Hydration index-a better parameter for explaining small molecule hydration in inhibition of ice recrystallization

Article abstract of DOI:10.1021/ja806284x

Several simple mono- and disaccharides have been assessed for their ability to inhibit ice recrystallization. Two carbohydrates were found to be effective recrystallization inhibitors. D-Galactose (1) was the best monosaccharide and D-melibiose (5) was the most active disaccharide. The ability of each carbohydrate to inhibit ice growth was correlated to its respective hydration number reported in the literature. A hydration number reflects the number of tightly bound water molecules to the carbohydrate and is a function of carbohydrate stereochemistry. It was discovered that using the absolute hydration number of a carbohydrate does not allow one to accurately predict its ability to inhibit ice recrystallization. Consequently, we have defined a hydration index in which the hydration number is divided by the molar volume of the carbohydrate. This new parameter not only takes into account the number of water molecules tightly bound to a carbohydrate but also the size or volume of a particular solute and ultimately the concentration of hydrated water molecules. The hydration index of both mono- and disaccharides correlates well with experimentally measured Rl activity. C-Linked derivatives of the monosaccharides appear to have Rl activity comparable to that of their O-linked saccharides but a more thorough investigation is required. The relationship between carbohydrate concentration and Rl activity was shown to be noncolligative and a 0.022 M solution of D-galactose (1) and C-linked galactose derivative (10) inhibited recrystallization as well as a 3% DMSO solution. The carbohydrates examined in this study did not possess any thermal hysteresis activity (selective depression of freezing point relative to melting point) or dynamic ice shaping. As such, we propose that they are inhibiting recrystallization at the interface between bulk water and the quasi liquid layer (a semiordered interface between ice and bulk water) by disrupting the preordering of water.

Full text of DOI:10.1021/ja806284x

Published on Web 11/20/2008
Hydration IndexsA Better Parameter for Explaining Small
Molecule Hydration in Inhibition of Ice Recrystallization
Roger Y. Tam, Sandra S. Ferreira, Pawel Czechura, Jennifer L. Chaytor, and
Robert N. Ben*
Department of Chemistry, D’Iorio Hall, 10 Marie Curie, UniVersity of Ottawa,
Ottawa, Ontario, Canada, K1N 6N5
Received August 8, 2008; E-mail: rben@uottawa.ca
Abstract: Several simple mono- and disaccharides have been assessed for their ability to inhibit ice
recrystallization. Two carbohydrates were found to be effective recrystallization inhibitors. D-Galactose (1)
was the best monosaccharide and D-melibiose (5) was the most active disaccharide. The ability of each
carbohydrate to inhibit ice growth was correlated to its respective hydration number reported in the literature.
A hydration number reflects the number of tightly bound water molecules to the carbohydrate and is a
function of carbohydrate stereochemistry. It was discovered that using the absolute hydration number of a
carbohydrate does not allow one to accurately predict its ability to inhibit ice recrystallization. Consequently,
we have defined a hydration index in which the hydration number is divided by the molar volume of the
carbohydrate. This new parameter not only takes into account the number of water molecules tightly bound
to a carbohydrate but also the size or volume of a particular solute and ultimately the concentration of
hydrated water molecules. The hydration index of both mono- and disaccharides correlates well with
experimentally measured RI activity. C-Linked derivatives of the monosaccharides appear to have RI activity
comparable to that of their O-linked saccharides but a more thorough investigation is required. The
relationship between carbohydrate concentration and RI activity was shown to be noncolligative and a
0.022 M solution of D-galactose (1) and C-linked galactose derivative (10) inhibited recrystallization as well
as a 3% DMSO solution. The carbohydrates examined in this study did not possess any thermal hysteresis
activity (selective depression of freezing point relative to melting point) or dynamic ice shaping. As such,
we propose that they are inhibiting recrystallization at the interface between bulk water and the quasi liquid
layer (a semiordered interface between ice and bulk water) by disrupting the preordering of water.
