Synthesis of fluorescein-labelled O-mannosylated peptides as components for synthetic vaccines: Comparison of two synthetic strategies

Article abstract of DOI:10.1039/b712926b

Mannose-binding proteins on the surface of antigen-presenting cells (APCs) are capable of recognizing and internalizing foreign agents in the early stages of immune response. These receptors offer a potential target for synthetic vaccines, especially vaccines designed to stimulate T cells. We set out to synthesize a series of fluorescein-labelled O-mannosylated peptides using manual solid phase peptide synthesis (SPPS) on pre-loaded Wang resin, in order to test their ability to bind mannose receptors on human APCs in vitro. A flexible and reliable method for the synthesis of fluorescein-labelled O-mannosylated glycopeptides was desired in order to study their lectin-binding properties using flow cell cytometry. Two synthetic strategies were investigated: incorporation of a fluorescein label into the peptide chain via a lysine side chain ε-amino group at the final stage of standard Fmoc solid phase peptide synthesis or attachment of the fluorescein label to the N^α- amino group of a lysine with further incorporation of a mannosylated peptide unit through the side chain N^ε-amino group. The latter strategy proved more effective in that it facilitated SPPS by positioning the growing mannosylated peptide chain further removed from the fluorescein label. The Royal Society of Chemistry 2008.

Full text of DOI:10.1039/b712926b

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Synthesis of fluorescein-labelled O-mannosylated peptides as components for
synthetic vaccines: comparison of two synthetic strategies
Margaret A. Brimble,*a,c Renata Kowalczyk,a,c Paul W. R. Harris,a,c P. Rod Dunbarb,c and Victoria J. Muira
Received 22nd August 2007, Accepted 5th October 2007
First published as an Advance Article on the web 26th October 2007
DOI: 10.1039/b712926b
Mannose-binding proteins on the surface of antigen-presenting cells (APCs) are capable of recognizing
and internalizing foreign agents in the early stages of immune response. These receptors offer a
potential target for synthetic vaccines, especially vaccines designed to stimulate T cells. We set out to
synthesize a series of fluorescein-labelled O-mannosylated peptides using manual solid phase peptide
synthesis (SPPS) on pre-loaded Wang resin, in order to test their ability to bind mannose receptors on
human APCs in vitro. A flexible and reliable method for the synthesis of fluorescein-labelled
O-mannosylated glycopeptides was desired in order to study their lectin-binding properties using flow
cell cytometry. Two synthetic strategies were investigated: incorporation of a fluorescein label into the
peptide chain via a lysine side chain e-amino group at the final stage of standard Fmoc solid phase
peptide synthesis or attachment of the fluorescein label to the Na-amino group of a lysine with further
incorporation of a mannosylated peptide unit through the side chain Ne-amino group. The latter
strategy proved more effective in that it facilitated SPPS by positioning the growing mannosylated
peptide chain further removed from the fluorescein label.
apart from the classically-described mannose receptor (also known
Introduction
as CD206) that are expressed on APCs.7 APC membrane lectins
The ability of chemical synthesis to provide glycopeptides in
that mediate efficient sugar-specific endocytosis could be poten-
homogeneous form plays a pivotal role for the advancement
tially exploited for glycotargeting tumour antigens to enhance
of glycobiology.1 For example there is current interest in the
design and synthesis of synthetic vaccine formulations capable
of stimulating the immune system for use against diseases caused
by infectious agents and for the treatment of cancer. In particular,
new vaccines are needed that are capable of strongly stimulating T
cells, which recognise unique peptides bound to MHC molecules
on the surface of pathogen-infected cells and tumour cells.
Peptides are attractive agents for incorporation into vaccines
because they can be synthesized and purified without the need for
biological processes and because they are flexible and inexpensive
to manufacture.2 Nevertheless, peptides often show poor stability
in biological fluids3 and are often not targeted efficiently to the cells
responsible for initiating an immune response, namely antigen-
presenting cells (APCs).
APCs capture pathogen- or tumour-derived protein antigens,
and process them into MHC-bound peptides that are trans-
ported to the cell surface for presentation to T cells.4 APCs
express receptors capable of recognizing and internalising foreign
agents. Several of these receptors are C-type lectins that bind
carbohydrates5 and mannose receptors in particular, and are
known to take up and present mannosylated antigens to T cells.6
Indeed within this lectin family there are a number of proteins
antigen presentation to different subsets of T cells. Burgdorf et al.8,9
have recently demonstrated that mannose-mediated endocytosis
of the model antigen soluble ovalbumin (OVA) targets early
endosomes enabling its cross-presentation to CD8+ T cells. In
order to direct a synthetic vaccine to these receptors, ligands
capable of binding to mannose receptors on APC should ideally
be incorporated into the vaccine construct. Many high affinity
glycopeptide binding ligands for mannose lectins have been
produced based on assembly of clustered patches of mannose
ligands to enhance binding to the protein receptors relying on
the cluster effect.10–12 Most of these constructs have relied on use
of mannosylation through the side chain Ne-amino group of a
lysine residue.13,14 An additional complication in the field is that
although most mannose receptors are probably multimeric, and
the highest affinity ligands will probably engage more than one
carbohydrate-recognition domain (CRD), the spacing between
˚
these CRDs is poorly defined. One high-resolution (1.8 A) crystal
structure15 of the head and neck part of rat serum mannose-
binding protein A, a soluble homologue of the mannose receptor,
is available. This structure showed that the individual CRDs are
˚
separated from each other by 53 A. This suggests that assembly of
˚
a polymannosylated scaffold with the mannose units spaced 53 A
apart provides an avenue for investigation of ligand binding.
We therefore postulate that placement of mannose units care-
fully positioned at defined distance on a linear peptide scaffold may
provide an alternative strategy for targeting the mannose receptor
on human APCs. With this idea in mind we have focused on
targeting mannose receptors through O-mannosylated serine units
dispersed at defined sites along a linear peptide scaffold. Our
aDepartment of Chemistry, University of Auckland, 23 Symonds St, Auck-
land 1142, New Zealand. E-mail: m.brimble@auckland.ac.nz; Fax: +64 9
3737422
bSchool of Biological Sciences, University of Auckland, Private Bag 92019,
Auckland 1142, New Zealand
cMaurice Wilkins Centre for Molecular Biodiscovery, University of Auck-
land, Private Bag 92019, Auckland 1142, New Zealand
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initial attention has therefore focused on the efficient synthesis of
O-mannosylated polyalanine peptide scaffolds of differing chain
length and mannose configurations in order to evaluate their
specificity for targeting human APCs in vitro. Thus, in the work
reported herein a set of glycopeptides containing O-mannosylated
serine residues was prepared with the aim of developing a suitable
APC-binding motif that can not only be conjugated to antigenic
peptides known to stimulate T cells,16 but also subsequently spaced
appropriately to allow multivalent binding to more than one
carbohydrate recognition domain of mannose receptor multimers.
Given that the exact binding requirements for the mannose
receptor are as yet unknown our initial intention was to produce
a series of fluorescently-labelled O-mannosylated peptides that
could be analysed in cellular immunology screens.
the lysine residue was deprotected and the 5(6)-carboxyfluorescein
label introduced.
Preparation of glycosyl amino acid Fmoc-[Man(OBz)4a1-]Ser
The initial synthetic strategy outlined above required access to
an Fmoc-protected a-mannosylated serine building block. A brief
screening of methods to effect mannosylation of Fmoc-protected
serine derivatives with several mannosyl donors led us to the con-
clusion that the optimum procedure involved mannosylation of
Fmoc-protected allyl serine (5) with tetra-O-benzoyl-a-D-mannose
trichloroacetimidate (3) using trimethylsilyl trifluoromethanesul-
fonate (20 mol%) in dichloromethane at −40 ◦C for 3 h affording
Fmoc-[Man(OBz)4a1-]Ser allyl ester (6) in 71% yield (Scheme 1).
