Antifreeze protein-induced selective crystallization of a new thermodynamically and kinetically less preferred molecular crystal

Article abstract of DOI:10.1002/chem.201302049

The formation of a new, dihydrate crystalline form of 5-methyluridine (m⁵U) was selectively induced by a protein additive, antifreeze protein (AFP) in a highly efficient manner (in 10^-6molar scale, whereas known kinetic additives nee

Full text of DOI:10.1002/chem.201302049

DOI: 10.1002/chem.201302049
Antifreeze Protein-Induced Selective Crystallization of a New
Thermodynamically and Kinetically Less Preferred Molecular Crystal
Sen Wang,*[a] Xin Wen,*[b] James A. Golen,[c] Josh F. Arifin,[b] and
Arnold L. Rheingold[c]
Abstract: The formation of a new, di-
hydrate crystalline form of 5-methyluri-
dine (m5U) was selectively induced by
a protein additive, antifreeze protein
(AFP) in a highly efficient manner (in
10ꢀ6 molar scale, whereas known kinet-
ic additives need 0.1 molar scale). The
hemihydrate form (form I, the only
previously known crystalline form of
m5U) and the dihydrate form of m5U
(form II) obtained herein were charac-
terized using X-ray crystallography
and differential scanning calorimetry
(DSC). Compared to form I, remarka-
bly, form II is thermodynamically and
kinetically less preferred. The presence
of AFP can selectively inhibit the ap-
pearance of form I and hence allows
the growth of form II, the pure form of
which cannot grow directly from m5U
supersaturated solutions under the
same conditions. An explanation sup-
ported by both experimental and theo-
retical results is provided for the AFP-
induced selection process. Implications
on AFP-induced ice shape changes are
also discussed. Control of crystalliza-
tion from supersaturated solutions is of
great interest in both fundamental re-
search and practical applications in
fields like chemistry, pharmacology and
materials science. These findings sug-
gest that crystallization processes with
AFPs could be valuable for selective
growth of hydrates and polymorphs of
important pharmaceutical compounds.
Keywords: crystal growth · 5-meth-
yluridine · nucleosides · proteins ·
X-ray crystallography
Introduction
compounds to select favored and/or discover new crystalline
forms. Although tremendous effort has been devoted to un-
derstanding the crystallization process and selective crystalli-
zation, crystallization control remains largely trial-and-error,
experiencing substantial difficulties in exclusive production
of the desired forms as well as the production of both ther-
modynamically and kinetically less-favored forms.[1a,5] More-
over, much less progress has been made in additive-con-
trolled organic crystallization than in additive-controlled in-
organic crystallization and the selective production of organ-
ic single crystals with defined crystal phase and morphology
is hard to achieve.[10]
Antifreeze proteins (AFPs) are a group of structurally di-
verse proteins that are known in many cold-adapted organ-
isms and are essential for their survival at subzero tempera-
tures.[11] AFPs can inhibit ice-crystal growth and modify the
ice-crystal habit by binding to specific faces of the ice crys-
tals, providing one of the most notable examples of crystalli-
zation control in nature.[12] The affinity of AFPs to ice de-
pends on hydrogen-bonding and/or hydrophobic interac-
tions, and is unlike most protein–mineral interactions in
which ionic interactions often dominate.[13] AFPs can also in-
hibit the growth of gas hydrates[14] as well as the growth of
other molecular crystals that are not ice-like.[15] Nucleosides
(and analogues) are important pharmaceutical compounds
and it is of practical significance to obtain different forms of
these compounds due to the various physicochemical prop-
erties and bioavailabilities of the diverse forms.[16] The nu-
cleoside, 5-methyluridine (m5U), is an important compound
in the pharmaceutical industry.[17] We have reported that
Controlling the crystallization of compounds is of great in-
terest in various fields of application, from chemistry to
pharmacology, and materials science to food science.[1] For
instance, changes in the size and shape of crystals of phar-
maceutical compounds can impact their bioavailability,
chemical stability, and production efficiency. Traditional
methods to control crystallization usually include alterations
in temperature, solvent, supersaturation, and seeding condi-
tions.[2] A number of recent methods, such as the use of
tailor-made additives,[3] ledge-directed epitaxy,[4] polymer
microgel,[5] polymer heteronuclei,[6] capillaries,[7] porous ma-
terials,[8] and laser-induced nucleation,[9] have been devel-
oped and employed in the crystallization process of certain
[a] Dr. S. Wang
Molecular Imaging Program, Stanford University
Stanford 94305 (USA)
E-mail: swang02@stanford.edu
[b] Prof. X. Wen, J. F. Arifin
Department of Chemistry and Biochemistry
California State University, Los Angeles
5151State University Drive, Los Angles 90032 (USA)
E-mail: xwen3@calstatela.edu
[c] Prof. J. A. Golen, Prof. A. L. Rheingold
Department of Chemistry and Biochemistry
University of California, San Diego
La Jolla, CA 92093 (USA)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201302049.
