Development of highly pure α-helical lipoglycopeptides as self-adjuvanting vaccines

Article abstract of DOI:10.1016/j.tet.2009.02.060

The incorporation of lipid moieties into synthetic peptide vaccines has been demonstrated to self-adjuvant otherwise poorly immunogenic peptides, whereas carbohydrates have emerged to be advantageous carriers for assembling these peptides. With the advent

Full text of DOI:10.1016/j.tet.2009.02.060

Tetrahedron 65 (2009) 3459–3464
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Development of highly pure
as self-adjuvanting vaccines
a-helical lipoglycopeptides
,
Wei Zhong a, Mariusz Skwarczynski a, Pavla Simerska a, Michael F. Good b, Istvan Toth a
*
a The University of Queensland, School of Chemistry and Molecular Biosciences, St. Lucia, Qld 4072, Australia
b Queensland Institute of Medical Research, Herston, Qld 4029, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
The incorporation of lipid moieties into synthetic peptide vaccines has been demonstrated to self-adjuvant
otherwise poorly immunogenic peptides, whereas carbohydrates have emerged to be advantageous car-
riers for assembling these peptides. With the advent of an efficient native chemical ligation method, which
is compatible with both peptides and carbohydrates, we have developed highly pure self-adjuvanting
tetravalent group A streptococcal vaccine candidates assembled on carbohydrate templates. The utility of
chemoselective ligation has overcome difficulties in the synthesis and purification of branched high
Received 25 November 2008
Received in revised form 27 January 2009
Accepted 13 February 2009
Available online 3 March 2009
molecular weight peptides. Circular dichroism measurements provided the evidence of
of the assembled peptide epitopes, which may have impact on their immunogenicity.
a-helix formation
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
vaccines, which have emerged as a potential approach for human
vaccination.2,3 The LCP delivery system features a non-microbial
lipopeptide adjuvant (synthetic lipidic amino acids and glycine
spacers) and a polylysine-based multiple antigenic peptide (MAP)8
dendrimer. The physiochemical properties of the LCP system may
be readily altered to improve vaccine efficacy. This includes
changing alkyl side-chain length or the number of lipoamino acids,
conjugation of same or different epitopes, or the use of different
carriers. One of the de novo designs of peptide immunogens in-
volves the application of a topological template predetermining
tertiary structure of the conjugated peptide epitopes. Carbohy-
drates have emerged as suitable templates9–12 due to (i) the mul-
tiple hydroxyl groups allowing for the attachment of several copies
of an epitope or possibly different epitopes, (ii) the rigid ring
directing antigens in a three-dimensional space, and (iii) the ready
availability of different carbohydrate entities facilitating an optimal
orientation for antigen recognition. Thus, adaptation of the carbo-
hydrate core in the LCP system, to produce conformation-defined
constructs, may have important implications for the development
of synthetic vaccines.
The LCP systemwasemployed to develop numerous experimental
vaccine candidates against group A streptococcus (GAS), which is
believed to be responsible for a range of common health problems
(e.g., sore throat) and even fatal diseases (e.g., rheumatic fever).13
Previous studies have shownthat LCP vaccine candidates synthesized
using stepwise solid phase peptide synthesis (SPPS) generally elicited
high serum IgG antibody titers against the incorporated epitope(s)
without the need for additional adjuvants.14–17 To produce highly
pure polylysine-based LCP vaccines, native chemical ligation (NCL)
has been successfully used.18–21
Vaccination, which has the potential to eradicate diseases, has
proved to be one of the most cost-effective medical interventions.
Problems associated with conventional vaccines, such as instability,
risks of infection, and autoimmunity, as well as manufacturing
difficulties, have called for the development of safe and effective
synthetic vaccines that contain minimal and optimal antigenic
determinant(s) rather than the whole pathogen. The major limi-
tation to the development of such vaccines has been the poor im-
munogenicity and the consequent necessity of co-administration
with effective adjuvants.1,2 Many powerful experimental adjuvants,
such as complete Freund’s adjuvant, are toxic and not suitable for
human use. Furthermore, in most cases, antigen fragments need to
be conjugated to or mixed with protein carriers to adopt T-helper
epitopes, which are necessary for stimulating immune responses in
outbreed human populations.1,3 Commonly used protein carriers
(e.g., bovine serum albumin, keyhole limpet hemocyanin, and tet-
anus toxoid) present significant problems including autoreactivity
and epitopic suppression.4–6 These problems have resulted in sig-
nificant interest in the development of potent and safe adjuvants to
overcome the very limited choice of adjuvant for human vaccina-
tion (mainly aluminum-based), and inert small molecular carriers
to minimize unfavorable effects on epitopic specificity.
