On the emerging role of chemistry in the fashioning of biologics: Synthesis of a bidomainal fucosyl GM1-based vaccine for the treatment of small cell lung cancer

Article abstract of DOI:10.1021/jo900918m

(Chemical Equation Presented) The synthesis of the novel small cell lung cancer (SCLC) fucosyl GM1-based vaccine construct, featuring insertion of the HLA-DR binding 15 amino acid sequence derived from Plasmodium falciparum, is described. The resultant gl

Full text of DOI:10.1021/jo900918m

pubs.acs.org/joc
On the Emerging Role of Chemistry in the Fashioning of Biologics:
Synthesis of a Bidomainal Fucosyl GM1-Based Vaccine for the Treatment
of Small Cell Lung Cancer
Pavel Nagorny,† Woo Han Kim,† Qian Wan,† Dongjoo Lee,† and
Samuel J. Danishefsky*,†,‡
†Laboratory for Bioorganic Chemistry, Sloan-Kettering Institute for Cancer Research, 1275 York Avenue,
New York, New York 10065 , and ‡Department of Chemistry, Columbia University, Havemeyer Hall, 3000
Broadway, New York, New York 10027
s-danishefsky@ski.mskcc.org
Received May 8, 2009
The synthesis of the novel small cell lung cancer (SCLC) fucosyl GM1-based vaccine construct,
featuring insertion of the HLA-DR binding 15 amino acid sequence derived from Plasmodium
falciparum, is described. The resultant glycopeptide has been synthesized in an efficient manner.
Finally, successful conjugation of the glycopeptide to the keyhole limpet hemocyanin (KLH) carrier
protein completed the preparation of the vaccine.
Introduction
MHC-II binding peptides bound to the epitope, thus helping
to present the carbohydrate to the T-cells for T-cell activa-
tion and initiation of the cellular response.3,4 Consequently,
one could imagine that the immunogenicity of a vaccine
might well be enhanced by providing MHC-II binding
peptides in the environs of the epitope, thereby serving to
increase the number of epitopes presented to the CD4 þ T
cell. In a sense, this rationale is related to the idea of
conjugating epitopes to carrier protein to create vaccines.
However, this approach of placing an MHC-II binding
sequence in a fixed relation to the antigen has been pursued
mostly for vaccines unconjugated to carrier protein.5 We
hope to explore the possibility that introduction of an
Among the large number of emerging anticancer strate-
gies, the prospect of mobilizing the immune system against
the disease is especially attractive. One can imagine employ-
ing a vaccine-based therapeutic approach against a number
of different primary tumors, as well as against metastatic
cells, in an adjuvant mode.1 Along these lines, we are
pursuing the idea of targeting as immune system markers
complex carbohydrate epitopes, which are overexpressed
on cancer cell surfaces. Though this concept has occurred
to others, our group has made a particularly strong commit-
ment to accessing these structures by total synthesis.2
A typical carbohydrate-based anticancer vaccine would
consist of a complex carbohydrate hapten, overexpressed
on the cancer cell, a carrier protein, and a linker attaching the
carbohydrate to the protein (Figure 1). Beside being a potent
immunogen, the carrier protein is known to provide the
(3) Zegers, N. D., Boersma, W. J. A., Claassen, E., Eds. Immunological
Recognition of Peptides in Medicine and Biology; CRC Press: Boca Raton,
1995; p 105.
(4) (a) Rudensky, A. Y.; Preston-Hurlbort, P.; Hong, S.-Ch.; Barlow, A.;
Janeway, C. A. Jr. Nature 1991, 353, 622–627. (b) Bona, C. A.; Casares, S.;
Brumeanu, T. D. Immunol. Today 1998, 19, 126–133. (c) Musselli, C.;
Livingston, P. O.; Ragupathi, G. J. Cancer Res. Clin. Oncol. 2001, 127,
20–26.
(1) Khleif, S. N., Ed. Tumor Immunology and Cancer Vaccines; Springer-
Verlag: New York, 2005.
(2) For an overview of our synthetic carbohydrate-based vaccine pro-
gram, see: (a) Danishefsky, S. J.; Allen, J. R. Angew. Chem., Int. Ed. 2000, 39,
836–863. (b) Keding, S. J.; Danishefsky, S. J. Proc. Natl. Acad. Sci. U.S.A.
2004, 101, 11937–11942.
(5) (a) Dziadek, S.; Hobel, A.; Shmitt, E.; Kunz, H. Angew. Chem., Int.
Ed. 2005, 44, 7630–7635. (b) Buskas, T.; Ingale, S.; Boons, G.-J. Angew.
Chem., Int. Ed. 2005, 44, 5985–5988. (c) Ingale, S.; Wolfert, M. A.; Gaekwad,
J.; Buskas, T.; Boons, G.-J. Nature, Chem. Biol. 2007, 3, 663–667.
DOI: 10.1021/jo900918m
r
Published on Web 06/25/2009
J. Org. Chem. 2009, 74, 5157–5162 5157
2009 American Chemical Society

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FIGURE 1. Next-generation bidomainal fucosyl GM1-based vaccine for the treatment of SCLC.
SCHEME 1a
aKey: (a) TBAF, AcOH, THF; (b) NaOMe, MeOH; (c) NaOH, THF; (d) Na, NH3, THF; (e) Ac2O, DMAP, Py; (f) DMAP, MeOH; (g) Ac2O,
DMAP, Py; 56% (7 steps); (h) A, B, CH2Cl2, rt; (i) H2, Pt/C, MeOH-H2O; 49%, two steps.
MHC-II binding sequence could also improve the immuno-
genicity of vaccines incorporating standard carriers such as
keyhole limpet hemocyanin (KLH).
The appendage of the fucosyl GM1 epitope to the peptide
portion could be accomplished using the norleucine linker
developed by our group.10 The long aliphatic chain of this
linker would be optimal in preventing potentially adverse
interactions between the epitope and the peptide backbone.