Deprotection of the allyl ester using Pd(PPh3)4 and phenylsilane
in dichloromethane afforded Fmoc-[Man(OBz)4a1-]Ser (7) in 70%
yield. Protection of the serine carboxyl group as an allyl ester
proved more effective than either the use of a benzyl ester or a
pentafluorophenyl ester in that the pentafluorophenyl ester proved
more difficult to purify and degraded upon prolonged storage
whilst deprotection of a benzyl ester by hydrogenolysis proved
unreliable in our hands and the lability of the Fmoc group under
hydrogenolysis conditions has been reported.19 Use of an allyl
ester offered the advantage of its facile preparation, stability under
a variety of glycosylation conditions20 and its small size that
does not hinder the glycosylation step.21 The use of a benzoyl-
protected mannosyl donor rather than an acetate-protected man-
nosyl donor was also advantageous in that the benzoyl groups
reduced the possibility of orthoester formation.22 Mannosyl
trichloroacetimidate 3 was readily prepared via perbenzoylation of
mannose to pentabenzoate 1,23 selective hydrolysis of the anomeric
benzoate group to tetrabenzoate 224 and finally conversion to
trichloroacetimidate 325 upon treatment with trichloroacetonitrile
and potassium carbonate. Fmoc-Ser allyl ester (5) was readily
prepared by treatment of serine with Fmoc-OSu affording Fmoc-
Ser (4)26 in 97% yield followed by allylation of the ester group
to Fmoc-Ser allyl ester (5)27 in 87% yield using allyl bromide in
dichloromethane under phase transfer conditions.
Results and discussion
The most efficient and versatile method for the preparation of
O-glycopeptides employs protected glycosylated amino acids as
building blocks in a sequential assembly of peptides.1 It was
decided to establish an O-linked mannosyl linkage by incorpo-
ration of a mannosylated serine residue into an alanine based
peptide scaffold. Previous reports on the synthesis of peptides
containing O-mannosylated serines are rare17 especially given the
additional requirement for introduction of a fluorescent label.
Moreover, a comparison of the O-mannosylation of serine residues
on resin-bound assembled peptides with the incorporation of O-
mannosylated serine units into SPPS concluded that the synthesis
of glycopeptides containing O-mannosylated serine units via this
latter method is in fact the method of choice.18 A fluorescent label
could be introduced in the final stages of SPPS via attachment
of 5(6)-carboxyfluorescein to a side chain Ne-amino group of
a lysine residue that is indirectly attached to Wang resin. Thus
our initial synthetic strategy focused on solid phase glycopeptide
assembly proceeding via coupling through the Na-amino group of
the lysine residue that was attached to pre-loaded alanine Wang
resin. After SPPS that included introduction of the mannosylated
serine residue, the orthogonally Mtt-protected Ne-amino group of
Scheme 1 Reagents and conditions: (a) BzCl, py, 66%; (b) ethanolamine, THF, 54%; (c) K2CO3, Cl3CCN, CH2Cl2, 94%; (d) Fmoc-OSu, Na2CO3, dioxane,
97%; (e) CH2 CHCH2Br, NaHCO3, Aliquot 336, H2O, CH2Cl2, 24 h, 87%; (f) SiMe3OTf (20%), CH2Cl2, −40 ◦C, 3 h, 71%; (g) Pd(PPh3)4, PhSiH3,
=
CH2Cl2, 1.5 h, 70%.
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Synthesis of the mannosylated peptides: H2N-[Man(OH)4a1-]Ser-
(Ala)n-[Lys{5(6)-carboxyfluorescein}]-Ala-OH (8) (n = 3), (9)
(n = 4) and (10) (n = 6)
agent HATU in the presence of HOAt, collidine and DMAP in
DMF with a longer reaction time (overnight).
The Mtt group was removed from the Ne-amino group of the
lysine residue using 2% TFA in dichloromethane and a 5(6)-
carboxyfluorescein label was then introduced via DIC-mediated
coupling in the presence of HOBt in DMF overnight. Aminolysis
of the additional 5(6)-carboxyfluorescein molecules bound to the
phenols of the labelled peptide using six cycles of the 20% piperi-
dine deprotection protocol also effected concomitant removal
of the Fmoc group from the newly introduced mannosylated
serine residue.28 Subsequent cleavage of the mannosylated peptide
from the resin using 95% TFA with triisopropylsilane and water
as scavengers, concentration in vacuo and trituration with cold
diethyl ether gave the crude benzoate-protected glycopeptides.
These crude glycopeptides were purified by reverse-phase HPLC
before final removal of the mannose benzoate protecting groups
The synthesis of the H2N-[Man(OH)4a1-]Ser-(Ala)n-[Lys{5(6)-
carboxyfluorescein}]-Ala-OH glycopeptides 8 (n = 3), 9 (n =
4) and 10 (n = 6) was initiated by deprotection of the Fmoc
group from Fmoc-Ala-Wang resin using 20% piperidine in DMF
followed by HBTU–HOBt mediated coupling of Fmoc-Lys(Mtt)-
OH (Scheme 2) and capping of unreacted amino groups using
acetic anhydride in NMP. The required number of alanine units
(n = 3, 4 or 6) were then introduced in a similar fashion also
using HBTU–HOBt as the coupling agent with a capping step
to minimize the formation of deletion sequences. Finally the
Fmoc-[Man(OBz)4a1-]Ser residue was introduced into the series
of alanine-based glycopeptides using the more powerful coupling
Scheme 2 Reagents and conditions: (a) 20% piperidine in DMF, rt; (a*) 6 cycles of 20% piperidine in DMF; (b) Fmoc-Lys(Mtt)-OH, HBTU, HOBt,
iPr2EtN, DMF, rt, 1.5 h; (c) cat. HOBt, Ac2O, iPr2EtN, NMP, 2 × 5 min, rt; (d) Fmoc-Ala-OH coupling, HBTU, HOBt, iPr2EtN, DMF, (e) repeat steps
(d), (c), (a) twice (n = 3), three times (n = 4) or five times (n = 6); (f) Fmoc-[Man(OH)4a1-]Ser-OH, HATU, HOAt, collidine, DMAP, DMF, rt, overnight;
(g) 2% TFA in CH2Cl2, rt; (h) 5(6)-carboxyfluorescein, DIC, HOBt, DMF, rt, overnight; (i) TFA, iPr3SiH, H2O, rt, 2 h; (j) cat. NaOMe in MeOH, rt,
pH 11.7–11.8, 3 h.
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with catalytic NaOMe in MeOH. A final purification step using
reverse-phase HPLC gave glycopeptides 8, 9 and 10 in 5%, 6% and
5% overall yield respectively.
With the above ideas in mind, the synthesis of trityl-protected
5(6)-carboxyfluorescein-Lys(Dde)-Gly-Wang resin was under-
taken as outlined (Scheme 3). The procedure was based on the
work of Brock et al.28 who extended the applicability of 5(6)-
carboxyfluorescein-labelled peptides in SPPS by on-resin intro-
duction of the trityl group as a protecting group strategy for 5(6)-
carboxyfluorescein. Introduction of O-trityl protecting groups to
the polymer-bound carboxyfluorescein prevented reaction of the
hydrazine used for removal of the lysine Dde group with the enone
tautomers of the phenolic groups.28 In the present work we have
extended this protocol to the synthesis of 5(6)-carboxyfluorescein-
labelled mannosylated peptides which can be readily evaluated
using flow cell cytometry based assays.
The structure of glycopeptide 9 was confirmed by MALDI-TOF
1
analysis (observed [MH + Na]+ 1132.4115) and H NMR spec-
troscopy thus supporting the assigned structure. Similar analyses
(ESI-MS) supported the successful formation of glycopeptides 8
and 10.
Synthesis of the mannosylated peptides: 5(6)-carboxyfluorescein-
Lys-[(Ala)n-{Man(OH)4a1-}Ser]-Gly-OH (11) (n = 3), (12) (n =
4) and (13) (n = 6)
The procedure described above in which the glycopeptide chain
is elaborated through the Na-amino group of lysine was found
to be problematic as complete removal of the Ne Mtt group
proved difficult even after prolonged treatment with 2% TFA in
dichloromethane. The overall yields of the glycopeptides were also
very disappointing. Hence, a second strategy was implemented
using Dde (1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl) as the
Ne protecting group of a lysine residue. In this revised strategy
it was also decided to introduce the fluorescein label by initial
coupling of 5(6)-carboxyfluorescein through the Na-amino group
of the lysine residue attached to Gly-Wang resin (Scheme 3).28
The mannosylated peptide chain was then attached to this pre-
labelled resin (Scheme 4) via the side chain Ne-amino group of the
same lysine residue. Using this improved method three additional
glycopeptides, with three (11), four (12), and six (13) alanine
residues in the peptide chain were successfully prepared.
