Synthesis of N-linked glycopeptides via solid-phase aspartylation

Article abstract of DOI:10.1039/c003673k

An efficient strategy for the preparation of N-linked glycopeptides is described. The method relies on the use of side chain protecting groups on aspartic acid residues, namely the allyl and Dmab esters, which are orthogonal to those utilised in Fmoc-strategy SPPS. After peptide assembly these protecting groups were selectively removed and the resulting free side chains derivatised with a glycosylamine to afford a resin bound glycopeptide bearing a native N-linkage. Initially, N-linked glycopeptides were successfully synthesised according to this strategy, however, yields varied substantially depending on the nature of the amino acid residue situated adjacent (C-terminal) to the putative glycosylation site. This was due to generation of substantial quantities of aspartimide by-products. Aspartimide formation was overcome by incorporation of a 2,4-dimethoxybenzyl (Dmb) backbone amide protecting group on the residue adjacent to an allyl- or Dmab-protected aspartic acid residue. N-linked glycopeptides were prepared in excellent yield after the solid-phase aspartylation reactions. The utility and orthogonality of the allyl and Dmab ester solid-phase approaches were exploited in the preparation of an N-linked glycodecapeptide bearing two different carbohydrate moieties. This exemplified the efficiency of the solid-phase methodology for the preparation of glycopeptides bearing various combinations of N-linked glycans.

Full text of DOI:10.1039/c003673k

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis of N-linked glycopeptides via solid-phase aspartylation†
Trent Conroy, Katrina A. Jolliffe and Richard J. Payne*
Received 26th February 2010, Accepted 24th May 2010
First published as an Advance Article on the web 21st June 2010
DOI: 10.1039/c003673k
An efficient strategy for the preparation of N-linked glycopeptides is described. The method relies on
the use of side chain protecting groups on aspartic acid residues, namely the allyl and Dmab esters,
which are orthogonal to those utilised in Fmoc-strategy SPPS. After peptide assembly these protecting
groups were selectively removed and the resulting free side chains derivatised with a glycosylamine to
afford a resin bound glycopeptide bearing a native N-linkage. Initially, N-linked glycopeptides were
successfully synthesised according to this strategy, however, yields varied substantially depending on
the nature of the amino acid residue situated adjacent (C-terminal) to the putative glycosylation site.
This was due to generation of substantial quantities of aspartimide by-products. Aspartimide
formation was overcome by incorporation of a 2,4-dimethoxybenzyl (Dmb) backbone amide protecting
group on the residue adjacent to an allyl- or Dmab-protected aspartic acid residue. N-linked
glycopeptides were prepared in excellent yield after the solid-phase aspartylation reactions. The utility
and orthogonality of the allyl and Dmab ester solid-phase approaches were exploited in the preparation
of an N-linked glycodecapeptide bearing two different carbohydrate moieties. This exemplified the
efficiency of the solid-phase methodology for the preparation of glycopeptides bearing various
combinations of N-linked glycans.
Ser/Thr consensus sequence. Despite the importance of glyco-
Introduction
proteins for a plethora of important biological processes, their
detailed study has been hampered by the fact that they are difficult
to access in pure form. This is a result of the glycosylation process
which, unlike protein synthesis, is not under templated control.
Instead, glycoproteins are generated as mixtures of glycoforms,
with the glycosylation patterns dependent on the relative activities
of a number of cellular glycosyltransferases. These glycoforms
are inseparable in most cases and, as such, recombinant methods
can not normally be used for the production of homogeneous
glycoproteins. It is currently accepted that synthetic chemistry can
be used to access homogeneous O- and N-linked glycopeptides
and glycoproteins.11–23
We were interested in the preparation of N-linked glycopep-
tides for use in the fragment-based assembly of homogeneous
glycoproteins. The most common method for the preparation of
N-linked glycopeptides is via the so-called cassette approach,
whereby preformed per-O-protected glycosylamino acid building
blocks are first synthesised and incorporated into the growing
peptide backbone during solid-phase peptide synthesis (SPPS)
(Scheme 1a).24–29 Synthesis of the glycopeptide is generally
achieved via the Fmoc-strategy due to the vulnerability of glyco-
sidic linkages in common oligosaccharides to the strongly acidic
conditions employed under the Boc-strategy. A major drawback
of the cassette approach is the greatly reduced efficiency of peptide
coupling reactions, both during glycosylamino acid insertion
and subsequent peptide elongation. This is due primarily to the
sterically hindered nature of amino acids bearing large glycans.30
The lack of convergence of the cassette-based approach can also
result in the loss of the precious building block over the iterative
coupling and deprotection steps. An alternative, more convergent
approach was developed by Lansbury and Cohen-Anisfeld.31,32
This method, dubbed the Lansbury Aspartylation, has been used
The majority of proteins in the human body are known to be
post-translationally modified. The most ubiquitous modification
is glycosylation, which introduces an enormous level of structural
diversity to proteins.1 It has been reported that over 50% of
all human proteins are glycosylated, a modification known to
be essential for a variety of key biological processes including
protein folding, transport, cell adhesion, cell differentiation and
cell growth.2-4 In addition, glycosylation plays a critical role in the
regulation of biological half life and the activity of many hormones
by maintaining structural integrity and by protecting the peptide
from proteolytic degradation.5,6 Aberrant glycosylation of proteins
is known to perturb intracellular recognition and, as such, has
been implicated in a number of serious illnesses including cancer
and autoimmune diseases. For example, in tumour progression
there are remarkable changes in cell surface carbohydrate profiles
which appears to be associated with the state of metastasis.7,8 As
they frequently serve as cell differentiation markers, glycoproteins
are involved in binding of pathogens to cells. Indeed, many
pathogens have evolved to use heavily glycosylated, membrane
bound proteins as a point of entry into host cells.9,10
Broadly speaking, almost all native protein glycosylation can
be classified into two types: O-glycosides, whereby a glycan is
a- or b-linked to the hydroxyl of serine, threonine or tyrosine, or
N-glycosides, in which N-acetylglucosamine is b-linked to the
amide side chain of asparagine present within an Asn-Xaa-
School of Chemistry, The University of Sydney, NSW 2006, AUSTRALIA.
E-mail: richard.payne@sydney.edu.au; Fax: +61 2 9351 3329; Tel: +61 2
9351 5877
† Electronic supplementary information (ESI) available: Additional ex-
perimental procedures and analytical data for all novel compounds and
N-linked glycopeptides. See DOI: 10.1039/c003673k
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were interested in the development of a convergent solid-phase
aspartylation strategy for the high yielding preparation of N-
linked glycopeptides which would completely avoid the formation
of aspartimide by-products.
Results and Discussion
Results of previous solid-phase aspartylation studies suggest that
neighbouring amino acids play a critical role in the outcome of
the reactions and the extent of unwanted by-products such as
aspartimides.39,40 To date there have been no detailed investigations
into the effect of amino acid substitution C-terminal to the
proposed aspartylation site (to be modified with a carbohydrate)
on the solid-phase. The initial goal of this study therefore entailed
the first detailed investigation of the role of neighbouring residues
on solid-phase aspartylation reactions by substituting with a range
of amino acids C-terminal to the putative aspartylation sites in a
model peptide system.
To this end, the protected pentapeptides 1a–1l were synthe-
sised from preloaded Wang resin 2 using Fmoc-strategy SPPS
(Scheme 2). After assembly of the protected resin bound tripeptide
(AYS) from 2, the resin was split and a variety of amino acids
coupled as the penultimate residues. Eleven amino acids were
chosen as a representative selection of the 20 proteinogenic build-
ing blocks. The final amino acid incorporated into the growing
peptides was commercially available Fmoc-Asp(OAll)–OH. This
residue represented the putative glycosylation site and, as such,
was incorporated with the b-carboxy side chain derivatised with an
orthogonal protecting group. It was proposed that chemoselective
removal of the allyl ester using Pd(0) would selectively liberate
the carboxylate side chain for the ensuing aspartylation reaction
on the solid phase.46,47 Following Fmoc deprotection of the fully
assembled pentapeptide, the N-terminus was acetylated with acetic
anhydride in pyridine to afford the fully protected, resin bound
pentapeptides 1a–1l.
