C-linked galactosyl serine AFGP analogues as potent recrystallization inhibitors

Article abstract of DOI:10.1021/ol050677x

(Chemical Equation Presented) A series of C-linked antifreeze glycoprotein analogues have been prepared to evaluate antifreeze activity as a function of distance between the carbohydrate moiety and polypeptide backbone. The building blocks for these analogues were prepared using either an olefin cross-metathesis or catalytic asymmetric hydrogenation. Analysis of antifreeze protein-specific activity revealed that only analogue 2a (n = 1) was a potent recrystallization inhibitor and thus has potential medical and industrial applications.

Full text of DOI:10.1021/ol050677x

ORGANIC
LETTERS
2005
Vol. 7, No. 12
2385-2388
C-Linked Galactosyl Serine AFGP
Analogues as Potent Recrystallization
Inhibitors
Suhuai Liu and Robert N. Ben*
Department of Chemistry, UniVersity of Ottawa, Ottawa, ON, Canada K1N6N5
robert.ben@science.uottawa.ca
Received March 30, 2005
ABSTRACT
A series of C-linked antifreeze glycoprotein analogues have been prepared to evaluate antifreeze activity as a function of distance between
the carbohydrate moiety and polypeptide backbone. The building blocks for these analogues were prepared using either an olefin cross-
metathesis or catalytic asymmetric hydrogenation. Analysis of antifreeze protein-specific activity revealed that only analogue 2a (n
a potent recrystallization inhibitor and thus has potential medical and industrial applications.
) 1) was
Antifreeze glycoproteins (AFGPs) are a subclass of biological
antifreezes found primarily in the plasma of deep sea polar
fish.1 These compounds have the ability to inhibit the growth
of ice, thus ensuring the survival of these organisms in sub-
zero environments. The typical AFGP structure is comprised
of a repeating L-threonine-L-alanine-L-alanine tripeptide unit
where the secondary hydroxyl group of the threonine is
glycosylated with a â-D-galactosyl-(1,3)-R-N-acetyl-D-ga-
lactosaminyl disaccharide (Figure 1).1g While this structure
is remarkably well-conserved among Teleost fish, lower
molecular weight AFGPs may have an L-arginine residue in
(1) (a) Wu, Y.; Banoub, J.; Goddard, S. V.; Kao, M. H.; Fletcher, G. L.
Comp. Biochem. Physiol. Part B 2001, 128, 265-273. (b) Chen, L.;
DeVries, A. L.; Cheng, C. C. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 3817-
3822. (c) Ben, R. N. ChemBioChem 2001, 2, 161-166. (d) Harding, M.
M.; Anderberg, P. I.; Haymet, A. D. J. Eur. J. Biochem. 2003, 270, 1381-
1392. (e) Feeney, R. E.; Burcham, T. S.; Yeh, Y. Annu. ReV. Biophys.
Biophys. Chem. 1986, 15, 59-78. (f) Cheng, C.-H.; Chen, L. Nature 1999,
401, 443-444. (g) Ananthanarayanan, V. S. Life Chem. Rep. 1989, 7, 1.
(h) DeVries, A. L. Comp. Biochem. Physiol. 1988, 90B, 611-621. (i)
Knight, C. A.; Wen, D.; Laursen, R. A. Cryobiology 1995, 32, 23-24.
Figure 1. Structures of native AFGP 8 (1) and the C-linked AFGP
analogues used in this study.
place of the L-threonine and/or an L-alanine residue substi-
tuted by L-proline. The AFGP mechanism of action is
10.1021/ol050677x CCC: $30.25
© 2005 American Chemical Society
Published on Web 05/12/2005