Introduction
last few decades.6 These studies have been limited to small
temperature and pressure ranges due to the fact that these molecules
Carbohydrates are prolific in biological systems and are involved
in many processes ranging from cellular adhesion, cell signaling,
infection, and regulation of the immune response.1 In addition,
carbohydrates are involved in the cryoprotection of organisms
inhabiting cold climates.2 While it is accepted that the hydration
of proteins plays an important role in modulating protein function,3
hydration of carbohydrates and oligosaccharides also tempers their
biological activity. Despite the undisputed importance of hydration,
assessing the degree of hydration is not a trivial process.4 While
NMR techniques have reportedly been used to assess hydration of
complex oligosaccharides, the results of these studies are often
subject to a large degree of uncertainty.5 In contrast, the hydration
of simple carbohydrates has been extensively studied during the
possess many different conformations in aqueous solution.6,7
Nonetheless, these studies demonstrate that carbohydrate hydration
is closely correlated to stereochemistry. While the exact reasons
for this are the subject of much debate, various hypotheses have
been proposed that rationalize hydration characteristics and the
subsequent influence of hydration on bulk water.8-15
During the last several years, our laboratory has successfully
designed and synthesized functional C-linked antifreeze glycopro-
tein (AFGP) analogues possessing custom-tailored antifreeze activ-
ity.16 These compounds are potent inhibitors of recrystallization
and do not possess thermal hysteresis (TH) activity. This is
significant as studies demonstrate that cellular damage as a result
of recrystallization is the major cause of decreased cellular viability
upon cryopreservation.17 As such, these compounds are novel lead
structures representing a new class of cryoprotectants where
improved cryoadjuvants and cryopreservation protocols are urgently
required to meet the steadily increasing demand for donor organs.18
We recently described how hydration of a C-linked carbohydrate
(1) (a) Cheng, C. C.; Bennett, D. Cell 1980, 19, 537–543. (b) Grabel,
L. B.; Rosen, S. D.; Martin, G. R. Cell 1979, 17, 477–484. (c) Stanley,
P.; Sudo, T. Cell 1981, 23, 763–769. (d) Neufeld, E.; Ashwell, G. In
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2008 American Chemical Society

Inhibiting Recrystallization with Small Molecules
A R T I C L E S
moiety in a C-linked AFGP analogue is a contributing factor to
antifreeze activity, specifically recrystallization-inhibition (RI)
activity.19 This work has not only shed new information on the
underlying mechanism of action of biological antifreezes, but also
highlights the importance of understanding hydration in all biologi-
cal processes. The current manuscript explores the relationship
between hydration of carbohydrates and carbohydrate derivatives
with recrystallization-inhibition activity. The ultimate goal of this
work is to enable accurate prediction of structures that function as
potent inhibitors of ice recrystallization for cryomedical and other
commercial applications.
camera (Nikon CoolPix 5000) fitted to the microscope. A total of
three images were taken from each wafer. During flash freezing,
ice crystals spontaneously nucleated from the supercooled solution.
These initial crystals were relatively homogeneous in size and quite
small. During the annealing cycle, recrystallization occurred,
resulting in a dramatic increase in ice crystal size. A quantitative
measure of the difference in recrystallization inhibition of two
compounds X and Y is the difference in the dynamics of the ice
crystal size distribution. Image analysis of the ice wafers was
performed using a novel domain recognition software (DRS)22
program that was developed at the Steacie Institute for Molecular
Sciences (SIMS) of the National Research Council of Canada
(NRCC). This processing employed the Microsoft Windows
Graphical User Interface to allow a user to visually demarcate and
store the vertices of ice domains in a digital micrograph. These
data were then used to calculate the domain areas. To eliminate
the need to fully process each micrograph, an algorithm was
developed to randomly display a number of x/y locations. The
algorithm made use of a built-in pseudo random number generator
(rand(x)) and was written so that no two locations were closer than
1/10th the field of view of the micrograph. The formula for the
area of a polygon that is not self-intersecting and contains no holes
is then given by eq 2,
Material and Methods
The monosaccharides used in this study were commercially
available and purchased from Sigma-Aldrich. All C-linked pyranose
derivatives were synthesized using standard literature procedures
(see Supporting Information and ref 19). Isentropic molar com-
pressibility (IMC) values were obtained from ultrasound density
measurements reported in the literature.8 Hydration numbers, nh,
were obtained using the Passynsky equation, eq 1,8,20
nh)(nw ⁄ns)(1 - ꢀs ⁄ ꢀso)
(1)
where nw and ns are the mole fractions of water and the solute,
respectively; and ꢀs and ꢀso are the isentropic coefficients of
compressibility of the solute and water, respectively.
1N-1
2 i)0
A )
(x yi + 1 - xi + 1 yi)
(2)
∑
i
Recrystallization-Inhibition (RI) Assay. Sample analysis for
RI activity was performed using the “splat cooling” method as
previously described.21 In this method, the analyte was dissolved
in phosphate buffered saline (PBS) solution and a 10 µL droplet
of this solution was dropped from a micropipette through a two-
meter high plastic tube (10 cm in diametre) onto a block of polished
aluminum precooled to approximately -80 °C. The droplet froze
instantly on the polished aluminum block and was approximately
1 cm in diameter and 20 µm thick. This wafer was then carefully
removed from the surface of the block and transferred to a cryostage
held at -6.4 °C for annealing. After a period of 30 min, the wafer
was photographed between crossed polarizing filters using a digital
where N is the number of vertices, and Σx0,Σy0 to ΣxN-1,ΣyN-1
are the vertices circumventing the polygon in a clockwise
direction. The point Σx0,Σy0is assumed to be equivalent to the
point ΣxN,ΣyN. The software was written in C using Microsoft
Visual Studio 6.0 on a Pentium class personal computer running
Microsoft Windows 2000 or XP. All data were plotted and
analyzed using Microsoft Excel.