Deprotection of the allyl ester from the fully assembled resin
bound pentapeptides 1a–1l was achieved by treating the resin
bound peptide with tetrakis(triphenylphosphine) palladium(0)
in the presence of phenylsilane as a scavenger to afford 3a–3l
(Scheme 2).48 Following deallylation, the resin was treated with Py-
BOP, 2-acetamido-2-deoxy-b-D-glucopyranosylamine (GlcNAc-
1-NH2) 4 (prepared from N-acetylglucosamine via Kotchetkov
amination49) and N-methylmorpholine (NMM) in DMF to
generate the desired resin bound N-linked glycopeptides 5a–5l.
Following this, deprotection and cleavage of the glycopeptides
from resin was achieved using an acidic cocktail. Purification was
accomplished by preparative reverse-phase HPLC to afford the
desired glycopeptides 6a–6l and the corresponding aspartimides
7a–7l which, as anticipated, were produced in highly variable yields
depending on the nature of the penultimate amino acid residue
(Table 1).
Scheme 1 Synthesis of N-linked glycopeptides via: a) coupling of
preformed glycosylamino acid cassettes; b) solution phase coupling of
glycosylamines (“Lansbury Aspartylation”); c) solid-phase coupling of
glycosylamines (solid-phase aspartylation).
for the synthesis of a number of complex glycopeptides.33–37 The
reaction involves coupling of a glycosylamine with a free aspartic
acid residue on a peptide backbone and is generally achieved
using standard peptide coupling reagents with a suitable base
(Scheme 1b). The major disadvantage of this method is the need for
extensive protection of the peptide backbone in order to prevent
unwanted side reactions such as the formation of aspartimides
during aspartic acid side chain activation.31 Additional protecting
groups orthogonal to Fmoc-strategy SPPS are also necessary to
allow for the selective modification of aspartic acid residues in the
presence of other aspartic acid and glutamic acid residues within
the peptide or protein sequence.21,33,34,37
The solid-phase preparation of N-linked glycopeptides utilising
a late stage aspartylation step represents a less common approach
(Scheme 1c).38–42 Although on-resin aspartylation represents a
more convergent approach toward the synthesis of glycopeptide
fragments, significant problems have been encountered with
regards to unwanted aspartimide formation during both peptide
elongation and aspartic acid side chain activation during the
glycan coupling step. Although aspartimide suppression has been
achieved with glycine C-terminal to the glycosylation site through
the use of the 2-hydroxy-4-methoxybenzyl (Hmb) backbone amide
protecting group,39,43,44 limitations arise from its use. While this
manuscript was in preparation, Tolbert and Chen reported an
elegant strategy for the solid-phase preparation of glycopeptides
bearing a high mannose N-linked glycan which could be achieved
with reduced aspartimide formation compared with previous
approaches.45 Specifically, this method relied on the use of a
2-phenylisopropyl-protected aspartic acid residue which could
be liberated selectively with weak acid and aspartylated on
the solid phase with a glycosylamine. In the present work, we
The results from this study can be rationalised by assessing the
steric and electronic characteristics of the amino acid preceding
the aspartic acid residue. For example, under standard conditions
(3 equivalents of NMM relative to the peptide) glycine, the
amino acid with lowest steric bulk in its side chain, gave rise
to the greatest levels of aspartimide formation (98%, entry 2,
Table 1). In contrast, incorporation of a penultimate valine
residue, bearing a sterically hindered side chain, led to much lower
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Scheme 2 Synthesis of N-linked glycopeptides 6a–6l via solid-phase aspartylation.
Table 1 Yields of N-linked glycopeptides 6a–l and aspartimides 7a–l
3 eq. NMM 1.5 eq. NMM
such, the corresponding glycopeptide 6i was only isolated in 42%
yield with 53% of the aspartimide byproduct 7i generated in the
process (entry 9, Table 1). Glycopeptide 6a was provided in high
yield (91%) from resin bound peptide 1a with no aspartimide
related byproducts observed (entry 1, Table 1). This result can
be explained by the presence of a tertiary amide adjacent to
the glycosylation site which renders the backbone amide non-
nucleophilic. Using the results of this study it is possible to
extrapolate likely outcomes for other amino acids based on their
steric and electronic properties. However, given the unacceptable
levels of aspartimides formed during the solid-phase aspartylation
reactions of most peptides studied here it was necessary to modify
the conditions in order to suppress aspartimide formation.
Aspartimide formation is known to be intimately linked with
the quantity of base used in the reaction.31 As such, it was
proposed that lowering the number of equivalents of base used
during the aspartylation reactions would reduce the propensity
of the peptides to form aspartimides, thereby improving the
efficiency of the solid-phase aspartylation reactions. To this end,
reactions were repeated for peptides 1a–1l, this time reducing
the amount of NMM to 1.5 equivalents relative to the resin
bound peptide (Table 1). Following solid-phase aspartylation
with glycosylamine 4, the desired glycopeptides were deprotected
and cleaved from the resin as previously described. Subsequent
purification by preparative reverse-phase HPLC then afforded the
desired glycopeptides. Gratifyingly, the reduction in base during
solid-phase aspartylation diminished aspartimide formation for
all amino acid substitutions. The effect was particularly profound
Product (6) Aspartimide (7) Product (6) Aspartimide (7)
Entry X
yield (%)
yield (%)
yield (%)
yield (%)
1
2
Pro 91
0
98
45
14
28
20
16
15
53
17
14
7
ND
48
82
77
45
83
84
77
87
65
92
90
ND
52
10
4
4
8
Gly
2
3
4
5
6
7
8
9
10
11
12
Ala 51
Ser 83
Cys 65
His 77
Lys 72
Arg 80
Asp 42
Gln 81
Phe 81
Val 87
6
11
11
20
4
8
levels of aspartimide formation (7%) and therefore the desired
glycopeptide 6l was provided in high yield (87%, entry 12, Table 1).
Resin bound peptides containing penultimate amino acid residues
including serine (1d), histidine (1f), lysine (1g), arginine (1h),
glutamine (1j) and phenylalanine (1k) gave good yields of N-linked
glycopeptides after solid-phase aspartylation and purification (77–
83%), however, unacceptable levels of aspartimides (14–20%)
were generated in all of these cases (Table 1). High levels of
aspartimide formation was observed when aspartic acid was
found adjacent to the glycosylation site (1i), which can perhaps
be attributed to increased acidity of the backbone amide.50 As
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in the case of glycine, alanine, and aspartic acid where almost
50% reduction in aspartimide formation was observed (entries 2,
3 and 9, Table 1). Resin bound peptides 1d–1g bearing penultimate
serine, cysteine, histidine, lysine, and 1k bearing a phenylalanine
residue exhibited less than 10% aspartimide formation during the
solid-phase aspartylation and, as such, these conditions represent
significant improvements to the methodology. However, although
these modified conditions minimised aspartimide formation, the
efficiency of the aspartylation reaction to install the glycan proved
to be less efficient. As a result, the yields of glycopeptides obtained
from these reactions were lower than those achieved previously
e.g. glycopeptides bearing a penultimate serine (6d) cysteine (6e)
or glutamine residue (6j) (45–77% yields cf. 65–83% using 3 eq.
of NMM). Finding an alternative method for the prevention
of aspartimides, whilst providing high solid-phase aspartylation
yields, was therefore essential for the efficient preparation of N-
linked glycopeptides as was the goal of this research.