The amino acid functionality makes this linker a powerful
handle for conjugation.
The synthesis of glycoprotein conjugates still presents a
challenge for numerous reasons. One of the issues is compat-
ibility and “collegiality” of the protecting groups required
for peptidic and carbohydrate assemblies. Thus, a complex
glycan is often unstable under the highly acidic conditions
that are required to remove peptide protecting groups.
Correspondingly, peptides may be unstable under the
basic conditions that are required for the deprotection and
retrieval of the oligosaccharide ensembles.11 However,
since the protecting groups of the selected peptide would
be limited to tert-butylcarboxy for lysine and tert-butyl for
tyrosines and threonine, there is, hopefully, no requirement
To test the notion of upgrading the immunogenicity of a
candidate carbohydrate-based vaccine in this way, we pur-
sued the synthesis of the construct illustrated in Figure 1.
Fucosyl GM1 is a carbohydrate epitope that is expressed on
the surface of small-cell lung cancer (SCLC) cells.6 This
carbohydrate has been previously synthesized by our group
as well as by others,7 and it was selected based on the
promising results demonstrated by its KLH conjugate
in recent clinical trials.8 A 15 amino acid peptide sequence
derived from Plasmodium falciparum and illustrated in
Figure 1 was chosen as the T-cell epitope. This sequence
has been shown to be general for binding up to nine different
genetic variants of human HLA-DR with binding capacity
prevalently in the nanomolar range.9
(6) Zhang, S.; Cordon-Cardo, C.; Zhang, H. S.; Reuter, V. E.; Adluri, S.;
Hamilton, Wm. B.; Lloyd, K. O.; Livingston, P. O. Int. J. Cancer 1997, 73,
42–49.
(7) (a) Allen, J. R.; Danishefsky, S. J. J. Am. Chem. Soc. 1999, 121,
10875–10882. (b) Mong, T. K.-K.; Lee, H.-K.; Duron, S. G.; Wong, C.-H.
Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 797–802.
(8) Dickler, M. N.; Ragupathi, G.; Liu, N. X.; Musselli, C.; Martino,
D. J.; Miller, V. A.; Kris, M. G.; Brezicka, F.-T.; Livingston, P. O.; Grant,
S. C. Clin. Cancer Res. 1999, 5, 2773–2779.
(9) Southwood, S.; Sidney, J.; Kondo, A.; del Guercio, M. S.; Appella, E.;
Hoffman, S.; Kubbo, R. T.; Chesnut, R. W.; Grey, H. M.; Sette, A. J. Immun.
1998, 160, 3363–3373.
(10) (a) Keding, S. J.; Atsushi, E.; Biswas, K.; Zatorski, A.; Coltart,
D. M.; Danishefsky, S. J. Tetrahedron Lett. 2003, 44, 3413–3416. (b) Wan,
Q.; Cho, Y. S.; Lambert, T. H.; Danishefsky, S. J. J. Carbohydr. Chem. 2005,
24, 425–440. (c) Ragupathi, G.; Koide, F.; Livingston, P. O.; Cho, Y. S.;
Endo, A.; Wan, Q.; Spassova, M. K.; Keding, S. J.; Allen, J.; Ouerfelli, O.;
Wilson, R. M.; Danishefsky, S. J. J. Am. Chem. Soc. 2006, 128, 2715–2725.
(11) For a comprehensive review on the subject, refer to: Herzner, H.;
Reipen, T.; Schulz, M.; Kunz, H. Chem. Rev. 2000, 100, 4495–4537.
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SCHEME 2a
aKey: (a) SPPS, then AcOH/CF3CH2OH/CH2Cl2 (1:1:8), 95%; (b) C, EDC, HOOBt, CHCl3/CF3CH2OH (3:1); (c) Piperidine, DMF; 71%, two
steps; (d) 5, EDCI, HOBt, DMF/CH2Cl2 (1:1), 81%.
SCHEME 3a
aKey: (a) piperidine, DMF; (b) Ac2O, Py; (c) Pd(PPh3)4, PhSiH3, CH2Cl2/DMF (1:1); (d) SAMA-OPbf, TEA, Py; (e) TFA/PhOH/H2O/TIPS, (71%
from 9, five steps); (f) NaOH in MeOH/H2O, pH = 10.5, 19%; (g) (i) TCEP gel; (ii) PBS buffer, pH = 7.2, 0.9 M NaCl, 0.1 M EDTA.
for prolonged treatment with acid in the deprotection
phase. Thus, the glycoside linkages of peracetylated fucose
and sialic acid moieties could be stable under the deprotec-
tion conditions. Of course, the cleavage of a methyl ester
and 17 acetate groups could prove to be a challenging
task that might well require careful selection of hydrolysis
conditions.
resultant product was debenzylated under Birch condi-
tions.12 The obtained acid 3 was exhaustively peracetylated
to provide a corresponding lactone that was subsequently
opened with methanol and DMAP. Acylation afforded 4
(56%, seven steps). Glycoside 4 was treated with Fmoc-L-
allylglycine benzyl ester (A) and Hoveyda-Grubbs catalyst
(B) under the previously developed conditions, and the
resultant olefin cross-metathesis product was subjected to
catalytic hydrogenation.10a,10b,13 The side-chain olefinic
linkage was reduced with concomitant selective removal of
Results and Discussion
The construction of the vaccine began from the known
Fuc-GM1 hexasaccharide 2, obtained through a previously
disclosed sequence.7a After deprotection of the triisopropyl-
silyl groups with TBAF/acetic acid and subsequent cleavage
of acetates, carbonate, and sialic acid methyl ester, the
(12) Wang, Z.-G.; Warren, J. D.; Dudkin, V. Y.; Zhang, X.; Iserloh, U.;
Visser, M.; Eckhardt, M.; Seeberger, P. H.; Danishefsky, S. J. Tetrahedron
2006, 62, 4954–4978.