This revised method provides a modular approach for the
construction of libraries of fluorescently-labelled glycopeptides.
The nature of the glycosylated amino acid can be varied as can
the composition of the basic peptide framework. For example,
introduction of a mannosylated hydroxyproline unit provides
access to a peptide chain in which the mannose residue is placed
within a turn. More importantly, it was hoped that by positioning
the large fluorescein label on the Na-amino group of the resin-
bound lysine unit, with the growing mannosylated peptide chain
extending from Ne lysine side chain amino group, the overall yield
of the glycopeptides would be improved.
Thus, pre-loaded Fmoc-Gly-Wang resin was Fmoc-deprotected
and coupled to Fmoc-Lys(Dde)-OH (HBTU, HOBt in DMF).
After a capping step and Na-Fmoc deprotection, the Na-amino
group of the lysine residue was coupled to 5(6)-carboxyfluorescein
by activation with DIC and HOBt in DMF overnight. The
additional phenol-linked carboxyfluorescein was then cleaved
using 20% piperidine in DMF (six cycles) and the resultant phenols
protected as trityl ethers upon treatment with trityl chloride and
diisopropylamine in dichloromethane overnight. The trityl groups
on the fluorescein were chemically inert towards treatment with
2% hydrazine hydrate that was required for removal of the Dde-
protecting group in order to elaborate SPPS through the side chain
Ne-amino group of the lysine residue.
The synthesis of mannosylated peptides 11, 12 and 13 was then
undertaken starting from the same trityl-protected fluorescein-
labelled resin (Scheme 4). Removal of the Dde group allowed
coupling to an Fmoc-protected alanine mediated by HBTU and
HOBt. After a capping step the SPPS procedure was then repeated
two, three or five times to introduce the appropriate number of
alanine residues. Finally the mannosylated serine building block
was introduced onto the peptide chain using the more powerful
coupling agent HATU–HOAt with collidine and DMAP in DMF
overnight. Cleavage of the glycopeptide from the resin using 95%
TFA, triisopropylsilane and water at room temperature for 2 h
also effected cleavage of the trityl groups. Concentration in vacuo
and trituration with cold diethyl ether gave the crude benzoate-
protected glycopeptides that were purified by reverse-phase
Scheme 3 Reagents and conditions: (a) 20% piperidine in DMF, rt; (a*) 6 cycles of 20% piperidine in DMF; (b) Fmoc-Lys(Dde)-OH, HBTU, HOBt,
iPr2EtN, DMF, rt, 1.5 h; (c) cat. HOBt, Ac2O, iPr2EtN, NMP, 2 × 5 min, rt; (d) 5(6)-carboxyfluorescein, DIC, HOBt, DMF, rt, overnight; (e) trityl
chloride, iPr2EtN, CH2Cl2, rt, overnight.
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Scheme 4 Reagents and conditions: (a) 2% hydrazine hydrate in DMF, rt, 2 × 3 min; (b) Fmoc-Ala-OH, HBTU, HOBt, iPr2EtN, DMF, rt, 1.5 h; (c) cat.
HOBt, Ac2O, iPr2EtN, NMP, 2 × 5 min, rt; (d) 20% piperidine in DMF, rt; (e) repeat steps (b), (c), (d) twice (n = 3), three times (n = 4) or five times (n =
6), (f) Fmoc-[Man(OBz)4a1-]Ser-OH, HATU, HOAt, collidine, DMAP, DMF, rt, overnight; (g) TFA, iPr3SiH, H2O, rt, 2 h; (h) NaOMe in MeOH, rt,
pH 11.7–11.8, 3 h.
HPLC. Finally deprotection of the benzoate esters on the mannose
residue using catalytic NaOMe in MeOH and final purification by
reverse-phase HPLC gave glycopeptides 11, 12 and 13 in 33%, 15%
and 20% overall yield respectively. Pleasingly, the yields obtained
for this set of mannosylated peptides (11, 12 and 13) were thus
substantially improved compared to the yields obtained for the
previous set of mannosylated peptides (8, 9 and 10).
their ability to bind to mannose receptors on human APC subsets.
It was found that incorporation of benzoyl-protected Fmoc-
[Man(OBz)4a1-]Ser into a polyalanine peptide chain was more
facile and proceeded in better overall yield when the peptide chain
was assembled through the side chain Ne-amino of lysine rather
than through the Na-amino of the lysine residue. It is postulated
that differential positioning of the fluorescein label relative to the
growing peptide chain in the two alternative synthetic strategies
together with the associated differences in solid phase reaction
dynamics, contributes to a more efficient synthetic method. This
strategy also makes use of Fmoc-Lys(Dde) in preference to Fmoc-
Lys(Mtt) thus reducing the exposure of the acid-sensitive Wang
resin bound glycopeptide to 2% TFA in dichloromethane that is
required for removal of the Mtt group. Furthermore, prolonged
treatment with 2% TFA can result in cleavage of the peptide from
the resin.29 More significantly, adopting this improved strategy
allows the initial synthesis of a fluorescein-preloaded Wang resin
that can be used for the modular synthesis of diverse peptide
scaffolds prepared in an analogous fashion to those reported
herein.
1
The structure of glycopeptide 11 was confirmed by H NMR
spectroscopy and MALDI-TOF analysis (observed [MH +
Na]+ 1047.7072) thus supporting the assigned structure. Similar
analyses supported the successful formation of glycopeptides
12 and 13.
Conclusion
In summary two sets of fluorescein-labelled peptides containing
mannosylated serine units incorporated into peptide scaffolds with
different numbers of alanines attached to an Na-amino group
or a side chain Ne-amino group of lysine have been successfully
synthesized. These mannosylated peptides will be tested in vitro for
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recorded on a Thermo Finnigan Surveyor MSQ Plus spectrometer.
Accurate mass spectra were recorded using Matrix-assisted laser
desorption/ionisation-time of flight mass spectrometry (MALDI-
TOF MS) on a Voyager-DE spectrometer using a-cyano-4-
hydroxycinnamic acid (CHCA) as matrix. Analytical RP HPLC
was performed using a Waters 600 System using an analytical
Experimental
General
All reagents were purchased as reagent grade and used without
further purification. Solvents were dried according to standard
methods.30 Solvents for RP HPLC were purchased as HPLC
grade and used without further purification. Fmoc-Gly-Wang
resin and Fmoc-Lys(Dde) were purchased from IRIS Biotech
GmbH; Fmoc-Ala-Wang resin, Fmoc-Ala, HOBt and HBTU
were purchased from Advanced ChemTech; Fmoc-Lys(Mtt)
was purchased from Biochem; HOAt was purchased from
Acros Organics; HATU, Fmoc-Ala-TentaGel S PHB resin and
5(6)-carboxyfluorescein (isomeric mixture) were purchased from
Fluka. Analytical thin layer chromatography was performed using
0.2 mm plates of Kieselgel F254 (Merck) and compounds were
visualised by ultraviolet fluorescence or by staining with 4%
sulfuric acid in ethanol or ethanolic ninhydrin solution (0.3%
ninhydrin in ethanol + 1% v/v acetic acid), followed by heating
the plate for a few minutes. Flash chromatography was carried out
on Kieselgel F254 S 0.063–0.1 mm (Riedel de Hahn) silica gel with
indicated solvents. Infrared spectra were obtained using a Perkin
Elmer Spectrum One Fourier Transform infrared spectrometer
as a thin film between sodium chloride plates. Absorption
maxima are expressed in wavenumbers (cm−1) with the following
abbreviations: s = strong, m = medium, w = weak, br = broad
and v = varying.