The introduction of a backbone amide protecting group on the
amino acid directly preceding the putative glycosylation site was
therefore proposed to prevent aspartimide formation and improve
the efficiency of the solid-phase aspartylation methodology for
the preparation of N-linked glycopeptides. We chose the 2,4-
dimethoxybenzyl (Dmb) protecting group as an alternative to
the commonly employed Hmb group for the protection of the
backbone amides. The Dmb group was chosen by virtue of its
acid lability allowing concomitant cleavage of the protecting group
after generation of the resin bound glycopeptides without further
modification (cf. Hmb). With the exception of Fmoc-(Dmb)Gly-
OH, Dmb-protected amino acids were not commercially available
and therefore needed to be synthesised. The Na-protection of
amino acids was accomplished via reductive amination of 2,4-
dimethoxybenzaldehyde with the appropriately side chain pro-
tected amino acids. To this end, treatment of amino acids 8a–8e
with 2,4-dimethoxybenzaldehyde and sodium triacetoxyborohy-
dride in the presence of acetic acid afforded the desired Dmb-
protected amino acids 9a–9e in 47–82% yield (Scheme 3).
In order to increase the convergency of our approach, we chose
to incorporate 9a–9e into the resin bound peptides as preformed
dipeptides. This would allow for a faster, more efficient solution-
phase coupling to the Dmb-secondary amine. In addition, it
would eliminate the need for Fmoc protection and deprotection
of the Dmb-protected derivatives in subsequent coupling steps
on the solid support. To this end, careful pre-activation of
Fmoc-Asp(OAll)-OH (10) with HATU and NMM followed by
addition of the Dmb-protected amino acids 9a–9e gave the desired
dipeptides 11a–11e. Unfortunately, in our hands the dipeptides
could not be easily purified by silica chromatography, however
reverse-phase HPLC afforded the desired building blocks in
moderate to high yields in most cases.
Scheme 3 Synthesis of Dmb-amino acids 9a–9e and Fmoc-Asp(OAll)-
(Dmb)-AA-OH dipeptides 11a–11e.
Following the complete assembly of 14a–14f, deallylation was
achieved via the palladium-catalysed conditions previously de-
scribed. The peptides were subsequently submitted to the solid-
phase aspartylation conditions (1.5 equiv. PyBOP, 3.0 equiv.
NMM) with glycosylamine 4 (1.5 equiv.) to afford 15a–15f. The
resin bound glycopeptides were then cleaved from the resin and
deprotected via acidolysis. Following purification by preparative
reverse-phase HPLC, the desired glycopeptides 6b, 6c, 6e, 6g, 6i
and 6j were obtained in high yield in all cases (Table 2). Indeed,
analysis of these reactions by analytical HPLC indicated that
preparation of the desired N-linked glycopeptides was highly
efficient with the backbone amide (Dmb) protection completely
eliminating aspartimide formation. Following the 14 linear steps
involved in the solid-phase synthesis, HPLC analysis indicated a
>90% yield of the desired N-linked glycopeptides in all cases. The
isolated yields were slightly lower due to loss during purification
of the products by HPLC. A representative analytical HPLC trace
for the crude alanine containing glycopeptide 6c clearly depicts
the efficiency of the solid-phase aspartylation of peptides bearing
Dmb protection on the amino acid residue C-terminal to the
glycosylation site (Fig. 1).
Having successfully established an efficient means for the
aspartimide-free solid-phase preparation of N-linked glycopep-
tides, our next goal was to extend the methodology to allow
Table 2 Preparation of N-linked glycopeptides via solid-phase asparty-
lation of backbone protected peptides
Due to the commercial availability of Fmoc-(Dmb)Gly-OH
(12) this was not incorporated as a dipeptide, but rather was
coupled as an individual amino acid followed by Fmoc-Asp(OAll)-
OH (10) to resin bound tripeptide 13 to give 14a (Scheme 4).
Dipeptides 11a–11e were coupled to 13 using HATU, to afford
the desired resin bound pentapeptides 14b–14f, possessing a Dmb
backbone amide protecting group on the penultimate residue
(Scheme 4). Gratifyingly the dipeptides could be coupled to 13
in quantitative yield in 2 h (as determined by measuring the
piperidine–fulvene adduct at 301 nm upon Fmoc deprotection).
HPLC
yield (%)
Isolated
yield (%)
Aspartimide
yield (%)
Entry
Amino Acid X
1
2
3
4
5
6
Gly
Ala
Cys
Lys
Asp
Gln
91
90
93
91
91
94
64
58
67
69
59
73
0
0
0
0
0
0
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Scheme 4 Synthesis of N-linked glycopeptides via solid-phase aspartylation from backbone amide protected resin bound peptides 14a–14f.
pre-activation of Fmoc-Asp(ODmab)-OH 17 with HATU and
NMM followed by treatment with Dmb-amino acids 9a, 9d
and 9e. Subsequent purification by preparative reverse-phase
HPLC afforded the desired dipeptides 16a–16c in 37–50% yield
(Scheme 5). Dmb protected dipeptides 16a–16c were next coupled
to tripeptide 13 using HATU to afford the desired resin bound
pentapeptides 18c–18e in quantitative yield (as determined by
measuring the UV absorbance of the piperidine–fulvene adduct at
301 nm upon Fmoc removal, Scheme 6). Fmoc-Pro-OH, Fmoc-
(Dmb)Gly-OH and Fmoc-Val-OH were incorporated as individ-
ual amino acids followed by coupling of Fmoc-Asp(ODmab)-
OH to afford 18a, 18b and 18f also in quantitative yield. Valine
was coupled without a backbone amide protecting group, as
previous observations indicated that the steric bulk of the side
chain would preclude significant aspartimide formation (Table 1).
Following assembly, the Dmab groups of pentapeptides 18a–18f
were removed by treatment with 2% hydrazine, followed by
5mM NaOH in 1 : 1 v/v MeOH–H2O to promote aminobenzyl
ester hydrolysis.54 The resulting peptides were then subjected to
the solid-phase aspartylation conditions (4, PyBOP and NMM
in DMF) to afford the resin bound N-linked glycopeptides.
Following acidolytic deprotection and cleavage from the resin, the
glycopeptides were purified by preparative reverse-phase HPLC
to afford 6a–6c, 6i, 6j and 6l in high yields in all cases (80–
96% yield, entries 1–6, Table 3). As expected, aspartimides were
not generated during the solid-phase aspartylation reactions,
with the exception of 18f bearing a penultimate valine residue
which, after aspartylation, provided glycopeptide 6l in 96% yield,
along with a 3% yield of the corresponding aspartimide which
was generated due to the lack of a backbone amide protecting
group.
Fig. 1 Crude analytical HPLC of a representative N-linked glycopeptide
6c after cleavage from the resin (l = 280 nm, gradient: 0–50%B over
30 min).
for the incorporation of two different sugars onto the same
resin bound peptide fragment. To this end, an alternative side
chain protecting group was sought which would be orthogonal
to the allyl ester and the acid labile protecting groups utilised in
Fmoc-strategy SPPS. In this regard, the 4-{N-[1-(4,4-dimethyl-
2,6-dioxocyclohexylidene)-3-methylbutyl]-amino} benzyl (Dmab)
ester has recently shown promise as an orthogonal protecting
group for the modification of peptides on resin.51-53 We recently
showed that the Dmab ester could be deprotected in quantitative
yield on the solid phase by treatment with 2% hydrazine to
remove the 1-(4,4-dimethyl-2,6-dioxo-cyclohexylidene)-3-methyl-
butyl (ivDde) moiety52 followed by treatment with dilute aqueous
hydroxide to facilitate elimination of the resultant p-aminobenzyl
ester.54 It was anticipated that this early work could be extended
in our solid phase aspartylation methodology for the facile
preparation of a range of N-linked glycopeptides.
Dmab protected dipeptides 16a–16c possessing Dmb protec-
tion of the backbone amide were synthesised in solution by
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Scheme 5 Synthesis of Fmoc-Asp(ODmab)(Dmb)-AA-OH dipeptides 16a–16c.
Scheme 6 Synthesis of N-linked glycopeptides via solid-phase aspartylation of Dmab ester protected resin bound peptides 18a–18f. * Fmoc-AA-OH
includes Fmoc-Pro-OH, Fmoc-(Dmb)Gly-OH (12) and Fmoc-Val-OH. # 18a, 18f, 19a and 19b were not Dmb-protected.