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DAFP-1, a beetle AFP from Dendroides canadensis, can ef-
ficiently inhibit the crystal growth of m5U hemihydrate, the
only previously known crystalline form of m5U, and that hy-
drogen-bonding interactions between DAFP-1 and specific
m5U crystal surfaces may play an important role.[15]
needle-like in appearance and there was no change (in the
appearance, single crystal X-ray diffraction pattern, or DSC
profile) of the crystals after being left in air at room temper-
ature for more than 24 months (Figure 1a). Form II crystals
Crystal growth at the molecular level can be modeled as
a process starting from prenucleation aggregate formation,
followed by crystal nuclei evolution and macroscopic crystal
development.[18] The arrangements and shapes of at least
some of these prenucleation aggregates and evolved nuclei
are thought to resemble the final crystal structure, and the
control of which is a decisive step in controlling the final
crystalline form. To further understand whether (and how)
DAFP-1 influences polymorphism or crystalline form(s) of
m5U, we have made significant efforts on altering and con-
trolling the crystallization conditions.
Figure 1. Optical micrographs of the final m5U crystals achieved:
a) needle-shaped m5U hemihydrates (form I); b) block-shaped m5U dihy-
drates (form II) obtained in the presence of DAFP-1; c) needle-shaped
m5U hemihydrate (form I) obtained in the presence of denatured DAFP-
1 (control).
Here, we present the first case of using DAFP-1 as
a novel, highly efficient additive to exclusively obtain a new
crystalline form of m5U, m5U dihydrate, from supersaturated
m5U aqueous solutions. The structures of the two crystalline
forms of m5U obtained herein, m5U hemihydrate (form I)
and m5U dihydrate (form II), were completely determined
using X-ray crystallography and the crystals were also char-
acterized with differential scanning calorimetry (DSC).
Form II crystals with a block-like morphology are thermody-
namically and kinetically less preferred than the needle-like
form I crystals. It is known that crystal shape changes can be
due to either the habit modification of the same crystalline
form or more frequently the formation of a new crystalline
form[1a,2] In this study, the formation of the new, thermody-
namically and kinetically less preferred m5U dihydrate crys-
tals could be induced by DAFP-1, demonstrating the latter
case.
Few physical or chemical processes can happen when the
product is neither thermodynamically nor kinetically fa-
vored.[2,19] Under the same conditions, pure form II cannot
grow directly from m5U supersaturated solutions, however,
the presence of DAFP-1 can selectively inhibit the nuclea-
tion of form I and hence allows the crystal growth of
form II. The AFP-induced selection process was rationalized
based on the experimental and theoretical results. Using
AFPs in crystallization processes may be valuable for selec-
tive growth of hydrates and polymorphs of hydroxyl com-
pounds.
are block-shaped (Figure 1b) and turned to white opaque
powders/blocks in 2 months. The resulting solid powders
were not suitable for single-crystal X-ray diffraction. By
using the denatured DAFP-1 as an additive (a control), no
effect on the crystallization of m5U was observed under the
same conditions, that is only the needle-like m5U form I
crystals were obtained (Figure 1c).
Structural aspects: The structures of both forms were deter-
mined by single-crystal X-ray diffraction (Figure 2). Both
forms crystallize in the orthorhombic crystal system. Form I
was solved in the space group P21212, whereas the new form
is in P212121 (Table 1). There is a high degree of torsional
Table 1. Crystallographic data for the hemihydrate (form I) and dihy-
drate (form II) of m5U.
Parameter
Form I
Form II
formula
Mr
C10H14N2O6·0.5H2O
267.24
C10H14N2O6·2H2O
294.26
T [K]
90(2)
100(2)
crystal system
space group
a [ꢁ]
b [ꢁ]
c [ꢁ]
orthorhombic
P21212
13.953(2)
17.201(3)
4.8017(7)
90
orthorhombic
P212121
6.75400(10)
8.83730(10)
20.9847(3)
90
a=b=g [8]
cell volume [ꢁ3]
1152.4(3)
1.540
1252.52(3)
1.560
1calcd [gcmꢀ3
]
Z
4
4
data/restraints/parameters
final R indices for I>2s(I)
1941/5/184
R1 =0.0274
wR2 =0.0730
2199/10/206
R1 =0.0282
wR2 =0.0741
Results and Discussion
The only previously known crystalline form of m5U is its
hemihydrate crystal, designated form I, which was crystal-
lized exclusively by evaporation from the supersaturated
aqueous solutions. By using DAFP-1 as an additive, remark-
ably, we were able to control the crystallization of m5U and
discover a novel, less thermodynamically and kinetically
preferred form, m5U dihydrate crystal, designated form II.
