Structurally diverse disaccharide analogs of antifreeze glycoproteins and their ability to inhibit ice recrystallization

Article abstract of DOI:10.1016/j.bmcl.2011.12.097

The β-D-galactosyl-(1,3)-α-N-acetyl-D-galactosamine disaccharide is present in antifreeze glycoproteins (AFGPs). Analogs of this disaccharide including the β-linked (1,3)-, (1,4)-, and (1,6)-galactosyl-N-acetyl galactosamine and the β-(1,3)-galactosyl-galactoside were synthesized and evaluated for ice recrystallization inhibition (IRI) activity. The results from this study demonstrate that the b-linked-(1,4) disaccharide exhibits more potent IRI activity than the native b-linked-(1,3) disaccharide. The C2 N-acetyl group of the disaccharide does not affect IRI activity but in monosaccharides, the presence of the C2 N-acetyl group decreases IRI activity. The current study will facilitate the design of potent small-molecule ice recrystallization inhibitors.

Full text of DOI:10.1016/j.bmcl.2011.12.097

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1719–1721
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structurally diverse disaccharide analogs of antifreeze glycoproteins and their
ability to inhibit ice recrystallization
⇑
Anna K. Balcerzak, Sandra S. Ferreira, John F. Trant, Robert N. Ben
Department of Chemistry, University of Ottawa, 10 Marie Curie, Ottawa, Ont., Canada K1N 6N5
a r t i c l e i n f o
a b s t r a c t
Article history:
The b-D-galactosyl-(1,3)-a-N-acetyl-D-galactosamine disaccharide is present in antifreeze glycoproteins
Received 3 November 2011
Revised 16 December 2011
Accepted 20 December 2011
Available online 30 December 2011
(AFGPs). Analogs of this disaccharide including the b-linked (1,3)-, (1,4)-, and (1,6)-galactosyl-N-acetyl
galactosamine and the b-(1,3)-galactosyl-galactoside were synthesized and evaluated for ice recrystalli-
zation inhibition (IRI) activity. The results from this study demonstrate that the b-linked-(1,4) disaccha-
ride exhibits more potent IRI activity than the native b-linked-(1,3) disaccharide. The C2 N-acetyl group
of the disaccharide does not affect IRI activity but in monosaccharides, the presence of the C2 N-acetyl
group decreases IRI activity. The current study will facilitate the design of potent small-molecule ice
recrystallization inhibitors.
Keywords:
Antifreeze glycoproteins
AFGP
Ice recrystallization inhibition
Carbohydrate hydration
Thermal hysteresis
Ó 2011 Elsevier Ltd. All rights reserved.
Antifreeze glycoproteins (AFGPs) protect polar fish inhabiting
subzero temperatures from cryo-injury and death.1 The typical
structure of an AFGP is comprised of a threonine–alanine–alanine
(TAA) tripeptide repeat in which the secondary oxygen of the thre-
inhibiting ice recrystallization has not been investigated. In fact,
previously synthesized C-linked AFGPs which are potent inhibitors
of ice recrystallization, do not possess this disaccharide.12 These
structures have only a carbon-linked galactopyranose moiety.
Based upon our previous work showing that simple mono- and
disaccharides exhibit weak to moderate IRI activity,15,16 we sought
to investigate the ice recrystallization inhibition (IRI) activity of al-
onine is glycosylated with
a b-D-galactosyl-(1,3)-a-D-N-acetyl
galactosamine disaccharide.2–5 These glycoproteins possess two
distinct types of antifreeze activity: thermal hysteresis (TH)6 and
ice recrystallization inhibition (IRI) activity.7 The latter is extre-
mely important for cryopreservation applications where prevent-
ing the re-organization of smaller ice crystals into larger crystals
results in an increase in cell viability post-thaw. Generally, AFGPs
are not effective cryoprotectants because the binding of the AFGP
to ice changes the shape of the ice crystal and this damages cells
at temperatures associated with cryopreservation.8–11 Further-
more, these are large glycoproteins; the synthesis of which is not
amenable to production in large quantities. Consequently, potent
small molecule inhibitors of ice recrystallization have tremendous
applications as novel cryoprotectants. Our laboratory has been ac-
tively engaged in the rational design and synthesis of small mole-
cule inhibitors of ice recrystallization. The structures of these
compounds have been based upon the novel C-AFGPs developed
in our laboratory which are potent inhibitors of ice recrystalliza-
tion.12 Previous structure–function studies have demonstrated that
lyl b-D-galactosyl-(1,3)-b-D-N-acetyl galactosamine (1), two closely
related structures (2–4), and several monosaccharide derivatives
(Fig. 1). The knowledge from this study will facilitate the rational
design of potent small molecule ice recrystallization inhibitors.
Disaccharide 1 is a close analog of b-D-galactosyl-(1,3)-b-D-N-
acetyl galactosamine found in the native AFGP but the C1 anomeric
oxygen is protected as an allyl ether. Earlier work by our laboratory
has demonstrated that simple alkyl groups do not affect the ability
of these structures to inhibit ice recrystallization.15 Disaccharides 2
and 3 are regioisomers of 1 where the terminal b-galactosyl pyra-
nose is linked to either the C4 or C6 hydroxyl group of allyl b-D-N-
acetyl galactosamine. These two compounds will allow us to study
how the position of the intersaccharidic linkage influences IRI
activity. Disaccharide 4 will elucidate whether the C2 N-acetyl
group is important for IRI activity. This is relevant since previous
studies have implicated this group as being important for TH activ-
ity.14 Finally, monosaccharides 5–8 were synthesized and tested to
quantify the IRI activity of the individual components that make up
disaccharides 1–4 (Fig. 1).
the b-D-galactosyl-(1,3)-a-D-N-acetyl galactosamine disaccharide
in native AFGPs is an essential structural feature for thermal hys-
teresis activity.13,14 However, the importance of this moiety for
N-Acetyl glucosamine was used as starting material to prepare
1 (Scheme 1). The anomeric hydroxyl group was alkylated with
allyl bromide to produce 10 in 75% yield. Treatment of 10 with
⇑
Corresponding author. Tel.: +1 613 562 5800x6231; fax: +1 613 562 5170.
E-mail address: rben@uottawa.ca (R.N. Ben).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.12.097