Table 3 Yields of N-linked glycopeptides from resin bound Dmab-
protected peptides 18a–18f
Having successfully demonstrated that the Allyl and Dmab
esters could be used interchangeably for the preparation of N-
linked glycopeptides via solid-phase aspartylation, the next goal
was to combine these two strategies to selectively aspartylate a
resin-bound peptide with two different carbohydrate moieties.
To demonstrate the feasibility of this approach, the model
decapeptide 20 was assembled on Wang resin using Fmoc-
strategy SPPS (Scheme 7). Dmb-protected glycine and alanine
residues were incorporated on the C-terminal side of two aspartic
acid residues which were side chain protected with Dmab and
Allyl esters respectively in 20. Peptide couplings until the final
aspartic acid residue proceeded with greater than 95% efficiency
HPLC
yield (%)
Isolated
yield (%)
Aspartimide
yield (%)
Entry
Amino Acid X
1
2
3
4
5
6
Proa
Gly
Ala
Asp
Gln
Vala
95
92
88
91
80
96
86
50
71
58
65
56
0
0
0
0
0
3
a No Dmb protection.
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Scheme 7 Synthesis of diglycosylated peptide 23 via a double solid-phase aspartylation reaction.
as determined by measuring the absorbance of the piperidine–
Conclusions
fulvene adduct after Fmoc deprotection of this residue. The final
cysteine residue was introduced as the N-Boc protected derivative
thereby allowing the final acidolytic deprotection and cleavage
step to afford a free N-terminus. Having successfully assembled
the fully protected peptide on resin, it was possible to commence
solid phase aspartylation. Accordingly, deallylation of the corre-
sponding aspartic acid residue was achieved with palladium(0)
in the presence of phenylsilane. Solid-phase aspartylation with
glycosylamine 4 then gave resin bound glycopeptide 21. The next
step involved deprotection of the Dmab ester which was achieved
in two steps using 2% hydrazine in DMF followed by a 5 mM
aqueous solution of NaOH. A second solid phase aspartylation
was then performed using the peracetylated glycosylamine 22
(prepared in three steps from N-acetylglucosamine55). The final
acidolytic side chain deprotection and resin cleavage step followed
by purification by preparative reverse phase HPLC afforded the
desired diglycopeptide 23 in an excellent 65% isolated yield over
26 linear steps (starting from preloaded Wang resin). Although
this first example was conducted using two monosaccharides,
it is anticipated that the double solid-phase aspartylation will
provide a useful means for the incorporation of large oligosac-
charides. Specifically, the selective incorporation of bridgehead
monosaccharides as described here should prove useful for enzy-
matic elaboration via the use of endo-b-N-acetylglucosaminidases
(Endo-A and Endo-M) to afford more complex N-linked
glycans on the peptide backbone.56-65 Alternatively, larger
oligosaccharides bearing anomeric amine moieties could be
incorporated to prepare glycopeptides bearing more complex
glycans.45
In summary, we have developed a rapid and efficient method
for the synthesis of N-linked glycopeptides via solid-phase as-
partylation. An extensive study was first conducted in order to
ascertain the extent to which amino acids adjacent (C-terminal)
to the putative glycosylation site affect aspartimide formation.
Peptides bearing allyl ester protected aspartic acid side chains
were deprotected with palladium(0) before aspartylation with an
glycosylamine. With the exception of peptides containing a penul-
timate glycine residue, the use of less molar equivalents of the base
NMM (1.5 equiv.) provided glycopeptides in satisfactory yields
(45–92%) with minimum aspartimide formation (4–20%). The
incorporation of a 2,4-dimethoxybenzyl (Dmb) backbone amide
protecting group adjacent to the putative glycosylation site com-
pletely prevented aspartimide formation during the solid-phase
aspartylation reaction, thus providing N-linked glycopeptides in
high yields in all cases. A second orthogonal protecting group,
the Dmab ester was also utilised as an aspartic acid side chain
protecting group within resin bound peptides. Deprotection using
hydrazine and dilute aqueous base followed by aspartylation with
a glycosylamine again provided the desired N-linked glycopeptides
in high yields. The presence of backbone amide protecting groups
in these peptides also prevented aspartimide by-products. To
demonstrate the scope of the methodology and the compatibility
of the allyl and Dmab esters, a resin bound peptide bearing both
of these protecting groups was prepared. Selective deprotection
and aspartylation at each putative glycosylation site was achieved
with two different glycans to afford a glycopeptide containing two
different N-glycans in high yield.
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Given the simplicity and efficiency of this methodology it
is anticipated that it will have far reaching applications in the
preparation of N-linked glycopeptides and N-linked glycopeptide
libraries. Future directions in our laboratory are focussed on the
use of the solid-phase aspartylation methodology developed here
for the synthesis of N-linked glycopeptides bearing native, complex
N-linked glycans. This will then be extended to facilitate the total
synthesis of a homogeneous glycoprotein by the use of convergent
ligation chemistry.
Rf [1 : 9 v/v MeOH–DCM containing 1% AcOH] = 0.18; m.p. =
146–148 ◦C; [a]D20 = -5.3 (c = 1.6, CHCl3); IR nmax = 3448, 3001,
1
2970, 2939, 2399, 1697, 1620; H NMR (400 MHz, CDCl3) 7.29
(d, 1H, J = 8.2 Hz, Ar H), 6.44–6.39 (m, 2H, Ar H), 4.13 (d, 1H,
J = 13.4 Hz, CHHN), 4.02 (d, 1H, J = 13.4 Hz, CHHN), 3.82 (s,
3H, OCH3), 3.74 (s, 3H, OCH3), 3.31 (m, 1H, a-H), 3.02 (m, 2H,
CH2), 1.82 (m, 2H, CH2), 1.43–1.38 (m, 13 H, 2 x CH2, 3 x CH3);
13C NMR (75 MHz, CDCl3) d 172.7, 162.4, 159.4, 156.6, 133.1,
112.2, 105.1, 98.9, 79.1, 61.0, 55.9, 55.8, 45.6, 40.3, 30.1, 29.9,
28.9, 23.3; MS (ESI) m/z 397.1 [(M+H)+, 100%]; HRMS Calcd
for C20H32N2O6Na: MNa+, 419.2153 found MNa+, 419.2153.
Experimental
H-(Dmb)-Asp(OtBu)-OH (9d). L-Aspartic acid-b-tert-butyl
ester 8d (1.51 g, 8.0 mmol) was Dmb protected using general
procedure A. The product was purified by column chromatogra-
phy (eluent: 1 : 9 MeOH–DCM) to afford 9d as a pale yellow solid
(1.90 g, 70%). Rf [1 : 9 v/v MeOH–DCM containing 1% AcOH] =
General procedure A: Na-Dmb protection of amino acids 9a–9e
To the appropriately side chain protected L-amino acid (8 mmol)
in DMF (20 mL) containing acetic acid (0.2 mL) was added 2,4-
dimethoxybenzaldehyde (1.40 g, 8.4 mmol) and sodium triacetoxy-
borohydride (1.87 g, 8.8 mmol) and the reaction stirred at 22 ◦C
for 14 h. Water (5 mL) and acetic acid (2 mL) were added and
the reaction warmed under reduced pressure until effervescence
ceased. The reaction was then concentrated in vacuo to afford a
viscous oil which was purified by column chromatography.
0.37; m.p. = 49–51 ◦C; [a]D20 = -6.65 (c = 1.5, CHCl3); IR nmax
=
1
3679, 3016, 3001, 2970, 2399, 1712, 1620; H NMR (400 MHz,
CD3OD) d 7.29 (d, 1H, J = 8.4 Hz, Ar H), 6.65 (d, 1H, J =
2.0 Hz, Ar H), 6.58 (dd, 1H, J = 8.4, 2.0 Hz, Ar H), 4.29 (d, 1H, J =
10.8 Hz, CHHN), 4.26 (d, 1H, J = 10.8 Hz, CHHN), 3.94 (s, 3H,
OCH3), 3.87 (dd, 1H, J = 7.2, 4.4 Hz, a-H), 3.84 (s, 3H, OCH3),
2.98 (dd, 1H, J = 18.0, 4.4 Hz, CHH), 2.87 (dd, 1H, J = 18.0,
7.2 Hz, CHH), 1.49 (s, 9H, CH3); 13C NMR (75 MHz, CD3OD)
d 172.1, 171.7, 164.6, 161.0, 134.0, 112.9, 106.7, 100.0, 84.0, 58.7,
56.6, 56.4, 48.5, 36.2, 28.7; MS (ESI) m/z 340.0 [(M+H)+, 100%];
HRMS Calcd for C17H25NO6Na: MNa+, 362.1574 found MNa+,
362.1573.