Thermal Hysteresis Assay. Nanoliter osmometry was performed
using a nanoliter osmometer (Clifton Technical Physics, Hartford,
NY) as described by Chakrabartty and Hew.23 All measurements
were made in doubly distilled water. Ice crystal morphology was
observed through a Leitz compound microscope equipped with an
Olympus 20X (infinity corrected) objective, Leitz Periplan 32X
photo eyepiece and a Hitachi KP-M2U CCD camera connected to
a Toshiba MV13K1 TV/VCR system. Still images were captured
directly using a Nikon CoolPix 5000 digital camera.
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Solution Chem. 1976, 5, 33–46.
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A.; Hauer, L. Org. Lett. 1999, 1, 1759–1762. (c) Eniade, A.; Ben,
R. N. Biomacromolecules 2001, 2, 557–561. (d) Liu, S.; Ben, R. N.
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Baust, J. M., Eds.; CRC Press: Boca Raton, 2007; pp 197-210, and
references therein.
Results and Discussion
The “hydration layer” is defined as water that encompasses
the carbohydrate and is often bound very tightly. Specific
hypotheses to rationalize the observed hydration characteristics
of a carbohydrate include: hydration number,10 anomeric
effect,11 hydrophobic index,12 hydrophilic volume,13 and com-
patibility with bulk water based upon the position of the next-
nearest-neighbor hydroxyl groups.8,9,14 Subsequent to the latter
hypothesis, a revised stereospecific hydration model has sug-
gested that hydration of a carbohydrate depends upon the ratio
of axial to equatorial hydroxyl groups.15 In an attempt to
generate a unifying hypothesis consistent with the influence of
carbohydrate stereochemistry, key thermodynamic parameters
thought to dictate hydration were measured by Galema et al.8,9
By using molecular dynamics simulations, kinetic experiments,
and density and ultrasound measurements, the partial molar
volumes, isentropic partial molar compressibilities, and hydra-
tion numbers of many commercially available hexoses have been
determined and correlated to carbohydrate stereochemistry.
(19) Czechura, P.; Tam, R. Y.; Murphy, A. V.; Dimitrijevic, E.; Ben, R. N.
J. Am. Chem. Soc. 2008, 130, 2928–2929.
(20) (a) Shiio, H. J. Am. Chem. Soc. 1958, 80, 70–73. (b) Moulik, S. P.;
Gupta, S. Can. J. Chem. 1989, 67, 356–364. (c) Bockris, J. O. M.;
Reddy, A. K. N. In Modern Electrochemistry 1sIonics; Bockris,
J. O. M., Ed.; Plenum Press: New York, 1977; pp 127-144. (d) Ernst,
S.; Jez´owska-Trzebiatowska, B. J. Phys. Chem. 1975, 79, 2113–2116.
(21) Knight, C. A.; Hallet, J.; DeVries, A. L. Cryobiology 1988, 25, 55–
60.
(22) Jackman, J.; Noestheden, M.; Moffat, D.; Pezacki, J. P.; Findlay, S.;
Ben, R. N. Biochem. Biophys. Res. Commun. 2007, 354, 340–344.
(23) Chakrabartty, A.; Hew, C. L. Eur. J. Biochem. 1991, 202, 1057–1063.
9
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A R T I C L E S
Tam et al.
Table 1. Isentropic Molar Compressibility (104 K2o(s), cm3 mol-1 bar-1) and Hydration Numbers of Various Monosaccharides, 1-4, and
Disaccharides, 5-9a
a Errors of molar compressibility values and hydration numbers are shown in parentheses.8,20b,44
Table 1 reports the isentropic molar compressibility values and
hydration numbers for various mono- and disaccharides.8,9
As an extension of the stereospecific hydration model,15
Furuki used differential scanning calorimetry (DSC) to measure
the heat of fusion of ice in carbohydrate solutions and
subsequently determined the amount of unfrozen water (Uw)
surrounding the carbohydrate molecule.24 These studies suggest
that larger amounts of unfrozen water are correlated with poorer
compatibility of the sugar with the three-dimensional hydrogen-
bonded network of bulk water. Thus, it was proposed that the
antifreeze activity of carbohydrates is a function of hydration,
which is in turn dependent upon carbohydrate stereochemistry.