A promising ‘one entity’ strategy, lipid core peptide (LCP) sys-
tem,7 has been developed based on the principle of lipopeptide
* Corresponding author. Tel.: þ61 7 3346 9892; fax: þ61 7 3365 1688.
E-mail address: i.toth@uq.edu.au (I. Toth).
0040-4020/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2009.02.060

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W. Zhong et al. / Tetrahedron 65 (2009) 3459–3464
In the current study, NCL was utilized to prepare highly pure
carbohydrate-based LCP vaccine candidates. To possibly optimize
the vaccine efficacy by altering the carbohydrate types, three dif-
CN
CN
OH
OH
O
O
O
O
acrylonitrile
DBU
HO
HO
NC
NC
O
N3
N3
O
ferent monosaccharides (D-glucose, D-galactose, and D-mannose)
1a
2a (37%)
were functionalized, incorporated to a lipopeptide, and conjugated
with four copies of B-cell peptide epitopes. In this project, a GAS
peptide epitope derived from the N-terminal region of the major
virulence factor (M protein) of GAS 8830 strain was selected as
a model epitope. However, other antigens can be incorporated into
this carbohydrate LCP system to produce vaccine candidates
against other diseases. Comparison of conformational properties of
these carbohydrate-based LCP vaccines was carried out by means of
circular dichroism (CD) spectroscopy.
CN
CN
O
O
O
O
i.H2/Pd/C
NC
H
O
N
O
OBn
ii.adipic acid mono-
benzyl ester
NC
O
3a (53%)
HBTU, DIPEA
NHBoc
NHBoc
O
NaBH4
O
O
CoCl2
O
H
N
BocHN
BocHN
O
(Boc)2O
O
OBn
O
4a (30%)
2. Results and discussion
2.1. Synthetic strategy
NHBoc
NHBoc
O
O
H2/Pd/C
O
O
H
BocHN
BocHN
O
N
O
OH
O
The designed vaccine candidates, which individually incorporate
a lipopeptide (C12–G–C12–C12–G; C12: 2-(R/S)-aminododecanoic
acid22), a carbohydrate template, and four copies of 8830 peptide
antigen (DNGKAIYERARERALQELGP),23 were synthesized using
NCL, which allows the branched lipopeptide–carbohydrate–antigen
to be generated from two building blocks by a single ligation re-
action. NCL is a chemoselective technique of coupling a C-terminal
peptide thioester and an N-terminal cysteine residue at physiolog-
ical pH in the presence of an added thiol catalyst. As there is no
cysteine residue in the 8830 sequence, the carbohydrate templates
were individually equipped with four N-terminal cysteine residues
and the 8830 peptide was synthesized as a thioester.
5a (83%)
Scheme 1. Synthesis of carbohydrate template 5a.
using standard hydrofluoric acid (HF)/scavenger conditions. Fol-
lowing purification by preparative RP-HPLC, 7a, 7b, and 7c were
obtained in 21%, 36%, and 24% yield, respectively.
