A chondroitin sulfate small molecule that stimulates neuronal growth

Article abstract of DOI:10.1021/ja0484045

Chondroitin sulfate glycosaminoglycans are sulfated polysaccharides involved in cell division, neuronal development, and spinal cord injury. Here, we report the synthesis and identification of a chondroitin sulfate tetrasaccharide that stimulates the grow

Full text of DOI:10.1021/ja0484045

Published on Web 06/02/2004
A Chondroitin Sulfate Small Molecule that Stimulates Neuronal Growth
Sarah E. Tully, Ross Mabon, Cristal I. Gama, Sherry M. Tsai, Xuewei Liu, and
Linda C. Hsieh-Wilson*
DiVision of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125
Received March 19, 2004; E-mail: lhw@caltech.edu
Chondroitin sulfate glycosaminoglycans are sulfated polysac-
charides implicated in cell division, neuronal development, and
spinal cord injury.1 While considerable attention has been focused
on heparan sulfate glycosaminoglycans, much less is known about
the chondroitin sulfate (CS) class. As with all glycosaminoglycans,
the complexity and heterogeneity of CS have hampered efforts to
understand its precise biological roles. For instance, CS has been
shown to prevent the growth of axons; yet it is also found in
developing, growth-permissive regions.1c,2 Synthetic access to CS
molecules of defined length and sulfation pattern, in combination
with biological studies, should enable a systematic examination of
structure-activity relationships. Toward this end, we report the
synthesis and identification of a CS tetrasaccharide that promotes
neuronal outgrowth. These studies represent the first biological
investigations of synthetic CS molecules and define a tetrasaccharide
as a minimal motif required for activity.
CS polysaccharides have been shown both to stimulate and to
attenuate the growth of cultured neurons.3,4 Notably, the molecules
used in those studies were ∼200 saccharides in length, poorly
defined, and heterogeneously sulfated, features that might account
for the contradictory observations. Among the sulfation patterns
implicated in the modulation of cell growth is the disulfated CS-E
motif (Figure 1). To investigate the biological properties of CS-E
and establish the minimal structural determinants for activity, we
sought to synthesize di- and tetrasaccharides bearing this sulfation
pattern.
We envisioned a convergent strategy to access various CS-E
molecules from a single disaccharide building block, 4. The sulfated
4- and 6-hydroxyls of D-galactosamine were masked with a
p-methoxybenzylidene acetal. This group was chosen with future
access to other sulfation motifs (e.g., CS-A, CS-C)5 and extension
to solid-phase methodologies in mind. In particular, oxidative
removal using DDQ6 or regioselective opening of the acetal ring7
was anticipated to permit selective deprotection of either or both
the 4- and 6-hydroxyls and circumvent the need for hydrogenolysis.
The orthogonal tert-butyldimethylsilyl group was installed at the
C-4 position on the nonreducing end of 4 to facilitate chain
elongation. To achieve stereoselective formation of â-glycosidic
linkages, we exploited well-precedented N-trichloroacetyl and
benzoyl participating groups.8 Finally, the anomeric hydroxyl of 4
was masked with an allyl group, which could be converted to
activated glycosyl donors and offers a convenient means to
conjugate CS to small molecules, proteins, or surfaces.
Figure 1. Chondroitin sulfate molecules 1-3 and retrosynthetic analysis.
conventional methods such as PdCl2, Pd(PPh3)4, [Ir(COD)(PMePh2)2]-
PF6, and Wilkinson’s catalyst did not yield the desired isomeriza-
tion, presumably due to interference by the neighboring trichloro-
acetamide.12 Fortunately, treatment with Grubbs’ second-generation
catalyst13 in the presence of H2 led to the desired outcome.
Hydrolysis of the enol ether and conversion to trichloroacetimidate
7 proceeded smoothly under standard conditions.14 Disaccharide 4
also provided ready access to glycosyl acceptor 8 via desilylation.
Subsequent glycosylation of 7 and 8 delivered the desired tetrasac-
charide with good stereoselectivity.
With the fully protected di- and tetrasaccharides in hand, we
embarked on the final deprotection-sulfation steps. Radical-
mediated conversion of the N-trichloroacetyl group to N-acetyl8
followed by oxidative cleavage of the p-methoxybenzylidene acetal6
afforded 9 and 10. Despite an earlier report suggesting that
simultaneous sulfation of the C-4 and C-6 hydroxyls might be chal-
lenging,7a treatment of 9 and 10 with SO3‚trimethylamine complex
in DMF delivered the sulfated compounds in 67% and 93% yields,
respectively. The target CS-E di- and tetrasaccharides 1 and 2 were
successfully obtained after silyl deprotection and saponification
utilizing NaOH or sequential LiOOH-NaOH treatment15 to mini-
mize â-elimination at the C-4 position. Deprotection of 9 under
similar conditions furnished the unsulfated tetrasaccharide 3.
To explore the ability of 1-3 to modulate neuronal growth,
primary hippocampal neurons were cultured on poly-DL-ornithine-
coated coverslips with or without each compound. After 48 h, the
neurons were fixed, immunostained with anti-tau antibodies, and
examined by confocal fluorescence microscopy. Sulfated tetrasac-
charide 2 exhibited striking effects on neuronal morphology and
growth (Figure 2). The number of neurites emanating from the cell
body was enhanced, and the growth of the major extension was
dramatically stimulated by 39.3 ( 3.6% relative to the poly-DL-
ornithine control. In contrast, sulfated disaccharide 1 and unsulfated
tetrasaccharide 3 had no significant effect on neuronal outgrowth.
These results indicate that a tetrasaccharide represents a minimum
structural motif and that sulfation is critical for CS activity.
The observed effects of 2 support previous studies implicating
the CS-E motif in the growth and development of neurons. For
example, CS-E is found on the protein appican, an isoform of the
The synthesis of the target compounds is illustrated in Scheme
1. Monosaccharides 5 and 6 were generated from known p-tolyl-
1-thio-â-D-glucopyranose9 or 1,3,4,6-tetra-O-acetyl-2-azido-2-
deoxy-D-galactopyranose,10 respectively (Supporting Information).
Coupling of 5 with 6 using the trichloroacetimidate procedure11
afforded exclusively the â-linked disaccharide 4 in 74% yield. At
this stage, activation of the disaccharide was envisioned to proceed
through conversion of the C-1 allyl group to the lactol. However,
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J. AM. CHEM. SOC. 2004, 126, 7736-7737
10.1021/ja0484045 CCC: $27.50 © 2004 American Chemical Society