(13) Biswas, K.; Coltart, D. M.; Danishefsky, S. J. Tetrahedron Lett.
2002, 43, 6107–6110.
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the benzyl protecting group in the presence of Fmoc-pro-
tected amine to afford the cassette 5 (49%, 2 steps) ready for
coupling (Scheme 1).
Experimental Section
Synthesis of Acetylated Glycoside 4 (Steps a-c, Scheme 1). To
a solution of the hexasaccharide 27a (335 mg, 0.131 mmol) in THF
(6.0 mL) were added glacial AcOH (0.12 mL) and TBAF (1.0 M in
THF, 1.31 mL). The reaction mixture was stirred at rt for
2 days, poured into ice-water (25 mL), and extracted with EtOAc.
The organic extracts were dried over MgSO4 and concentrated.
The resulting triol was dissolved in anhydrous MeOH (6 mL), and
sodium methoxide was added (25% solution in MeOH, 0.6 mL).
The contents were stirred at rt for 3 days, and then water (6.0 mL)
and THF (6.0 mL) were added. Stirring at rt for an additional 2
days was followed by neutralization with Dowex-Hþ, filtration
with MeOH washing, and concentration. The crude material was
allowed to dry under high vacuum for 1 day.
The synthesis of peptide 7 was accomplished by standard
Fmoc solid-phase peptide synthesis (SPPS), starting from
the protected tyrosine, 6, preloaded on NovaSyn TGT resin.
Peptide 7 was obtained in 95% yield after cleavage from the
resin, in more than 95% purity, as judged by LC/MS and 1H
NMR analysis. The elaboration of 7 to peptide 8 was
executed by first conjugating 7 to the linker C14 using the
standard EDCI/HOBt protocol.10c The Fmoc protecting
group was next removed by treatment with piperidine,
providing fragment 8 in 71% yield (two steps). The attach-
ment of 8 to carbohydrate epitope 5 proceeded in 81% yield,
thereby providing glycopeptide 9 (see Scheme 2).
Synthesis of Acetylated Glycoside 4 (Steps d-g, Scheme 1). To
a blue solution of sodium (160 mg) in liquid NH3 (50 mL) was
added a solution of the white solid from above in THF (5.0 mL),
and the resulting mixture was stirred at -78 °C for 2 h. The
reaction was quenched by the addition of anhydrous MeOH
(20 mL), warmed to rt, and concentrated with a stream of dry
argon. The residue was diluted with MeOH (70 mL) and treated
with Dowex 50wX8-400 until pH was nearly 5-6. The mixture
was filtered and concentrated to provide a solid. This solid was
dissolved in a mixture of pyridine (12.0 mL) and Ac2O (6.0 mL)
at rt. To the solution of tetrasaccharide was added DMAP
(10 mg), and the mixture was stirred for an additional 2 days.
The reaction mixture was cooled to 0 °C and treated with MeOH
(24 mL). To this solution was added DMAP (15 mg), and the
resultant mixture was stirred at rt for an additional 4 days. The
reaction mixture was then concentrated and coevaporated with
toluene (4 ꢀ 100 mL). The residue was dissolved in pyridine
(5.0 mL) and Ac2O (1.0 mL) at rt. The mixture was stirred for
1 day and then concentrated. The resultant oil was dissolved in
MeOAc (10.0 mL) and MeI (0.2 mL). To the solution was added
cesium carbonate (33 mg), and the mixture was stirred for 1 h
and then diluted with methyl acetate (250 mL). The organic
phase was washed with brine/NH4Cl(satd) (1:1, 100 mL), NaH-
CO3(satd) (100 mL), and brine (100 mL) and dried over MgSO4.
Concentration followed by flash chromatography (silica, 5%
methanol/dichloromethane) provided the acetylated glycoside
4 (142 mg, 56% from 2): [R]24D=-40.4 (c 1.00, CHCl3); IR (film
CHCl3) 2969, 1746, 1689, 1530, 1371, 1231, 1131, 1058 cm-1; 1H
NMR (CDCl3, 600 MHz) δ 7.22 (d, J=5.9 Hz, 1H), 5.76-5.69
(m, 1H), 5.61-5.58 (m, 1H), 5.43 (d, J=3.5 Hz, 1H), 5.34-5.30
(m, 4H), 5.21-5.10 (m, 5H), 5.00 (d, J = 8.2 Hz, 1H), 4.97-4.89
(m, 4H), 4.82 (t, J=8.8 Hz, 1H), 4.74 (td, J=11.4, 3.6 Hz, 1H),
4.65 (d, J=7.7 Hz, 1H), 4.52 (d, J=7.8 Hz, 1H), 4.49-4.45 (m,
1H), 4.39-4.33 (m, 3H), 4.21-4.17 (m, 2H), 4.14-4.02 (m, 6H),
4.00-3.89 (m, 3H), 3.85-3.70 (m, 9H), 3.58-3.56 (m, 2H),
3.45-3.40 (m, 2H), 3.01 (dt, J=12.7, 5.6 Hz, 1H), 2.81 (dd, J=
12.9, 4.1 Hz, 1H), 2.19 (s, 3H), 2.12 (s, 3H), 2.10 (s, 3H), 2.09
s, 3H), 2.03-1.97 (m, 33H), 1.95 (s, 3H), 1.94 (s, 3H), 1.93 (s,
3H), 1.80 (s, 3H), 1.70 (t, J=12.8 Hz, 1H), 1.64-1.57 (m, 2H),
1.22-1.20 (m, 2H), 1.11 (d, J=6.4 Hz, 3H); 13C NMR (CDCl3,
150 MHz) δ 173.6, 171.0, 170.9, 170.9, 170.4, 170.4, 170.4, 170.3,
170.3, 170.3, 170.3, 170.3, 170.2, 170.2, 170.0, 169.7, 169.6,
169.5, 169.4, 169.2, 168.2, 137.7, 114.9, 102.0, 100.4, 98.7,
97.3, 94.4, 75.6, 73.7, 73.5, 73.3, 73.2, 72.8, 72.5, 72.0, 71.8,
71.7, 71.3, 70.9, 70.3, 70.2, 69.8, 69.4, 69.2, 69.1, 68.7, 68.1, 67.7,
67.4, 67.2, 67.0, 65.0, 63.3, 62.4, 62.4, 62.4, 60.6, 60.3, 55.5, 53.7,
52.5, 49.3, 37.1, 31.6, 29.7, 29.2, 28.5, 23.5, 23.0, 21.3, 20.9, 20.8,
20.7, 20.7, 20.7, 20.7, 20.6, 20.6, 20.5, 20.4, 20.4, 15.9, 14.1;
ESI/MS exact mass calcd for C83H116N2O50 [M þ Na]þ 1963.7,
[M þ Cl]-1975.6, found 1963.9, 1977.0.