2.3. Chemical ligation
Given the high hydrophobicity of the Cys-containing lip-
opeptide–carbohydrate templates and the high hydrophilicity of
the peptide thioester, reaction conditions needed to be identified
under which both building blocks could be maintained in the li-
gation buffer throughout the course of reaction, thus allowing an
efficient NCL. Published successful conditions involve the addition
of organic co-solvents,30–32 liposomes,33 detergents,34 and lipid
bilayers.35 However, little success was reported for the ligations of
hydrophobic lipopeptide constructs and hydrophilic peptide thio-
esters when organic co-solvents such as isopropyl alcohol, aceto-
nitrile (ACN), dimethyl sulfoxide (DMSO), N,N-dimethylformamide
(DMF), trifluoroethanol or dioxane were used.18,19,21 The failure of
those reactions was most likely due to the very poor water solu-
bility and long lipid chains of the lipopeptide, which may have
hampered the Cys residue from reacting with the thioester. In
contrast, the use of sodium dodecyl sulfate (SDS) was demonstrated
to be a successful alternative,18,19,21 presumably due to the lipid part
entering SDS micelles, and leaving the Cys residue faced toward the
aqueous solution. It was envisaged that the presence of SDS would
facilitate ligation of the lipopeptide–carbohydrate templates and
the thioester. Thus, peptide 7a, 7b or 7c in an SDS aqueous solution
was lyophilized and redissolved in 0.1 M phosphate buffer (pH 7.6),
with tris(2-carboxyethyl)phosphine (TCEP) added to suppress
disulfide formation. Peptide thioester 820 was assembled by mi-
crowave-assisted SPPS using Boc-protected amino acids on
p-MBHA resin. Thioester 8 was dissolved in 0.1 M phosphate buffer
(pH 7.6) and pre-activated for 1 h using 2-mercaptoethane sulfo-
nate (MESNA) to give 8a. This solution was added to the phosphate
buffer containing 7a, 7b or 7c and the mixture was incubated at
37 ꢀC (Scheme 2). The ligation was monitored by HPLC and initially
peaks corresponding to partially substituted lipopeptide–carbo-
hydrates were observed, followed by the appearance of a peak with
a lower retention time corresponding to the tetra-substituted
compound. The three vaccine candidates 9a, 9b, and 9c were
obtained in high yields (71%, 76%, and 72%, respectively), with pu-
rity and identity confirmed by RP-HPLC and ESI-MS (see Fig. 1 and
Experimental section).
2.2. Synthesis of Cys-containing lipopeptide–carbohydrate
templates
b-D
-Mannopyranosyl azide 1a24 was cyanoethylated with acry-
lonitrile in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene
(DBU) to give tetranitrile 2a in 37% yield. Reduction of the azido
group of 2a was achieved by catalytic hydrogenation, and the cor-
responding amine was reacted directly with adipic acid monobenzyl
ester25 using O-benzotriazole-N,N,N0,N0-tetramethyluronium hex-
afluorophosphate (HBTU)/N,N-diisopropylethylamine (DIPEA) ac-
tivation method to afford 3a in 53% overall yield. The nitrile groups
of 3a were reduced to primary amines using sodium borohydride
and cobalt(II) chloride26 and in situ converted to Boc-protected
amines in the presence of di-tert-butyl dicarbonate (Boc2O), giving
compound 4a in 30% yield. Following removal of the benzyl group
by catalytic hydrogenation, compound 5a was obtained in 83% yield
(Scheme 1). The glucose derivative 5b and the galactose derivative
5c were synthesized according to reported procedures.13,27
The conjugation of lipopeptide and cysteines to compound 5a,
5b or 5c is shown in Scheme 2. Glycine and aminododecanoic acid
(C12) residues were sequentially (C12–G–C12–C12–G) attached to
p-methylbenzhydrylamine (p-MBHA) resin using Boc-based SPPS
according to previously reported procedures.28 To improve the
coupling efficiency, either the bulky lipoamino acids were pre-ac-
tivated for 5 min or the reaction was performed in the presence of
N-methyl pyrrolidinone. To minimize the consumption of the
synthesized carbohydrate templates, a modified version of in situ
neutralization protocol29 was utilized in the coupling of 5a, 5b or 5c
to the C12–G–C12–C12–G–resin. This modification involved using
2 equiv (instead of 4 equiv) of the acid (5a, 5b or 5c) and a longer
coupling time (6 h instead of 10 min). The obtained peptidyl resin
6a, 6b or 6c was Boc-deprotected using trifluoroacetic acid (TFA)
and four copies of the Cys residue were coupled to the amine
moieties of the compound to give resin-bound 7a, 7b, and 7c.
