A new homobifunctional p-nitro phenyl ester coupling reagent for the preparation of neoglycoproteins

Article abstract of DOI:10.1021/ol048614m

(Chemical equation presented) A new linker system has been designed and applied to neoglycoprotein synthesis. Reaction of oligosaccharide ω-aminoalkyl glycosides with homobifunctional adipic acid p-nitrophenyl diesters in dry DMF gave the corresponding am

Full text of DOI:10.1021/ol048614m

ORGANIC
LETTERS
2004
Vol. 6, No. 24
4407-4410
A New Homobifunctional p-Nitro Phenyl
Ester Coupling Reagent for the
Preparation of Neoglycoproteins
Xiangyang Wu, Chang-Chun Ling, and David R. Bundle*
Alberta Ingenuity Centre for Carbohydrate Science, Department of Chemistry,
UniVersity of Alberta, Edmonton, AB T6G 2G2 Canada
daVe.bundle@ualberta.ca
Received July 19, 2004
ABSTRACT
A new linker system has been designed and applied to neoglycoprotein synthesis. Reaction of oligosaccharide
ω
-aminoalkyl glycosides with
homobifunctional adipic acid p-nitrophenyl diesters in dry DMF gave the corresponding amide half ester in good yields and of sufficient
stability to permit chromatographic purification. Subsequent conjugation with bovine serum albumin under very mild conditions afforded the
corresponding neoglycoproteins with good efficiency. The method is well suited for the coupling of very small amounts (mg) of oligosaccharide
and protein.
Oligosaccharides covalently attached to proteins, insoluble
matrixes, and surfaces find applications as vaccines,1 antigens
in solid-phase assays,2 and immunoadsorption columns3 and
in biosensors.4 A wide variety of conjugation reactions5 have
been employed to effect the covalent linkage of oligosac-
charides to proteins and solid supports, and the majority of
these methods rely upon the use of a large excess of
derivatized oligosaccharide to achieve acceptable degrees of
substitution.6,7 Since most complex oligosaccharides are
derived from challenging multistep syntheses, conjugation
methods that achieve modest yields are wasteful of precious
oligosaccharide sample. Lemieux introduced a very effective
method based on acyl azide conjugation methodology, but
this approach requires the construction of complex oligosac-
charide on a nine-carbon linker arm that is most often carried
through the entire synthetic scheme, thereby imposing
orthogonal protection strategies that can become limiting.8
Synthesis of complex oligosaccharides as simple allyl or
pentenyl glycosides affords oligosaccharide final products
that can be easily derivatized for different applications.
Consequently, there remains a need for improved coupling
reactions that would ideally proceed in near quantitative yield
with oligosaccharide glycosides of this type.
(1) (a) Sabbatini, P. J.; Kudryashov, V.; Ragupathi, G.; Danishefsky, S.
J.; Livingston, P. O.; Bornmann, W.; Spassova, M.; zatorski, A.; Spriggs,
D.; Aghajanian, C.: Soignet, S.; Peyton.; O’Flaherty, C.; Curtin, J.; O.
Lloyd, K. O. Int. J. Cancer 2000, 87, 79. (b) Kuduk, S. D.; Shwarz, J. B.;
Chen X. T.; Glunz, P. W.; Sames, D.; Ragupathi, G.; Livingston, P. O.;
Danishefsky, J. S. J. Am. Chem. Soc. 1998, 120, 12474. (c) Danishefsky,
S. J.; Allen J. R. Angew. Chem., Int. Ed. 2000, 39, 836.
(2) Houseman, B. T.; Mrksich, M. Chem. Biol. 2002, 9, 443. (b) Wang,
D.; Liu, S.; Trummer, B. J.; Deng, C.; Wang, A. Nat. Biotechnol. 2002,
20, 275. (c) Bryan, M. C.; Plettenburg, O.; Sears, P.; Rabuka, D.;
Wacowichsgarbi, S.; Wong, C. H. Chem. Biol. 2002, 9, 713.
(3) Mendez, R.; Sakhrani, L.; Aswad, S.; Minasian, R.; Obispo, E.;
Mendez, R. G.; Transplant Proc. 1992, 24, 1738. (b) Bensinger, W. I.;
Baker, D. A.; Buckner, C. D.; Clift, R. A.; Thomas, E. D. Transfusion 1981,
21, 335. (c) Andersen, S. M.; Ling, C. C.; Zhang, P.; Townson, K.; Willison,
H. J.; Bundle, D. R. Org. Biomol. Chem. 2004, 2, 1199.
One of the most efficient coupling methods involves the
use of the homobifunctional reagent, diethyl squarate,9-11
(6) Gray, G. R. Arch. Biochem. Biophys. 1974, 163, 426-428.
(7) Nilsson, U. J.; Heerze, L. D.; Liu, Y.-C.; Armstrong, G. D.; Palcic,
M. M.; Hindsgaul, O. Bioconjugate Chem. 1997, 8, 466.
(8) Hindsgaul, O.; Norberg, T.; Le Pendu, J.; Lemieux, R. U. Carbohydr.
Res. 1982, 109, 109.
(4) Mrksich, M. Chem. Soc. ReV. 2000, 29, 267-273.
(5) Stowel, C. P.; Lee, Y. C. AdV. Carbohydr. Chem. Biochem. 1980,
37, 255.
10.1021/ol048614m CCC: $27.50
© 2004 American Chemical Society
Published on Web 10/26/2004