It should be noted that the technique for determining thermal
hysteresis or antifreeze activity in this study was DSC. Although
DSC has been utilized to study thermal hysteresis (TH) activity
in AFPs and AFGPs, the standardized technique is nanoliter
osmometry.2 However, neither of these techniques assesses
recrystallization-inhibition (RI) activity.
difference between these two temperatures is known as a thermal
hysteretic gap. The significance of this gap is that within this
temperature range, a seeded ice crystal is stable and does not
continue to grow, resulting in a heterogeneous solution contain-
ing suspended microcrystals. Thermal hysteresis is always
preceded by dynamic ice shaping (DIS), which is a direct result
of a solute binding at the interface of the ice lattice and the
quasi-liquid layer (QLL). Alternatively, recrystallization inhibi-
tion activity prevents (or slows down) the enthalpically driven
re-organization of individual ice crystals in an already frozen
sample.2,21,25 While the mechanism of TH activity remains the
source of much debate,26,27 it is ice recrystallization inhibition
activity that is the most desirable property of a cryoprotectant,
as the majority of cellular damage occurs during the holding
and thawing phase of cryopreservation, where recrystallization
is a dominant process.17
TH is defined as a selective depression of the freezing point
of a solution relative to a static melting point, whereby the
(25) (a) Knight, C. A.; Duman, J. G. Cryobiology 1986, 23, 256–262. (b)
McKown, R. L.; Warren, G. J. Cryobiology 1991, 28, 474–482. (c)
Yeh, Y.; Feeney, R. E.; McKown, R. L.; Warren, G. J. Biopolymers
1994, 34, 1495–1504.
(24) (a) Furuki, T. Carbohydr. Res. 2000, 323, 185–191. (b) Furuki, T.
Carbohydr. Res. 2002, 337, 441-450.
(26) Hew, C. L.; Yang, D. S. C. Eur. J. Biochem. 1992, 203, 33–42.
(27) Kristiansen, E.; Zachariassen, K. E. Cryobiology 2005, 51, 262–280.
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Table 2. Ice Crystal Morphology and Melting Points of 10 mg/mL Solutions of D-Galactose (1), D-Glucose (2), D-Talose (4), D-Melibiose (5),
D-Trehalose (7), and D-Sucrose (9) in Double-Distilled Water, Determined Using Nanoliter Osmometry23
Assessing the Carbohydrate-Ice Interaction. Recent reports
have suggested that the antifreeze activity of simple mono- and
disaccharides is based on complimentarity of hydroxyl groups
with the ice lattice. It has been proposed that the optimal distance
between hydroxyl groups is 4.2-4.5 Å.28 To investigate this
possibility, we examined three monosaccharides (galactose,
glucose, and talose) and three disaccharides (melibiose, treha-
lose, and sucrose) for antifreeze activity as a function of thermal
hysteresis using nanoliter osmometry.23 From Table 2, it is
evident that none of these carbohydrates exhibit dynamic ice
shaping, and therefore do not have thermal hysteresis activity.
The slight freezing point depressions are due to the colligative
properties of each carbohydrate. More interesting is the fact that
the ice crystal morphologies of the carbohydrate solutions are
consistent with samples of distilled water. This is significant,
as it indicates that no direct interaction with the ice lattice is
occurring as previously proposed,28 and that a specific distance
between hydroxyls is not the only factor responsible for
imparting antifreeze activity. While it has been suggested that
the protein-ice interaction has an element of surface compli-
mentarity with antifreeze proteins,29 recent molecular dynamic
simulations have implied that the QLL plays an important role
in the binding of biological antifreezes to ice.30 However, it is
still unclear how these biological antifreezes recognize the
pseudo-ordered QLL. We propose that a key contributing factor
for antifreeze activity is hydration of the carbohydrate residue.
To explore this hypothesis, the RI activity of a variety of
carbohydrates was examined as a function of their hydration
number.
Effect of Concentration on Recrystallization-Inhibition Acti-
vity. To determine the optimal working concentration for our
RI assay, a concentration scan was performed. Figure 1 shows
that a 0.22 M solution of galactose in PBS is an effective
inhibitor of recrystallization.
Although this concentration results in reasonable RI activity,
the viscosity of this solution was quite high and posed technical
difficulties that adversely affected assay performance. However,
a 0.022 M solution showed RI activity nearly identical31 to that
of the 0.044 M solution, but did not present viscosity problems.
Consequently, concentrations of 0.022 M were employed for
all saccharides. The overall relationship between carbohydrate
concentration and RI activity appears to be a logarithmic
relationship for the five highest concentrations tested, as shown
in Figure 2. At concentrations lower than 0.00022 M, there is
a limiting effect, whereby the size of the ice crystals in more-
dilute galactose solutions approached the size of ice crystals in
PBS solution. This suggests that at higher concentrations, the
RI activity of galactose is non-colligative, as has been observed
for biological antifreezes.32 This non-colligative relationship has
also been reported by Uchida, who utilized field-emission type
transmission electron microscopy (FE-TEM) to study ice crystal
size as a function of trehalose concentration.33
(28) Baruch, E.; Belostotskii, A. M.; Mastai, Y. J. Mol. Struct. 2008, 874,
170–177.