Cleavage of peptide 7a, 7b, and 7c from the resin was accomplished

W. Zhong et al. / Tetrahedron 65 (2009) 3459–3464
3461
NHBoc
O
R1
R4
O
H
N
O
R2
BocHN
O
OH
R3
O
5a: R1 = R3 = H; R2 = R4 = O(CH2)3NHBoc
5b: R1 = R4 = H; R2 = R3 = O(CH2)3NHBoc
5c: R1 = R3 = O(CH2)3NHBoc; R2 = R4 = H
pMBHA resin
Boc-Gly-OH
Boc-C12-OH
SPPS
NHBoc
O
R1
R4
O
O
H
N
O
H
N
O
H
R2
N
NH
BocHN
O
N
H
N
H
N
H
R3
O
O
O
O
6a: R1 = R3 = H; R2 = R4 = O(CH2)3NHBoc
6b: R1 = R4 = H; R2 = R3 = O(CH2)3NHBoc
6c: R1 = R3 = O(CH2)3NHBoc; R2 = R4 = H
i. Boc-Cys(MBzl)-OH ii. HF cleavage
SPPS
NH-Cys
O
R1
R4
O
O
H
O
H
N
O
H
N
R2
N
NH2
Cys-HN
O
N
N
H
N
R3
H
O
O
H
O
O
7a: R1 = R3 = H; R2 = R4 = O(CH2)3NHCys (21%)
7b: R1 = R4 = H; R2 = R3 = O(CH2)3NHCys (36%
7c: R1 = R3 = O(CH2)3NHCys; R2 = R4 = H (24%)
Ac-[8830]-Gly
MESNA
S
Leu NH2
8
O
0.1 M phosphate buffer
pH 7.6
0.1 M phosphate buffer pH 7.6
1% (w/v) SDS, TCEP, 37 °C
O
O
O
Na
S
Ac-[8830]-Gly
S
8a
NH-Cys-Gly-[8830]-Ac
O
R1
R4
O
O
H
O
H
N
O
H
N
R2
N
NH2
Ac-[8830]-Gly-Cys-HN
O
N
H
N
H
N
R3
O
O
H
O
O
9a: R1 = R3 = H; R2 = R4 = O(CH2)3NHCysGly[8830]Ac (71%)
9b: R1 = R4 = H; R2 = R3 = O(CH2)3NHCysGly[8830]Ac (76%)
9c: R1 = R3 = O(CH2)3NHCysGly[8830]Ac; R2 = R4 = H (72%)
8830: DNGKAIYERARERALQELGP
Scheme 2. Synthesis of lipopeptide–carbohydrate template assembled peptides 9a, 9b, and 9c.
2.4. Conformational studies
Our interest was in investigating whether the formation of 4-
a
-
helix bundles of the 8830 antigen could have impact on its immu-
nogenicity. Therefore, the helical characteristics of vaccine
candidates 9a, 9b, and 9c were studied by CD measurements. Helix-
inducing features of carbohydrate template assembled peptides
have been previously reported,9,10,12 as such, similar carbohydrate
influence on peptide topology was envisaged in our study. Indeed,
spectra typical for a-helices were observed for 9a, 9b, and 9c (in
phosphate buffer, pH 7.3), showing double minima at 208 nm and
222 nm (Fig. 2). The free 8830 peptide 1020 was found to exist in
phosphate buffer (pH 7.3) in an almost random structure (Fig. 2),
demonstrating that the helical character of the attached peptides is
Figure 1. RP-HPLC and ESI-MS spectra of 9b.

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W. Zhong et al. / Tetrahedron 65 (2009) 3459–3464
d6 at room temperature and were internally referenced to CHCl3 (dH
7.24 and dC 77.0) or DMSO (dH 2.49 and dC 39.5). Positive low res-
olution ES-MS (LRESMS) was performed on either a Perkin Elmer-
Sciex API 3000 instrument (Applied Biosystems, Foster city, CA,
USA) or an API QSTAR pulsar i ES-MS (Applied Biosystems, Foster
city, CA, USA) equipped with Agilent 1200 HPLC (Waldbronn, Ger-
many). High resolution ES-MS (HRESMS) was performed on
a MicroTof Q instrument (Bruker Daltonics, Bremen, Germany).
Analytical RP-HPLC was performed using Shimadzu (Tokyo, Japan)
instrumentation (Class Vp 6.12 software, SCL-10AVp controller,
SIL-10A autoinjector, LC-10AT pump, LC-10AD pump, Waters 486
tunable absorbance detector or SPD-6A UV detector). Peptide pu-
rification was performed on either a Vydac preparative C18 column
or a Vydac preparative C4 column. Analytical RP-HPLC was per-
formed on either a Vydac analytical C18 column or a Vydac ana-
lytical C4 column. Separation was achieved in a gradient mode
using 0.1% TFA/H2O as solvent A and 90% ACN/0.1% TFA/H2O (B1) or
90% MeOH/0.1% TFA/H2O (B2) as solvent B. CD spectra were mea-
sured on a JASCO (Tokyo, Japan) J-710 spectropolarimeter using
quartz cuvette of 1 mm path length at 23 ꢀC.
4.2. Synthesis of mannose derivatives 2a–5a
Figure 2. CD spectra of peptides 7a, 7b, 7c, 9a, 9b, 9c, and 10.