regarded as an absorption-inhibition process1a-d,h in which
AFGPs bind tightly to the surface of an existing ice crystal.
This binding ultimately inhibits the addition of other water
molecules to the ice lattice resulting in a localized freezing
point depression. The temperature difference between the
melting and freezing point is referred to as thermal hysteresis
(TH). While the protein-ice interactions of native AFGP
have not been definitively identified, hydrogen-bonding
interactions involving hydrophilic hydroxyl groups on the
disaccharide2 and possible hydrophobic interactions with the
â-methyl group of the threonine residue are thought to be
essential interations.3 Furthermore, recent studies have identi-
fied the N-acetyl group, R-configuration of glycoside, and
â-methyl group on the threonyl residue as important for
activity.4
Scheme 1. Synthesis of Building Blocks 5, 6, 8, and 10
for OCM
AFGPs are also potent recrystallization inhibitors. While
the mechanism by which this re-organization of ice crystals
is not known, this property has many potential applications
in cryomedicine and the prevention of cellular damage during
freezing and thawing cycles.1i
Unfortunately, two factors have precluded the com-
mercialization of native AFGP for medical and industrial
applications. These are the limited bioavailability and the
inherent instability of the C-O glycosidic bond. Conse-
quently, rationally designed carbon-linked or C-linked AFGP
analogues are very attractive.5 Toward this end, we have
previously reported on the preparation of C-linked AFGP
analogues bearing an amide bond in the side chain that
demonstrated antifreeze protein-specific activity.6 In this
paper, we report the synthesis of a series of “simplified”
C-linked AFGP analogues (general structure 2) lacking the
amide bond and correlate the distance between the carbo-
hydrate moiety and peptide backbone to antifreeze protein-
specific activity. Many of the structural features in the first-
generation analogues have been incorporated into AFGP
analogues 2a-c. For instance, the native disaccharide has
been truncated and replaced by a single galactose residue
and the alanine residues replaced with glycines.
Preparation of the requisite building blocks for olefin cross-
metathesis are outlined in Scheme 1. C-Allylated galactose
pentaacetate 5 was prepared via a photochemical-mediated
allylation in 90% yield.9a C-Glycoside 8 was obtained by
reducing 5 with borane followed by PCC oxidation and
Wittig olefination with methyl triphenyl phosphonium
bromide.9b,c C-(1-Propenyl) glycoside 6, a key intermediate
in the preparation of AFGP analogue 2a, was generated via
a palladium-mediated isomerization of 5.
Vinyl glycine derivative 10 was obtained in 34% yield
from the orthogonally protected glutamic acid derivative 9
by oxidative decarboxylation.10 With the requisite building
blocks in hand, olefin cross-metathesis of 10 with 5, 6, and
8 was conducted using the second- generation Grubbs
catalyst (Scheme 2). As anticipated, building blocks 12 and
Recently, several methodologies have been developed to
prepare C-glycosyl amino acids including olefin cross
metathesis (OCM) and catalytic asymmetric hydrogenation.7
The former approach is amenable to preparing analogues
such as 2 as it requires the readily available vinyl glycine
and C-alkenyl galactose derivatives as starting mate-
rials.8
Scheme 2. Preparation of C-Linked Building Blocks 14 and 15
(2) (a) Chao, H.; Houston, M. E.; Hodges, R. S.; Kay, C. M.; Sykes, B.
D.; Loewen, M. C.; Davies, P. L.; So¨nnichsen, F. D. Biochemistry 1997,
36, 14652-14660. (b) Haymet, A. D. J.; Ward, L. G.; Harding, M. M. J.
Am. Chem. Soc. 1999, 121, 941-948. (c) Knight, C. A.; Driggers, E.;
DeVries, A. L. Biophys. J. 1996, 71, 8-18.
(3) Lavalle, P.; DeVries, A. L.; Cheng, C.-C. C.; Scheuring, S.; Ramsden,
J. J. Langmuir 2000, 16, 5785-5789.
(4) Tachibana, Y.; Fletcher, G. L.; Fujitani, N.; Tsuda, S.; Monde, K.;
Nishimura, S.-I. Angew. Chem., Int. Ed. 2004, 43, 856-862.
(5) (a) Ravishankar, R.; Surolia, A.; Vijayan, M.; Lim, S.; Kishi, Y. J.
Am. Chem. Soc. 1998, 120, 11297-11303. (b) Wang, J.; Kovac, P.; Sinay,
P.; Gluademans, C. P. J. Carbohydr. Res. 1998, 308, 191-193.
(6) (a) Ben, R. N.; Eniade, A. A.; Hauer, L. Org. Lett. 1999, 1, 1759-
1762. (b) Eniade, A. A.; Murphy, A. V.; Landreau, G.; Ben, R. N.
Bioconjugate Chem. 2001, 12, 817-823. (c) Enaide, A. A.; Purushotham,
M.; Ben, R. N.; Wang, J. B.; Horwath, K. Cell Biochem. Biophys. 2003
38, 115-124.
2386
Org. Lett., Vol. 7, No. 12, 2005