Form I and II crystals are readily distinguished from each
other by their morphology and stability. Form I crystals are
5
ꢀ
freedom in the N1 C6 bond in the m U molecule. The over-
lap of m5U molecules in form I and form II reveals a signifi-
cant conformational difference in the ribose moiety
(Figure 3). Such change may allow optimization of hydrogen
bonds between the hydroxyl groups in the ribose moiety
and neighboring groups in m5U and water. Water molecules
are hydrogen bound to m U molecules through N H···O
5
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S. Wang, X. Wen et al.
Figure 3. Overlay of unique molecules in the crystal structure of m5U
form I (all in gray) and form II (all in gray, except hydrogen atoms in
light gray).
moiety (O4), and water (O7). Two oxygen atoms from a hy-
droxyl group in the ribose moiety (O5) and water (O7) are
on the face (010) (Figure 4a and Table 2). The oxygen atom
in the thymine moiety (O1) is on the face of (001) (Fig-
ure 4b and Table 2). In contrast to form I, the three growing
faces of form II are more hydrophobic. No potential hydro-
gen-bond donor or acceptor atom is observed on the face of
(010) or (001); and the oxygen atoms (O7 and O8) of the
two water molecule are on the (100) face (Figure 4c and d;
Table 2).
Hydrogen-bonding interactions are known to be essential
in some AFPs for recognizing ice crystals and the distance
of the side chain hydroxyl groups in the conserved repeat
residues in the AFPs matches that of repeating oxygen
atoms on the prism face of the normal ice crystal.[15,20]
DAFP-1 is a repeat protein with conserved threonines as pu-
tative ice-binding residues in each repeat unit.[21] This sug-
gests DAFP-1 may recognize these faces of m5U form I
through hydrogen-bonding interactions, whereas it may not
interact with any face of m5U form II crystal due to the lack
of match of possible hydrogen-bond donor/acceptor atoms
in DAFP-1 and the growing faces of form II (Supporting In-
formation, Figure S3). Therefore, DAFP-1 can selectively in-
hibit form I crystallization, but allow the growth of form II
crystal.
Figure 2. Representative molecules in: a) m5U form I (hemihydrate), and
b) m5U form II (dihydrate).
ꢀ
and O H···O interactions. Compared to those in form I, the
number of strong intermolecular hydrogen bonds between
two m5U molecules decreases, whereas the number of hy-
drogen bonds between m5U and water molecules increases
in form II (Table 2). The strong intermolecular hydrogen
bonds between two m5U molecules in form I are disrupted
upon the introduction of additional water molecules. Conse-
quently, the packing motifs of the two forms of m5U are dif-
ferent (Figure 4).
The interactions between DAFP-1 and m5U form I are
highly efficient since only a tiny amount of DAFP-1 as addi-
tives is needed (the additive/m5U molar ratio was 5ꢂ10ꢀ6).
Moreover, such interactions need to be weak in order to
guide the crystal growth. The selective crystallization of
form II by using DAFP-1 cannot be obtained if the interac-
tions between DAFP-1 and m5U are strong. This is because
if the interactions between DAFP-1 and m5U were strong,
All the growing faces in the form I crystal contain poten-
tial hydrogen-bond donor or acceptor atoms (Figure 4a and
b). On the (100) face, there are three oxygen atoms from
the thymine moiety (O1), a hydroxyl group in the ribose
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Antifreeze Protein-Induced Crystallization
FULL PAPER
with water loss, is at 139.078C,
whereas the second, ascribed to
the melting, is at 185.658C.
Form II exhibits two endo-
therms at 137.04 and 153.728C
for the loss of the two water
molecules, followed by a recrys-
tallization
exotherm
at
157.668C and then a melting
endotherm at 183.418C. Form II
starts losing water and then
melts at relatively lower tem-
peratures than those of form I.
The enthalpy profiles for both
forms are compared in Figure 6.
Form I is more thermodynami-
cally stable, which is in accord-
ance with the previous observa-
tions.