4.2.1. 2,3,4,6-Tetra-O-(cyanoethyl)-b-D-mannopyranosyl azide (2a)
not intrinsic to the sequence itself, but due to the template topol-
ogy-controlling nature. -Helical content was calculated based on
the mean residue ellipticity (MRE, [ 222) according to the published
equation.36 The contents of
-helix for 9a, 9b, and 9c were found to
be 44%, 45%, and 45%, respectively. The accurate determination of
the content relied on the quantification by UV absorption at 280 nm
as each tetravalent peptide contains four tyrosine residues.37 Under
the same conditions, the lipopeptide–carbohydrate templates were
recorded with very low MRE values, suggesting that the templates
have an insignificant contribution to the CD spectra of the tetrava-
lent peptides. It was also found that the CD spectra of 9a, 9b, and 9c
Acrylonitrile (6.4 mL, 5.2 g, 97 mmol) and DBU (0.6 mL, 0.6 g,
3.9 mmol) were added to a suspension of compound 1a (1.7 g,
8.28 mmol) in ACN (50 mL) with stirring. After 3 h, the next portion
of acrylonitrile (6.4 mL, 5.2 g, 97 mmol) and DBU (0.6 mL, 0.6 g,
3.9 mmol) was added, and the mixture was stirred overnight. The
solution was evaporated under reduced pressure and the residue
was purified by flash column chromatography on silica (EtOAc/
hexane 3:1) to give 2a as a colorless oil (1.26 g, 37%). nmax (ATR)
a
q]
a
2252, 2116, 1108 cmꢁ1 1H NMR (CDCl3, 500 MHz)
; d 4.41 (1H, d,
J¼0.9 Hz, H-1), 4.07–3.82 (10H, m, 4ꢂCH2O, H-2, H-4), 3.73–3.69
(2H, m, CH2O), 3.41–3.37 (2H, m, H-3, H-5), 2.76–2.54 (8H, m,
were concentration independent in the range of 14–32 mM (see
Supplementary data), implying there was no oligomerization or
aggregation of the peptide within the concentration range.
4ꢂCH2CN); 13C NMR (CDCl3, 125 MHz)
d 118.6, 118.3, 118.0, 117.9,
86.2, 82.7, 77.8, 77.7, 73.4, 69.2, 67.8, 67.3, 66.5, 65.8, 19.4, 19.3, 19.3,
19.1; LRESMS m/z 440.2 [MþNa]þ; HRESMS m/z 440.1654 [MþNa]þ
(calcd for C18H23N7O5Na, 440.1653).
3. Conclusion
4.2.2. 6-[2,3,4,6-Tetra-O-(cyanoethyl)-b-D-mannopyranosylamino]-
6-oxohexanoic acid, benzyl ester (3a)
Compound 2a (650 mg, 1.55 mmol) was dissolved in THF
(40 mL) and hydrogenated over 10% Pd/C (50 mg) overnight. Upon
completion, adipic acid monobenzyl ester (400 mg, 1.7 mmol),
Subunit peptide vaccines display significant advantages over the
conventional whole pathogen vaccines. Research on subunit pep-
tide vaccines for human vaccination has led to the development of
self-adjuvanting lipopeptide vaccines as a potentially effective and
safe approach. Covalent attachment of antigenic units of the viru-
lent protein on carbohydrate molecules has produced multivalent
immunogens with predetermined tertiary structure. Herein, we
have provided techniques, using chemoselective ligation, for the
synthesis of lipopeptide–carbohydrate assembled multivalent
peptides in a highly pure state. The synthesized monosaccharide
HBTU (640 mg, 1.7 mmol), and DIPEA (324 mL, 239 mg, 1.86 mmol)
were added, and the mixture was stirred under an inert atmo-
sphere overnight. The solution was filtered through CeliteÒ and the
filtrate was evaporated under reduced pressure. The residue was
dissolved in EtOAc (100 mL) and washed with 5% HCl (3ꢂ50 mL),
saturated NaHCO3 (2ꢂ50 mL), and saturated NaCl (50 mL). The or-
ganic layer was dried (MgSO4), filtered, and concentrated. The
obtained residue was purified by flash column chromatography on
silica (EtOAc) to yield 3a as a colorless oil (499 mg, 53%). nmax (ATR)
molecules have induced the formation of 4-a-helix bundles of the
attached peptides, which may induce better immune responses. The
presented carbohydrate-based LCP system, together with the syn-
thetic strategy, opens the way for the development of highly pure,
self-adjuvanting, and topologically defined synthetic vaccines.
