Assessing the ability of a short fluorinated antifreeze glycopeptide and a fluorinated carbohydrate derivative to inhibit ice recrystallization

Article abstract of DOI:10.1016/j.bmcl.2010.06.148

A short fluorinated antifreeze glycopeptide (2) was synthesized and evaluated for ice recrystallization inhibition (IRI) activity. The activity of 2 was compared to native biological antifreeze AFGP 8 and a rationally designed C-linked AFGP analogue (OGG-Gal, 1). In addition, a simple fluorinated galactose derivative was prepared and its IRI activity was compared to non-fluorinated compounds. The results from this study suggest that the stereochemistry at the anomeric position in the carbohydrate plays a role in imparting ice recrystallization inhibition activity and that incorporation of hydrophobic groups such as fluorine atoms cause a decrease in IRI activity. These observations are consistent with the theory that fluorine atoms increase ordering of bulk water resulting in a decrease of IRI activity, supporting our previously proposed mechanism of ice recrystallization inhibition.

Full text of DOI:10.1016/j.bmcl.2010.06.148

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5251–5254
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Assessing the ability of a short fluorinated antifreeze glycopeptide and
a fluorinated carbohydrate derivative to inhibit ice recrystallization
*
Jennifer L. Chaytor, 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:
A short fluorinated antifreeze glycopeptide (2) was synthesized and evaluated for ice recrystallization
inhibition (IRI) activity. The activity of 2 was compared to native biological antifreeze AFGP 8 and a ratio-
nally designed C-linked AFGP analogue (OGG-Gal, 1). In addition, a simple fluorinated galactose deriva-
tive was prepared and its IRI activity was compared to non-fluorinated compounds. The results from this
study suggest that the stereochemistry at the anomeric position in the carbohydrate plays a role in
imparting ice recrystallization inhibition activity and that incorporation of hydrophobic groups such as
fluorine atoms cause a decrease in IRI activity. These observations are consistent with the theory that
fluorine atoms increase ordering of bulk water resulting in a decrease of IRI activity, supporting our pre-
viously proposed mechanism of ice recrystallization inhibition.
Received 25 May 2010
Revised 25 June 2010
Accepted 29 June 2010
Available online 23 July 2010
Keywords:
Antifreeze glycoprotein
AFGP 8
Ice recrystallization inhibition
Fluorine
Ó 2010 Elsevier Ltd. All rights reserved.
Carbohydrate hydration
The design of novel carbohydrates that inhibit ice recrystalliza-
tion has been an ongoing initiative in our laboratory. Ice recrystal-
lization inhibition (IRI) activity is associated with biological
antifreezes such as antifreeze proteins (AFPs) and antifreeze glyco-
proteins (AFGPs). IRI activity has also been reported in carbohy-
drates and various polymers.1–5 Recent work in our laboratory
has suggested that carbohydrate hydration is important in IRI
activity4,5 and that IRI activity is correlated to cryopreservation
ability. In other words, compounds able to inhibit the recrystalliza-
tion of ice were also able to protect against cell damage and death
during cryopreservation. With this knowledge, we have been
studying how structural modifications to carbohydrates and carbo-
hydrate derivatives modulate IRI activity. These structure–function
studies will facilitate the rational design and synthesis of novel
cryoprotectants required for various medical applications, includ-
ing cryopreservation and cryosurgery.6–10
crystal.5 For example, galactose (one of the most potent monosac-
charide inhibitors of ice recrystallization) is known to fit very
poorly into the hydrogen bonded network of water resulting in a
large disruption in the order of these bulk water molecules.5 In
contrast, talose, which is poorly hydrated has a very good fit into
the hydrogen bonded network of water, and consequently exhibits
very little IRI activity.5 Based upon this precedent, we expect com-
pounds that are poorly hydrated (those that cause bulk water mol-
ecules to become more ordered) to be poor inhibitors of ice
recrystallization. To test this hypothesis, we have prepared galact-
ose derivatives that contain poorly hydrated substituents and
examined the effect on IRI activity.
It has been demonstrated that fluorine atoms mediate the bio-
logical and chemical properties of carbohydrates.12–14 Conse-
quently, glycosyl fluorides are often used as both substrates15,16
and inhibitors15–17 of carbohydrate enzymatic reactions. A fluorine
atom has a similar size to that of a hydroxyl group, which makes
the fluorine atom an appropriate replacement for the hydroxyl
group in binding studies.18,19 Furthermore, the difluoromethylene
group is known to be isopolar and isosteric to oxygen.17 The hydra-
tion of fluorine atoms has been studied and it has been observed
that fluorine atoms behave like hydrocarbons in that they facilitate
the ordering of water molecules.13 For instance, Nishi et al., have
examined the effects of 4-hydroxyproline and 4-fluoroproline on
the stability of the collagen triple helix and confirmed that fluorine
is more hydrophobic than oxygen ensuring that fluorinated proline
residues are poorly hydrated relative to proline in the native triple
helix.20 The trend reported here suggests that fluorine atoms order
We have previously reported that the best inhibitors of ice
recrystallization are highly hydrated carbohydrates known to fit
poorly within the three dimensional hydrogen bonded network
of water.5,11 We hypothesize that these compounds disorder bulk
water molecules, thus slowing the transfer of bulk water molecules
to the quasi liquid layer (QLL) and subsequently to the growing ice
Abbreviations: AFGP, antifreeze glycoprotein; AFP, antifreeze protein; IRI, ice
recrystallization inhibition; OGG, Ornithine–glycine–glycine; TH, thermal hystere-
sis; PBS, phosphate buffered saline.
* Corresponding author. Tel.: +1 613 562 5800x6321; fax: +1 613 562 5170.
E-mail address: rben@uottawa.ca (R.N. Ben).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.06.148