The crystallization processes
of both forms were carried out
at ambient conditions. If we
assume that the reaction of
form II becoming form I by
losing water at room tempera-
ture, that is, m5U form II!m5U
form I+1.5H2O, then heat is
needed to cause the dihydate
form II to lose water and
become the hemihydrate form I
Figure 4. Packing diagrams of: a,b) m5U form I, and c,d) form II. Dotted lines represent intermolecular hydro-
gen bonding in the crystals.
Table 2. Geometrical parameters for hydrogen-bonding interactions in forms I and II.
(Figure 6). Therefore, the DH for the reaction is
positive, that is:
[a]
ꢀ
ꢀ
D H···A
d(D H)
d(H···A)
d(D···A)
a(DHA)
[8]
[ꢁ]
[ꢁ]
[ꢁ]
[i]
form I[b]
O(4) H(4O)···O(5)
0.886(15) 1.881(15) 2.7658(15) 175.3(17)
0.840(15) 1.887(15) 2.7158(15) 168.7(18)
0.833(15) 1.890(15) 2.7097(14) 167.6(19)
0.860(14) 2.023(14) 2.8722(13) 169.2(18)
0.889(14) 2.106(15) 2.9764(13) 166.0(17)
DH > 0
ð1Þ
ꢀ
[ii]
[iii]
ꢀ
O(5) H(5O)···O(2)
ꢀ
At room temperature, T:[19a]
O(6) H(6O)···O(1)
ꢀ
O(7) H(7O)···O(6)
[iv]
ꢀ
N(2) H(2N)···O(7)
dðDG=TÞdT ¼ ꢀDH=T2
ð2Þ
form II[c] O(4) H(4O)···O(8)
0.838(16) 1.853(16) 2.6902(15) 177(2)
ꢀ
[i]
ꢀ
O(5) H(5)···O(7)
0.86(2)
2.09(2)
2.8868(15) 154.2(19)
For a tiny temperature increase, dT, the resulting
temperature, T’ can be expressed as T’=T+dT
and:
[ii]
ꢀ
O(6) H(6O)···O(2)
0.865(16) 1.957(17) 2.7954(16) 163(2)
0.846(15) 2.073(15) 2.9103(17) 170.0(19)
0.848(15) 2.041(16) 2.8510(16) 159(2)
0.862(15) 1.926(15) 2.7783(16) 169.9(19)
0.854(14) 2.049(15) 2.9016(16) 177.2(19)
[iii]
[iv]
[v]
ꢀ
O(7) H(7WB)···O(5)
O(8) H(8WA)···O(5)
O(8) H(8WB)···O(1)
ꢀ
ꢀ
T0 > T
ð3Þ
ꢀ
N(2) H(2N)···O(7)
[a] D=donor, A=acceptor. [b] Symmetry transformations used to generate equiva-
lent atoms: [i] ꢀx+1,ꢀy+1,z; [ii] ꢀx+1/2,yꢀ1/2,ꢀz+1; [iii] xꢀ1/2,ꢀy+3/2,ꢀz;
[iv] ꢀx+1/2,y+1/2,ꢀz+1. [c] Symmetry transformations used to generate equivalent
atoms: [i] ꢀx+3/2,ꢀy+1,zꢀ1/2; [ii] ꢀx,y+1/2,ꢀz+1/2; [iii] ꢀx+1,y+1/2,ꢀz+1/2;
[iv] x+1/2,ꢀy+1/2,ꢀz; [v] ꢀx+2,yꢀ1/2,ꢀz+1/2.
Assuming that DG is a constant for such a tiny
change, we obtain:
DGðd1=TÞ ¼ ꢀDH=T2dT
ð4Þ
some m5U molecules would bind to DAFP-1 completely in
solution, leaving only pure m5U and finally resulting in
form I only.
By integrating the above equation, we obtain:
DGðlnT0=TÞ ¼ DH ð1=T0ꢀ1=TÞ
ð5Þ
Thermodynamic stability analysis: Both forms of m5U were
analyzed by DSC (Figure 5). Form I displays two endother-
mic events upon heating. The first endotherm, associated
Combining Equatons (1), (3) and (5), we obtain DG<0.
Therefore, the free energy change of the form II-to-I transi-
tion at room temperature is negative. That is the form I
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S. Wang, X. Wen et al.
Figure 5. DSC thermograms (exo up) of: a) form I, and b) form II of
m5U.
Figure 7. Comparison of crystallization kinetics of form I and form II of
m5U. Mass increase of crystals during the crystallization processes was
used to estimate relative crystallinity.
