Synthesis of O-Glycolyl-Linked Neuraminic Acids through a Spirocyclic Intermediate

Article abstract of DOI:10.1021/ol026317g

(Equation Presented) The neuraminic acid derivative 5 is readily converted in several steps to the neuraminic acid dimer 12, linked through the hydroxyl of a 5-N-glycolyl group in an α-2,5 glycosidic linkage. The sequence is shown to proceed through a spi

Full text of DOI:10.1021/ol026317g

ORGANIC
LETTERS
2
002
Vol. 4, No. 18
067-3069
Synthesis of O-Glycolyl-Linked
Neuraminic Acids through a Spirocyclic
Intermediate
3
c
Joseph C. M Auliffe, David Rabuka, and Ole Hindsgaul*
The Burnham Institute, 10901 North Torrey Pines Road, San Diego, California 92037
ole.hindsgaul@ualberta.ca
Received June 6, 2002
ABSTRACT
The neuraminic acid derivative 5 is readily converted in several steps to the neuraminic acid dimer 12, linked through the hydroxyl of a
-N-glycolyl group in an r-2,5 glycosidic linkage. The sequence is shown to proceed through a spirocyclic intermediate 9 by in situ NMR
5
experiments. Similar derivatives of N-glycolylneuraminic acid (Neu5Gc), including polymers, have been identified from marine sources, including
starfish and sea urchins, often as sulfated derivatives and are thought to mediate sperm−egg recognition.
O-Glycolyl-linked oligosialic acids are novel carbohydrate
the dimer and trimer, are produced by the starfish Asterias
rubens as their 8-O-methylated derivatives.
3
polymers initially identified as components of glycoproteins
1
associated with the egg jelly coat of sea urchins. The
Despite the unique structures of these molecules, little
effort has been directed toward their chemical synthesis. This
challenge led us to examine methods for the assembly of
O-glycolyl-linked sialic acids that could be readily extended
to the synthesis of oligomers. Rather than employing a
glycosylation-based approach for the assembly of the O-
glycolyl-linkage, we adapted a strategy first developed by
Gervay et al. for the synthesis of neuraminic acid oligomers
repeating unit 1 (Figure 1) consists of a 5-N-glycolyl-
4
linked through the C-1 carboxylate group.
(1) Kitazume, S.; Kitajima, K.; Inoue, S.; Troy, F. A.; Cho, J.-W.;
Lennarz, W. J.; Inoue, Y. J. Biol. Chem. 1994, 269, 22712-22718. (b)
Karamanos, N. K.; Manouras, A.; Anagnostides, S.;. Makatsori, E.;
Tsegenidis, T.; Antonopoulos, C. A. Biochimie 1996, 78, 171-182. (c)
Kitazume, S.; Kitajima, K.; Inoue, S.; Haslam, S. M.; Morris, H. R.; Dell,
A.; Lennarz, W. J.; Inoue, Y. J. Biol. Chem. 1996, 271, 6694-6701.
Figure 1. Repeating unit of poly-N-glycolylneuraminic acid.
(
2) Kitazume-Kawaguchi, S.; Inoue, S.; Inoue, Y.; Lennarz, W. J. Proc.
Natl. Acad. Sci. 1997, 94, 3650-3655.
3) (a) Bergwerff, A. A.; Hulleman, S. H. D.; Kamerling, J. P.;
neuraminic acid unit (Neu5Gc) linked to adjacent sugars
through the glycolyl hydroxyl in an R-2,5 glycosidic linkage.
Subsequent studies have suggested that 9-O-sulfated Neu5Gc
oligomers are involved in sperm-egg recognition in a
(
Vliegenthart, J. F. G.; Shaw, L.; Reuter, G.; Schauer, R. In Polysialic Acid;
Roth, J., Rutishauser, U., Troy, F., II, Eds.; Birkhaeuser: Basel, 1993; pp
2
01-272. (b) Kitazume-Kawaguchi, S. Trends Glycosci. Glycotechnol.
2
number of sea urchin species. Smaller oligomers, including
1998, 10, 383-392.
1
0.1021/ol026317g CCC: $22.00 © 2002 American Chemical Society
Published on Web 08/16/2002