Compound 9 was treated sequentially with piperidine/
DMF and Ac2O/Py. The allylcarboxy protecting group
was exchanged to the acetate-protected 2-sulfhydrylacetate
linker by reduction with Pd(PPh3)4/PhSiH3 followed by
acylation with SAMA-OPbf.10c Finally, three tert-butyl
and one tert-butylcarboxy groups were removed by treating
10 with TFA/PhOH/H2O/TIPS providing the correspond-
ing deprotected product in 71% yield (five steps) from
9 following purification by HPLC. This compound was
treated with a degassed solution of NaOH in MeOH/H2O
(pH=10.5), providing the desired deprotected product 11 in
19% yield following HPLC purification.15 Minor amounts
of a dehydration side product (ca. 5% yield after HPLC)
were also isolated.
Next, the conjugation of construct 11 to maleimide-acti-
vated KLH 12 was examined (Scheme 3). Thus, 11 was
pretreated with TCEP gel for 2 h and then treated with freshly
prepared 12 at pH=7.2. The efficiency of the coupling was
estimated by a combination of Bradford protein assay16 and
neuraminic acid determination according to Svennerholm17
to be 210 epitopes per molecule of KLH (MW=8 MDa).
Conclusion
In summary, a new kind of fucosyl GM1 epitope-based
vaccine has been efficiently prepared through the coupling of
the fucosyl GM1 cassette with a promiscuous HLA-DR
binding peptide. The resultant construct was further func-
tionalized and deprotected to provide the glycopeptide,
which was next conjugated to the carrier protein (KLH).
The results of immunological evaluations of the vaccine will
be forthcoming. This synthetic accomplishment is in keeping
with an important theme in our laboratory, to the effect that
there have emerged exciting opportunities for chemistry in
the fashioning of structure types previously perceived as
strictly “biologics”. It goes without saying that chemistry
provides vast opportunities (and challenges) in de novo
design, thus offering far greater flexibility than is available
though strictly biology-driven routes to biologics.
(14) Pittelkow, M.; Lewinsky, R.; Christensen, J. B. Synthesis 2000, 15,
2195–2202.
(15) Glunz, P. W.; Hintermann, S.; Williams, L. J.; Schwarz, J. B.;
Kuduk, S. D.; Kudryashov, V.; Lloyd, K. O.; Danishefsky, S. J. J. Am.
Chem. Soc. 2000, 122, 7273–7279.
(16) Bradford, M. Anal. Biochem. 1976, 72, 248–254.
(17) Svennerholm, L. Biochim. Biophys. Acta 1957, 24, 604–611.
(18) (a) Frechet, J. M. J.; Haque, K. E. Tetrahedron Lett. 1975, 35, 3055–
3056. (b) Novabiochem catalog, 2006/2007.
Synthesis of Amino Acid 5 (Step h, Scheme 1). The first-
generation Hoveyda-Grubbs catalyst (B, 9.6 mg, 0.016 mmol)
was added to a solution of acetylated glycoside 4 (124 mg,
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Nagorny et al.
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0.064 mmol) and allylglycine A (273 mg, 0.640 mmol) in CH2Cl2
(1 mL) at rt. The reaction mixture was stirred for 12 h and
exposed to air for 3 h. The mixture was concentrated, and the
resultant residue was purified by flash chromatography (100%
ethyl acetate) to provide the coupled product.
(m, 1H), 4.54-4.47 (m, 3H), 4.46-4.41 (m, 2H), 4.41-4.37
(m, 2H), 4.30 (m, 2H), 4.29-4.23 (m, 2H), 4.00-3.85 (m, 4H),
3,77 (d, J= 16.7 Hz, 1H), 3.70 (m, 2H), 3.52 (br t, J=8.8 Hz, 2H),
3.40 (q, J=8.5 Hz, 1H), 3.23 (d, J=10.6 Hz, 1H), 3.19-3.08 (m,
3H), 3.08-2.96 (m, 3H), 2.96 (s, 2H), 2.79 (s, 2H), 2.18-2.05
(m, 4H), 2.02-1.93 (m, 1H), 1.92-1.83 (m, 3H), 1.77-1.72 (m,
1H), 1.71-1.66 (m, 2H), 1.63 (m, 2H), 1.54 (m, 1H), 1.40 (s, 9H),
1.42-1.33 (m, 6H), 1.31 (s, 9H), 1.30 (s, 9H), 1.26 (s, 9H), 1.3-1.23
(m, 2H), 1.23 (s, 6H), 1.19 (s, 3H), 1.12 (d, J=6.2 Hz, 6H), 1.05 (d, J
=6.2 Hz, 3H), 0.99 (d, J=6.8 Hz, 3H), 0.97 (d, J=6.6 Hz, 3H), 0.92
(d, J=6.2 Hz, 6H), 0.90 (d, J=5.2 Hz, 6H); LC/MS (ESI) Rf=16.6
min (C4 column, 50-95% MeCN in H2O, 30 min); exact mass calcd
for C108H154N16O23 [M þ H]þ 2045.2, [M þ Na]þ 2067.1, [M þ
2H]2þ, 1023.1, found 2044.7, 2066.6, 1022.9.
