washing with dichloromethane (10×) and DMF
(10×), by treatment with the active ester 38 (3 mol equiv.), dis-
solved in DMF (0.6 cm3), for 6 h. After Fmoc cleavage with
20% piperidine in DMF the peptide synthesis was continued
by utilizing the Fmoc amino acid OPfp esters (3 mol equiv.) and
DHBT-OH (3 mol equiv.) in DMF (0.6 cm3) for 3 h, respect-
ively. After the final Nα-Fmoc deprotection, the resins were
washed successively with DMF and diethyl ether and dried. The
cleavage of the glycopeptides from the linker and simultaneous
side-chain deprotection were performed by treatment with 95%
aq. TFA (2 cm3) for 2 h at room temperature, followed by filtra-
1
76% yield of the azido group-containing glycopeptide 30. H
NMR data of compound 30 are presented in Table 3.
General procedure for N-Dts and azido reduction utilizing DTT/
DIPEA on the solid phase
The appropriate Macrosorb SPR resin (100 mg) with the N-
Dts- or the azido group-containing glycopeptides was washed
thoroughly with dichloromethane (5×). The thiolytic reduction
was then performed by treatment of the resin with a solution of
DTT (0.2 )–DIPEA (0.1 m) in dichloromethane (1 × 2 min,
2 × 3 h). In the case of the N-Dts-containing building blocks
the thiolysis procedure could be reduced to 2 × 10 min. The
resin was then washed thoroughly with dichloromethane (5×)
and DMF (10×) because traces of thiols left on the resin could
lead to the undesired reduction of the azido function of the
photoaffinity label 34 after coupling to the glycopeptide. This
882
J. Chem. Soc., Perkin Trans. 1, 1997

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Table 5 1H NMR data of the photoaffinity labelled glycopeptides 39–45 measured in CD3CO2D–D2O (1:1) at 600 MHz at 300 K
39
40
41
42
43
44
45
Val
NH
α-H
β-H
γ-H3
γ-H3
3.95
2.22
1.00
1.00
3.94
2.23
1.00
1.00
3.95
2.23
1.01
1.00
3.93
2.22
1.00
0.99
3.95
2.23
1.00
1.01
3.93
2.23
1.01
1.00
3.95
2.22
1.01
1.00
Ile
NH
8.37
8.38
8.39
8.38
8.39
8.39
8.40
α-H
4.36
4.33
4.33
4.32
4.34
4.32
4.33
β-H
1.81
1.82
1.82
1.80
1.80
1.80
1.81
γ-H2
γ-H3
δ-H3
1.54/1.15
0.89
0.86
1.51/1.14
0.87
0.85
1.52/1.17
0.85
0.87
1.49/1.12
0.88
0.84
1.51/1.14
0.86
0.85
1.50/1.14
0.86
0.88
1.50/1.13
0.86
0.87
Thr
NH
α-H
β-H
γ-H3
8.06
4.48
4.23
1.17
8.14
4.40
4.15
1.09
8.11
4.39
4.19
1.14
8.14
4.40
4.17
1.10
8.14
4.38
4.19
1.12
8.14
4.32
4.19
1.11
8.15
4.41
4.18
1.12
Ala
NH
α-H
β-H3
β-H3
8.06
4.44
1.24
7.96
4.39
1.26
8.01
4.30
1.25
7.99
4.31
1.26
8.03
4.30
1.24
8.01
4.33
1.26
8.02
4.30
1.25
Phe
NH
7.83
8.01
7.90
8.00
7.88
7.95
7.99
α-H
β-H2
4.45
2.91/2.67
4.66
3.11/2.96
4.60
3.06/2.91
4.67
3.07/2.91
4.61
3.04/2.89
4.69
3.05/2.91
4.66
3.05/2.92
Ser or Thr
NH
α-H
β-H/β-HЈ
γ-H3
7.92
4.60
4.19/4.02
7,85
4.54
4.35
1.13
8.13
4.66
3.98/3.84
7.85
4.51
4.28
1.20
7.88
4.65
4.01/3.87
7.92
4.51
4.28
1.21
7.85
4.51
4.30
1.20
Glu
NH
8.01
8.00
8.11
7.85
8.10
7.84
8.05
α-H
4.39
4.43
4.38
4.17
4.34
4.20
4.40
β-H2
γ-H2
2.16/1.98
2.47
2.17/2.01
2.52
2.05/1.89
2.41
1.88/1.62
2.25
2.04/1.86
2.40
1.90/1.61
2.27
2.14/1.98
2.47
Gly
NH
α-H2
8.01
3.97
8.10
3.97
8.12
3.91/3.86
8.08
3.93
8.11
3.89
8.03
3.95
8.08
3.94
Leu
NH
7.79
7.84
7.87
7.92
7.88
7.94
7.91
α-H
β-H2
γ-H
4.39
1.59
1.60
4.39
1.58
1.59
4.39
1.59
1.62
4.41
1.59
1.60
4.38
1.59
1.62
4.42
1.60
1.60
4.40
1.60
1.59
δ-H3
0.88/0.82
0.84/0.89
0.90/0.85
0.92/0.87
0.88/0.84
0.87/0.91
0.87/0.91
Lys
NH
8.08
8.11
8.14
8.15
8.16
8.18
8.15
α-H
4.41
4.41
4.40
4.40
4.42
4.42
4.43
β-H2
γ-H2
δ-H2
ε-H2
NH2
1.91/1.75
1.44
1.69
3.02
7.45
1.91/1.72
1.42
1.68
3.01
7.46
1.90/1.75
1.45
1.70
3.03
7.48
1.90/1.73
1.43
1.69
3.02
7.48
1.90/1.76
1.44
1.68
3.02
7.48
1.91/1.71
1.44
1.69
3.02
7.48
1.90/1.72
1.44
1.70
3.03
7.48
GalNHR
1-H
5.08
(3.14)
4.47
4.02
4.04
4.31
4.39
4.39
5.17
(3.4)
4.41
4.08
4.08
4.33
4.40
4.39
5.08
5.19
2-H
3-H
4-H
5-H
6-H
6-HЈ
4.62
4.13
4.02
4.31
4.35
4.35
4.58
4.20
4.35
GlcNHR
1-H
4.77
4.77
5.16
(8.28)
(8.26)
(3.24)
J. Chem. Soc., Perkin Trans. 1, 1997
883

View Online
Table 5 (contd.)