˚
column (Phenomenex Jupiter C4, 300 A, 150 mm × 4.6 mm, 5 lm
˚
or Phenomenex Jupiter C18, 300 A, 150 mm × 4.6 mm, 5 lm) at a
flow rate of 1 mL min−1, using a linear gradient of 10% B to 100% B
over 35 min with detection at 254 nm; buffer A = 0.1% TFA–H2O,
buffer B = 0.1% TFA–CH3CN. Semi-preparative RP HPLC was
performed using a Waters 600 System using a semi-preparative
R
column (Waters XTerraꢀ Prep. C18, 300 mm × 19 mm, 10 lm, or
˚
Phenomenex Jupiter C4, 300 A, 250 mm × 10 mm, 5 lm) at a flow
rate of 10 mL min−1 or 13 mL min−1, using a linear gradient of
0.1% TFA–H2O (buffer A) and 0.1% TFA–CH3CN (buffer B) with
detection at 254 nm. Gradient systems were adjusted according to
the elution profiles and peak profiles obtained from the analytical
RP HPLC chromatograms.
Synthesis of mannosylated building blocks
1,2,3,4,6-Penta-O-benzoyl-a-D-mannopyranose
(1)23. D-
Mannose (1.00 g, 5.55 mmol) and a catalytic amount of DMAP
were dissolved in pyridine (12 mL). After cooling the reaction
mixture to 0 ◦C in an ice bath, benzoyl chloride (4.8 mL,
41.63 mmol) was added and the mixture was left stirring
overnight at room temperature and concentrated in vacuo. The
residue was dissolved in CH2Cl2 (ca. 150 mL) and H2O was added
carefully with cooling and vigorous stirring to decompose excess
benzoyl chloride. The product was extracted with CH2Cl2, the
organic phase was washed with brine (1×), NaHCO3(sat) (2×),
and brine (1×), dried (MgSO4) and concentrated in vacuo. The
crude product was dissolved in a mixture of acetone–methanol
(1 : 1, ca. 14 mL), and left overnight. The solution was then
diluted with methanol (7 mL), left for 2 h, further diluted with
methanol (14 mL) and left for two days at 4 ◦C. Precipitated
crystals were collected by filtration, washed with a small amount
of methanol and dried in air to give the title compound (1) as
a white solid (◦2.58 g, 66%); mp 152–153 ◦C (from methanol)
(lit.,23 152–153 C); [a]2D0 −19.4 (c 1.78, CHCl3) (lit.,23 −18.6); Rf
0.46 (ethyl acetate–hexane, 3 : 5); mmax(film)/cm−1 3064 w (Ar–H),
R
Melting points were determined on an Electrothermalꢀ melting
point apparatus and are uncorrected. Optical rotations were
determined at the sodium D line (589 nm), at 20 ◦C with a Perkin-
Elmer 341 polarimeter and are given in units of 10−1 deg cm2 g−1.
Nuclear magnetic resonance (NMR) spectra were recorded as
indicated on either a Bruker AVANCE DRX300 (1H, 300 MHz,
13C, 75 MHz), a Bruker AVANCE DRX400 spectrometer (1H,
400 MHz, 13C, 100 MHz) or on a Bruker AVANCE DRX600
spectrometer (1H, 600 MHz). Chemical shifts are reported in
parts per million (ppm) relative to the tetramethylsilane signal
recorded at dH 0.00 ppm in CDCl3–SiMe4 solvent, or the residual
methanol signal at dH 3.34 ppm in CD3OD solvent, or the residual
water signal at dH 4.79 ppm in D2O solvent. The 13C values
were referenced to the residual chloroform signal at dC 77.0 ppm
in CDCl3–SiMe4 solvent or the residual methanol signal at dC
49.15 ppm in CD3OD solvent. 1H NMR shift values are reported
as chemical shift (dH), relative integral, multiplicity (s, singlet; d,
doublet; t, triplet; q, quartet; m, multiplet; br s, broad singlet; dd,
doublet of doublets; ddt, doublet of doublet of triplets), coupling
constant (J in Hz) and assignments. 13C values are reported
as chemical shift (dC), degree of hybridisation and assignment.
Assignments were made with the aid of DEPT135, COSY, HSQC
and HMBC experiments. The ratio of 5-carboxyfluorescein and
6-carboxyfluorescein regioisomers was assumed to be 60 : 40 for
=
1728 s (C O), 1601 m, 1584 m (C–C, Ar), 1271 s (C–O), 756 s
(C–H, Ar), 713 s (C–C, Ar); dH (400 MHz; CDCl3) 4.52 (1H, dd,
JAB 12.3, J6A,5 3.6, H-6A), 4.59–4.63 (1H, m, H-5), 4.72 (1H, dd,
JAB 12.3, J6B,5 2.5, H-6B), 5.94 (1H, dd, J2,3 3.1, J2,1 2.0, H-2), 6.10
(1H, dd, J3,4 10.2, J3,2 3.1, H-3), 6.32 (1H, t, J4,3 = J4,5 10.2, H-4),
6.65 (1H, d, J1,2 1.9, H-1), 7.26–7.69 (15H, m, Ph), 7.86–8.22
(10H, m, Ph); dC (100 MHz; CDCl3) 62.3 (CH, C-6), 66.1 (CH,
C-4), 69.4 (CH, C-2), 69.9 (CH, C-3), 69.9 (CH, C-5), 91.3 (CH,
d, JC-1,H-1 178.7, C-1), 128.3, 128.4, 128.6, 128.7 (CH, Ph), 129.7,
129.9, 130.1 (quat., Ph), 133.0, 133.3, 133.5, 133.6, 134.0 (CH,
Ph), 163.7, 165.1, 165.2, 165.6, 165.9 (quat., COOBz). NMR data
were in agreement with those reported in the literature.31
1
the assignment of H NMR data. Low resolution mass spectra
were recorded on a VG-70SE mass spectrometer operating at a
nominal accelerating voltage of 70 eV (FAB). High resolution mass
spectra were recorded using a VG-70SE spectrometer at a nominal
resolution of 5000 to 10 000 as appropriate. Major and significant
fragments are quoted in the form x (y%), where x is the mass
to charge ratio and y is the percentage abundance relative to the
base peak. Electrospray ionization mass spectra (ESI-MS) were
2,3,4,6-Tetra-O-benzoyl-a-D-mannopyranose
(2). 1,2,3,4,6-
Penta-O-benzoyl-a-D-mannopyranose (1) (1.00 g, 1.43 mmol)
was dissolved in dry THF (8 mL) and ethanolamine (0.094 mL,
1.57 mmol) was added dropwise. The mixture was stirred
overnight, during which time a white precipitate appeared. The
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precipitate was filtered, the solvent was removed in vacuo and
the crude mixture was purified by flash chromatography (ethyl
acetate–hexane, 3 : 5) to yield the title compound (2) as white
foam (0.46 g, 54%); [a]D20 −79.5 (c 1.02, CHCl3) (lit.,24 −81.0); Rf
0.29 (ethyl acetate–hexane, 3 : 5); mmax(film)/cm−1 3447 s br (O–H),
was recrystallized from CH2Cl2–light petroleum (bp 60–80 ◦C) to
give the title compound (4) as a white solid (6.04 g, 97%); mp
88–◦90 ◦C (from CH2Cl2–light petroleum (bp 60–80 ◦C))(lit.,35 86–
88 C); [a]2D0 +14.7 (c 0.95, ethyl acetate) (lit.,36 +14.8); Rf 0.31
(ethyl acetate–hexane + 10% v/v acetic acid, 3 : 1); mmax(Nujol
1728 s (C O), 1601 m, 1584 m (C–C, Ar), 1267 s (C–O), 708 s
mull)/cm−1 3363 m, 3304 m (N–H, O–H), 1728 s, 1687 s (C O),
=
=
(C–C, Ar); dH (400 MHz; CDCl3) 3.50 (1H, br s, OH), 4.45 (1H,
dd, JAB 12.2, J6A,5 3.7, H-6A), 4.65–4.69 (1H, m, H-5), 4.77 (1H,
dd, JAB 12.2, J6B,5 2.6, H-6B), 5.54 (1H, d, J1,2 1.7, H-1), 5.74–5.75
(1H, m, H-2), 6.01 (1H, dd, J3,4 10.1, J3,2 3.2, H-3), 6.18 (1H, t,
J4,3 = J4,5 10.1, H-4), 7.26–7.60 (12H, m, Ph), 7.84–8.13 (8H, m,
Ph); dC (100 MHz; CDCl3) 62.7 (CH2, C-6), 66.8 (CH, C-4), 68.9
(CH, C-5), 69.7 (CH, C-3), 70.8 (CH, C-2), 92.4 (CH, d, JC-1,H-1
173.1, C-1), 128.3, 128.5, 128.6 (CH, Ph), 129.0, 129.1, 129.3,
129.8 (quat., Ph), 129.8, 133.1, 133.2, 133.5 (CH, Ph), 165.5,
165.6, 166.3 (quat., COOBz). NMR data were in agreement with
those reported in the literature.32
1261 s, 1056 s (C–O), 759 s, 737 s (C–H, Ar); dH (400 MHz; MeOD)
3.84 (1H, dd, JAB 11.3, JSerb-HA,Sera-H 4.0, Serb-HA), 3.90 (1H, dd, JAB
11.3, JSerb-HB,Sera-H 5.0, Serb-HB), 4.20 (1H, t, JCH,CH2 6.9, CH Fmoc),
4.27–4.38 (3H, m, CH2 Fmoc and Sera-H), 7.28 (2H, t, J 7.4, CH
Fmoc Ph), 7.36 (2H, t, J 7.4, CH Fmoc Ph), 7.65 (2H, d, J 6.8,
CH Fmoc Ph), 7.76 (2H, d, J 7.8, CH Fmoc Ph); dC (100 MHz;
MeOD) 48.3 (CH, Fmoc), 57.8 (CH, Sera), 63.1 (CH2, Serb), 68.2
(CH2, Fmoc), 120.9, 126.3, 128.2, 128.8 (CH, Fmoc), 142.6, 145.2,
145.3 (quat., Fmoc), 158.6 (quat., CONH), 173.8 (quat., COOH).