Synthesis of Compound 8 (Step b, Scheme 2). To compound
7 (30 mg, 0.0147 mmol) were added linker C (5.6 mg, 0.0352
mmol), HOOBt (5.7 mg, 0.0352 mmol) in 1:3 trifluoroethanol/
CHCl3, and EDC (6.2 mL, 0.0352 μmol). After 2 h, LC/MS indi-
cated completion of the reaction. The mixture was concentrated
under reduced pressure and purified via flash chromatography
(silica, 2%f10% MeOH/ CH2Cl2), and the appropriate fractions
were concentrated (Rf 0.5, 10% MeOH/ CH2Cl2) to afford 30 mg
of product in 94% yield. This material was found to be >95% pure
as judged by reversed-phase LC/ESI (C4 column): MS exact
mass calcd for C115H166N18O24 [M þ H]þ 2185.2, [M þ Na]þ
2207.2, [M þ 2H]2þ 1093.1, found 2184.8, 2206.8, 1093.2.
Synthesis of Amino Acid 5 (Step i, Scheme 1). Pt/C (10% w/w,
15 mg) was added to a solution of the metathesis adduct from
above in MeOH (3 mL) and H2O (0.2 mL), and the hydrogen
atmosphere was established. The reaction mixture was stirred
for 4 days at rt, filtered through a short pad of silica gel, and
concentrated. The residue was purified by flash chromatogra-
phy (10% MeOH in CH2Cl2) to give the amino acid 5 (70 mg,
49% over two steps): [R]24D=-30.2 (c 1.00, CHCl3); IR (film
CHCl3) 3470, 2928, 2854, 1746, 1429, 1370, 1232, 1057 cm-1; 1H
NMR (CDCl3, 600 MHz) δ 8.16 (d, 1H, J=6.4 Hz), 7.79 (d, 2H,
J=7.4 Hz), 7.67-7.65 (m, 2H), 7.39-7.37 (m, 2H), 7.30 (br.s,
2H), 5.62-5.60 (m, 1H), 5.48-5.47 (m, 2H), 5.39 (d, 1H, J=10
Hz), 5.25-5.23 (m, 3H), 5.14-5.05 (m, 3H), 4.98-4.78 (m, 6H),
4.68 (d, 1H, J=7.5 Hz), 4.64 (d, 1H, J=7.8 Hz), 4.54-4.53 (m,
2H), 4.47 (d, 1H, J=11.2 Hz), 4.40-4.32 (m, 3H), 4.29-4.10 (m,
7H), 4.07-3.76 (m, 15H), 3.72-3.65 (m, 3H), 3.57 (s, 1H), 3.48-
3.44 (m, 1H), 3.25-3.17 (m, 1H), 2.86 (dd, 1H, J=3.9 and 12.5
Hz), 2.27 (s, 3H), 2.16 (s, 3H), 2.14 (s, 3H), 2.12 (s, 6H), 2.11
(s, 3H), 2.08 (s, 3H), 2.06 (s, 6H), 2.03 (s, 6H), 2.02 (s, 6H), 2.00
(s, 3H), 1.98 (s, 6H), 1.95 (s, 6H), 1.82 (s, 3H), 1.61 (t, 2H, 12.5
Hz), 1.52 (br.s, 2H), 1.37-1.25 (m, 7H), 1.18 (d, 3H, J= 6.2 Hz);
13C NMR (CDCl3, 150 MHz) δ 180.2, 175.4, 173.6, 172.4, 172.4,
172.4, 172.3, 172.2, 172.2, 172.1, 171.8, 171.7, 171.7, 171.5,
171.4, 171.1, 169.7, 158.2, 145.5, 145.4, 142.6, 128.8, 128.2,
126.3, 126.3, 121.0, 102.6, 102.3, 101.7, 101.1, 98.7, 97.3, 77.6,
75.2, 75.0, 74.8, 74.4, 74.3, 74.2, 73.8, 73.3, 73.2, 73.0, 72.7, 72.4,
72.2, 71.9, 71.3, 71.0, 70.6, 69.7, 69.2, 68.9, 68.4, 67.7, 66.2, 65.0,
63.8, 63.6, 63.5, 62.6, 57.6, 55.8, 53.8, 50.0, 49.6, 48.6, 38.7, 34.1,
30.6, 30.2, 27.0, 26.8, 24.2, 22.8, 21.7, 21.7, 21.3, 21.0, 21.0, 20.9,
20.9, 20.8, 20.8, 20.8, 20.7, 20.6, 20.6, 20.6, 16.3; ESI/MS exact
mass calcd for C101H133N3O54 [M þ Na]þ 2275.8, [M þ 2Na]2þ
1149.4, found 2275.5, 1149.3.
Synthesis of Compound 8 (Step c, Scheme 2). The product
from above (26.8 mg, 0.0123 mmol) was dissolved in 1.0 mL of
DMF, and to this solution was added piperidine (0.25 mL).