39
40
41
42
43
44
45
GlcNHR
2-H
3-H
4-H
5-H
6-H
6-HЈ
4.08
4.00
4.07
3.79
3.55
3.55
3.97
3.97
3.97
3.78
3.52
3.52
4.16
3.99
3.61
3.79
3.89
3.83
Gal
1-H
2-H
3-H
4-H
5-H
6-H
6-HЈ
4.57
3.62
3.61
3.93
3.64
3.81
3.78
4.63
3.67
3.62
3.95
3.65
3.81
3.76
NH
NHAc
Phenyl
7.25,
7.22,
7.06
7.26,
7.22,
7.16
7.28,
7.23,
7.15
7.29,
7.24,
7.16
7.28,
7.26,
7.13
7.28,
7.24,
7.15
7.28,
7.24,
7.15
Azido-
benzoyl
7.75,
7.54,
7.29,
7.22
7.70,
7.57,
7.33,
7.25
7.75,
7.56,
7.29,
7.25
7.77,
7.56,
7.31,
7.26
7.78,
7.57,
7.29,
7.24
7.78,
7.55,
7.30,
7.25
7.77,
7.57,
7.31,
7.26
6 S. Hakamori, Adv. Cancer Res., 1989, 52, 257.
tion and washing of the resin successively with 95% aq. TFA
and acetic acid. After concentration the acetylated glycopep-
tides were freeze dried with water. The final removal of the
acetyl groups from the glycopeptides was performed with
sodium methoxide in methanol at a pH of 8.5. After 4 h the
solutions were neutralized by addition of solid CO2, concen-
trated, and lyophilized from water. The glycopeptides 39–45
were purified by semipreparative HPLC, using a linear gradient
of 0–50% solvent B in 50 min and then 50–100% in 10 min. The
pure compounds were lyophilized from water and fully charac-
terized by ESMS and 1H NMR spectroscopy. Yields and ESMS
7 R. M. Chicz, W. S. Lane, R. A. Robinson, M. Trucco, J. L.
Strominger and J. C. Gorga, Int. Immunol., 1994, 6, 1639.
8 (a) C. V. Harding, J. Kihlberg, M. Elofsson, G. Magnusson and
E. R. Unanue, J. Immunol., 1993, 151, 2419; (b) S. Mouritsen,
M. Meldal, I. Christensen-Brams, H. Elsner and O. Werdelin,
Eur. J. Immunol., 1994, 24, 1066; (c) J. S. Haurum, G. Arsequell,
A. C. Lellouch, S. Y. Wong, R. A. Dwek, A. J. McMichael and
T. Elliott, J. Exp. Med., 1994, 180, 739; (d) T. Jensen, L. Galli-
Stampino, S. Mouritsen, K. Frische, S. Peters, M. Meldal and
O. Werdelin, Eur. J. Immunol., 1996, 26, 1342; (e) T. Jensen,
P. Hansen, L. Galli-Stampino, S. Mouritsen, K. Frische, E. Meinjo-
hanns, M. Meldal and O. Werdelin, J. Immunol., 1996, in the press;
(f ) L. Galli-Stampino, E. Meinjohanns, K. Frische, M. Meldal,
T. Jensen, O. Werdelin and S. Mouritsen, Cancer Res., 1996,
in the press.
1
data are presented in Table 4, H NMR data are presented in
Table 5.
9 B. D. Evavold, S. G. Williams, B. L. Hsu, S. Buus and P. M. Allen,
J. Immunol., 1992, 148, 347.
Acknowledgements
10 K. Frische, M. Meldal, O. Werdelin, S. Mouritsen, T. Jensen,
L. Galli-Stampino and K. Bock, J. Pept. Sci., 1996, 2, 2.
11 M. Meldal, Curr. Opin. Struct. Biol., 1994, 4, 710; Glycopeptide
Synthesis, in Neoglycoconjugates: Preparation and Applications,
ed. Y. C. Lee and R. T. Lee, Academic Press, San Diego, 1994,
p. 145; M. Meldal and K. Bock, Glycoconjugate J., 1994, 11, 59.
12 M. Meldal and K. J. Jensen, J. Chem. Soc., Chem. Commun., 1990,
483.
The present work was supported by a grant from the Danish
Cancer Society and in part by the EU-Science Program (grant
SCI* CT92-0765).
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Paper 6/06725E
Received 2nd October 1996
Accepted 29th November 1996
884
J. Chem. Soc., Perkin Trans. 1, 1997