N-(9-Fluorenylmethoxycarbonyl)-L-serine allyl ester (5)27.
A
solution of N-(9-fluorenylmethoxycarbonyl)-L-serine (4) (2.00 g,
6.11 mmol) and sodium bicarbonate (0.52 g, 6.17 mmol) in H2O
(20 mL) was added to a solution of tricaprylmethylammonium
chloride (aliquot 336) (1.93 g, 5.99 mmol) and allyl bromide
(3.90 g, 32.23 mmol) in CH2Cl2 (38 mL). The suspension was
stirred vigorously at room temperature for 24 h under N2. H2O
(50 mL) was added to the reaction mixture and the suspension
was extracted with CH2Cl2 (3×). The combined organic layers
were dried (Na2SO4), filtered, concentrated in vacuo and the crude
product was purified by flash chromatography (ethyl acetate–
hexane, 2 : 3) to yield the title compound (5) as a white solid
2,3,4,6-Tetra-O-benzoyl-a-D-mannopyranosyl trichloroacetim-
idate (3)25. 2,3,4,6-Tetra-O-benzoyl-a-D-mannopyranose (2)
(13.86 g, 23.23 mmol) was dissolved in dry CH2Cl2 (140 mL) under
N2, and potassium carbonate (4.62 g, 63.87 mmol) was added. The
reaction mixture was stirred for 10 min and trichloroacetonitrile
(9.2 mL, 91.74 mmol) was added dropwise. The mixture was
vigorously stirred overnight under N2, filtered, concentrated
in vacuo and the crude mixture was purified by flash column
chromatography (ethyl acetate–hexane, 1 : 2) to yield the title
compound (3) as white foam (16.15 g, 94%); [a]2D0 −36.9 (c
1.3, CHCl3) (lit.,33 −37); Rf 0.66 (ethyl acetate–hexane, 1 : 2);
◦
◦
(2.24 g, 87%); mp 76–78 C (lit.37 82.5–84 C); [a]D20 +0.4 (c 7.5,
ethyl acetate) (lit.,37 +0.3), Rf 0.77 (ethyl acetate–hexane, 1 : 1);
mmax(Nujol mull)/cm−1 3448 m, 3377 m, 3303 m (N–H, O–H),
mmax(film)/cm−1 3441 m, 3337 m ( NH), 3054 w, 3032 w (Ar–H),
=
=
=
1730 s (C O), 1679 v (C N), 1601 m, 1584 m (C–C, Ar), 1265 s
(C–O), 1106 s (O–C–C), 756 s (C–H, Ar), 708 s (C–C, Ar); dH
(400 MHz; CDCl3) 4.51 (1H, dd, JAB 12.3, J6A,5 4.0, H-6A), 4.64
(1H, ddd, J5,4 10.2, J5,6A 4.0, J5,6B 2.3, H-5), 4.74 (1H, dd, JAB 12.3,
J6B,5 2.3, H-6B), 5.95 (1H, dd, J2,3 3.3, J2,1 1.9, H-2), 5.99 (1H, dd,
=
1741 s, 1675 s (C O), 1213 s, 1052 s (C–O), 763 s, 740 s (C–H,
Ar); dH (400 MHz; CDCl3) 2.75 (1H, br s, OH), 3.88–4.01 (2H,
m, Serb-H2), 4.20 (1H, t, JCH,CH2 6.9, CH Fmoc), 4.34–4.47 (3H,
=
m, CH2 Fmoc and Sera-H), 4.66 (2H, m, OCH2CH CH2), 5.23
=
J
3,4 10.2, J3,2 3.3, H-3), 6.24 (1H, t, J4,3 = J4,5 10.2, H-4), 6.58 (1H,
(1H, dd, J 10.5, JAB 1.1, OCH2CH CHAHB), 5.32 (1H, dd, J 17.2,
=
=
d, J1,2 1.9, H-1), 7.25–7.63 (12H, m, Ph), 7.83–8.11 (8H, m, Ph),
8.87 (1H, s, NH); dC (100 MHz; CDCl3) 60.3 (CH2, C-6), 66.0
(CH, C-4), 68.8 (CH, C-2), 69.8 (CH, C-3), 71.5 (CH, C-5), 90.6
JAB 1.1, OCH2CH CHAHB), 5.83–5.93 (2H, m, OCH2CH CH2
and NH), 7.29 (2H, t, J 7.4, CH Fmoc Ph), 7.38 (2H, t, J 7.4,
CH Fmoc Ph), 7.58 (2H, d, J 6.7, CH Fmoc Ph), 7.74 (2H, d,
J 7.4, CH Fmoc Ph); dC (100 MHz; CDCl3) 47.0 (CH, Fmoc),
=
(quat., CCl3C( NH)), 94.6 (CH, C-1), 128.3, 128.4, 128.6, 128.7,
=
128.8, 128.9 (CH, Ph), 129.7, 129.8, 129.9 (quat., Ph), 133.1, 133.3,
56.1 (CH, Sera), 60.4 (CH2, Serb), 66.2 (CH2, OCH2CH CH2),
=
=
133.5, 133.7 (CH, Ph), 159.8 (quat., CCl3C( NH)), 165.1, 165.3,
67.1 (CH2, Fmoc), 118.8 (CH2, OCH2CH CH2), 119.9, 125.0,
=
165.4, 166.0 (quat., COOBz). NMR data were in agreement with
127.0, 127.7 (CH, Fmoc Ph), 131.3 (CH, OCH2CH CH2), 141.2,
those reported in the literature.25
143.6, 143.7 (quat., Fmoc), 156.2 (quat., CONH), 170.2 (quat.,
COOAllyl). NMR data were in agreement with those reported in
the literature.37
N-(9-Fluorenylmethoxycarbonyl)-L-serine (4). For the prepa-
ration of N-(9-fluorenylmethoxycarbonyl)-L-serine (4), the same
procedure as for N-(9-fluorenylmethoxycarbonyl)-L-tyrosine34
was employed. L-Serine (2.00 g, 19.03 mmol) was suspended in
a mixture of 10% aqueous Na2CO3 (35 mL) and 1,4-dioxane
(15 mL) and cooled in an ice bath. N-Fluorenylmethoxycarbonyl
succinimide (6.74 g, 19.98 mmol) was dissolved in 1,4-dioxane
(25 mL) by gentle heating and the solution was added over 30 min
via a dropping funnel with efficient stirring. The reaction mixture
was stirred overnight and the solvent was removed in vacuo. The
suspension was diluted with H2O (150 mL), washed with Et2O
(2×) and the aqueous phase was acidified with citric acid to
pH 3.5 (pH paper). The aqueous phase was extracted with ethyl
acetate (3×), the organic phases were combined, dried (MgSO4),
filtered, the solvent was removed in vacuo and the crude product
N-(9-Fluorenylmethoxycarbonyl)-O-(2,3,4,6-tetra-O-benzoyl-a-
D-mannopyranosyl)-L-serine allyl ester (6). 2,3,4,6-Tetra-O-
benzoyl-a-D-mannopyranosyl trichloroacetimidate (3) (1.78 g,
2.40 mmol) and N-(9-fluorenylmethoxycarbonyl)-L-serine allyl
ester (5) (0.90 g, 2.44 mmol) were dried together under high
˚
vacuum for 8 h prior to the reaction. Activated powdered 4 A
molecular sieves were added and the mixture was stirred in dry
CH2Cl2 (10 mL) under N2 for 30 min. The suspension was cooled
to −40 ◦C and trimethylsilyl trifluoromethanesulfonate (0.09 mL,
0.48 mmol) was quickly added. The reaction mixture was stirred
under N2 for 3 h, triethylamine was added to neutralise the mixture
(pH paper) and the temperature of the solution was slowly raised to
room temperature. The mixture was filtered, the yellowish solution
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was washed with NaHCO3(sat) (2×), brine (2×), dried (Na2SO4),
filtered, the solvent was removed in vacuo and the crude product
was purified by flash chromatography with gradient elution (ethyl
acetate–hexane, 1 : 3 to 1 : 2) to yield the title compound (6) as
white foam (1.62 g, 71%); [a]2D0 −26.3 (c 1.02, CHCl3); Rf 0.59
(ethyl acetate–hexane, 2 : 3); mmax(film)/cm−1 3425 m, 3351 m (N–
906.2762), 906 (MH+, 3%), 579 (12), 178 (24), 120 (11), 105 (66)
and 89 (22).