(31) Statistically significant differences are defined as p < 0.05 based on
a two-sample unequal variance Student’s T-test.
(32) Bouvet, V.; Lorello, G.; Ben, R. N. Biomacromolecules 2006, 7, 565–
571.
(29) (a) Leinala, E. K.; Davies, P. L.; Jia, Z. Structure 2002, 10, 619–627.
(b) Baardsnes, J.; Davies, P. L. Biochim. Biophys. Acta 2002, 1601,
49–54.
(30) Madura, J. D.; Baran, K.; Wierzbicki, A. J. Mol. Recognit. 2000, 13,
(33) Uchida, T.; Nagayama, M.; Shibayama, T.; Gohara, K. J. Cryst.
Growth 2007, 299, 125–135.
101–113.
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Figure 3. RI activity of various monosaccharides (1-4) and disaccharides
(5-9) at 0.022 M in PBS solution, plotted against their respective hydration
numbers.
Figure 1. Recrystallization-inhibition (RI) activity of various concentrations
of D-galactose (1) solutions in PBS.
hertz spectroscopic measurement,39 and viscosity and acoustic
measurements36,40 to molecular dynamic simulations.41 The
density ultrasound technique is regarded as one of the best
experimental methods in that it delineates subtle differences in
solution structure, such as water that is tightly bound to a solute
versus water that is only loosely associated with a solute.42
Although this technique is routinely used to obtain accurate
adiabatic compressibility coefficients, recent studies have shown
that hydration numbers calculated from these coefficients
represent a more accurate summary of contributions influencing
the hydration of the solute molecule.43 The hydration number
differs from isentropic molar compressibility values in that
hydration numbers accurately predict the total number of water
molecules hydrogen-bonded to the sugars. However, this is still
regarded as a dynamic measurement because it really predicts
the number of water molecules that have a relatively long
residence time and hence move in solution with the sugars.
Given that hydration numbers are a more accurate representation
of solute hydration, we plotted the RI activity as a function of
hydration numbers8 (calculated from isentropic molar compress-
ibility coefficients) for simple mono- and disaccharides with
varying hydroxyl stereochemistry, Figure 3.
From this data, each series of carbohydrates (mono- and
disaccharides) show a strong linear trend between hydration
numbers and their respective RI activity. However, the general
increase in hydration number for disaccharides relative to the
monosaccharides did not necessarily correlate to an increase in
RI activity. For example, the RI activity of galactose (hydration
number ) 8.7) and melibiose (15.5) are very similar (mean grain
size ) 0.0082 mm2), despite the large difference between their
respective hydration numbers. More surprisingly, the difference
in hydration numbers between galactose (8.7) and sucrose (13.9)
resulted in a decrease in RI activity for the latter. We believe
this is due to the difference in steric volume between monosac-
charides and disaccharides and have corrected for it in the
following manner. Hydration numbers were obtained from the
Figure 2. RI activity of various concentrations of D-galactose (1) in PBS
solution. The dashed line represents the mean grain size of PBS control.
X-axis represents the log10(concentration) of D-galactose solution.
Carbohydrate Stereochemistry. Literature reports have sug-
gested that the compatibility of a carbohydrate with the three-
dimensional hydrogen-bonded network of water is governed by
carbohydrate stereochemistry.8,9,34 This observation correlates
well with experimentally derived molar compressibility values
in that these also vary as a function of hydroxyl stereochemistry
at C2 and C4 (Table 1).
Our laboratory previously reported the RI activity of C-linked
AFGP analogues.19 It was observed that analogues containing
carbohydrates with low isentropic molar compressibilities (such
as galactose) fit poorly into the three-dimensional hydrogen
bonded network of bulk water and hence are effective inhibitors
of recrystallization. While this hypothesis was based upon
isentropic molar compressibilities, this measurement of solute
hydration may not be truly representative of the actual hydration
state.
Many experimental methods have been reported to study the
structure of solutions. These range from near-infrared spectros-
copy,35 density and density ultrasound,8,36 nuclear magnetic and
dielectric-relaxation,37 quasi elastic neutron scattering,38 tera-
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A R T I C L E S
Figure 5. C-allylated derivatives of galactose (10, 14), glucose (11, 15),
mannose (12), and talose (13).
determine the influence of a carbon substituent at C1 and its
stereochemistry on carbohydrate hydration and RI activity.