Scheme 1. Strategy for the Assembly of the Neu5Gc-R2,5-Oglycolyl-Neu5Gc Linkage
A similar approach was recently applied by Ren et al. and
led to the synthesis of a Neu5GcR2,5-OglycolylNeu5Gc dimer.
We now describe the synthesis of a novel spirocyclic
sialoside intermediate 9, which undergoes a regioselective
ring opening with a minimally protected neuraminic acid
derivative to give an R-2,5-linked Neu5Gc dimer set up for
further extension (Scheme 1).
aqueous methanol over 2 h, followed by acid resin, gave
5
the diacid 4 in near quantitative yield. Further treatment of
compound 4 with 2 M NaOH at 95 °C gave the amino acid
5 (89%) following neutralization with acid resin and
purification on Sephadex LH-20. The amino acid 5 was
subsequently converted to both the N-Fmoc 6 and methyl
ester 7 derivatives (Scheme 3).
A direct route to pure R-glycolyl sialosides was crucial to
our approach. Previous studies have shown that high yields
of simple aliphatic R-sialosides could be obtained from
treatment of sialyl donors with lower alcohols in the presence
Scheme 3a
6
of insoluble promoters such as silver zeolite. In our hands,
conversion of N-acetylneuraminic acid to the 2-chloro
7
derivative 2, followed by treatment with methyl glycolate
and silver zeolite in toluene, gave the R-glycolyl glycoside
3
in 69% overall yield. Stereoselectivity was judged as being
better than 15:1 in favor of the R-anomer (Scheme 2).
Scheme 2a
a
O then H+ resin,
Reaction conditions: (i) NaOH, MeOH/H
2
+
9
3%. (ii) 2 M NaOH, 95 °C, 10 h then H resin, 89%. (iii) HCl/
MeOH (1:99), 12 h, 71%. (iv) Fmoc-Cl, NaHCO
then H resin, 51%.
3
, dioxane/H
2
O,
+
1
The H NMR spectrum of the diacid 4 in DMF-d
7
contained two doublets at δ 4.24 and 4.35 with a coupling
constant of 16.1 Hz corresponding to the glycolyl methylene
protons. Upon addition of diisopropylcarbodiimide (DIC) (1.2
equiv) to the NMR tube, these signals were replaced by a
singlet at δ 4.94, attributed to the formation of a spirocyclic
anhydride 8 (Scheme 4). A similar experiment was performed
in acetonitrile (ACN) and monitored by electrospray MS in
the negative mode. Addition of N-(3-dimethylaminopropyl)-
N′-ethyl carbodiimide (EDAC) to a solution of 4 (m/z 367.1)
in ACN resulted in the appearance of new signals at m/z
348.1 and 384.0 corresponding to the expected values for
a
+
Reaction conditions: (i) MeOH, H . (ii) AcOH, AcCl. (iii) Me
glycolate, Ag zeolite, toluene, rt, 30 h, 69% over three steps.
We next directed our attention to the synthesis of a suitably
protected intermediate that could act as both acceptor and
donor. Thus, treatment of compound 3 with 1 M NaOH in
(4) (a) Gervay, J.; Flaherty, T. M.; Nguyen, C. Tetrahedron Lett. 1997,
3
6
8, 1493-1496. (b) Ramamoorthy, P. S.; Gervay, J. J. Org. Chem. 1997,
+ -
- -
[M - H ] and [M + Cl ] of the anhydride 8.
2, 7801-7805.
The anhydride 8 was also observed following treatment
(
5) Ren, C.-T.; Chen, C.-S.; Wu, S.-H. J. Org. Chem. 2002, 67, 1376-
1
2
379.
of 4 with a resin-bound carbodiimide (NovaBiochem) and
showed no signs of decomposition after standing at room
temperature for 30 min. Addition of benzylamine (1.2 equiv)
(
50.
6) Ossowski, P.; Garegg, P. J. Acta Chem. Scand. 1983, B 37, 249-
(7) Roy, R.; Laferreire, C. Can. J. Chem. 1990, 68, 2045-2054.
3068
Org. Lett., Vol. 4, No. 18, 2002