AFP-induced control of crystallization rests on the assump-
tion that supersaturated solutions contain nuclei adopting
similar arrangements to the final crystal structure. This as-
sumption has been successful in designing tailor-made inhib-
itors, remarkably however, the efficiency of AFP is superior
to most known additives.[22] That is, the amount of AFP
needed is significantly smaller than other additives. The
molar ratio of AFP and m5U is in the range of 10ꢀ6; in con-
trast 5–10% of additives are usually needed for crystalliza-
tion control.[22]
In the supersaturated solution of m5U, form I nuclei the
structures of which are similar to those of form I crystals are
evolved at around day 2 (Figure 7). In the absence of
DAFP-1, form I crystals grow. As suggested by the match
between the hydroxyl groups in the conserved threonines in
DAFP-1 and the hydrogen-bond donor/acceptor atoms on
the growing faces of (100) and (010) in form I of m5U,
DAFP-1 can recognize the fast-growing face (100) and the
(010) face of form I through hydrogen-bonding interactions.
Hence, form I nuclei can be completely inhibited in the
presence of DAFP-1 and then the extent of supersaturation
increases during the following days until the crystallization
of form II at around day 5 (Figure 7 and Figure 8). That is
the higher degree of supersaturation attained in the pres-
ence of DAFP-1 is required for the formation of form II
nuclei. In addition, due to the mismatch between the hydro-
gen-bond donor/acceptor atoms in DAFP-1 and in the grow-
ing faces of form II, DAFP-1 cannot interact with the newly
Figure 6. Comparison of relative enthalpies for the phase transitions of
form I and form II m5U.
crystal is more thermodynamically stable. The results are
consistent with the observations that form I crystals were
stable in air for at least 2 years, whereas form II crystals
became white powders/blocks in 2 months in air.
Crystallization kinetics analysis: In the absence of DAFP-1,
form I starts growing on day 3 and the crystallization com-
pletes on day 9 (Figure 7). DAFP-1 can inhibit the crystalli-
zation of form I. In the presence of DAFP-1, only form II
can be grown and the crystallization starts on day 6 and fin-
ishes on day 15. The crystallization of form II cannot
happen in the absence of DAFP-1, since the crystallization
of form I has much faster kinetics than that of form II. How-
ever, the presence of DAFP-1 completely inhibits the fast
growing form I and hence results in the exclusive formation
of the new, slow growing form II. The average crystal
growth rate of form II is estimated to be 10.5% per day,
which is slower than that of form I, 13.3% per day.
Figure 8. Suggested mechanism for crystallization of: a) m5U, and b) se-
lective crystallization of m5U form II using DAFP-1.
Mechanism of AFP-induced selective nucleation and
growth: Our approach with regard to the mechanism of the
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Antifreeze Protein-Induced Crystallization
FULL PAPER
formed form II nuclei. Consequently, only form II grows,
even though it is a thermodynamically and kinetically less
preferred phase (Figure 8).
1.[11b,21,23] Although the ice-binding site of some other types
of AFPs are reported to be more hydrophobic than the rest
of the protein,[11b,24] the ice-binding site of TmAFP an
DAFP-1 are suggested to be more hydrophilic.[11b,21,23] Due
to the remarkable diverse structures of AFPs, it would be
possible that different ice-binding mechanisms may occur in
different AFPs and both hydrophobic and hydrogen-bond-
ing interactions can be important for the binding of AFPs to
the ice–water interface.[25] Therefore, we speculate that the
hydrophilic face of DAFP-1 can bind to the thermodynami-
cally preferred form I nuclei of m5U, whereas the hydropho-
bic face of DAFP-1 is thus exposed to bulk solution[15,20,21,23a]
resulting in large repulsive interactions against incoming
m5U molecules and hence the inhibition of form I crystalli-
zation, which leads F>1. However, DAFP-1 cannot bind to
m5U form II nuclei. The total contributions from F, interfa-
cial tension and the radius of nuclei, finally lead to the nu-
cleation of the metastable crystalline. At this point, the free
energy of form I nuclei with AFP attachment is still higher
than that of crystalline form II. This finally allows the ther-
modynamically less preferred dihydrate crystalline form to
fully grow.
Theoretical analysis: Both thermodynamic analysis and ki-
netics analysis show the possibility of the AFP-induced se-
lective crystallization of the less thermodynamically and ki-
netically preferred m5U dihydrate crystal and give insights
into this process.