H-(Dmb)-Ala-OH (9a). L-Alanine 8a (0.71 g, 8.0 mmol) was
Dmb protected using general procedure A. The product was
purified by column chromatography (eluent: 3 : 20 v/v MeOH–
DCM) to afford 9a, as a pale yellow solid (0.90 g, 47%). Rf [3 : 20
MeOH–DCM containing 1% AcOH] = 0.24; m.p. = 216–218 ◦C;
[a]2D0 = -0.41 (c = 2.2, MeOH); IR (thin film) nmax = 2993, 2939,
2831, 2360, 2337, 1612; 1H NMR (400 MHz, CD3OD) d 7.28 (d,
1H, J = 8.2 Hz, Ar H), 6.61 (d, 1H, J = 2.0 Hz, Ar H), 6.57 (dd,
1H, J = 8.2, 2.0 Hz, Ar H), 4.15 (d, 1H, J = 16.0 Hz, CHHN),
4.13 (d, 1H, J = 16.0 Hz, CHHN), 3.89 (s, 3H, OCH3), 3.81 (s,
3H, OCH3), 3.52 (q, 1H, J = 7.2 Hz, a-H), 1.49 (d, 3H, J =
7.2 Hz, CH3); 13C NMR (75 MHz, CD3OD) d 174.7, 164.4, 161.0,
133.9, 113.3, 106.6, 99.9, 58.9, 56.6, 56.4, 47.0, 16.5; MS (ESI) m/z
239.9 [(M+H)+, 100%]; HRMS Calcd for C12H17NO4Na: MNa+,
262.1050 found MNa+, 262.1052.
H-(Dmb)-Gln(Trt)-OH
(9e). N-g-Trityl-L-glutamine
8e
(3.10 g, 8.0 mmol) was Dmb protected using general procedure
A. The product was purified by column chromatography (eluent:
1 : 9 v/v MeOH–DCM containing 1% AcOH) to afford 9e as
a pale yellow solid (3.53 g, 82%). Rf [1 : 9 v/v MeOH–DCM
containing 1% AcOH] = 0.36; m.p. = 119–121 ◦C; [a]D20 = -2.26
(c = 1.5, CHCl3); IR nmax = 3687, 3425, 3085, 3031, 3008, 2977,
2399, 1650, 1620; 1H NMR (400 MHz, CD3OD) d 7.25-7.12 (m,
15H, Trt Ar H), 6.99 (d, 1H, J = 8.8 Hz, Dmb Ar H), 6.36-6.32
(m, 2H, 2 x Ar H), 3.88 (d, 1H, J = 12.8 Hz, CHHN), 3.80 (d, 1H,
J = 12.8 Hz, CHHN), 3.76 (s, 3H, OCH3), 3.58 (s, 3H, OCH3),
3.33 (dd, 1H, J = 5.2, 5.2 Hz, a-H), 2.52-2.33 (m, 2H, CH2),
2.16-2.06 (m, 1H, CHH), 1.99-1.89 (m, 1H, CHH); 13C NMR
(75 MHz, CD3OD) d 173.8, 170.7, 162.5, 159.3, 144.8, 132.8,
129.2, 128.2, 127.2, 111.8, 104.9, 98.9, 71.1, 61.1, 55.8, 51.1, 48.2,
31.3, 25.4; MS (ESI) m/z 539.1 [(M+H)+, 100%]; HRMS Calcd
for C33H34N2O5Na: MNa+, 561.2360 found MNa+, 561.2364.
H-(Dmb)-Cys(Trt)-OH (9b). S-Trityl-L-Cysteine 8b (2.90 g,
8.0 mmol) was Dmb protected using general procedure A. The
product was purified by column chromatography (eluent: 1 : 9 v/v
MeOH–DCM) to afford 9b as a pale yellow solid (2.34 g, 57%).
Rf [1 : 9 v/v MeOH–DCM containing 1% AcOH] = 0.40; m.p. =
108–110 ◦C; [a]D20 = -0.3 (c = 1.5, CHCl3); IR nmax = 3726, 3679,
3610, 3031, 3016, 3001, 2970, 2893, 2399, 2360, 1751, 1666, 1620;
1H NMR (400 MHz, CDCl3) d 7.36 (d, 6H, J = 7.6 Hz, Ar H),
7.23–7.14 (m, 9H, Ar H), 7.03 (d, 1H, J = 8.0 Hz, Ar H), 6.38 (dd,
1H, J = 1.5, 8.0 Hz, Ar H), 6.37 (br. s, 1H, Ar H), 3.95 (d, 1H,
J = 13.2 Hz, CHHN), 3.80 (d, 1H, J = 13.2 Hz, CHHN), 3.78 (s,
3H, OCH3), 3.65 (s, 3H, OCH3), 2.98 (m, 1H, CHHS), 2.77 (m,
2H, a-H + CHHS); 13C NMR (75 MHz, CDCl3) d 169.8, 162.5,
159.2, 144.6, 133.0, 129.9, 128.5, 127.3, 112.5, 105.0, 99.0, 67.9,
59.4, 55.9, 55.7, 47.6, 32.9; MS (ESI) m/z 514.1 [(M+H)+, 100%];
HRMS Calcd for C31H31NO4SNa: MNa+, 536.1866 found MNa+,
536.1875.
General procedure B: Synthesis of Fmoc-Asp(OAll)-(Dmb)AA-
OH dipeptides 11a–11e. A solution of Fmoc-Asp(OAll)-OH 10
(0.20 g, 0.34 mmol), HATU (0.13 g, 0.34 mmol) and NMM
(110 mL, 1 mmol) in DMF (5 mL) was stirred for 30 min
before the addition of Dmb amino acid 9a–9e (0.50 mmol). The
reaction was stirred for 3 h before dilution with EtOAc (20 mL)
and saturated aqueous NH4Cl solution (20 mL). The aqueous
layer was separated and re-extracted with EtOAc (20 mL). The
combined organic layers were washed with brine (20 mL), dried
over Na2SO4, concentrated in vacuo and the resulting residue
purified by preparative HPLC, gradient: 30–100%B over 45 min
(solvent A: water + 0.1% TFA, solvent B: MeCN + 0.1%TFA).
H-(Dmb)-Lys(Boc)-OH (9c). N-e-Boc-L-Lysine 8c (1.97 g,
8.0 mmol) was Dmb protected using general procedure A. The
product was purified by column chromatography (eluent: 1 : 9 v/v
MeOH–DCM) to afford 9c as a pale yellow solid (1.96 g, 62%).
3730 | Org. Biomol. Chem., 2010, 8, 3723–3733
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Fmoc-Asp(OAll)-(Dmb)Ala-OH (11a). H-(Dmb)-Ala-OH 9a
(0.12 g, 0.50 mmol) was coupled to Fmoc-Asp(OAll)-OH 10 using
general procedure B◦to afford dipeptide 11a as a white solid (0.14 g,
144.2, 141.7, 132.3, 131.5, 128.1, 127.5, 125.7, 120.3, 118.9, 115.9,
104.4, 99.1, 79.5, 67.7, 66.0, 61.1, 60.6, 55.8, 55.7, 49.3, 47.5,
40.6, 37.9, 29.7, 29.0, 28.8, 23.7; MS (ESI) m/z 796.3 [(M+Na)+,
100%]; HRMS Calcd for C42H51N3O11Na: MNa+, 796.3416 found
MNa+, 796.3407.