which affords reproducible conjugation in high yields under
mild conditions with small amounts of oligosaccharide and
protein at low concentrations. However, its use in conjugate
vaccine applications has been correlated with a reduced
immune response to the oligosaccharide epitope12 and with
potential immune response to the squarate residue itself.13
Reductive amination is a favored method employed in the
preparation of conjugate vaccines but suffers from the major
drawback of the large excess of oligosaccharide that is
required to achieve respectable degrees of conjugation.14
Homobifunctional succinimide esters, while commercially
available and very effective for amide bond formation, are
too reactive to permit chromatographic workup of the
oligosaccharide half ester intermediate. We conclude on the
basis of our data15 as well as recent reports12,16 that the
development of an aliphatic straight-chain coupling reagent
is highly desirable for vaccine applications.
would modulate this reactivity to acceptable levels, while
preserving sufficient reactivity to permit efficient coupling
with free amino groups present in the target proteins.17 The
literature describing the use of 1 was consistent with this
expectation. Although it has not been used to create
glycoconjugates, 1 has been used to effectively create
bivalent ligands18 and cyclic peptides19 and as a bivalent
affinity labeling reagent for antibody specific for the nitro-
phenyl hapten.20
To confirm the feasibility of this strategy, ω-aminoalkyl
glycosides (I-IV in Figure 1) were employed. 6-Amino-
Here we apply a simple, efficient, bifunctional linker for
the preparation of neoglycoproteins that fulfils the following
demands: (i) chemoselective reaction of the linker with an
oligosaccharide ω-aminoalkyl glycoside without affecting
unprotected hydroxy groups; (ii) the activated intermediate
(half ester) should be sufficiently stable to permit purification
of the activated oligosaccharide; (iii) coupling of the purified,
activated intermediate with proteins such as bovine serum
albumin (BSA) should proceed with good to high efficiency.
To achieve this objective we selected the homobifunctional
reagent, adipate 4-nitrophenyl diester 1, which was readily
synthesized from commercially available adipoyl chloride
2. Reaction of 2 with 4-nitrophenol 3 in pyridine gave 1 in
85% yield, as shown in Scheme 1. The corresponding
Figure 1. Oligosaccharide amines I-IV.
hexyl-â-D-glucopyranoside I,21 6-aminohexyl-â-D-galacto-
pyranoside II,21 and 6-aminohexyl-4-O-(â-D-galactopyranosyl)-
â-D-glucopyranoside III22 were synthesized according to
published procedures.
6-Aminohexyl 2-acetamido-2-deoxy-â-D-galactopyrano-
side IV was synthesized from 6-azido alcohol 4, prepared
in 96% yield by substitution of 6-bromo-hexanol 5. Glyco-
sylation of 4 with the 3,4,6-tri-O-acetyl-2-deoxy-2-phthal-
imido-â-D-galactopyranosyl trichloroacetimidate 623 pro-
moted by trimethylsilyl trifluoromethane sulfonate (TMSOTf)
in dichloromethane afforded the intermediate glycoside 7 in
excellent yield (93%). Treatment of 7 in methylamine (33%
in ethanol) overnight, followed by selective acetylation with
acetic anhydride in dry methanol, gave compound 8 in 97%
yield, which was hydrogenated to afford free amine IV in
70% yield, as shown in Scheme 2.
Scheme 1. Synthesis of Homobifunctional Linker 1
The synthetic mono- or disaccharides used in this paper
contained the 6-aminohexyl aglycone employed by Andersen
(15) Lemieux, R. U.; Bundle, D. R.; Baker, D. A. J. Am. Chem. Soc.
1975, 97, 4076. Pinto, B. M.; Bundle, D. R. Carbohydr. Res. 1983, 124,
313.
(16) Buskas, T.; Li, Y.; Boons, G.-J. Chem. Eur. J. 2004, 10, 3517.
(17) Mukerjee, H.; Pal, P. R. J. Org. Chem. 1970, 35, 2042.
(18) Graminski, G. F.; Carlson, C. L.; Ziemer, J. R.; Cai, F.; Vermeulen,
N. M. J.; Scott, M.; Burns, M. R. Bioorg. Med. Chem. Lett. 2002, 12, 35.
(19) Kanaoka, Y.; Okumura, K.; Itoh, H.; Hatanaka, Y.; Tanizawa, K.
Peptide Chem. 1984, 221.
(20) Plotz, P. H.; Kimberly, R. P.; Guyer, R. L.; Segal, D. M. Mol.
Immunol. 1979, 16, 721.
(21) Paul, H. W.; Makoto, N.; Saul, C. L.; Yuan, C. L. Carbohydr. Res.
1979, 70, 83.
N-succiminide diester was found to be too reactive to allow
purification of the activated oligosaccharide, but we reasoned
that the electron-withdrawing effect of the 4-nitrophenyl ester
(9) Tietze L.; Arlt, M.; Beller, M.; Glusenkamp, K. H.; Jahde, E.;
Rajewsky, M. F. Chem. Ber. 1991, 124, 1215.
(10) Kamath, P.; Diedrich, P.; Hindsgaul, O. Glycoconjugate J. 1996,
13, 315.
(11) Nitz, M.; Bundle, D. R. J. Org. Chem. 2001, 66, 8411.
(12) Mawas, F.; Niggemann, J.; Jones, C.; Corbel, M. J.; Kamerling, J.
P.; Vliegenthart, Johannes F. G. Infect. Immun. 2002, 70, 5107.
(13) (a) Nitz, M. Ph.D. Thesis, University of Alberta, Edmonton, Canada,
2002.
(14) Pozsgay V.; Chu, C.; Pannell, L.; Wolfe, J.; Robbins, J. B.;
Schneerson, R. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 5194-5197.
(22) Limberg, G.; Slim, G. C.; Compston, C. A.; Stangier, p.; Palcic,
M. M.; Furneaux, R. H. Liebigs. Ann. Org. Bioorg. Chem. 1996, 1773.
(23) Seeventer, P. B.; Corsten, M. A.; Sanders, M. P.; Kamerling, J. P.;
Vliegenthart, J. F. G. Carbohydr. Res. 1997, 299, 171.
4408
Org. Lett., Vol. 6, No. 24, 2004