General method for the synthesis of glycopeptides38
Glycopeptides were assembled manually using Fmoc solid phase
peptide synthesis (SPPS) using a fritted glass reaction vessel.
Pre-loaded Fmoc-Ala-Wang resin (0.7 mmol g−1) (for com-
pounds 8 and 9), pre-loaded Fmoc-Ala-TentaGel S PHB resin
(0.2 mmol g−1) (for compound 10†) or 5(6)-carboxyfluorescein-
Lys-Gly-Wang resin (0.8 mmol g−1) (for compounds 11, 12, and
13) was shaken in DMF for 30 min prior to the synthesis, followed
by filtration. Na-Fmoc protecting group was deprotected with 20%
piperidine solution in DMF (1 × 5 min, 1 × 20 min). Coupling
of Fmoc-AA was performed using method 1, coupling of Fmoc-
[Man(OBz)4a1-]Ser was performed using method 2 and coupling
of 5(6)-carboxyfluorescein was performed using method 3.
=
H), 1731 s (C O), 1601 m, 1584 m (C–C, Ar), 1266 s (C–O),
759 s, 741 s (C–H, Ar), 710 s (C–C Ar); dH (400 MHz; CDCl3)
4.18 (2H, m, Serb-H2), 4.25 (1H, t, JCH,CH2 6.9, CH Fmoc), 4.40–
4.52 (4H, m, CH2 Fmoc and H-6A and H-5), 4.69–4.85 (4H, m,
=
Sera-H and H-6B and OCH2CH CH2), 5.12 (1H, m, H-1), 5.31
=
(1H, dd, J 10.3, JAB 1.0, OCH2CH CHAHB), 5.41 (1H, dd, J 17.1,
=
J
AB 1.0, OCH2CH CHAHB), 5.69 (1H, m, H-2), 5.88 (1H, dd, J3,4
=
10.1, J3,2 3.0, H-3), 5.95–6.05 (1H, m, OCH2CH CH2), 6.09–6.16
(2H, m, H-4 and NH), 7.26–8.12 (28H, m, Ph); dC (100 MHz;
CDCl3) 47.0 (CH, Fmoc), 54.4 (CH, Sera), 62.6 (CH2, C-6), 66.5
=
(CH2, OCH2CH CH2), 66.7 (CH, C-4), 67.4 (CH2, Fmoc), 69.4
(CH, C-5), 69.6 (CH2, Serb), 69.7 (CH, H-3), 70.1 (CH, H-2),
Coupling method 1 (Fmoc-AA)
=
98.5 (CH, d, JC-1,H-1 172.8, C-1), 119.6 (CH2, OCH2CH CH2),
Na-Protected amino acid (4 eq.) was dissolved in DMF, HOBt
(4 eq.) and HBTU (3.9 eq.) were then added and shaken until
dissolved. The solution was transferred to the reaction vessel and
shaken for 2 min, followed by the addition of iPr2EtN (8 eq.). The
mixture was shaken for 1.5 h, filtered, washed with DMF (3×),
ethanol (3×) and CH2Cl2 (3×).
119.9, 125.1, 127.0, 127.6, 128.3, 128.4, 128.6, 128.8, 128.9, 129.0,
=
129.6, 129.7 (CH, Ph), 131.2 (CH, OCH2CH CH2), 133.0, 133.2,
133.4, 133.5 (quat., Ph), 141.2, 143.7 (quat., Fmoc), 156.0 (quat.,
CONH), 163.6, 165.2, 165.4, 166.1 (quat., COOBz), 169.4 (quat.,
COOAllyl); m/z (FAB) 946.3080 (MH+, C55H48NO14 requires
946.3075), 946 (MH+, 3%), 579 (18), 178 (39), 154 (60), 136 (43)
120 (8), 105(100) and 89 (14).
Coupling method 2 (Fmoc-[Man(OBz)4a1-]Ser)
N-(9-Fluorenylmethoxycarbonyl)-O-(2,3,4,6-tetra-O-benzoyl-a-
Fmoc-[Man(OBz)4a1-]Ser (7) (1.5 eq.) was dissolved in NMP,
HOAt (1.5 eq.) and HATU (1.45 eq.) were then added and shaken
until dissolved. The solution was transferred to the reaction vessel
andshakenfor 2min, followedbythe additionofcollidine(4.5 eq.).
The mixture was shaken overnight, filtered, washed with DMF
(3×), ethanol (3×) and CH2Cl2 (3×).
D-mannopyranosyl)-L-serine
(7). N-(9-Fluorenylmethoxycar-
bonyl)-O-(2,3,4,6-tetra-O-benzoyl-a-D-mannopyranosyl)-L-serine
allyl ester (6) (1.53 g, 1.62 mmol) was dissolved in CH2Cl2 (15 mL)
and the mixture was degassed under an argon atmosphere.
Pd(PPh3)4 (0.08 g, 0.06 mmol) was added and argon was bubbled
through the yellowish solution for 10 min. PhSiH3 (0.8 mL,
6.48 mmol) was then added and the mixture was stirred under
argon for 1.5 h during which time the colour of the mixture
turned black. Upon completion of the reaction (TLC, ethyl
acetate–hexane, 2 : 1 + 10% v/v acetic acid) the solvent was
removed in vacuo and the crude product was purified twice by
flash chromatography (CH2Cl2–methanol, 9 : 1). The resultant
brown solid was lyophilised from tert-butanol to yield the title
compound (7) as a pale yellow amorphous powder (1.03 g, 70%);
[a]2D0 −11.7 (c 1.44, CHCl3); Rf 0.29 (ethyl acetate–hexane, 1 : 1 +
10% v/v acetic acid); mmax(film)/cm−1 3427 m, 3348 m (N–H),
Coupling method 3 (5(6)-carboxyfluorescein)
5(6)-Carboxyfluorescein (2.5 eq.) was dissolved in DMF. HOBt
(2.5 eq.) was added, the mixture was shaken until dissolved and
the solution was transferred to the reaction vessel which was then
shaken for 2 min, followed by the addition of DIC (2.5 eq.).
The mixture was shaken overnight, filtered, washed with DMF
(3×) and treated with 20% piperidine solution in DMF 6 times
(1 × 5 min, 1 × 20 min) to remove additional ester-bound 5(6)-
carboxyfluorescein, washed with DMF (3×), ethanol (3×) and
CH2Cl2 (3×).