After 1 h, LC/MS analysis indicated the completion of the
reaction. The mixture was concentrated under reduced pressure
and purified via flash chromatography (silica, 10% f 12%
MeOH/ CH2Cl2), and the appropriate fractions were concen-
trated (Rf 0.15, 10% MeOH/CH2Cl2) to afford 18 mg of product
8 in 75% yield. This material was found to be >95% pure
as judged by reversed-phase LC/ESI analysis: Rf = 15.7 (C4
column, 40-85% MeCN in H2O, 30 min); exact mass calcd for
Synthesis of Peptide 7 (Step a, Scheme 2). NovaSyn TGT resin
(purchased from NovaBiochem) was chlorinated, esterified with
Fmoc-Tyr(tBu)-OH for 3 h, and then immediately Fmoc-depro-
tected according to the literature procedure.18 A 0.21 g (ca. 0.05
mmol) portion of this resin was subjected to continuous flow
automated peptide synthesis. For coupling steps, the resin was
treated with a 4-fold excess of HATU and Fmoc amino acids in 1
M DIEA/DMF, and for deblocking, a solution of 2% piperidine/
2% DBU in DMF was used. The amino acids used were, in order of
synthesis: Fmoc-Pro-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ala-OH,
Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Val-OH,
Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Lys(Boc)-OH, Fmoc-Tyr
(tBu)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Gly-OH. The
resin was then transferred to a manual peptide synthesis vessel
and treated with a cleavage solution of 5 mL of 1:1:8 trifluoroetha-
nol/acetic acid/dichloromethane for 1.5 h. The beads were filtered,
rinsed with another 10 mL of cleavage solution, filtered again, and
then treated for another 1 h with 10 mL of the cleavage solution.
This process was repeated for a total of three 2-h cleavage cycles,
and the combined organic phase was concentrated in vacuo to
afford 97 mg of peptide after cleavage (ca. 95% yield). This material
was found to be >95% pure as judged by reversed-phase LC/ESI
C
100H156N18O22 [M þ H]þ 1963.2, [M þ Na]þ 1985.2, [M þ
2H]2þ 982.1, found 1962.9, 1984.9, 982.1.
Synthesis of Compound 9 (Step d, Scheme 2). Amine 8 (9.5 mg,
0.048 mmol) was combined with acid 5 (6.6 mg, 0.029 mmol),
EDCI (1.8 mg, 0.093 mmol), and HOBt (1.3 mg, 0.0093 mmol),
and this mixture was dissolved in 0.30 mL of 1:1 DMF/CH2Cl2.
After 3 h ofstirring under argon, the solventswereremovedunder
high vacuum, the resultant oil was purified via flash chromatog-
raphy (silica, 5%f10% MeOH/CH2Cl2), and the appropriate
fractions were concentrated (Rf 0.5, 10% MeOH/CH2Cl2) to
afford 10 mg of product 9 in 81% yield. This material was found
to be >90% pure as judged by reversed-phase LC/ESI MS (C4
column) and 1H NMR analysis: 1H NMR (500 MHz, CD3OD)19
selected peaks δ 8.12 (d, 1H), 7.95 (m, 1H), 7.80 (d, J =9.1 Hz,
2H), 7.74 (d, J =7.6 Hz, 2H), 7.72 (m, 1H), 7.67 (d, J =8.2 Hz,
2H), 7.66 (m, 1H), 7.62 (d, J =7.4 Hz, 1H), 7.57 (d, J =7.5 Hz,
1H), 7.56 (m, 1H), 7.45 (t, J=7.2 Hz, 1H), 7.39(t, J=7.7 Hz, 1H),
7.33 (t, J=7.4 Hz, 1H), 7.24 (dt, J=7.3, 4.1 Hz, 1H), 7.18 (d, J=
7.3 Hz, 1H), 7.14 (t, J=7.6 Hz, 1H), 7.08 (d, J=5.5 Hz, 4H), 6.97
(m, NH), 6.93 (m, NH), 6.85 (d, J=7.9 Hz, 2H), 6.80 (d, J=8.0
Hz, 4H), 5.85 (ddd, J=16.2, 10.5, 5.5 Hz, 1H), 5.55 (dt, J=9.5,
4.1 Hz, 1H), 5.42 (dd, J=9.5, 2.8 Hz, 2H), 5.32 (dd, J=9.6, 2.1
Hz, 1H), 5.19 (m, 4H), 5.07 (m, 4H), 4.97 (m, 2H), 4.75 (m, 1H),
4.60 (d, J=7.5 Hz, 3H), 4.49 (dt, J=16.3, 8.1 Hz, 1H), 4.47-4.37
(m, 8H), 4.33-4.24 (m, 8H), 4.20-4.08 (m, 7H), 4.06 (dd, J =
1
1
(C4 column) MS and H NMR analysis: H NMR (500 MHz,
DMF-d7)19 δ 8.44 (t, J=5.5 Hz, NH), 8.34 (br, NH), 8.27 (br, NH),
8.13 (t, J=8.6 Hz, NH), 8.00 (m, NH), 7.94 (d, J=7.6 Hz, 2H), 7.89
(d, J=6.5 Hz, NH), 7.86 (d, J=7.1 Hz, NH), 7.79 (br, NH), 7.73 (t,
J=8.4 Hz, 2H, NH), 7.62 (d, J=7.9 Hz, NH), 7.60 (d, J=7.4 Hz,
NH), 7.55 (d, J=6.0 Hz, NH), 7.45 (t, J = 7.4 Hz, 2H), 7.34 (t, J=
7.6 Hz, 2H), 7.31 (d, J =7.6 Hz, 2H), 7.25 (m, 2H), 7.22-7.17
(m, 5H), 6.93 (t, J =8.3 Hz, 2H), 6.88 (d, J = 8.2 Hz, 2H), 6.63
(m, NH), 5.12 (t, J =4.7 Hz, NH), 4.71-4.67 (m, 2H), 4.66-4.59
(19) Due to the high degree of the NH exchange, the presence of the
multiple peptide rotomers in the solution, as well as the high overlap, there is
an ambiguity associated in the tabulation and interpretation of the 1H NMR
data. Refer to the attached 1H NMR spectrum in the Supporting Informa-
tion for additional details.
J. Org. Chem. Vol. 74, No. 15, 2009 5161

Nagorny et al.