Thermodynamic analysis: According to the Gibbs–Volmer
theory of homogeneous nucleation, the overall free energy
change, DG, between a small nucleus of m5U and m5U in
aqueous solution is equal to the sum of the surface excess
free energy, DGs, and the volume excess free energy, DGv.[2]
Therefore:
DGiðriÞ ¼ DGsi þ DGvi
ð6Þ
where i=I or II, representing form I or form II of m5U. DGsi
is a positive quantity, whereas DGvi is a negative quantity.[2]
For simplicity, a nucleus of m5U is assumed to be a sphere
with a radius, r. Then:
Kinetic analysis: For a system with two forms of potential
crystals, that is, m5U crystalline forms I and II, the following
condition must hold in order to obtain m5U crystalline
form II as the only crystallization product:[26]
DGiðriÞ ¼ 4pri2gi þ ð4pri3DGviÞ=3
ð7Þ
where, gi is the interfacial tension between the developing
crystalline surface and the supersaturated solution in which
the nucleus is located. The critical nucleus, rci =ꢀ2g/DGvi, is
obtained at dDGi(ri)/dri =0. At the critical m5U form I nu-
cleus, we obtain:
3
JIIkI3I ꢁ JI0 kI0
ð11Þ
where JIꢃ and JII are the nucleation rates for the form I crys-
tal in the presence of additives and the form II crystal, re-
spectively, and the kinetic coefficients for form I crystal
growth in the presence of additives and for form II are kIꢃ
and kII , respectively. This suggests that the appearance of
different structures may be influenced by additives designed
to interfere selectively with either the nucleation, growth
rates of a particular phase or both. According to the Arrhe-
nius equation,[2] the rate of nucleation can be expressed as:
DGcI ¼ ð4pgIrc2IÞ=3
ð8Þ
DAFP-1 can bind to m5U form I nucleus.[15] For the AFP-at-
tached form I nuclei:
DGcI0 ¼ Fð4pgIrc2IÞ=3
ð9Þ
where I’ is m5U form I nucleus with AFP attached, and F is
a correct factor resulting from the AFP attachment of m5U
form I nucleus. To inhibit m5U form I nucleation and crystal
growth, we need F>1 and hence DGcI’ >DGcI. At the criti-
cal m5U form II nucleus, we obtain:
2
Ji ¼ AiexpðꢀDGci=kTÞ ¼ Aiexp½ꢀð16pgi3vi2Þ=ð3k3T3flnSig Þꢂ
ð12Þ
where Ai is Arrhenius pre-exponential factor for species i, k
is the Boltzmann constant, T is the temperature in Kelvin, vi
is the molecular volume for species i, Si is supersaturation of
the species i. In the absence of AFPs, only m5U form I crys-
tals can form. Thus:
DGcII ¼ ð4pgIIrc2IIÞ=3
ð10Þ
To make the less-preferred m5U form II nucleation and crys-
tal growth occur before m5U form I nucleation and crystal
growth, we need DGcI’ >DGcII, that is, F>(VIIrc2II)/(VIrc2I).
JIkI3 ꢁ JIIkI3I
ð13Þ
Similar to the known structure of a beetle AFP from Ten-
ebrio molitor (TmAFP), the two rows of solvent-accessible
repeated threonines in DAFP-1 are putative ice-binding res-
idues and compared to the side chain methyl groups of the
threonines, the side chain hydroxyl groups play a more im-
portant role in the ice binding of TmAFP and DAFP-
In the presence of AFPs, AFPs inhibit the form I nuclea-
tion.[15] Therefore, JI’!!0 and JII @JI’, and we obtain:
3
JIIkI3I ꢁ JI0 kI0 !! 0
ð14Þ
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S. Wang, X. Wen et al.
Under the kinetic condition described in the above equa-
tion, we can exclusively obtain the kinetically less preferred
m5U crystalline form II.
AFPs can be used as novel scaffolds to design effective
additives for crystallization control, which are valuable in
many fields including chemistry, materials science, and the
pharmaceutical industry. Future studies on controlling crys-
tallization by different types of AFPs and engineered AFPs,
and on extending the findings to other crystal growth pro-
cesses, should be of great interests to both basic research
and practical applications.