1720
A. K. Balcerzak et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1719–1721
Figure 1. Structures of disaccharides containing the b-(1,3), -(1,4), or -(1,6)
glycosidic linkages and monosaccharides.
Scheme 3. Synthesis of disaccharide 3.
The azide was reduced and the amine was acetylated furnishing
24 (48% yield over two steps). Removal of the acetate protecting
groups and reaction with 2,2-dimethoxy propane and p-toluene
sulfonic acid furnished glycosyl acceptor 25 which was glycosyl-
ated at the C6 oxygen by reaction with glycosyl bromide 18 and
Hg(CN)2 to produce disaccharide 26. Finally, deprotection of the
acetate groups and the isopropylidene protecting group resulted
in 3 (85% yield over two steps).
The synthesis of disaccharide 4 is outlined in Scheme 4. Stan-
dard protecting group manipulations ultimately furnished diol 30
which was selectively protected resulting in glycosyl acceptor 31
(97% yield).18,20,21 Glycosyl donor 32 was reacted with 31 in the
presence of triflic acid to produce 33 in 23% yield. Subsequent
deprotection furnished 4 (88% yield).
Scheme 1. Synthesis of disaccharide 1.
pivaloyl chloride furnished 11 which, when treated with triflic
anhydride followed by water and heat, resulted in 12 (60%
yield).17,18 Orthogonally protected glycosyl acceptor 12 was gly-
cosylated with trichloroacetimidate 13 using boron trifluoride
diethyl etherate as a promoter, to provide disaccharide 14 in 50%
yield. Deprotection with sodium methoxide in methanol resulted
in disaccharide 1 in 30% yield.
The synthesis of 2 was similar to that of 1 but intermediate 11
was treated with triflic anhydride and sodium nitrate to furnish 15
in 67% yield (Scheme 2). Glycosyl acceptor 15 was reacted with tri-
chloroacetimidate 13 furnishing 16 in 20% yield. Removal of the
protecting groups with sodium methoxide yielded 2 (50% yield).
Disaccharide 3 was synthesized using an azido nitration of gly-
cal 19 to install the C2 acetamide group (Scheme 3).19 Bromination
of 17 followed by treatment with zinc furnished glycal 19 in 77%
yield. Azido-nitration with sodium azide and ceric ammonium ni-
trate provided azide 20. Reduction with PhSH and DIPEA produced
21 which was reacted with trichloroacetonitrile and catalytic diaz-
abicycloundec-7-ene (DBU) forming 22. Displacement of the tri-
chloroacetimidate with allyl alcohol resulted in 23 (88% yield).
Scheme 2. Synthesis of disaccharide 2.
Scheme 4. Synthesis of disaccharide 4.