Scheme 3. Preparation of C-Linked Serine Building Block 28
Table 1. Olefin Cross-Metathesis To Prepare C-Linked
Building Blocks 11-13
entry
C-alkene glycoside
product
yield, %
1
2
3
5
6
8
12
11
13
98
trace
100
13 were obtained in quantitative yield (Table 1). Unfortu-
nately, cross-metathesis between 10 and 6 produced furnished
building block 11 in only trace quantities. Presumably, this
is due to the fact that the carbon-carbon double bond in 6
is too close to the pyranose ring resulting in significant steric
interactions during the OCM. A similar effect has been
observed by McGarvey et al.8a The carbon-carbon double
bonds in enamide esters 12 and 13 were reduced by
hydrogenation with palladium over carbon. Under these
conditions, the Cbz and benzyl protecting groups were
simultaneously removed necessitating reprotection of the
amino terminus as an Fmoc carbamate to afford building
blocks 14 and 15 in 55% and 85% yield, respectively.
To prepare the building block for AFGP analogue 2a, we
adopted a catalytic asymmetric hydrogenation of C-glycosyl
enamide esters first reported by Toone and co-workers.11 The
substrate for this hydrogenation (25) was constructed via
Horner-Emmons olefination12 with phosphonate 2313 and
C-linked pyranosyl aldehyde 24, as shown in Scheme 3.
Catalytic asymmetric hydrogenation of the C-glycosyl ena-
mide 25 was accomplished using cationic rhodium-Du-
PHOS catalyst under hydrogen atmosphere (100 psi). Hy-
drogenolysis of the benzyl ester followed by conversion of
the Boc carbamate to a Fmoc carbamate furnished building
block 28.
Assembly of building blocks 14, 15, and 28 into C-linked
AFGP analogues was accomplished by using standard Fmoc-
(7) (a) Urban, D.; Skrydstrup, T.; Beau, J. Chem. Commun. 1998, 9,
955-956. (b) Dondoni, A.; Marra, A. Chem. ReV. 2000, 100, 4395-4421.
(c) Palomo, C.; Oiarbide, M.; Landa, A.; Gonza´lez-Rego, M. C.; Garc´ıa, J.
M.; Gonza´lez, A.; Odriozola, J. M.; Mart´ın-Pastor, M.; Linden, A. J. Am.
Chem. Soc. 2002, 124, 8637-8643. (d) Paterson, D. E.; Griffin, F. K.;
Alcaraz, M.-L.; Taylor, R. J. K. Eur. J. Org. Chem. 2002, 67, 1323-1336.
(e) Dondoni, A.; Mariotti, G.; Marra, A. J. Org. Chem. 2002, 67, 4475-
4486. (f) Dondoni, A.; Mariotti, G.; Marra, A.; Massi, A. Synthesis 2001,
2129-2137. (g) Dondoni, A.; Mariotti, G.; Marra, A. Tetrahedron Lett.
2000, 41, 3483-3486. (h) Nolen, E. G.; Watts, M. M.; Fowler, D. J. Org.
Lett. 2002, 4, 3963-3965. (i) Vincent, S. P.; Schleyer, A.; Wong, C.-H. J.
Org. Chem. 2000, 65, 4440-4443.
based solid-phase synthesis protocols (Scheme 4). The
protected glycopeptides were cleaved from the resin using
TFA, and the acetate protecting groups on the pyranose were
removed by treatment with sodium in methanol to afford
Scheme 4. Synthesis of C-Linked AFGP Analogues 2a-c
(8) (a) McGarvey, G. J.; Benedum, T. E.; Schmidtmann, F. W. Org.
Lett. 2002, 4, 3591-3594. (b) Nolen, E. G.; Kurish, A. J.; Wong, K. A.;
Orlando, M. D. Tetrahedron Lett. 2003, 44, 2449-2453. (c) Dondoni, A.;
Giovannini, P. P.; Marra, A. J. Chem. Soc., Perkin Trans. 1 2001, 2380-
2388.
(9) (a) Ponten, F.; Magnusson, G. J. Org. Chem. 1996, 61, 7463.(b)
Perkins, M. V.; Sampson, R. A. Org. Lett. 2001, 3, 123-126. (c) Abad,
A.; Agullo´, C.; Cun˜at, A. C.; Garc´ıa, A. B.; Gime´nez-Saiz, C. Tetrahedron
2003, 59, 9523-9536.
(10) Bacha, J. D.; Kochi, J. K. Tetrahedron 1968, 24, 2215-2226.
(11) Debenham, S. D.; Debenham, J. S.; Burk, M. J.; Toone, E. J. J.
Am. Chem. Soc. 1997, 119, 9897-9898.
(12) (a) Schmidt, U.; Lieberknecht, A.; Wild, J. Synthesis 1984, 53-60.
(b) Schmidt, U.; Lieberknecht, A.; Schanbacher, U.; Benttler, T.; Wild, J.
Angew. Chem., Int. Ed. Engl. 1982, 21, 776-777. (c) Schmidt, U.; Griesser,
H.; Leitenberger, V.; Lieberknecht, A.; Mangold, R.; Meyer, R.; Riedl, B.
Synthesis 1992, 487-490.
Org. Lett., Vol. 7, No. 12, 2005
2387