Implications on AFP-induced ice-habit changes: AFPs can
inhibit ice growth by binding to specific faces of ice crystals
and changing the ice crystal shape. For example, a Ca2+ de-
pendent AFP[27] and TmAFP[23a] can change the ice crystal
shape to hexagonal plates; type I fish AFP,[24a,28] type III fish
AFP,[29] and type IV fish AFP[30] can change ice shape to pyr-
amids; and LpAFP[31] and winter flounder AFP[32] can alter
the ice shape to hexahedrons. The resulting ice shapes by
the same AFP can be different, which depend on the con-
centration of the AFP, the cooling rate, and other experi-
mental conditions.[33] Ice is known to have many poly-
morphs[34] and studies have shown various ice crystal habits
induced by AFPs, but few structures of these ice crystals
with altered habits have been determined by X-ray diffrac-
tion or neutron diffraction. To date, only the ice crystal with
a changed shape by a fish glycoprotein has been reported to
be ice Ih.[12] Ice Ih is the most common form of ice, which is
highly stable and the most dangerous to life.[35] Some carbo-
hydrates are known to be able to induce the crystallization
of water into unstable cubic ice under certain conditions.[36]
Are these newly shaped ice crystals induced by AFPs new
ice polymorphs or just habit modifications of the common
ice Ih? How does their stability compare to ice Ih? Structure
determination of these shape-changed ice crystals can be im-
portant to address the above questions and the answers will
lead to a better understanding of the mechanism of anti-
freeze action of AFPs implied by this study.
Experimental Section
Materials: Chemicals were purchased from Sigma–Aldrich (St. Louis,
MO) at ACS grade or better and used as received. Milli-Q water pro-
duced from a Synergy water system (Millipore) with a minimum resistivi-
ty of 18 MWcm was used for making solutions. All the sample solutions
were filtered through 0.2 mm filters before use. Sample vials (10 mL, Na-
tional Scientific) were used for crystallization. Glassware and stir bars
were cleaned as previously described.[15]
Synthesis: The syntheses of o,o’-bis(trimethylsilyl)thymine and 5-methyl-
uridine used the following procedures modified from previous meth-
ods.[38] Trimethylchlorosilane (6.51 g, 0.06 mol) and thymine (3.78 g,
0.03 mol) were suspended in dry benzene (100 mL). The suspension was
quickly stirred under N2 and 8.25 mL triethylamine in benzene was
added dropwise. The mixture was then refluxed for 2 h. O,O’-Bis(trime-
thylsilyl)thymine crystallized upon standing at room temperature. After
recrystallization three times, the pure o,o’-bis(trimethylsilyl)thymine
(7.55 g, yield 93%) appeared as slightly yellow crystals. Then, the ob-
tained o,o’-bis(trimethylsilyl)thymine (11.90 g, 0.044 mol, 10% excess)
and b-d-ribofuranose 1-acetate 2,3,5-tribenzoate (20.18 g, 0.04 mol) were
first mixed in CH3CN (80 mL) under N2. The mixture temperature was
kept at 08C and stirred. SnCl4 (1.0m, 41.34 mL) in methylene chloride
was added dropwise. The reaction was run at 08C for 2 h and warmed to
room temperature for 1 h. KHCO3 (60 g) in water (60 mL) was added to
remove the trimethylsilyl-protected group. The solvents were removed
under reduced pressure at 608C. The product was a white solid or crystals
after silica gel chromatography using EtOAc in CH2Cl2 as eluent and the
formation was revealed by TLC analysis (Rf =0.60) using 20% EtOAc in
CH2Cl2. Sodium ethoxide in ethanol (668 mL, 21% w/v) was used to
remove the Bz protecting group. The solvents were removed under re-
duced pressure at 508C. The product was a white solid or crystals after
silica gel chromatography using ethanol in CH2Cl2 as eluent and the for-
mation was revealed by TLC analysis (Rf =0.70) using 25% EtOAc in
CH2Cl2 and LC/ESI-MS and NMR spectroscopy (Supporting Informa-
tion, Figures S1–S3).
Conclusion
An AFP-induced selective nucleation and growth of a new
crystalline form has been demonstrated for the first time.
Significantly, the newly identified form of m5U is thermody-
namically and kinetically less preferred and its exclusive
crystallization is extremely difficult and could not be ach-
ieved previously. Moreover, compared to other known addi-
tives for crystallization control, the AFP-induced selective
crystallization is highly efficient, and has great potential in
applications, such as new crystalline form screening.[2,37]
The mechanism of AFP-induced crystallization is dis-
cussed and analyzed based on both theoretical and experi-
mental aspects. This study provides essential hints for the so-
lution of the long-standing problem of the antifreeze mecha-
nism of AFPs. Do AFPs have similar actions on m5U and on
ice in terms of selectively controlling crystallization? That is,
could AFPs induce the exclusive crystallization of thermo-
dynamically and kinetically less preferred ice polymorphs?
Structure determination of the ice crystals with altered
habits achieved by AFPs under different experimental con-
ditions, particularly, under those close to physiological con-
ditions, may improve our understanding of AFP action.