Scheme 4a
Scheme 5a
a
Reaction conditions: (i) pyBOP, DIEA, NMP, 3 h, 57%. (ii)
MeI, DIEA, DMF, 24 h, 62%
a
2
Reaction conditions: (i) DIC, DMF. (ii) BnNH (1.2 equiv), 5
min.
assignment, so this compound was further converted to the
1
methyl ester 13 with methyl iodide and DIEA. The H NMR
rapidly (ca. 2-3 min) produced the benzyl amide 10, derived
from attack at the less hindered glycolyl carbonyl. This
assignment was confirmed by an examination of long-range
carbon-proton coupling through the acquisition an HMBC
spectrum. The glycolyl carbonyl signal at δ 172.3 correlated
to the glycolyl methylene protons at δ 4.37 and 4.23, as well
as the benzyl methylene protons at δ 4.42.
spectrum of compound 13 exhibited singlets at δ 3.83 and
3.81, corresponding to methoxyl groups at C-1 and C-1′, in
addition to a singlet at δ 3.70 arising from the glycolyl
methyl ester functionality at C-2. This evidence, combined
with the outcome of the model experiments and comparison
3
a,5
to published spectra of similar compounds,
led us to
conclude that the ring opening of the spirocyclic anhydride
9 occurred preferentially through attack at the less hindered
glycolyl carbonyl functionality.
In conclusion, we have successfully demonstrated the
formation of the naturally occurring Neu5GcR2,5-Oglycolyl-
Repetition of this sequence with the N-Fmoc diacid 6
resulted in a similar outcome, producing a compound upon
treatment with carbodiimide resin that was identified as the
8
spirocyclic anhydride 9 on the basis of NMR spectra.
Treatment of the anhydride 9 with a slight excess of
benzylamine gave the benzyl amide 11 in good yield in under
Neu5Gc linkage by an efficient procedure that should be
amenable to solid-phase synthesis and the construction of
1
0
5
min. Selectivity for the desired regioisomer was judged as
higher oligomers. In addition, the spirocyclic sialosides 8
and 9 derived from the amino acid 5 could serve as versatile
reagents for the formation of sialoconjugates under very mild
conditions.
being 8:1 or better from the HMBC NMR spectrum. In this
case, the signal at δ 172.1 (glycolyl CONH) was found to
correlate to signals at δ 4.34 and 4.12 (glycolyl CH
.42 (benzyl CH ).
Having obtained favorable results in the above model
2
) and δ
4
2
Acknowledgment. This work was supported in part by
a grant from the National Institutes of Health.
systems, we turned our attention to the synthesis of O-
glycolyl-linked sialyl dimers. Treatment of the diacid 6 with
a slight excess of a coupling reagent was followed after 2
min with a solution of the amine 7. Best results were obtained
with the coupling agent pyBOP and diisopropylethylamine
Supporting Information Available: Experimental and
analytical details for compounds 3-13, including NMR and
mass spectra. This material is available free of charge via
the Internet at http://pubs.acs.org.
(
DIEA) in N-methylpyrrolidone (NMP). Solid-phase extrac-
tion followed by chromatography on silica gel gave the dimer
2 in 57% yield. In all instances the desired product was
OL026317G
1
1
(
9) Analytical data for compound 12 H NMR (300 MHz, CD3OD): δ
formed swiftly with little evidence of the unwanted regio-
7
.26-7.80 (m, 8H, ArH); 4.41, 4.31 (AB pattern, Jgem ) 16.5 Hz, OCH2-
9
isomer.
CO2Me); 4.32, 4.16 (AB pattern, Jgem ) 16.4 Hz, OCH2CONH); 3.81, 3.70
In this case, the complexity of the HMBC NMR spectrum
of compound 12 did not allow unambiguous structural
(2s, 6H, CO2Me); 3.48-3.94 (m, 12H); 3.10-3.24 (m, 2H), 2.76, 2.84 (2dd,
+
2
H-3ax); 1.74 (bt, 2H, H-3eq). Found, m/z 905.3; calcd, 905.4 [M + Na] .
1
For compound 13 H NMR (300 MHz, CD3OD): δ 7.24-7.80 (m, 8H,
ArH); 4.39, 4.31 (AB pattern, Jgem ) 16.2 Hz, OCH2CO2Me); 4.35, 4.05
(AB pattern, Jgem ) 15.8 Hz, OCH2CONH); 3.83, 3.81 (2s, 6H, CO2Me);
3.70 (s, 3H, glycolyl CO2Me); 3.45-3.90 (m, 14H); 2.68, 2.76 (2dd, 2H-
3ax); 1.75, 1.92 (2dd, 2H, H-3eq). Found, m/z 919.2; calcd, 919.5 [M +
1
1
(
8) H NMR for compound 8 H NMR (300 MHz, DMF-d7): δ 8.13 (d,
NH); 4.94 (s, 2H, OCH2CO); 2.58 (dd, J3,3 ) 12 Hz, J3,4 ) 5 Hz, H-3ax);
.97 (s, 3H, COMe); 1.72 (dd, J3,4 ) 10 Hz, H-3eq). For compound 9 (300
1
+
MHz, DMF-d7): δ 7.27-7.98 (m, 9H, ArH, NH); 4.93 (s, 2H, OCH2CO);
.24-4.36 (m, 3H); 3.40-3.95 (m, 7H); 2.59 (dd, J3,3 ) 13.4 Hz, J3,4 )
.4 Hz, H-3ax); 1.81 (dd, J3,4 ) 11.2 Hz, H-3eq).
Na] .
4
5
(10) Szabo, L.; Smith, B. L.; McReynolds, K. D.; Parrill, A. L.; Morris,
E. R.; Gervay, J. J. Org. Chem. 1998, 63, 1074-1078.
Org. Lett., Vol. 4, No. 18, 2002
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