C O M M U N I C A T I O N S
Scheme 1. Synthesis of 1-3a
a Conditions: (a) TMSOTf, CH2Cl2, -40 f -15 °C, 74%. (b) RuCl2(dCHPh)(PCy3)L, L ) 1,3-dimesityl-4,5-dihydroimidazolylidene (20 mol %),
CH2Cl2, H2, 77%. (c) I2, H2O, pyr/THF, 81%. (d) DBU, CCl3CN, CH2Cl2, 90%. (e) HF‚pyr, pyr/THF, 0 °C, 85%. (f) TMSOTf, CH2Cl2, -15 °C, 31%. (g)
TBTH, AIBN, DMA/benzene, 25 f 80 °C, 85%. (h) DDQ, H2O/CH2Cl2, 75%. (i) SO3‚Me3N, DMF, 50 °C, 67%. (j) HF‚pyr, pyr/THF/H2O, 0 °C. (k) LiOH,
H2O2, THF/H2O, then NaOH, MeOH/H2O, 25% over three steps. (l) TBTH, AIBN, benzene, 25 f 80 °C, 85%. (m) DDQ, H2O/CH2Cl2, 62%. (n) SO3‚Me3N,
DMF, 50 °C, 93%. (o) HF‚pyr, pyr/THF/H2O, 0 °C. (p) NaOH, MeOH/H2O, 55% over two steps. (q) HF‚pyr, pyr/THF/H2O, 0 °C. (r) LiOH, H2O2, THF/
H2O, then NaOH, MeOH/H2O, 52% over three steps.
Caltech for instrumentation. We also thank J. Chen, J. Ho, B. Ling,
and C. Wang for experimental assistance. This research was
supported by the Beckman Young Investigator Program, the Human
Frontier Science Program, and the Alfred P. Sloan Foundation.
Note Added after ASAP Posting. After this paper was posted
ASAP on 06/02/2004, a correction was made to structure 10 in
Scheme 1. The corrected version was posted 06/04/2004.
Supporting Information Available: Syntheses and experimental
procedures. This material is available free of charge via the Internet at
http://pubs.acs.org.
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Figure 2. CS-E tetrasaccharide 2 stimulates the outgrowth of hippocampal
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Acknowledgment. We thank Dr. R. H. Grubbs for the catalyst
and helpful discussions and the Biological Imaging Center at
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