RI activity suggests that an optimal distance between the
two moieties exists and plays a key role in RI activity of
our C-linked analogues. Analogue 2a possesses the same
number of atoms between the carbohydrate and peptide
moieties as native AFGP. While this analogue is not as potent
as AFGP8, it appears to be a more effective recystallization-
inhibitor than type III AFP from the ocean pout (Macrozo-
arces americanus) which has an effective concentration for
RI activity at 7.10 × 10-7 M compared with 5.0 × 10-8
M
in 2a.14
In summary, we have utilized olefin cross-metathesis and
catalytic asymmetric hydrogenation to prepare a series of
novel C-linked AFGP analogues with different distances
between the carbohydrate and peptide backbone moieties.
The analogue with the shortest distance between these
moieties is an potent recrystallization inhibitor. This result
suggests that the rational design of potent recrystallization
inhibitors for medical, industrial and commercial applications
is a feasible and worthwhile goal. The effectiveness of AFGP
analogue 2a in preventing cryo-injury in mammalian cell
culture is currently under investigation.
Figure 2. RI activity of C-linked AFGP Analogues
the C-linked AFGP analogues 2a-c (73% to 93% isolated
yield) ranging in molecular weight from 1.5 to 1.6 KDa.
AFGP analogues 2a-c were assayed for antifreeze protein-
specific activity using nanoliter osmometry and a recrystal-
lization-inhibition assay.6c In contrast to our first-generation
AFGP analogues, 2a-c did not possess any thermal hys-
teresis or exhibit any dynamic ice-shaping ability.
Acknowledgment. This work was supported by grants
from the NIH (RO1GM60319), Natural Science and Engi-
neering Research Council (NSERC) of Canada, and Canada
Research Chair Program (CRC). R.N.B. holds a Tier 2
Canada Research Chair (CRC) in medicinal chemistry.
However, all three analogues demonstrated recrystalliza-
tion inhibition (RI) activity (Figure 2) relative to the
phosphate-buffered saline (PBS) control. The Y-axis in Figure
2 represents the mean largest grain size (MLGS) which is
an average ice crystal surface area for each sample.
Supporting Information Available: Experimental pro-
cedures and spectroscopic data for 4-31. This material is
available free of charge via the Internet at http://pubs.acs.org.
Different concentrations of each analogue in PBS were
assayed to rule out the nonspecific effects.6c AFGP analogue
2a was the most potent with activity close to that of native
AFGP8. Interesingly, when the side chain length is increased
by one or two additional carbon-carbon bonds (2b and 2c),
these analogues showed very limited RI activity. The
correlation between increased side chain length and decreased
OL050677X
(13) Shankar, R.; Scott, A. I. Tetrahedron Lett. 1993, 34, 231-234.
(14) Tomczak, M. M.; Marshall, C. B.; Gilbert, J. A.; Davies, P. L
Biocem. Biophys. Res. Commun. 2003, 311, 1041-1046.
2388
Org. Lett., Vol. 7, No. 12, 2005