=
3300–2500 br (O–H), 2958 s, 2898 s (C–H), 1728 s (C O), 1601
m, 1584 m (C–C, Ar), 1515 w (N–H), 1451 m (C–O–H), 1265 s
(C–O), 1109 s (O–C–C), 1095 s (C–O–C), 759 s, 742 s (C–H,
Ar); dH (400 MHz; CDCl3) 4.17–4.29 (3H, m, CH Fmoc and
Serb-H2), 4.34–4.38 (2H, m, CH2 Fmoc), 4.45–4.55 (2H, m, H-5
and H-6A), 4.65–4.73 (2H, m, H-6B and Sera-H), 5.17 (1H, m,
H-1), 5.74 (1H, m, H-2), 5.99 (1H, dd, J3,4 9.9, J3,2 2.7, H-3), 6.13
(1H, t, J4,3 = J4,5 9.9, H-4), 6.56 (1H, br s, NH), 7.20–8.08 (28H,
m, Ph); dC (100 MHz; CDCl3) 47.0 (CH, Fmoc), 54.6 (CH, Sera),
62.7 (CH2, C-6), 66.6 (CH, C-4), 67.3 (CH2, Fmoc), 69.1 (CH2,
Serb), 69.3 (CH, C-5), 70.3 (CH, C-2 and C-3), 98.2 (CH, C-1),
119.8, 125.1, 127.5, 128.3, 128.4, 128.5, 128.6, 128.7, 129.0, 129.7
(CH, Ph), 132.0, 132.2 (quat., Ph), 141.1, 143.8 (quat., Fmoc),
156.2 (quat., CONH), 165.4, 166.0, 166.1 (quat., COOBz), 171.3
(quat., COOH); m/z (FAB) 906.2792 (MH+, C52H44NO14 requires
Unreacted amino groups were capped after each coupling step
by treatment of the resin with Ac2O (0.5 M in NMP), iPr2EtN
(0.125 M in NMP) and a catalytic quantity of HOBt in NMP. For
removal of the Mtt group from the Ne-amino group of the lysine
residue the glycopeptidyl resin (compounds 8, 9 and 10) was
treated with 2% TFA in CH2Cl2. The mixture was washed with
2% TFA in CH2Cl2 (5×), shaken with 2% TFA in CH2Cl2 for
5 min, filtered, and the cycle was repeated (3×), followed by
washing with CH2Cl2 (5×). The procedure was repeated until the
yellow colour of the filtrate almost disappeared (12×). Removal
† Synthesis of glycopeptide 10 using Fmoc-Ala-Wang (loading
0.66 mmol g−1) was unsuccessful hence Fmoc-Ala-TentaGel S PHB resin
(0.2 mmol g−1) was used.
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of the Dde protecting group from the Ne-amino group of the
lysine residue (for compounds 11, 12 and 13) was performed using
2% hydrazine hydrate solution in DMF (2 × 3 min). The Kaiser
test39 was performed after each coupling and deprotection step.
Cleavage of the resin and simultaneous removal of the protecting
groups from the glycopeptides was achieved by treatment of the
glycopeptidyl resin with 95 : 2.5 : 2.5 (v/v/v) TFA : TIS : H2O
for 2 h. Glycopeptides were precipitated from cold Et2O, isolated
by centrifuge, lyophilised from CH3CN–H2O and purified by RP
HPLC prior to final removal of the mannose benzoate protecting
groups.
flow rate of 10 mL min−1, using a linear gradient of 10% B to
100% B over 35 min. Removal of benzoate protecting groups of
the mannose moiety, followed by purification by RP HPLC on
Phenomenex Jupiter C4 column, at a flow rate of 10 mL min−1,
using a linear gradient of 10% B to 100% B over 35 min, yielded
glycopeptide 10 as a yellow amorphous solid (2.72 mg, 5%); Rt
13.40 min (Phenomenex Jupiter C18); m/z (ESI-MS) 1251.5 ([M +
H]+ requires 1251.5).
5(6)-Carboxyfluorescein(Trt)2-Lys(Dde)-Gly-Wang resin28
Fmoc-Gly-Wang resin (0.8 mmol g−1) was shaken in DMF for
30 min prior to the synthesis. The Na-Fmoc protecting group was
removed using 20% piperidine solution in DMF (1 × 5 min, 1 ×
20 min) and 5(6)-carboxyfluorescein was coupled using method
3 as described above, washed with DMF (3×), CH2Cl2 (5×)
and dried under a flow of N2. Trityl chloride (12 eq.) was then
added, followed by addition of CH2Cl2 and iPr2EtN (12 eq.),
the reaction mixture was shaken overnight, filtered, washed with
CH2Cl2 (5×), dried under a flow of N2 and a fresh portion of the
tritylation reagents was added and the procedure was repeated
to yield 5(6)-carboxyfluorescein(Trt)2-Lys(Dde)-Gly-Wang resin
which was used for further synthesis (compounds 11, 12 and 13).
A sample of the trityl-protected fluorescein-labelled resin (25.0 mg)
was Dde deprotected using 2% hydrazine hydrate solution in DMF
(2 × 3 min), cleaved using 95 : 2.5 : 2.5 (v/v/v) TFA : TIS : H2O
for 2 h, precipitated from cold Et2O, isolated by centrifuge and
lyophilised from CH3CN–H2O to yield 5(6)-carboxyfluorescein-
Lys-Gly-OH; Rt 13.08 min (Phenomenex Jupiter C18); m/z (ESI-
MS) 562.3 ([M + H]+ requires 562.2).
Removal of benzoate protecting groups
The benzoate groups were removed by dissolution of the glycopep-
tides in methanol (3 mL) and treatment with 1 M NaOMe solution
in methanol in pH 11.7–11.8 for 3 h. When all starting material
had disappeared as judged by analytical RP HPLC, the solution
was neutralised with a portion of dry ice, the solvent was removed
in vacuo, and the crude product was purified by RP HPLC and
lyophilised from CH3CN–H2O.
H2N-[Man(OH)4a1-]Ser-(Ala)3-[Lys{5(6)-carboxyfluorescein}]-
Ala-OH (8). Using the standard peptide synthesis procedure
as outlined above, the crude benzoyl-protected glycopeptide was
R
purified by RP HPLC on a Waters XTerraꢀ Prep. C18 column at
a flow rate of 13 mL min−1, using a linear gradient of 10% B to
100% B over 35 min. Removal of benzoate protecting groups of
the mannose moiety, followed by purification by RP HPLC on a
Phenomenex Jupiter C4 column, at a flow rate of 10 mL min−1,
using a linear gradient of 10% B to 100% B over 35 min, yielded
glycopeptide 8 as a yellow amorphous solid (3.20 mg, 5%); Rt
14.54 min (Phenomenex Jupiter C18); m/z (ESI-MS) 1038.4 ([M +
H]+ requires 1038.4).
5(6)-Carboxyfluorescein-Lys[(Ala)3 -{Man(OH)4a1-}Ser]-Gly-
OH (11). Using the standard peptide synthesis procedure as
outlined above, crude benzoyl-protected glycopeptide was purified
R
by RP HPLC on a Waters XTerraꢀ Prep. C18 column at a flow
H2N-[Man(OH)4a1-]Ser-(Ala)4-[Lys{5(6)-carboxyfluorescein}]-
Ala-OH (9). Using the standard peptide synthesis procedure
as outlined above, the crude benzoyl-protected glycopeptide was
purified by RP HPLC on a Phenomenex Jupiter C4 column, at
a flow rate of 10 mL min−1, using a linear gradient of 10% B to
100% B over 35 min. Removal of benzoate protecting groups of
the mannose moiety, followed by purification by RP HPLC on a
Phenomenex Jupiter C4 column, at a flow rate of 10 mL min−1,
using a linear gradient of 10% B to 100% B over 35 min, yielded
glycopeptide 9 as a yellow amorphous solid (5.22 mg, 6%);
Rt 14.40 min (Phenomenex Jupiter C4); dH (600 MHz; D2O)
1.19–1.28 (15H, m, 5 × Alab-H3), 1.41–1.89 (6H, m, 3 × Lys-H2),
3.11–3.41 (2H, m, Lys-H2), 3.49–3.55 (2H, m, H-5 and H-4),
3.62–3.70 (2H, m, H-3 and H-6A), 3.75–3.83 (3.2H, m, Serb-H2
and H-6B and H-2), 4.05–4.28 (7.8H, m, Sera-H and Serb-H2 and
5 × Alaa-H and Lysa-H), 4.73–4.84 (H-1 signal obscured by HOD
signal), 6.67–6.69 (2H, m, CH Fluoro Ph), 6.81 (2H, br s, CH
Fluoro Ph), 6.95–6.96 (2H, m, CH Fluoro Ph), 7.27–7.28 (0.6H,
m, CH Fluoro Ph), 7.47 (0.4H, br s, CH Fluoro Ph), 7.92–8.02
(1.6H, m, CH Fluoro Ph), 8.27 (0.4H, br s, CH Fluoro Ph); m/z
(MALDI-TOF) 1132.4115 ([MH + Na]+ requires 1132.4203).