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J. L. Chaytor, R. N. Ben / Bioorg. Med. Chem. Lett. 20 (2010) 5251–5254
Figure 1. Image A: fluorinated AFGP analogues reported by Quirion and Castelot–Deliencourt–Godefroy. Image B: C-linked AFGP analogue OGG-Gal (1) reported by Ben et al.
Image C: proposed analogue (2).
water molecules to the same degree as hydrocarbons. Based upon
our proposed mechanism of IRI,5 we would therefore expect fluo-
rinated carbohydrates and those containing hydrocarbon chains
to be equally poor inhibitors of ice recrystallization due to their en-
hanced ability to order bulk water molecules and facilitate the
transfer of these water molecules to the ice lattice.
Introduction of fluorine is also a prudent choice as it has been
previously shown that fluorinated AFGP analogues (Fig. 1, image
A) were able to preserve human embryonic kidney cells, erythro-
cytes, blood platelets, and skin fibroblasts at physiological and
sub-zero temperatures.21 However, these compounds were not
evaluated for antifreeze-specific activity such as IRI and thermal
hysteresis (TH). Thermal hysteresis is defined as the selective
depression of the freezing point of a solution below that its melting
point. This property ultimately prevents the seeding of ice crystals
in organisms inhabiting sub-zero temperatures and prevents sub-
sequent cryo-injury and death.22,23
during cryopreservation,10,25–27 potent inhibitors of ice recrystalli-
zation have potential to be used as novel cryoprotectants. Addi-
tional details of this assay are described in the Supplementary data.
Figure 2 shows that 2 did not exhibit any IRI activity, as ice crys-
tals had the same mean grain size as PBS (a positive control for ice
recrystallization). These results indicate that this short fluorinated
glycopeptide is not an effective inhibitor of ice recrystallization.
The reported cryopreservation ability21 of the structurally similar
compound shown in Figure 1 (having a hydroxyl group instead of
a methoxy group at the anomeric position) may therefore be due
to some other mechanism that does not involve ice recrystalliza-
tion inhibition.
We have previously synthesized C-linked AFGP analogue 1 that
exhibited potent IRI activity but had no TH activity (Fig. 1, image
B). To properly investigate the effect of fluorine incorporation into
this analogue, we have prepared 2 (Fig. 1, image C) and evaluated
its IRI activity. Fluorinated tripeptide 2 was synthesized (Supple-
mentary data) and assessed for IRI activity relative to native AFGP
8 and C-AFGP analogue OGG-Gal (1). IRI activity was assessed
using a splat cooling assay developed by Horwath et al.24 In this as-
say, ice crystals are measured after a 30 min annealing time at
À6.4 °C and compared to a positive control for ice recrystallization
to give a quantitative measure of ice grain size. In this case, the po-
sitive control is phosphate buffered saline (PBS). Since mechanical
damage to cells as a result of large ice crystals formed by ice
recrystallization has been proposed as a major cause of cell death
Figure 2. Ice recrystallization inhibition activity of native AFGP 8 (positive control),
OGG-Gal (1), and fluorinated tripeptide 2. The fluorinated tripeptide solution
formed ice crystals that were identical in size to those in a phosphate buffered
saline (PBS) solution (negative control).