Despite the absence of hydration numbers, the RI activity of
C-linked analogues and their respective O-linked monosaccha-
rides is shown in Figure 6. Of all the R-C-allyl pyranoses
(10-13), galactose derivative 10 possesses the most RI activity
with mean grain size (MGS) statistically identical31 to native
D-galactose. In fact, the RI activity trend of 10-13 is identical
to their corresponding native monosaccharides 1-4, respec-
tively. This is consistent with the hypothesis that the nature of
the substituent at C1 may have little influence on hydration and
hence RI activity. In contrast, ꢀ-anomer 14 exhibits a marked
decrease in RI activity relative to native O-linked galactose, 1,
and R-C-linked derivative, 10, suggesting that the anomeric
configuration of C-linked carbohydrates may play some role in
hydration and therefore RI activity. Comparatively, ꢀ-C-allyl
glucose, 15, displayed the same amount of RI activity as its
R-anomer, 11, with no statistically significant difference31 in
MGS. Further studies are required to ascertain the exact effect
of anomeric configuration on RI activity and its subsequent
relationship to hydration.
Figure 4. RI activities of carbohydrates (1-9), plotted against their
respective hydration index (hydration number/partial molar volume) (mol1
cm-3).
literature and were derived according to the Passynsky equation
(eq 1) using molar compressibility coefficients, ꢀ, which were
obtained from ultrasound and density measurements (eq 3),8
ꢀ)1 ⁄ u2d
(3)
where u is the speed of sound and d is the density of the solution.
Dividing hydration numbers by partial molar volumes8,44 results
in a description of the number of tightly bound water molecules
per molar volume of carbohydrate. We refer to this value as a
hydration index. Similar correlations have been reported by
Parke et al. in relating the taste properties of solutes of various
masses, volumes, and hydrophobicity to the hydration per unit
volume of the solute.45 Figure 4 shows that plotting the
hydration index against RI activity of carbohydrates 1-9 results
in a single trend for all carbohydrates, compared to Figure 3
which had two distinct correlations.
Comparison to DMSO. The ability of D-galactose 1 and R-C-
allyl galactose derivative 10 to function as cryoprotectants was
assessed. This was accomplished using dimethylsulfoxide
(DMSO) as a standard. DMSO is regularly used as an additive
to cellular suspensions prior to freezing and storage at subzero
temperatures.47 Despite the routine use of DMSO, studies have
shown it is cytotoxic and capable of eliciting apoptosis in many
different cell types.47 The generally accepted mechanism by
which DMSO imparts cryopreservation is two-fold. First, it is
thought to replace water in the cell membrane and thus prevent
fractionation of the cell membrane during thermatropic phase
transitions.48,49 Second, molecular dynamics simulations have
shown that individual DMSO molecules may cooperatively form
extensive ion channels in the cell membrane and thus facilitate
the transport of water into and out of the cell to relieve osmotic
stress during the freezing event.48,49 Regardless of the nature
of the mechanism, the use of DMSO does result in improved
viability of many cell types post freeze-thaw as compared to
other protocols, and thus it is routinely employed in cryopreser-
vation.50 Typically, DMSO concentrations employed in routine
cryopreservation range between 1-20 mol%, but recent studies
have shown that very little additional cryoprotection is conferred
This is significant in that while the absolute number of water
molecules surrounding a solute is important for predicting RI
activity, the number of water molecules per unit volume of
solute is also important; thus, a higher concentration of water
molecules around a solute will increase disorder of the sur-
rounding bulk water29 which consequently leads to increased
RI activity.
RI Activity Comparison between C-Glycosides and O-Gly-
cosides. The AFGP analogues previously synthesized by our
laboratory are C-linked in nature.19 While the conformation of
C-linked carbohydrate analogues is generally accepted to be
similar to O-linked systems,46 hydration numbers of C-linked
carbohydrates have not been reported and the relationship
between hydration and RI activity has not been previously
studied. It has been suggested that hydration is largely dictated
by substituents at C2 and C4 of the pyranose ring.8,34 To test
this hypothesis, C-allylated derivatives of galactose, glucose,
mannose, and talose (10-15, Figure 5), were synthesized to
(44) (a) Høiland, H.; Holvik, H. J. Solution Chem. 1978, 7, 587–596. (b)
Shahidi, F.; Farrell, P. G.; Edward, J. T. J. Solution Chem. 1976, 5,
807–816.
(45) Parke, S. A.; Birch, G. G.; Dijk, R. Chem. Senses 1999, 24, 271–279.
(46) (a) Espinosa, J. F.; Montero, E.; Vian, A.; Garc´ıa, J. L.; Dietrich, H.;
Schmidt, R. R.; Mart´ın-Lomas, M.; Imberty, A.; Can˜ada, F. J.;
Jime´nez-Barbero, J. J. Am. Chem. Soc. 1998, 120, 1309–1318. (b)
Wang, J.; Kova´cˇ, P.; Sinay¨, P.; Glaudemans, C. P. J. Carbohydr. Res.