rate of 10 mL min−1, using a linear gradient of 10% B to 100%
B over 35 min. Removal of benzoate protecting groups of the
mannose moiety, followed by purification by RP HPLC on a
R
Waters XTerraꢀ Prep. C18 column at a flow rate of 10 mL min−1,
using a linear gradient of 1% B to 50% B over 35 min, yielded
glycopeptide 11 as a yellow amorphous solid (5.75 mg, 33%);
Rt 13.45 min (Phenomenex Jupiter C18); dH (600 MHz; MeOD)
1.30–1.38 (9H, m, 3 × Alab-H3), 1.39–1.62 (4H, m, 2 × Lys-H2),
1.83–2.01 (2H, m, Lys-H2), 3.14–3.16 (1.2H, m, Lys-H2), 3.21–
3.23 (0.8H, m, Lys-H2), 3.50–3.54 (1H, m, H-5), 3.58–3.61 (1H,
m, H-4), 3.67–3.72 (2H, m, H-3 and H-6A), 3.77–3.81 (1.2H, m,
Serb-H2), 3.82–4.03 (4H, m, H-6B and H-2 and Glya-H2), 4.12–
4.16 (2.8H, m, Serb-H2 and Sera-H and Alaa-H), 4.23–4.29 (1H,
m, Alaa-H), 4.38–4.44 (1H, m, Alaa-H), 4.53–4.56 (0.6H, m, Lysa-
H), 4.65–4.68 (0.4H, m, Lysa-H), 4.82 (1H, br s, H-1), 6.54–6.57
(2H, m, CH Fluoro Ph), 6.59–6.63 (2H, m, CH Fluoro Ph), 6.67–
6.70 (2H, m, CH Fluoro Ph), 7.30–7.32 (0.6H, m, CH Fluoro
Ph), 7.72 (0.4H, br s, CH Fluoro Ph), 8.08–8.26 (1.6H, m, CH
Fluoro Ph), 8.53 (0.4H, br s, CH Fluoro Ph); m/z (MALDI-TOF)
1047.7072 ([MH + Na]+ requires 1047.3674).
H2N-[Man(OH)4a1-]Ser-(Ala)6-[Lys{5(6)-carboxyfluorescein}]-
Ala-OH (10). Using the standard peptide synthesis procedure
as outlined above, crude benzoyl-protected glycopeptide was
purified by RP HPLC on Phenomenex Jupiter C4 column, at a
5(6)-Carboxyfluorescein-Lys[(Ala)4-{Man(OH)4a1-}Ser]-Gly-
OH (12). Using the standard peptide synthesis procedure as
outlined above, crude benzoyl-protected glycopeptide was purified
R
by RP HPLC on a Waters XTerraꢀ Prep. C18 column at a flow
120 | Org. Biomol. Chem., 2008, 6, 112–121
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rate of 10 mL min−1, using a linear gradient of 10% B to 100%
B over 35 min. Removal of benzoate protecting groups of the
mannose moiety, followed by purification by RP HPLC on a
2 S. Kaech, J. Wherry and R. Ahmed, Nat. Rev. Immunol., 2002, 2, 251.
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R
Waters XTerraꢀ Prep. C18 column at a flow rate of 10 mL min−1,
using a linear gradient of 1% B to 50% B over 35 min, yielded
glycopeptide 12 as a yellow amorphous solid (3.74 mg, 15%); Rt
11.80 min (Phenomenex Jupiter C18); dH (400 MHz; D2O) 1.26–
1.42 (12H, m, 4 × Alab-H3), 1.53–2.04 (6H, m, 3 × Lys-H2), 3.09–
3.32 (2H, m, Lys-H2), 3.67–3.71 (2H, m, H-5 and H-4), 3.77–3.86
(2H, m, H-3 and H-6A), 3.89–4.08 (5.2H, m, Serb-H2 and H-6B
and H-2 and Glya-H2), 4.21–4.30 (3.8H, m, 2 × Alaa-H and Sera-
H and Serb-H2), 4.33–4.34 (1H, m, Alaa-H), 4.39–4.44 (1H, m,
Alaa-H), 4.48–4.52 (0.6H, m, Lys-Ha), 4.64–4.67 (0.4H, m, Lys-
Ha), 4.91 (1H, br s, 1 × H-1), 6.72–6.76 (2H, m, CH Fluoro Ph),
6.86 (2H, br s, CH Fluoro Ph), 6.93–7.00 (2H, m, CH Fluoro Ph),
7.31–7.33 (0.6H, m, CH Fluoro Ph), 7.67 (0.4H, br s, CH Fluoro
Ph), 8.13–8.22 (1.6H, m, CH Fluoro Ph), 8.51 (0.4H, br s, CH
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1094.4080).
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5(6)-Carboxyfluorescein-Lys[(Ala)6-{Man(OH)4a1-}Ser]-Gly-
OH (13). Using the standard peptide synthesis procedure as
outlined above, crude benzoyl-protected glycopeptide was purified
R
by RP HPLC on a Waters XTerraꢀ Prep. C18 column at a flow
rate of 10 mL min−1, using a linear gradient of 10% B to 100%
B over 35 min. Removal of benzoate protecting groups of the
mannose moiety, followed by purification by RP HPLC on a
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R
Waters XTerraꢀ Prep. C18 column at a flow rate of 10 mL min−1,
using a linear gradient of 1% B to 50% B over 35 min, yielded
glycopeptide 13 as a yellow amorphous solid (15.9 mg, 20%); Rt
13.68 min (Phenomenex Jupiter C18); dH (600 MHz; D2O) 1.09–
1.36 (18H, m, 6 × Alab-H3), 1.36–1.89 (6H, m, 3 × Lys-H2), 3.03–
3.18 (2H, m, Lys-H2), 3.51–3.56 (2H, m, H-5 and H-4), 3.61–3.66
(2H, m, H-3 and H-6A), 3.69–3.72 (1.2H, m, Serb-H2), 3.74–3.84
(2H, m, CH-6B and H-2), 3.90–3.97 (2H, m, Glya-H2), 4.04–4.39
(7.8H, m, Serb-H2 and Sera-H and 6 × Alaa-H), 4.46–4.54 (1H,
m, Lysa-H), 4.84 (1H, br s, H-1), 6.54–6.58 (2H, m, CH Fluoro
Ph), 6.67 (2H, br s, CH Fluoro Ph), 6.75–6.80 (2H, m, CH Fluoro
Ph), 7.13–7.15 (0.6H, m, CH Fluoro Ph), 7.52 (0.4H, br s, CH
Fluoro Ph), 7.97–8.03 (1.6H, m, CH Fluoro Ph), 8.34 (0.4H, br s,
CH Fluoro Ph); m/z (MALDI-TOF) 1298.8071 ([M + Na + K]+
requires 1298.4347).
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Acknowledgements
We thank the Maurice Wilkins Centre for Molecular Biodiscovery
for the award of a Doctoral Scholarship to RK.
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References
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tion, Topics in Current Chemistry, ed. V. Wittmann, Springer-Verlag,
Berlin and Heidelberg, 2007, vol. 267.
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The Royal Society of Chemistry 2008
Org. Biomol. Chem., 2008, 6, 112–121 | 121
©