2251, 1730, 1687, 1503, 1107 cmꢁ1 1H NMR (CDCl3, 500 MHz)
;
d
7.35–7.29 (5H, m, 5ꢂAr–H), 6.88 (1H, d, J¼9.5 Hz, NH), 5.24 (1H,
dd, J¼9.5, 1.2 Hz, H-1), 5.08 (2H, s, CH2Ph), 4.42–4.38 (1H, m, 1H of
CH2O), 4.01–3.79 (8H, m, 7H of CH2O, H-4), 3.77 (1H, dd, J¼2.6,
1.2 Hz, H-2), 3.73–3.62 (2H, m, CH2O), 3.46 (1H, dd, J¼9.7, 2.7 Hz, H-
3), 3.36 (1H, m, J¼1.4 Hz, H-5), 2.84–2.52 (8H, m, 4ꢂCH2CN), 2.37–
2.33 (2H, m, CH2CON), 2.26–2.23 (2H, m, CH2COO), 1.68–1.62 (4H,
4. Experimental
4.1. General experimental procedures
Microwave-assisted SPPS was performed using a CEM (Mat-
thews, NC, USA) Discovery reactor equipped with an external FO
temperature sensor. 1H NMR and 13C NMR spectra were acquired
using a Bruker Avance 400 Hz or 500 MHz spectrometer (Bruker
Biospin, Germany). NMR spectra were obtained in CDCl3 or DMSO-
m, CH2CH2CH2CH2); 13C NMR (CDCl3, 125 MHz)
d 173.2, 172.7, 136.0,
128.5, 128.1, 128.1, 119.3, 118.6, 118.5, 118.0, 84.2, 76.8, 76.5, 73.8,
69.3, 68.0, 67.3, 66.4, 66.2, 66.1, 35.7, 33.8, 24.6, 24.2, 19.8, 19.4, 19.3,
19.0; LRESMS m/z 632.3 [MþNa]þ, 648.3 [MþK]þ; HRESMS m/z
632.2701 [MþNa]þ (calcd for C31H39N5O8Na, 632.2691).

W. Zhong et al. / Tetrahedron 65 (2009) 3459–3464
3463
4.2.3. 6-[2,3,4,6-Tetra-O-(3-tert-butoxycarbonylaminopropyl)-
mannopyranosylamino]-6-oxohexanoic acid, benzyl ester (4a)
b-
D-
4.3. Synthesis of Cys-containing lipopeptide–carbohydrate
templates 7a, 7b, and 7c
Sodium borohydride (3.84 g, 101 mmol) was added portionwise
at 0 ꢀC to
a
solution of compound 3a (3.09 g, 5.07 mmol),
Peptides 7a, 7b, and 7c were synthesized on a p-MBHA resin
using the standard Boc-based in situ neutralization SPPS.29 Boc–
C12–G–C12–C12–G–resin was synthesized (0.45 mmol NH2/g,
0.5 mmol scale) according to the reported procedure.28 Each amino
acid coupling cycle consisted of Boc-deprotection with neat TFA
(2ꢂ1 min), a 1 min DMF flow wash, followed by a 1–4 h coupling
with the pre-activated amino acid. Amino acid activation was
achieved by dissolving the amino acid (2.2 mmol) in 0.5 M HBTU/
CoCl2$6H2O (9.67 g, 40.6 mmol), and Boc2O (6.65 g, 30.4 mmol) in
MeOH (170 mL) with stirring. After 4 h, MeOH was evaporated
under reduced pressure and CHCl3 (100 mL) was added. The mix-
ture was filtered through CeliteÒ and the filtrate was concentrated
under reduced pressure. The crude residue was purified by flash
column chromatography on silica (EtOAc/hexane 4:1) to give 4a as
a colorless oil (1.54 g, 30%). nmax (ATR) 3347, 1688, 1515, 1112 cmꢁ1
1H NMR (CDCl3, 400 MHz)
;
d
7.35–7.26 (5H, m, 5ꢂAr-H), 5.22 (1H, br
DMF solution (4 mL, 2 mmol) to which DIPEA (700 mL, 4 mmol) was
s, H-1), 5.06 (2H, s, CH2Ph), 3.82–3.12 (22H, m, 4ꢂCH2N, 5ꢂCH2O,
4ꢂCHO), 2.36–2.27 (m, 4H, 2ꢂCH2CO), 1.82–1.58 (12H, m,
4ꢂCH2CH2CH2, CH2CH2CH2CH2), 1.40 (36H, s, 4ꢂC(CH3)3); 13C NMR
added, and the activation proceeded for 1 min (Boc–Gly–OH) or