1998, 308, 191. (c) Wang, Y.; Barbirad, S. A.; Kishi, Y. J. Org. Chem.
1992, 57, 468–481. (d) Ravishankar, R.; Surolia, A.; Vijayan, M.; Lim,
S.; Kishi, Y. J. Am. Chem. Soc. 1998, 120, 11297–11303. (e) Ma, B.;
Schaefer, H. F., III.; Allinger, N. L. J. Am. Chem. Soc. 1998, 120,
3411–3422.
(47) (a) Heng, B. C.; Ye, C. P.; Liu, H.; Toh, W. S.; Rufiahah, A. J.; Yang,
Z.; Bay, B. H.; Ge, Z.; Ouyang, H. W.; Lee, E. H.; Cao, T. J. Biomed.
Sci. 2006, 13, 433–445. (b) Ji, L.; de Pablo, J. J.; Palecek, S. P.
Biotechnol. Bioeng. 2004, 88, 299–312.
(48) Gurtovenko, A. A.; Anwar, J. J. Phys. Chem. B 2007, 111, 10453–
10460.
(49) Notman, R.; Noro, M.; O’Malley, B.; Anwar, J. J. Am. Chem. Soc.
2006, 128, 13982–13893.
(50) McGann, L. E.; Walterson, M. L Cryobiol. 1987, 24, 11–16.
9
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A R T I C L E S
Tam et al.
Figure 6. RI activity of native O-linked monosaccharides (1-4) and their C-glycoside derivatives (10-15) at 0.022 M in PBS solution.
inorganic composites.52 In ice, there are two key issues that
need to be considered. First, a small amount of bulk water is
present between adjacent ice crystals. Second, the interface of
the ice lattice and bulk water is not an abrupt transition.
Independent studies have proven that a layer of semi-ordered
ice (quasi-liquid layer, QLL) exists between the highly ordered
ice lattice and bulk water.53 During the last several years, the
nature and properties of this interfacial domain have been studied
extensively and implicated in the ability of antifreeze proteins
to recognize ice.30 Consequently, a mechanism of action for
inhibitors of ice recrystallization must account for the QLL. On
the basis of the results of the current study, a direct interaction
between the carbohydrate and the ice lattice is unlikely since
no thermal hysteresis or dynamic ice shaping is observed (Table
2).
In the splat-cooling RI assay, the wafer is frozen and contains
very little unfrozen water. Furthermore, as solutes are excluded
from the ice lattice, the carbohydrate will be concentrated at
the interface between two adjacent ice crystals and their QLLs.
The QLL is expected to be approximately 3-10 Å in thickness,
although this thickness is known to be temperature-dependent.53
Taking all of this into account, a representation of a carbohydrate
solvated in bulk water between two adjacent ice crystals and
their QLLs is shown in Figure 8.
Figure 7. RI activity of various concentrations of DMSO and 0.022 M
solutions of compounds 1 and 10 in PBS solution.
at concentrations above 5 mol%.51 To the best of our knowledge,
the ability of DMSO to function as an inhibitor of recrystalli-
zation has not yet been explored. Consequently, an initial
concentration scan was performed using our RI assay. The
results of this study are shown in Figure 7.
As illustrated in Figure 7, concentrations of 0.1 to 4% (v/v)
DMSO were assessed. In practice, concentrations above 6%
(v/v) were very difficult to use in this assay and produced
inconsistent results due to large portions of unfrozen solution
in the ice wafer. Consequently, only concentrations less than
that of 6% were reliably assessed. A 0.1% DMSO solution failed
to inhibit recrystallization and yielded ice crystals identical in
size to the PBS control. In contrast, a 4% solution of DMSO
exhibited RI activity higher than a 0.022 M galactose solution.
As a benchmark for comparison between DMSO concentration
and galactose concentration, a 0.022 M solution of galactose is
as efficient at inhibiting recrystallization as a 3% (v/v) DMSO
solution, Figure 7. These results suggest that galactose may be
a suitable cryoprotectant.
The question of where the carbohydrate localizes with respect
to the QLL is a difficult question to answer given current
inabilities to study the QLL. However, there are two possibilities.
In the first, the carbohydrate is concentrated at the bulk
water-QLL interface while in the second, the carbohydrate is
actually incorporated into the QLL. The latter does not seem
probable, as we are not aware of any precedent for incorporation
of a carbohydrate into the QLL. Furthermore, Uchida and co-
workers have studied the RI activity of trehalose using TEM
and proposed that trehalose functions at the bulk water-QLL
interface.33
Our studies are consistent with others24,28,34 in that they
suggest that carbohydrate stereochemistry does play some role
(52) (a) Pronk, P.; Infante Ferreira, C. A.; Witkamp, G. J. J. Cryst. Growth
2005, 275, e1355-e1361. (b) Huige, N. J. J.; Thijsenn, H.A. C. J.