J. L. Chaytor, R. N. Ben / Bioorg. Med. Chem. Lett. 20 (2010) 5251–5254
5253
Figure 3. Fluorinated and non-fluorinated carbohydrate analogues 3–5.
Figure 4. Galactose (6), a-C-allyl-galactose (7), b-C-allyl-galactose (8), and a-methyl galactopyranoside (9) were evaluated for IRI activity and compared to compounds 3–5.
It is important to note that although the fluorinated tripeptide 2
was tested at a higher concentration than OGG-Gal (1), it still ap-
peared to have no IRI activity. There are several plausible explana-
tions for this observation. The lack of activity in this short
glycopeptide may indicate that more than one tripeptide unit is re-
quired for successful inhibition of ice recrystallization. A similar ef-
fect has been observed for thermal hysteresis (TH) activity in AFGP
analogues.28 Alternatively, the lack of IRI activity may be due to the
change in anomeric stereochemistry compared to OGG-Gal (1). Our
laboratory has previously observed that small structural variations
can result in changes in the presentation of the carbohydrate moi-
ety of short glycopeptides and this can affect IRI activity.29 Never-
theless, this was an interesting result that we felt was worthy of
further study.
Earlier work in our laboratory has demonstrated that IRI activity
of complex glycoconjugates can be assessed using only the carbo-
hydrate component of the glycoconjugate.5 While the magnitude
activity of IRI active carbohydrates was generally lower than for
the glycoconjugates, trends in IRI activity were the same. This is
important as it allowed us to assess the potential for IRI activity
of a proposed glycoconjugate before expending the effort to syn-
thesize it. Utilizing this approach, we chose to further examine
the effect of fluorine atoms on IRI activity by synthesizing a galact-
ose derivative containing fluorine atoms that was structurally sim-
ilar to the fluorinated carbohydrate moiety in the Quirion AFGP
analogue.21 The structure of this fluorinated compound and the
corresponding non-fluorinated monosaccharides are illustrated in
Figure 3.
TheIRIactivityof thesecarbohydrateswas compared togalactose
(6) and the galactose derivatives (7 –9) shown in Figure 4. Com-
pounds 3–5 were prepared as reported in Supplementary data, and
exhibited poor IRI activity as shown in Figure 5. In fact, these com-
pounds were much less active than galactose (6), which is the most
active monosaccharide tested to date. Interestingly, a-C-allyl galact-
ose (7) is as active as galactose itself, yet we have previously ob-
served a decrease in activity when the anomeric stereochemistry
was changed from a
- to the b-anomer (8).5 This supports our theory
that IRI activity is linked to the hydration of the carbohydrate, as b-
linked carbohydrates have been reported to order bulk water to a
greater degree than
served decrease in activity of 3–5 relative to
a
-linked compounds.30,31 Consequently, the ob-
a-C-allyl galactose (7)
was not surprising due to the change in anomeric stereochemistry.
The activity of non-fluorinated carbohydrate derivatives 4 and 5
was statistically identical21 to the activity of fluorinated 3, suggest-
ing that fluorine atoms have no effect on the IRI activity of mono-
saccharides. Our observation that the non-fluorinated compounds
had equally poor IRI activity to 3 was consistent with our hypoth-
esis that carbohydrates containing hydrocarbon chains will be poor
inhibitors of ice recrystallization. Incorporation of a methoxy group
at the anomeric position of 3 and 5 did not result in a change in IRI
activity relative to 4 which contains a free hydroxyl group even
though the methoxy group is more hydrophobic. This increased
hydrophobicity is supported by the fact that 9 was much less active
than galactose and suggests that the overall hydration of com-
pounds 3–5 and 9 is similar. Based upon this trend, it is plausible
that the hydrophobic groups (methoxy groups, CF2 groups, CH2
groups and/or b-linked esters) in 3–5 result in poorer hydration
relative to galactose itself. However, it should be noted that the
hydration of these compounds has not been quantified in the liter-
ature and was not assessed in this study. Irrespective of this, these
preliminary results indicate that fluorine atoms and methoxy
groups do not improve the IRI activity of monosaccharides.
In conclusion, a short fluorinated glycopeptide displayed no IRI
activity relative to a PBS negative control. Studies of fluorinated
and non-fluorinated carbohydrates showed that these compounds
were poor inhibitors of ice recrystallization. We have observed that
increasing the hydrophobicity of carbohydrate derivatives causes a
decrease in their IRI activity. These results support our proposed
mechanism of IRI activity which is dependent on the hydration
of the carbohydrate.
Acknowledgements
Figure 5. IRI activity of fluorinated monosaccharide 3, non-fluorinated control
monosaccharides 4 and 5, galactose (6),
(8), and -methyl galactopyranoside (9). All compounds are relative to the PBS
negative control. MGS = mean grain size.
a-C-allyl galactose (7), b-C-allyl galactose
The authors acknowledge NSERC, CIHR, and CFI for financial
support. J.L.C. thanks NSERC for a PGS-D award.
a

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