reaction is readily monitored by TLC or by UV spectroscopy,
and the half esters are stable to both silica gel chromatograph
and reverse-phase isolation under acidic conditions. Excess
linker could be removed by washing with dichloromethane,
and the yields of this reaction were in the range of 65-
82%. The half esters were then coupled to BSA by 18 h
incubation in buffer (PH ) 7.5) at ambient temperature. The
BSA conjugates were obtained as white powders after
dialysis against deionized water and then freeze-dried.
Scheme 2. Synthesis of Compound IV
The degree of incorporation of the oligosaccharides on
BSA was established by MALDI-TOF MS using sinapinic
acid as the matrix. The mass spectra (positive ion mode)
obtained are shown in Figure 2. The spectrum obtained for
et al. as a linking arm for the attachment to proteins and
sepharose gel.3c Previously, coupling of such compounds to
bovine serum albumin (BSA) protein was accomplished
through a squarate linker.7-9 Diethyl squarate, 3,4-diethoxy-
3-cyclobutene-1,2-dione, conveniently permits reaction with
the tether amino group in methanol or aqueous carbonate
solution to afford the corresponding squarate half esters,
which were subsequently coupled to BSA through the
terminal amine group of exposed lysine residues of the
protein. Under these conditions the percentage of the
oligosaccharide attached to BSA varied in the range 50-
80%. In the present method, the oligosaccharide amines were
treated with 5 equiv of homobifunctional linker in dry DMF
at room temperature for 5 h, affording the corresponding
half esters 9-12 in good yields, as shown in Table 1. The
Figure 2. MALDI-TOF spectra of (A) BSA calibration standard,
(B) lactose-BSA (n ) 8.9), (C) glucose-BSA (n ) 12.6), (D)
galactose-BSA (n ) 9.6), and (E) GalNAc-BSA (10.6).
Table 1. Synthesis of Half Esters 9-12
BSA itself (panel A) shows a sharp peak for the singly
charged protein (m/z 66478). Panels B-E (Figure 2) show
the corresponding MALDI-TOF mass spectra obtained from
the neoglycoprotein. The peaks become broader compared
with that of BSA, because each neoglycoprotein sample is
made up of a collection of oligosaccharide-protein conju-
gates with a range of different incorporation values centered
around the average. The average molecular weight and this
distribution are obtained from the spectra in Figure 2. This
average molecular weight allows the calculation of the
average number of oligosaccharide residues attached to BSA.
Table 2 summarizes the results obtained for the preparation
of neoglycoproteins. When a ligand/protein ratio of 10 was
employed, the average number of ligands per BSA ranged
from 4.8 to 7.5 as determined by MALDI-TOF-MS. When
this ratio was increased to 20/1, the average number of ligand
per BSA increased to 8.9-12.6, and when this ratio was
Org. Lett., Vol. 6, No. 24, 2004
4409