68%). m.p. = 45–47 C; [a]2D0 = -28.97 (c = 0.9, CHCl3); IR nmax
=
3687, 3610, 3047, 3001, 2970, 2900, 2437, 2408, 2355, 1735, 1720,
1650, 1612; 1H NMR (400 MHz, CD3OD, major rotamer) d 7.53
(d, 2H, J = 7.2 Hz, 2x Ar H), 7.38 (m, 2H, 2 x Ar H), 7.17 (t,
2H, J = 7.2 Hz, 2 x Ar H), 7.07 (t, 2H, J = 7.2 Hz, 2 x Ar H),
7.02 (d, 1H, J = 8.2 Hz, Ar H), 6.22 (br. s, 1H, Ar H), 6.16 (d,
1H, J = 8.2 Hz, Ar H), 5.71-5.61 (m, 1H, CH=CH2), 5.06 (d, 1H,
Fmoc-Asp(OAll)-(Dmb)Asp(OtBu)-OH (11d). H-(Dmb)Asp-
(OtBu)-OH 9d (0.17 g, 0.50 mmol) was coupled to Fmoc-
Asp(OAll)-OH 10 using general procedure B to afford dipeptide
◦
11d as a white solid (0.17 g, 70%). m.p. = 50–52 C; [a]D20
=
=
=
J = 17.2 Hz, CH CHH), 4.97 (m, 2H, CH CHH, CH), 4.44
(d, 1H, J = 16.4 Hz, CHHN), 4.34 (m, 3H, CH2, OCHH), 4.29
(d, 1H, J = 16.4 Hz, CHHN), 4.06 (d, 1H, J = 7.2 Hz, OCHH),
3.96 (m, 1H, CH), 3.84 (q, 1H, J = 7.2 Hz, CH), 3.55 (s, 3H,
OCH3), 3.48 (s, 3H, OCH3), 2.66 (dd, 1H, J = 16.2, 7.2 Hz, CHH),
2.46 (dd, 1H, J = 16.2, 6.8 Hz, CHH), 1.02 (d, 3H, J = 7.2 Hz,
CH3); 13C NMR (75 MHz, CD3OD, major rotamer) d 173.8, 171.1,
170.8, 161.3, 161.2, 158.8, 144.1, 141.6, 132.2, 130.2, 128.0, 127.4,
125.4, 120.2, 118.5, 116.7, 104.5, 98.8, 67.4, 65.9, 56.1, 55.9, 55.5,
55.4, 49.1, 47.4, 37.7, 14.3; MS (ESI) m/z 639.3 [(M+Na)+, 100%]
HRMS Calcd for C34H36N2O9Na: MNa+, 639.2313 found MNa+,
639.2301.
-26.97 (c = 0.7, CHCl3); IR nmax = 3621, 3448, 3024, 3001, 2893,
2399, 2360, 1720, 1650, 1612; 1H NMR (400 MHz, CDCl3, major
rotamer) d 7.77 (m, 2H, 2 x Ar H), 7.62 (m, 2H, 2 x Ar H), 7.41
(m, 2H, 2 x Ar H), 7.33 (m, 3H, 3 x Ar H), 6.47 (br. s, 1H, Ar H),
6.42 (m, 1H, Ar H), 5.95-5.83 (m, 1H, CH=CH2), 5.43 (m, 1H,
=
CH), 5.34-5.18 (m, 2H, CH CH2), 4.74 (m, 1H, OCHH), 4.66-
4.51 (m, 3H, OCHH, CH2), 4.42-4.18 (m, 4H, CH2N, 2 x CH),
3.80 (s, 3H, OCH3), 3.78 (s, 3H, OCH3), 3.22-2.72 (m, 3H, CH2,
CHH), 2.35-2.10 (m, 1H, CHH), 1.39 (s, 9H, CH3); 13C NMR
(75 MHz, CDCl3, major rotamer) d 173.1, 171.8, 170.5, 170.2,
161.6, 158.9, 156.0, 144.0, 141.3, 131.9, 131.1, 127.8, 127.2, 125.4,
120.0, 118.7, 104.3, 104.0, 98.8, 81.2, 67.5, 65.1, 65.0, 58.0, 55.4,
49.0, 47.1, 37.7, 35.3, 31.0, 28.0; MS (ESI) m/z 716.8 [(M+H)+,
100%]; HRMS Calcd for C39H44N2O11Na: MNa+, 739.2837 found
MNa+, 739.2836.
Fmoc-Asp(OAll)-(Dmb)Cys(Trt)-OH
(11b). H-(Dmb)Cys-
(Trt)-OH 9b (0.26 g, 0.50 mmol) was coupled to Fmoc-Asp(OAll)-
OH 10 using general procedure B to afford dipeptide 11b as a
◦
white solid (0.21 g, 70%). m.p. = 79–81 C [a]2D0 = -4.61 (c =
Fmoc-Asp(OAll)-(Dmb)Gln(Trt)-OH
(11e). H-(Dmb)Gln-
0.7, CHCl3); IR nmax = 3687, 3417, 3294, 3055, 3036, 3024, 3008,
2947, 2839, 1720, 1650, 1612; 1H NMR (400 MHz, 1 : 1 v/v
CDCl3/CD3OD, major rotamer) d 7.76 (d, 2H, J = 7.2 Hz, 2 x
Ar H), 7.60 (d, 2H, J = 7.2 Hz, 2 x Ar H), 7.38 (t, 2H, J = 7.2 Hz,
2 x Ar H), 7.35-7.07 (m, 18H, 18 x Ar H), 6.39 (m, 1H, Ar H),
6.37 (br. s, 1H, Ar H), 5.90-5.75 (m, 1H, CH=CH2), 5.34-5.15
(Trt)-OH 9e (0.27 g, 0.50 mmol) was coupled to Fmoc-Asp(OAll)-
OH 10 using general procedure B to afford dipeptide 11e as a
◦
white solid (0.11 g, 36%). m.p. = 59–61 C; [a]2D0 = -23.40 (c =
0.5, CHCl3); IR nmax = 3687, 3425, 3047, 2977, 2360, 2337, 1720,
1689, 1650, 1612; 1H NMR (400 MHz, 2 : 1 v/v CDCl3/CD3OD,
major rotamer) d 7.77 (d, 2H, J = 7.6 Hz, 2 x Ar H), 7.60 (t,
2H, J = 6.8 Hz, 2 x Ar H), 7.41 (t, 2H, J = 7.2 Hz, 2 x Ar H),
7.36-7.15 (m, 17H, 17 x Ar H), 7.06 (d, 1H, J = 8.0 Hz, Ar H),
6.40 (br. s, 1H, Ar H), 6.32 (d, 1H, J = 8.4 Hz, Ar H), 5.88-5.74
=
(m, 3H, CH CH2, CH), 4.58-4.50 (m, 3H, CH2, CH), 4.37 (m,
1H, CH), 4.26 (d, 1H, J = 10.4 Hz CHHN), 4.22 (d, 1H, J =
10.4 Hz, CHHN), 4.09 (m, 1H, CH), 3.77 (s, 3H, OCH3), 3.71
(s, 3H, OCH3), 3.57 (m, 1H, CH), 3.01-2.72 (m, 4H, 2 x CH2);
13C NMR (75 MHz, 1 : 1 v/v CDCl3/CD3OD, major rotamer) d
172.6, 172.2, 170.4, 161.3, 160.7, 155.8, 144.4, 143.9, 141.3, 132.0,
131.9, 129.6, 127.9, 127.8, 127.2, 126.8, 125.3, 120.0, 118.6, 115.4,
103.9, 98.7, 67.4, 67.3, 65.8, 60.9, 55.4, 55.2, 49.5, 47.2, 46.7, 38.0,
30.3; MS (ESI) m/z 913.7 [(M+Na)+, 100%]; HRMS Calcd for
C53H50N2O9SNa: MNa+, 913.3129 found MNa+, 913.3126.