5 min (Boc–C12–OH22). Coupling efficiency was monitored by
quantitative ninhydrin test.38 Where necessary, couplings were
repeated to give coupling efficiency greater than 99.7%. Following
removal of the N-terminal Boc group, carbohydrate template 5a, 5b
or 5c was C-terminally coupled to the Boc–C12–G–C12–C12–G–
resin (0.1 mmol scale) to give 6a, 6b or 6c. Carbohydrate template
activation was achieved by dissolving the compound (5a, 5b or 5c)
(0.2 mmol) in 0.5 M HBTU/DMF solution (0.38 mL, 0.19 mmol) to
(CDCl3, 100 MHz) d 173.2, 156.1, 156.0, 146.7, 136.0, 128.5, 128.1, 85.1,
79.1, 79.0, 78.9, 77.2, 77.1, 74.1, 71.1, 69.7, 68.4, 66.0, 38.3, 38.0, 37.3,
35.5, 33.9, 30.3, 30.2, 29.6, 28.4, 28.4, 28.1, 27.3, 24.8, 24.4; LRESMS
m/z 1048.6 [MþNa]þ; HRESMS m/z 1048.6003 [MþNa]þ (calcd for
C51H87N5O16Na, 1048.6040).
4.2.4. 6-[2,3,4,6-Tetra-O-(3-tert-butoxycarbonylaminopropyl)-
b-
D-
which DIPEA (70 mL, 0.4 mmol) was added and the activation pro-
mannopyranosylamino]-6-oxohexanoic acid (5a)
ceeded for 5 min. Coupling proceeded for 6 h with coupling effi-
ciency greater than 99.7%. Following removal of the N-terminal Boc
groups, four Boc–Cys(MBzl) residues were then coupled to 6a, 6b or
6c. Boc–Cys(MBzl)–OH activation was achieved by dissolving the
amino acid (1.76 mmol) in 0.5 M HBTU/DMF solution (3.16 mL,
Compound 4a (1.54 g, 1.5 mmol) was dissolved in THF (100 mL)
and hydrogenated over 10% Pd/C (200 mg) overnight. After com-
pletion, the mixture was filtered through CeliteÒ. Evaporation of
the filtrate under reduced pressure without further purification
gave 5a as a colorless foam (1.17 g, 83%). nmax (ATR) 3345, 1688,
1.58 mmol) to which DIPEA (522 mL, 3 mmol) was added and the
1515, 1103 cmꢁ1
;
1H NMR (DMSO-d6, 500 MHz)
d
8.10 (1H, d,
activation proceeded for 1 min. Following removal of the N-ter-
minal Boc groups, the peptidyl resin was then washed with DMF
(3ꢂ10 mL), DCM (3ꢂ10 mL), and MeOH (3ꢂ10 mL) and then dried
under vacuum prior to cleavage with HF. HF cleavage (10 mL HF/g
resin) was performed for 2 h at 0 ꢀC in the presence of 5% (v/v)
p-cresol and 5% (v/v) thiocresol as scavengers. The crude peptides
were purified by preparative RP-HPLC (40–70% B1 over 40 min, C4
column). The yields and characteristic data for the Cys-containing
lipopeptide–carbohydrate templates 7a, 7b and 7c are shown in
Table 1.
J¼8.6 Hz, NHCO), 6.75–6.65 (4H, m, 4ꢂNHCOO), 5.04 (1H, br d,
J¼8.9 Hz, H-1), 3.67–2.93 (22H, m, 4ꢂCH2N, 5ꢂCH2O, 4ꢂCHO),
2.17–2.16 (4H, m, 2ꢂCH2CO), 1.63–1.45 (12H, m, 4ꢂCH2CH2CH2,
CH2CH2CH2CH2), 1.35 (36H, s, 4ꢂC(CH3)3); 13C NMR (DMSO-d6,
125 MHz) d 174.3, 171.6, 155.6, 155.5, 82.8, 77.4, 77.3, 76.2, 76.1, 74.3,
70.1, 69.9, 69.6, 69.4, 67.3, 59.7, 37.3, 37.2, 37.1, 36.8, 34.5, 33.4, 30.2,
30.1, 30.0, 29.6, 28.2, 24.5, 24.0; LRESMS m/z 958.6 [MþNa]þ.