JOCFeatured Article
11.2, 6.0 Hz, 2H), 4.05 (dt, J=9.1, 4.0 Hz, 2H), 3.95 (m, 2H),
3.94-3.81 (m, 8H), 3.83 (s, 3H), 3.80 (m, 3H), 3.75 (m, 1H),
3.71 (m, 1H), 3.68 (m, 1H), 3.64 (m, 3H), 3,58 (m, 1H), 3.50
(s, 1H), 3,42 (m, 1H), 3.15 (dd, J =13.3, 6.2 Hz, 2H), 3.12 (m, 1H),
3.11-3.03 (m, 4H), 2.97 (t, J=6.2 Hz, 2H), 2.93-2.83 (m, 4H),
2.80 (dd, J=12.7, 4.4 Hz, 1H), 2.21 (s, 3H), 2.20-2.13 (m, 2H),
2.11 (s, 3H), 2.09 (s, 3H), 2.06 (s, 9H), 2.01 (s, 3H), 2.00 (s, 3H),
1.99 (s, 3H), 1.97 (s, 6H), 1.96 (s, 6H), 1.94 (s, 3H), 1.92 (s, 3H),
1.91 (s, 3H), 1.89 (s, 3H), 1.89 (s, 3H), 2.13-1.77 (m, 13H), 1.75 (s,
3H), 1.58-1.48 (m, 12H), 1.36 (s, 3H), 1.36 (s, 3H), 1.34-1.24 (m,
8H), 1.23 (s, 18H), 1.21 (s, 9H), 1.15 (s, 6H), 1.15-1.09 (m, 6H),
1.05 (d, J=6.2 Hz, 3H), 0.91 (d, J = 6.7 Hz, 3H), 0.89 (d, J = 8.6
Hz, 6H), 0.87 (d, J = 7.0 Hz, 6H), 0.85 (d, J = 6.6 Hz, 3H), 0.75
(m, 3H); Rf = 22.5 (C4 column, 50-95% MeCNinH2O, 30min);
exact mass calcd for C201H287N21O75 [M þ 2H]2þ 2099.5, [M þ
2Na]2þ 2121.5, [Mþ3H]3þ 1400.0, found 2099.4, 2121.6, 1400.2.
Synthesis of Compound 10 (Step a, Scheme 3). Compound 9
(10.5 mg, 0.00250 mmol) was dissolved in 1.0 mL of DMF, and
piperidine (0.25 mL) was added. After 1 h, LC/MS analysis
indicated the completion of the reaction: Rf=15.3 (C4 column,
50-95% MeCN in H2O, 30 min); exact mass calcd for
(C4 column, 35-75% MeCN in H2O, 30 min) to afford pure
product (6.7 mg, 71% yield from 9). The product was >95%
pure as judged by LC/MS: exact mass calcd for C171
-
H247N21O72S [M þ 2H]2þ 1891.3, [M þ 3H]3þ 1261.2, found
1891.4, 1261.5.
Synthesis of Compound 11 (Step f, Scheme 3). The pro-
duct from above was redissolved in degassed 1:1 MeOH/water
(10 mL), and degassed 0.03 M NaOH (0.75 mL) was added. The
reaction mixture was stirred for 40 h before being neutralized
with MAC-3 Dowex resin to pH = 5, filtered, and purified by
HPLC: Rf=14.5 (C18 column, 10-85% MeCN in H2O, 30 min)
to afford 11 (1.0 mg, 19% yield from 11a, 13% from 9). The
product was found to be >95% pure as judged by LC/MS and
1
1H NMR: H NMR (500 MHz, D2O)19 δ 7.46 (d, J =9.7 Hz,
1H), 7.38 (d, J=7.4 Hz, 1H), 7.37 (d, J=7.6 Hz, 2H), 7.32 (t, J =
7.4 Hz, 2H), 7.26 (d, J =7.0 Hz, 2H), 7.07 (d, J =8.5 Hz, 2H),
6.87 (d, J =8.6 Hz, 2H), 6.81 (d, J =7.4 Hz, 2H), 5.28 (s, 1H),
5.26(d, J=3.9Hz, 1H), 4.70 (dd, J=5.2, 1.5Hz, 2H), 4.66 (d, J =
6.9 Hz, 2H), 4.62 (d, J =8.0 Hz, 1H), 4.60 (d, J =5.5 Hz, 2H),
4.47 (t, J=7.5 Hz, 2H), 4.16 (t, J=6.1 Hz, 2H), 4.10 (at, J=5.5
Hz, 4H), 4.07 (d, J =8.5 Hz, 2H), 3,48 (m, 1H), 3.42 (dd, J =
10.0, 2.0 Hz, 1H), 3.28 (t, J=8.9 Hz, 1H), 3.20 (dt, J=13.1, 6.4
Hz, 1H), 3.12 (dd, J=13.5, 6.8 Hz, 1H), 2.58 (dd, J=12.1, 4.4
Hz, 1H), 2.31 (m, 1H), 2.24 (s, 3H), 2.05 (s, 3H), 2.04 (s, 3H),
2.01 (s, 3H), 1.87 (t, J=13.2 Hz, 1H), 1.81 (dd, J=6.5 Hz, 1H),
1.42 (d, J =8.5 Hz, 3H), 1.26 (d, J =6.1 Hz, 3H), 1.24 (d, J =
6.6 Hz, 3H), 1.01 (d, J =6.8 Hz, 3H), 0.99 (d, J =6.8 Hz, 3H),
0.94 (d, J= 6.3 Hz, 3H), 0.91 (d, J =6.7 Hz, 3H), 0.89 (d, J =
6.3 Hz, 3H), 0.87 (d, J=6.7 Hz, 3H); exact mass calcd for sodium
salt C134H208N21NaO54S [Mþ2H]2þ 1516.7, [Mþ3H]3þ 1011.5,
C
186H277N21O71 [M þ 2H]2þ 1988.4, [M þ Na þ H]2þ 1999.4,
[Mþ2Na]2þ 2010.4, [Mþ3Na]3þ 1326.0, found 1988.4, 1999.37,
2010.45, 1326.1.