A. K. Balcerzak et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1719–1721
1721
100
80
60
40
20
0
PBS
1
4
2
3
5
6
7
8
2.5%
DMSO
5% AFGP-8
(5.5 µM)
Disaccharides
Monosaccharides
22 mM Carbohydrate in PBS Standard
Chart 1. IRI activity of disaccharides and monosaccharides (1–8). Asterisks indicate P <0.05 within each set. Error bars indicate standard error of the mean (SEM).
All compounds were analyzed for IRI activity using the splat-
cooling assay15,22 with domain recognition software to quantify
mean ice crystal size.23 The IRI activity of all carbohydrates is pre-
sented in Chart 1. The y-axis represents ice crystal mean grain size
(MGS) relative to a phosphate-buffered saline (PBS) control. Conse-
quently, large bars indicate large ice crystals while small bars indi-
cate small crystals (inhibition of ice recrystallization). 2.5% and 5%
(350 mM and 700 mM, respectively)24 solutions of dimethyl sulfox-
tical IRI activity suggesting that a group at C1 modulates IRI activity.
Additional studies to thoroughly investigate these effects are cur-
rently being conducted in our laboratory. The results of these studies
will be reported in due course and will facilitate the design of potent
small-molecule ice recrystallization inhibitorsfor medical, commer-
cial, and industrial applications.
Acknowledgments
ide (DMSO), a common cryoprotectant, and native AFGP-8 (5.5 lM)
are included as positive controls for ice recrystallization inhibition.
These results show that allylated galactose derivative 6 is less active
than galactose (5). This is not completely surprising as we have pre-
viously demonstrated that C-linked allyl-galactose analogs with the
The authors acknowledge the Natural Sciences and Engineering
Research Council of Canada (NSERC), Canadian Blood Services
(CBS), Canadian Institutes of Health Research (CIHR) and Canadian
Foundation Innovation (CFI) for financial support.
b stereochemistry are less active than the
a
anomers.15,16 Similarly,
N-acetyl galactosamine (7) is also less active than galactose. It is
interesting to see that the addition of the acetamide group to C2
actually decreases IRI activity perhaps suggesting that the role of
this group in IRI activity is different that with TH activity. Finally,
the presence of both the C1 O-allyl group and C2 acetamide on gal-
actose (8) resulted in very weak IRI activity similar to that observed
with monosaccharides 6 and 7. Ultimately, monosaccharides 6–8
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.12.097.
References and notes
1. Yeh, Y.; Feeney, R. E. Chem. Rev. 1996, 96, 601.
2. DeVries, A. L. Science 1971, 172, 1152.
exhibited less IRI activity than the parent D-galactose.
Disaccharides 1–4 exhibit similar levels of IRI activity (Chart 1)
comparable to galactose itself. The activities of 1 and 3 are statis-
tically identical to galactose. However, disaccharide 2 is more ac-
tive than 1, 3, and galactose (5). This is interesting as the native
disaccharide in AFGPs possesses a b-(1,3) linkage but disaccharide
2 possesses the b-(1,4) linkage. It is important to note that 2 is not
conjugated to the TAA polypeptide backbone that is found in native
AFGPs. It is unknown whether the corresponding TAA glycoconju-
gate containing disaccharide 2 would exhibit this same trend. Re-
moval of the C2 acetamide group from disaccharide 1 does not
have an effect on IRI activity as the activity of disaccharide 4 is
identical to that of 1. In contrast, monosaccharide 7 containing
the C2 acetamide group is less active that galactose (5).
3. DeVries, A. L.; Wohlschlag, D. E. Science 1969, 163, 1073.
4. DeVries, A. L.; Komatsu, S. K.; Feeney, R. E. J. Biol. Chem. 1970, 245, 2901.
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2008, 130, 17494.
In summary, we have quantified the IRI activity of several disac-
charides and monosaccharides closely related to the b-
D-galactosyl-
16. Chaytor, J. L.; Ben, R. N. Bioorg. Med. Chem. Lett. 2010, 20, 5251.
17. Bouvet, V. R.; Ben, R. N. J. Org. Chem. 2005, 71, 3619.
18. Deslongchamps, P. Stereoelectronic Effects in Organic Chemistry; Pergamon Press
Ltd: Oxford, 1983. p 83.
19. Lemieux, R. U.; Ratcliffe, R. M. Can. J. Chem. 1979, 57, 1244.
20. King, J. F.; Allbutt, A. D. Can. J. Chem. 1969, 47, 1445.
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(1,3)- -N-acetyl galactosamine disaccharide found in the native
a
-D
AFGP. Interestingly, the b-(1,4) disaccharide (2) was a more potent
inhibitor of ice recrystallization than the native AFGP disaccharide
(1). In contrast to prior work with the AFGP glycoconjugates where
the C2 acetamide group was important for TH activity, this acetam-
ide group appears not to be an essential structural feature for IRI
activity in disaccharide 1 suggesting that the structural features
for TH and IRI may be different. However in reducing sugars (mono-
saccharides 5 and 7), the presence of the acetamide group actually
reduced the IRI activity. Allylated derivatives 6 and 8 exhibited iden-
24. Chaytor, J. L.; Tokarew, J. M.; Wu, L. K.; Leclère, M.; Tam, R. Y.; Capicciotti, C. J.;
Guolla, L.; von Moos, E.; Findlay, C. S.; Allan, D. S.; Ben, R. N. Glycobiology 2012,
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