=
(m, 1H, CH=CH2), 5.37 (m, 1H, CH), 5.22 (m, 2H, CH CH2),
4.67 (d, 1H, J = 14.8 Hz, CHHN), 4.45-4.22 (m, 5H, CHHN,
OCH2, CH2), 4.20 (t, 1H, J = 7.2 Hz, CHCH2) 3.92 (m, 1H,
CH), 3.71 (s, 6H, OCH3), 3.00 (dd, 1H, J = 16.0, 7.6 Hz, CHH),
2.77 (dd, 1H, J = 16.0, 4.0 Hz, CHH), 2.50-2.23 (m, 4H, 2 x
CH2); 13C NMR (75 MHz, CDCl3, major rotamer) d 173.5, 173.0,
172.9, 170.8, 161.7, 159.3, 155.9, 144.7, 144.5, 141.7, 132.1, 129.0,
128.4, 128.3, 128.1, 127.7, 127.5, 125.6, 120.4, 118.9, 115.4, 104.5,
99.1, 81.0, 71.1, 67.7, 65.9, 59.8, 55.7, 49.2, 48.8, 47.4, 38.2, 34.1,
25.1; MS (ESI) m/z 938.4 [(M+Na)+, 100%]; HRMS Calcd for
C55H53N3O10Na: MNa+, 938.3623 found MNa+, 938.3620.
Fmoc-Asp(OAll)-(Dmb)Lys(Boc)-OH
(11c). H-(Dmb)Lys-
(Boc)-OH 9c (0.20 g, 0.50 mmol) was coupled to Fmoc-
Asp(OAll)-OH 10 using general procedure B to afford dipeptide
11c as a white solid (0.84 g, 32%). m.p. = 54–56 ◦C; [a]D20 = -11.17
(c = 0.7, CHCl3); IR nmax = 3687, 3101, 3024, 3016, 2931, 2399,
1720, 1650, 1612; 1H NMR (400 MHz, 2 : 1 v/v CDCl3/CD3OD,
major rotamer) d 7.74 (d, 2H, J = 6.0 Hz, 2 x Ar H), 7.60 (m, 2H,
J = 7.2 Hz, 2 x Ar H), 7.38 (m, 2H, 2 x Ar H), 7.29 (m, 2H, 2 x Ar
H), 7.19 (m, 1H, Ar H), 6.43 (br. s, 1H, Ar H), 6.41 (m, 1H, Ar
General procedure C: Synthesis of Fmoc-Asp(ODmab)-
(Dmb)AA-OH dipeptides 16a–16c.
A solution of Fmoc-
Asp(ODmab)-OH 17 (0.10 g, 0.15 mmol), HATU (57 mg,
0.15 mmol) and NMM (33 mL, 0.30 mmol) in DMF (5 mL) were
stirred for 30 min before the addition of Dmb amino acid 9a, 9d or
9e (0.23 mmol). The reaction was stirred for 3 h before diluting with
EtOAc (20 mL) and saturated aqueous NH4Cl solution (20 mL).
The aqueous layer was separated and re-extracted with EtOAc
(20 mL). The combined organic layers were washed with brine
(20 mL), dried over NaSO4, and concentrated in vacuo to afford a
viscous oil which was purified by preparative reverse-phase HPLC,
=
H), 5.94-5.81 (m, 1H, CH=CH2), 5.50-5.16 (m, 3H, CH CH2,
CH), 4.69 (m, 1H, CHHN), 4.60-4.55 (m, 3H, CHHN, CH2), 4.36
(m, 1H, OCHH), 4.28 (m, 1H, OCHH), 4.21 (t, 1H, J = 6.4 Hz,
CHCH2), 3.83-3.67 (m, 7H, 2 x OCH3, CH), 3.10-2.62 (m, 4H, 2
x CH2), 2.10-1.60 (m, 2H, CH2), 1.40 (s, 9H, 3 x CH3), 1.31-1.02
(m, 4H, 2 x CH2); 13C NMR (75 MHz, 2 : 1 v/v CDCl3/CD3OD,
major rotamer) d 173.9, 172.3, 170.8, 161.7, 159.3, 156.1, 156.0
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gradient: 30–100%B over 45 min (solvent A: water + 0.1% TFA,
solvent B: MeCN + 0.1% TFA).
(75 MHz, 1 : 1 v/v CDCl3/CD3OD, major rotamer) d 198.8,
176.6, 173.0, 172.9, 170.7, 162.3, 160.9, 158.7, 156.2, 144.1, 143.7,
143.4, 136.1, 129.6, 129.2, 128.4, 127.6, 126.9, 126.7, 126.4, 126.4,
124.9, 119.7, 115.7, 112.8, 107.4, 103.8, 98.2, 78.0, 70.1, 66.9, 65.3,
55.5, 54.9, 53.2, 52.5, 46.8, 38.2, 36.1, 33.6, 33.4, 29.8, 29.4, 27.8,
24.7, 22.2; MS (ESI) m/z 1188.4 [(M+H)+, 100%]; HRMS Calcd
for C72H74N4O12Na: MNa+, 1209.5201 found MNa+, 1209.5193.
Fmoc-Asp(ODmab)-(Dmb)-Ala-OH (16a). H-(Dmb)Ala-OH
9a (0.05 g, 0.23 mmol) was coupled to Fmoc-Asp(ODmab)-OH 17
(0.10 g, 0.15 mmol) using general procedure C to afford dipeptide
16a as a yellow oil (0.07 g, 50%). [a]2D0 = -5.32 (c = 0.5, CHCl3); IR
nmax = 3692, 3448, 3016, 3001, 2939, 2893, 2677, 2553, 2229, 2067,
1789, 1720, 1643; 1H NMR (400 MHz, 1 : 1 v/v CDCl3/CD3OD,
major rotamer) d 7.76 (d, 2H, J = 7.6 Hz, 2 x Ar H), 7.61 (d,
2H, J = 6.8 Hz, 2 x Ar H), 7.44-7.35 (m, 4H, 4 x Ar H), 7.31 (m,
2H, 2 x Ar H), 7.21 (m, 1H, Ar H), 7.10 (m, 2H, 2 x Ar H), 6.43
(br. s, 1H, Ar H), 6.38 (m, 1H, Ar H), 5.27 (m, 1H, CH), 5.17 (s,
2H, OCH2), 4.68 (m, 1H, NCHH), 4.56 (m, NCHH), 4.37-4.14
(m, 3H, CH2, CH), 4.08 (t, 1H, J = 7.2 Hz, CHCH2), 3.77 (s, 3H,
OCH3), 3.71 (s, 3H, OCH3), 2.98-2.72 (m, 4H, 2 x CH2), 2.46 (s,
4H, 2 x CH2), 1.86-1.77 (m, 1H, CH), 1.26 (d, 3H, J = 7.2 Hz,
Solid-phase peptide assembly (25 lmol scale). Fmoc Deprotec-
tion: A solution of piperidine/DMF (5 mL, 1 : 9 v/v) was added
to pre-loaded Wang resin, shaken for 3 min and the procedure
repeated. The resin was subsequently washed with DMF (5 ¥
3 mL), DCM (5 ¥ 3 mL), and DMF (5 ¥ 3 mL). Amino Acid
Coupling: A solution of protected amino acid (100 mmol), PyBOP
(52 mg, 100 mmol) and NMM (22 mL, 200 mmol) in DMF (1 mL)
was added to the resin and shaken. After 1 h the resin was washed
with DMF (5 ¥ 3 mL), DCM (5 ¥ 3 mL), and DMF (5 ¥ 3 mL).
Capping: Acetic anhydride/pyridine (1 : 9 v/v) was added to the
resin and shaken. After 3 min the resin was washed DMF (5 ¥
3 mL), DCM (5 ¥ 3 mL), and DMF (5 ¥ 3 mL). Final N-terminal
deprotection: When the peptide had been fully assembled the resin
was treated with piperidine/DMF (5 mL, 1 : 9 v/v) and shaken
for 3 min (x 2). The N-terminus was then acetylated by treatment
with Ac2O/pyridine (5 mL, 1 : 9 v/v) for 5 min and subsequently
washed with DMF (10 ¥ 3 mL) then DCM (10 ¥ 3 mL).