HRESMS m/z 958.5534 [MþNa]þ (calcd for C44H81N5O16Na,
958.5571).
Table 1
Yields and analytical data of synthesized peptides
Cys
carbohydrate template -C12-Gly-C12-C12-Gly-NH2
Cys Cys
Compound
Cys
Yield (%)
tR (min)
ES-MS (MW: 1653.31)
7a
7b
7c
21
36
24
22.1a
22.1a
22.0a
1653.9 (z¼1), 827.2 (z¼2)
1653.7 (z¼1), 827.3 (z¼2)
1653.2 (z¼1), 827.3 (z¼2)
Ac-[8830]-Gly
S
Leu NH2
O
Compound
Yield (%)
14
tR (min)
17.0b
ES-MS (MW: 2585.89)
8
1294.4 (z¼2), 863.3 (z¼3)
Ac-[8830]-Gly-Cys Ac-[8830]-Gly-Cys
carbohydrate template -C12-Gly-C12-C12-Gly-NH2
Ac-[8830]-Gly-Cys Ac-[8830]-Gly-Cys
Compound
Yield (%)
tR (min)
ES-MS (MW: 11124.23)
9a
9b
9c
71
76
72
19.1,a 27.8c
19.2,a 26.9c
19.0,a 27.1c
1391.5 (z¼8), 1237.0 (z¼9), 1113.4 (z¼10), 1012.3 (z¼11), 928.0 (z¼12), 856.7 (z¼13)
1590.1 (z¼7), 1391.4 (z¼8), 1236.9 (z¼9), 1113.3 (z¼10), 1012.2 (z¼11), 927.9 (z¼12), 856.6 (z¼13)
1590.1(z¼7), 1391.5 (z¼8), 1237.0 (z¼9), 1113.4 (z¼10), 1012.3 (z¼11), 928.0 (z¼12)
a
Column: C4; Gradient: 0–100% B1 over 30 min.
Column: C18; Gradient: 0–100% B1 over 30 min.
Column: C4; Gradient: 0–100% B2 over 30 min.
b
c

3464
W. Zhong et al. / Tetrahedron 65 (2009) 3459–3464
4.4. Chemical ligation
Supplementary data
Peptide 7a (2.2 mg, 1.33
aqueous SDS (3 mL), frozen, and lyophilized. The powder was
then dissolved in 0.1 M phosphate buffer pH 7.6 (1.5 mL), to
m
mol) was dissolved in 1% (w/v)
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.tet.2009.02.060.
which TCEP (4.57 mg; 15.9
adjusted to 7.3 with 0.1 M sodium phosphate dibasic (300
Peptide 8 (27.5 mg; 10.6 mol) was dissolved in 0.1 M phosphate
buffer pH 7.6 (1 mL) with addition of MESNA (26.2 mg;
m
mol) was added and the pH was
References and notes
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(ES-MS: [Mþ2H]2þ m/z 1256.6 (calcd 1255.4), [Mþ3H]3þ m/z
837.5 (calcd 837.3), MW 2508.77 g/mol). This solution was then
transferred into the vessel containing lipopeptide 7a with extra
0.1 M phosphate buffer pH 7.6 (200 mL) and the reaction mix-
ture was incubated at 37 ꢀC. The ligation was monitored by
analytical RP-HPLC (0–100% B1 over 30 min, C4 column) and
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opeptide–carbohydrates were observed (19.7 min and 20.3 min
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ligation of 7b and 8a, and 19.7 min and 20.1 min for the ligation
of 7c and 8a). The ligation appeared to be completed within
24 h. The reaction mixture was separated by preparative RP-
HPLC on a C4 column using a gradient of 30–70% B1 over
40 min. The fractions were analyzed by analytical RP-HPLC,
combined where appropriate and lyophilized to give 9a. Com-
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the procedure as for compound 9a. Compound 9c was synthe-
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compound 9a. The yields and characteristic data for 9a, 9b, and
9c are shown in Table 1.
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Acknowledgements
This work was supported by the National Health and Medical
Research Council (NHMRC) of Australia and the Australian National
Heart Foundation (NHF). The authors would like to acknowledge
Chris Wood (The University of Queensland, SCMB) for LC–ESI-MS
analysis.