Table 2. Summary of the Coupling Efficiency to BSA
monoester
concentration
(µM)
BSA
concentration
(µM)
incorporation
efficiency
(%)
saccharide
(mg)
molar ratio of
BSA:monoester
hapten
incorporated
product
Lac-BSA
1
2
3
1:10
1:20
1:30
1:10
1:20
1:30
1:10
1:20
1:30
1:10
1:20
1:30
724
1448
2172
756
1512
2268
756
1512
2268
790
1580
2370
72.4
72.4
72.4
75.6
75.6
75.6
75.6
75.6
75.6
79.0
79.0
79.0
5
9
16
7
13
19
5
10
13
5
50
45
53
70
65
63
50
50
43
50
50
50
Gal-BSA
0.8
1.6
2.4
0.8
1.6
2.4
0.9
1.8
1.7
Glc-BSA
GalNAc-BSA
10
15
increased to 30/1, the average number of ligands per BSA
increased to 14.8-18.4.
Acknowledgment. This work was funded by the Alberta
Ingenuity Centre for Carbohydrate Science and the Canadian
Institutes of Health Research (CIHR).
In summary, a novel, simple, linear homobifunctional
linker has been developed for coupling 6-aminohexyl gly-
cosides to BSA with high efficiency under very mild
conditions. The method is also suitable for coupling of very
small amounts of oligosaccharide. The strong UV absorption
of the intermediates I-IV allows ready monitoring of the
purification by chromatography. Preliminary data show that
this linker is also suitable for coupling larger oligosaccharides
to BSA.
Supporting Information Available: Experimental pro-
cedures and 1H NMR and other spectral data. This material
is available free of charge via the Internet at http://pubs.acs.org.
OL048614M
4410
Org. Lett., Vol. 6, No. 24, 2004