CH3), 1.09 (s, 6H, 2 x CH3), 0.77 (d, 6H, J = 6.8 Hz, 2 x CH3); 13
C
NMR (75 MHz, 1 : 1 v/v CDCl3/CD3OD, major rotamer) d 198.6,
176.4, 173.1, 172.9, 170.2, 160.6, 160.5, 158.1, 155.6, 143.2, 140.8,
135.9, 135.4, 129.5, 128.6, 127.3, 126.8, 126.2, 126.1 124.7, 119.6,
116.2, 115.9, 107.1, 103.6, 98.0, 66.7, 65.4, 55.4, 55.2, 54.8, 52.3,
46.8, 38.1, 37.2, 37.1, 29.6, 29.2, 27.6, 25.2, 22.0, 13.6; MS (ESI)
m/z 910.1 [(M+Na)+, 100%]; HRMS Calcd for C51H57N3O11Na:
MNa+, 910.3885 found MNa+, 910.3884.
Coupling of dipeptides. Following assembly of tripeptide 13 on
Wang resin using the solid-phase assembly procedure described
above, a solution of dipeptide 11a–11e or 16a–16c (37.5 mmol),
HATU (14.3 mg, 37.5 mmol) and NMM (8.25 mL, 75 mmol) in
DMF (0.5 mL) were added to the resin and placed on a shaker for
2 h. The resin was subsequently washed with DMF (5 ¥ 3 mL),
DCM (5 ¥ 3 mL), and DMF (5 ¥ 3 mL).
Fmoc-Asp(ODmab)-(Dmb)Asp(OtBu)-OH (16b). H-(Dmb)-
Asp(OtBu)-OH 9d (0.08 g, 0.23 mmol) was coupled to Fmoc-
Asp(ODmab)-OH 17 (0.10 g, 0.15 mmol) using general procedure
C to afford dipeptide 16b as a yellow oil (0.06 g, 43%). [a]D20
=
-16.56 (c = 0.5, CHCl3); IR nmax = 3680, 3634, 3433, 3008, 2978,
2677, 2399, 2252, 2068, 1720, 1604; 1H-NMR (400 MHz 3 : 1 v/v
CD3OD/CDCl3 major rotamer) d 7.77 (d, 2H, J = 7.6 Hz, 2 x
Ar H), 7.63 (m, 2H, 2 x Ar H), 7.46-7.05 (m, 9H, 9 x Ar H),
6.61-6.39 (m, 2H, 2 x Ar H), 5.40-5.15 (m, 1H, CH), 4.92-4.16
(m, 7H, 3 x CH2, CH), 3.17-2.80 (m, 4H, 2 x CH2), 2.75-2.63
(m, 6H, 3 x CH2), 2.49-2.32 (m, 3H, CH2, CHH), 2.18-2.06 (m,
1H, CHH), 1.82-1.70 (m, 1H, CH), 1.38 (s, 9H, 3 x CH3), 1.08
(s, 6H, 2 x CH3), 0.96 (d, 6H, J = 6.4 Hz, 2 x CH3); 13C NMR
(100 MHz, CDCl3, major rotamer) d 199.3, 177.6,172.5, 171.7,
170.5, 170,3, 161.5, 161.1, 158.8, 155.7, 143.7, 141.2, 136.3, 135.6,
131.3, 130.9, 129.1, 127.7, 127.1, 126.5, 126.4, 125.2, 120.0, 115.1,
114.8, 107.3, 104.1, 103.9, 98.7, 81.0, 67.4, 65.9, 57.0, 56.9, 55.3,
52.3, 49.0, 47.0, 38.8, 37.6, 35.2, 30.1, 29.7, 28.1, 27.9, 22.5. MS
(ESI) m/z 988 [(M+H)+, 100%]; HRMS Calcd for C56H66N3O13:
MH+, 988.4590 found MH+, 988.4589.
Allyl ester deprotection. A solution of tetrakis(triphenyl-
phosphine)palladium(0) (20 mg, 18 mmol) and phenylsilane
(123 mL, 1 mmol) in dry DCM (1 mL) was added to the fully
assembled resin bound peptide (25 mmol). The resin was placed
on a shaker for 1 h and the procedure repeated. The resin was
subsequently washed with DMF (10 ¥ 3 mL), DCM (10 ¥ 3 mL),
and DMF (5 ¥ 3 mL).
Dmab ester deprotection. A solution of hydrazine monohy-
drate in DMF (2% v/v, 3 mL) was added to the resin and
placed on a shaker for 3 min. Removal of the ivDde moiety was
monitored by measuring the UV absorbance at 290 nm and the
above procedure repeated until no further indolone adduct could
be detected. Following ivDde removal, the resin was washed with
H2O/Methanol (3 mL, 1 : 1 v/v) containing 5 mM NaOH for 3 h.
Fmoc-Asp(ODmab)-(Dmb)-Gln(Trt)-OH
(16c). H-(Dmb)-
Gln(Trt)-OH 9e (0.12 g, 0.23 mmol) was coupled to Fmoc-
Asp(ODmab)-OH 17 (0.10 g, 0.15 mmol) using general procedure
Solid-phase aspartylation.
A solution of 2-acetamido-2-
deoxy-b-D-glucopyranosylamine 4 (8.3 mg, 37.5 mmol), PyBOP
(19.5 mg, 37.5 mmol) and NMM (8.3 mL, 75 mmol) in DMF
(0.5 mL), was added to the deprotected resin bound peptide and
placed on a shaker for 16 h. The resin was subsequently washed
with DMF (10 ¥ 3 mL) then DCM (10 ¥ 3 mL).
C to afford dipeptide 16c as a yellow oil (0.07 g, 37%). [a]D20
=
-14.23 (c = 0.5, CHCl3); IR nmax = 3618, 3448, 3031, 3001,
2977, 2893, 2461, 1720, 1650; 1H NMR (400 MHz, 1 : 1 v/v
CDCl3/CD3OD, major rotamer) d 7.74 (m, 2H, 2 x Ar H), 7.60
(m, 2H, 2 x Ar H), 7.39-7.04 (m, 24 H, 24 x Ar H, Dmb Ar H),
6.39-6.36 (m, 2H, 2 x Ar H), 5.38 (m, 1 H, CH), 5.09-4.40 (m, 4H,
2 x CH2), 4.30 (d, 2H, J = 7.2 Hz, CH2), 4.17 (t, 1H, J = 7.2 Hz,
CH), 3.76-3.60 (m, 7H, 2 x OCH3, CH), 3.02-2.74 (m, 6 H, 3 x
CH2), 2.54 (s, 4H, 2 x CH2), 2.35-2.13 (m, 2H, CH2), 1.87-1.80 (m,
1H, CH), 1.11 (s, 6H, 2 x CH3), 0.76 (m 6H, 2 x CH3); 13C NMR
Deprotection and cleavage. A mixture of TFA/thioanisole/
triisopropylsilane/water (17 : 1 : 1 : 1 v/v/v/v) was added to the
resin and placed on a shaker for 1.5 h. The resin was then washed
with TFA (3 ¥ 3 mL), and the combined cleavage and washing
solutions were concentrated in vacuo. The resulting residue was
3732 | Org. Biomol. Chem., 2010, 8, 3723–3733
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dissolved in DMSO (1.5 mL), purified by preparative HPLC and
lyophilised to afford the desired glycopeptides.
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33 B. Wu, Z. Tan, G. Chen, J. Chen, Z. Hua, Q. Wan, K. Ranganathan
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Acknowledgements
35 I. J. Krauss, J. G. Joyce, A. C. Finnefrock, H. C. Song, V. Y. Dudkin, X.
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We would like to thank the Australian Research Council (ARC) for
funding this project (DP0986632) and an Australian Postgraduate
Award for PhD funding (TC). We would also like to thank Dr Ian
Luck and Dr Kelvin Picker for technical support.
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