Preparation of antifreeze protein and control: DAFP-1 was expressed
and purified as described previously.[39] The purified protein was charac-
terized using SDS-PAGE, MALDI-TOF MS, CD spectroscopy, and dif-
ferential scanning calorimetry (DSC), as previously described[21] and the
identity of DAFP was confirmed. The concentration of stock DAFP-1 so-
lution was determined using a Cary 100 Bio UV/Vis spectrophotometer
(Varian) and the extinction coefficient of 5.47ꢂ103 mꢀ1 cmꢀ1 at 280 nm
was used.[40] The denatured DAFP-1 with completely reduced disulfide
bonds was prepared following the previously reported methods[41] and
used as a control in this study. Purified DAFP-1 at around 1 mm was in-
cubated in sodium citrate (0.10m), pH 3.0, and tris(2-carboxyethyl)phos-
phine hydrochloride (15.0 mm; TCEP) at 608C for 30 min. The denatured
DAFP-1 was then purified using a Sephacryl S-100 gel filtration column
connected to an ꢄKTA Purifier 10 (GE Healthcare).
Crystallization: Supersaturated m5U aqueous solutions (0.6m) were made
at 308C and filtered as quickly as possible. Crystals did not form in the
first few days after the temperature of the supersaturated solution was
lowered to room temperature. On day 1, 600 mL of 0.60m m5U solution
was first added to each sample vial, and then 5 mL of water, 0.36 mm
DAFP-1, or 0.40 mm denatured DAFP-1 solution was added into the spe-
cific vial, respectively. The vials were gently swirled after the additions.
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Antifreeze Protein-Induced Crystallization
FULL PAPER
The resulting m5U concentration was 595 mm in each vial and additive/
m5U molar ratios (ꢂ10ꢀ6) were either 0 or 5. The sample vials were left
open in the air at room temperature and three observations at least were
recorded per day (every 8 h) until the solutions in all the vials were dry.
The above experiments were repeated five times. Optical micrographs
were taken under Nikon SMZ800 microscope with a Nikon Coolpix 5400
when the crystallization was complete.
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Crystallization kinetics: Under the above conditions, only form I crystal-
line m5U was able to grow in the absence of DAFP-1; however, in the
presence of DAFP-1, form II crystalline m5U was grown exclusively. The
kinetics of crystallization of m5U form I and form II was estimated based
on the rate of the weight of the occurring crystals and the initial weight
of m5U in the specific vial. All the liquid in the vial was collected gently
and the weight of the vial or the vial with crystals was measured. After
the measurement, the collected liquid was put back into the vial and the
crystallization was continued. The weights were measured on an Ohaus
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three vials and the average values are reported.
Single crystal X-ray diffraction: Colorless crystal of m5U form I was
mounted on a Cryoloop with Paratone-N oil and data were collected at
90 K with a Bruker APEX I CCD using CuKa radiation generated from
a rotating anode. In a similar fashion, a colorless crystal of m5U form II
was mounted on a Cryoloop with Paratone-N oil and data were collected
at 100 K with a Bruker APEX II CCD using CuKa radiation generated
from a rotating anode. For both crystals data were corrected for absorp-
tion with SADABS and structures were solved by direct methods. All
non-hydrogen atoms were refined anisotropically by full matrix least
squares on F2. Hydrogen atoms on O and N atoms were found from
Fourier difference maps and were refined isotropically with distances of
ꢀ
ꢀ
O H and 0.85(0.02) or 0.86 (0.02) ꢁ and N H distance 0.87(0.02) ꢁ and
at 1.20 or 1.50 Ueq parent atom. All other hydrogen atoms were placed
ꢀ
in calculated positions and refined as riding models with C H distances
of 0.950 (CHar), 1.000, (CH), 0.990 (CH2), 0.980 ꢁ (CH3), and at 1.20 or
1.50 Ueq of parent C atom. Flack parameters for m5U forms I and II
were ꢀ0.0187 and ꢀ0.1358. Bruker suite of X-ray data collection
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(SHELXS97, SHELXL97, SHELXTL) were used for these structural de-
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supplementary crystallographic data for this paper. These data can be ob-
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via www.ccdc.cam.ac.uk/data_request/cif.
Differential scanning calorimetry (DSC): All experiments were per-
formed with a DSC 1 (Mettler Toledo, OH). An indium standard (Met-
tler Toledo, OH) was used to calibrate the instrument. Samples weighed
3–4 mg and were hermetically encapsulated in standard aluminum
sample pans with pierced lids to release any pressure build up during the
experiments. The samples were heated from 25 to 2008C at a rate of
58Cminꢀ1
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Received: May 29, 2013
Revised: August 9, 2013
Published online: October 9, 2013
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