Cryst. Growth 1972, 13/14, 483–487. (c) Inada, T.; Modak, P. R.
Chem. Eng. Sci. 2006, 61, 3149–3158. (d) Kingery, W. D. J. Appl.
Phys. 1959, 30, 301–306.
Proposed Recrystallization-Inhibition Mechanism of Action
with Ice. The mechanism of recrystallization has been studied
extensively in the metallurgical literature within the context of
(53) (a) Karim, O.; Haymet, A. D. J. Chem. Phys. Lett. 1987, 138, 531–
534. (b) Sadtchenko, V.; Ewing, G. E. J. Chem. Phys. 2002, 116, 4686–
4697.
(51) Leseth, K.; Abrahamsen, J. F.; Bjorsvik, S.; Grottebo, K.; Bruserud,
O. Cryotherapy 2005, 4, 328–333.
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Inhibiting Recrystallization with Small Molecules
A R T I C L E S
molecules by increasing the number of water-carbohydrate
hydrogen bonds that can interact with water. Consequently,
carbohydrates with larger hydration indices function as better
ice recrystallization inhibitors. Larger absolute hydration num-
bers for disaccharides relative to smaller-sized monosaccharides
do not necessarily translate to an increased inhibition of ice
growth, Figure 3; a disaccharide with a small hydration index,
such as sucrose, has little effect upon ice recrystallization despite
a larger absolute hydration number and volume than that of a
monosaccharide such as galactose. The ultimate significance of
this relationship is that there needs to be a large number of water
molecules in the hydration shell, and these must be highly
concentrated around the solute, helping to increase the entropy
of surrounding bulk water.29
Conclusions
In summary, we have demonstrated that the hydration of a
carbohydrate is correlated to RI activity. The relationship
between carbohydrate concentration and RI activity is non-
colligative, and the hydration index modulates the ability of a
sugar to function as a potent inhibitor of recrystallization.
Among the monosaccharides examined in this study, galactose
possesses the most RI activity, whereas in the disaccharides,
melibiose is the most potent. While C-linked derivatives of
monosaccharides appear to parallel the RI activity of their
O-linked native structures, the importance of anomeric stereo-
chemistry in C-linked derivatives requires further investigation.
A 0.022 M solution of D-galactose, 1, and C-linked galactose
derivative, 10, inhibit recrystallization as well as a 3% DMSO
solution. On the basis of the fact that the carbohydrates examined
in this study did not possess any TH activity or dynamic ice
shaping, we propose that they are inhibiting recrystallization at
the bulk water-QLL interface by disrupting the pre-ordering
of water. The concept of a hydration index will greatly facilitate
the rational design of novel inhibitors of recrystallization for
medical, commercial, and industrial applications.
Figure 8. Schematic representation of the proposed mechanism for
inhibition of ice recrystallization. Carbohydrates reside at the QLL–bulk
water interface between two adjacent ice crystals. Water molecules
surrounding a carbohydrate with a high hydration index will be more
disordered than in cases with a low hydration index carbohydrate or in the
absence of solute. The area shaded red represents hydrated solute, QLL )
quasi-liquid layer.
in inhibiting the process of ice recrystallization, but we propose
that due to the interaction with the QLL, hydration of the
carbohydrate is a significant factor for inhibiting recrystallization
of ice.
Our previous work tentatively linked isentropic molar com-
pressibilities and carbohydrate stereochemistry to RI activity.19
The work in the present paper suggests that hydration index
(hydration number per molar volume of carbohydrate) consti-
tutes a better predictor of RI activity, Figure 4. Carbohydrates
with larger hydration indices (such as galactose and melibiose)
positioned at the interfacial domain cause a greater disruption
on the ordering of bulk water surrounding the hydration shell,
which leads to slightly increased energies associated with the
transfer of bulk water to the QLL. Given that the thickness of
the QLL is small and the entropy of the first hydration shell
can extend out to the third hydration shell,39 this energy
difference would be significant. Heyden et al. have recently
confirmed this long-range effect on bulk water using terahertz
absorption measurements.39 This study demonstrates that sol-
vated carbohydrates alter the long-range motion of water
Acknowledgment. The authors acknowledge NSERC, CIHR,
and CFI for financial support. R.N.B. holds a Tier 2 Canada
Research Chair (CRC) in medicinal chemistry. J.L.C. holds an
NSERC PGS-D award.
Supporting Information Available: Procedures and charac-
terization of all new compounds. This material is available free
of charge via the Internet at http://pubs.acs.org.
JA806284X
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