Synthesis of Compound 10 (Step b, Scheme 3). The mixture
from above was concentrated under reduced pressure, redis-
solved in pyridine (1.0 mL), and treated with acetic anhydride
(0.5 mL). After 4 h, the reaction mixture was concentrated and
purified via flash chromatography (silica, 10% MeOH/
CH2Cl2), and the appropriate fractions were concentrated
(Rf 0.2, 10% MeOH/CH2Cl2) to afford 10 mg (quantitative
yield) of product 9a. This material was found to be >85% pure
as judged by reversed-phase LC/ESI analysis: Rf = 17.8 (C4
column, 50-95% MeCN in H2O, 30 min); exact mass calcd for
[M þ CF3CO2
]
- 2- 1572.2, found 1517.7, 1012.3, 1572.9.
Preparation of Conjugate 1 (Step g, Scheme 3). A solution
of sulfo-SMCC (10 mg/mL, 0.10 mL) in 0.1 M sodium
phosphate, 0.9 M NaCl (pH=7.2), was added tothe reconstituted
with water solution of KLH (Aldrich, H7017, 10 mg/mL,
1.0 mL). The resultant solution was stirred for 1 h and then
purified over a G-25 Sephadex column using 0.1 M sodium
phosphate, 0.9 M NaCl, 0.1 M EDTA, pH = 7.2 for elution.
The fractions containing KLH were collected and combined,
givinga total volume of 3.0 mL. Compound11(2mg, 0.665μmol)
in 0.2mLofthe pH=7.2 buffer was treatedwithTCEPgel for 2 h,
filtered, combined with the solution of KLH (0.6 mL), and
reacted under argon for 2 h. The resultant solution was purified
by repetitive centrifugation over a molecular filter (30 kDa cut
off) resulting in ca. 1 mL of the final solution of the vaccine
construct. The degree of the epitope incorporation was estimated
to be 210 epitopes per molecule of KLH using Bradford protein
assay with KLH as a standard and Svennerholm sialic acid assay
to determine the carbohydrate concentration.16,17
C
188H279N21O74 [M þ 2H]2þ 2009.5, [M þ 2Na]2þ 2031.4, [M þ
3H]3þ 1340.0; found 2009.4, 2032.0, 1340.0.
Synthesis of Compound 10 (Step c, Scheme 3). A solution of Pd
(PPh3)4 (14 mg, 0.0125 mmol) and phenylsilane (46 μL, 0.373
mmol) in 3.5 mL of CH2Cl2 was prepared, and 0.35 mL of this
solution was added to the solution of compound 9a (10 mg,
0.00249 mmol) in DMF (0.35 mL). After 30 min, LC/MS
analysis indicated the completion of the reaction. Pyridine
(0.1 mL) was added, and the resultant mixture was concentrated
under vacuum to provide crude product: Rf=16.4 (C4 column,
50-95% MeCN in H2O, 30 min); exact mass calcd for
C
184H275N21O72 [M þ 2H]2þ 1966.9, [M þ Na þ H]2þ 1977.9,
[M þ 2Na]2þ 1988.9, [M þ 3Na]3þ 1311.6, found 1967.5, 1978.6,
1989.7, 1312.6.
Synthesis of Compound 10 (Step d, Scheme 3). The residue was
redissolved in pyridine (0.35 mL) and triethylamine (0.15 mL),
and SAMAOPbf (11.2 mg, 0.0373 mmol) was added to this
solution. The reaction mixture was stirred for 3 h, concentrated,
and purified by flash chromatography (silica, 5% f 10%
MeOH/CH2Cl2), and the appropriate fractions were concen-
trated (Rf 0.3, 10% MeOH/CH2Cl2) to afford product 10
contaminated with SAMAOPbf decomposition products
(70% purity as judged by LC/MS): Rf = 16.5 (C4 column,
50-95% MeCN in H2O, 30 min); exact mass calcd for
Acknowledgment. This work was supported by the Na-
tional Institutes of Health (Grant No. CA28824 to S.J.D.).
P.N. thanks the National Institutes of Health for a Ruth L.
Kirschstein postdoctoral fellowship (CA125934-02), W.H.K.
is grateful for a Korea Research Foundation Grant funded
by the Korean government (KRF-2007-357-c00060), and
D.L. is thankful for support provided by the Terri Brodeur
Breast Cancer Foundation. We thank Yu Rao, Yu Yuan,
and Jennifer Stockdill for fruitful discussions during the
preparation of this manuscript.
C
188H279N21O74S [M þ 2Na]2þ 2026.4, [M þ 3Na]3þ 1351.3,
found 2026.0, 1350.8. This product was advanced to the next
step without further purification.
Synthesis of Compound 11 (Step e, Scheme 3). Phenol (60 mg),
triisopropylsilane (0.15 mL), and water (0.2 mL) were added to
trifluoroacetic acid (3.0 mL). The resultant solution (1.0 mL)
was added to a vial with compound 10 from above (ca. 10 mg,
0.00249 mmol). The reaction mixture was stirred for 40 min
before being diluted with dichloromethane (3 mL), con-
centrated, and purified by reversed-phase HPLC: Rf = 18.7
Supporting Information Available: 1H and 13C NMR
spectra for compounds 4 and 5, LC/MS data for compounds 7,
8a, 8, 9, 10b, 10, 11a, 11, and selected intermediates, and 1H NMR
spectra for compounds 7, 8a, 9, 10b, 11a, and 11. This material is
available free of charge via the Internet at http://pubs.acs.org.
5162 J. Org. Chem. Vol. 74, No. 15, 2009