Facially Amphipathic Glycopolymers Inhibit Ice Recrystallization

Article abstract of DOI:10.1021/jacs.8b02066

Antifreeze glycoproteins (AFGPs) from polar fish are the most potent ice recrystallization (growth) inhibitors known, and synthetic mimics are required for low-temperature applications such as cell cryopreservation. Here we introduce facially amphipathic glycopolymers that mimic the three-dimensional structure of AFGPs. Glycopolymers featuring segregated hydrophilic and hydrophobic faces were prepared by ring-opening metathesis polymerization, and their rigid conformation was confirmed by small-angle neutron scattering. Ice recrystallization inhibition (IRI) activity was reduced when a hydrophilic oxo-ether was installed on the glycan-opposing face, but significant activity was restored by incorporating a hydrophobic dimethylfulvene residue. This biomimetic strategy demonstrates that segregated domains of distinct hydrophilicity/hydrophobicity are a crucial motif to introduce IRI activity, which increases our understanding of the complex ice crystal inhibition processes.

Full text of DOI:10.1021/jacs.8b02066

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Facially Amphipathic Glycopolymers Inhibit Ice Recrystallization
Ben Graham, Alice E. R. Fayter, Judith E. Houston, Rachel C. Evans, and Matthew I. Gibson
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b02066 • Publication Date (Web): 16 Apr 2018
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Facially Amphipathic Glycopolymers Inhibit Ice Recrystallization
Ben Graham,1 Alice E. R. Fayter,1 Judith E. Houston,3 Rachel C. Evans,4 and Matthew I. Gibson1,2*
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1 Department of Chemistry, University of Warwick, Coventry, UK, CV4 7AL.
2 Warwick Medical School, University of Warwick, Coventry, UK, CV4 7AL.
3 Jülich Centre for Neutron Science, Forschungszentrum Jülich GmbH, Lichtenbergstr. 1, Garching, 85747, Germany.
4 Department of Materials Science & Metallurgy, University of Cambridge, Cambridge, UK, CB3 0FS.
Supporting Information Placeholder
alcohol) and poly(ampholytes) which have been found
to enhance the cryopreservation of blood,10–12 and nucleꢀ
ated cells.4,13
ABSTRACT: Antifreeze glycoproteins from polar fish
are the most potent ice recrystallization (growth) inhibiꢀ
tors known, and synthetic mimics are required for low
temperature applications such as cell cryopreservation.
Here we introduce facially amphipathic glycopolymers
which mimic the 3ꢀdimensional structure of AFGPs.
Glycopolymers featuring segregated hydrophilic and
hydrophobic faces were prepared by ringꢀopening meꢀ
tathesis polymerization and their rigid conformation was
confirmed by smallꢀangle neutron scattering. Ice recrysꢀ
tallization inhibition (IRI) activity was reduced when a
hydrophilic oxoꢀether was installed on the glycanꢀ
opposing face, but significant activity was restored by
incorporating a hydrophobic dimethylfulvene residue.
This biomimetic strategy demonstrates that segregated
domains of distinct hydrophilicity/hydrophobicity are a
crucial motif to introduce IRI activity, and increases our
understanding of the complex ice crystal inhibition proꢀ
cesses.
In the case of AFPs, defined iceꢀbinding faces have been
identified using structural biology methods.14 Converseꢀ
ly, there is no crystal structure available of AFGPs, and
the exact structural motifs required for IRI are unknown,
although the glycan unit is essential for ice shaping.7
Solution NMR studies suggest that AFGPs form a polyꢀ
proline II type helix, with the glycans on one face, and
peptides on the opposite, forming a facially amphipathic
structure.15 It is emerging that this segregated display of
hydrophobic/phillic groups, rather than a ‘binding site’ is
the essential feature for IRI activity.8,16,17 Molecular
modelling recently revealed that the hydrophobic face,
not the glycans, of AFGP interact with the ice, and that
the spatial segregation along the polyproline II helix is
essential.18 Gibson and coworkers have shown that hoꢀ
moꢀpolyproline has weak IRI,4 and that selfꢀassembled
metallohelicies with ‘patchy’ amphipathy are potent
IRIs,19 which supports a hypothesis that well defined ice
binding domains are not essential for IRI.20 Amphipathy
has also been seen to be important in ice nucleation.21
This evidence suggests that IRI, but not ice shaping,22
could be selectively introduced into new and emerging
(bio)materials, if precise control over hydrophilic/phobic
domains is possible. The design of polymers with solꢀ
ventꢀexposed hydrophobic domains is, however, nonꢀ
trivial. Block copolymeric amphiphiles spontaneously
selfꢀassemble into micelles/vesicles to reduce hydrophoꢀ
bic domain contact with water, and hence only ‘water
loving’ surfaces are exposed.23 Tew and coworkers have
developed facially amphipathic cationic polymers, with
opposing positive charges and lipophilic domains to
mimic the function of antimicrobial peptides.24,25 A cruꢀ
cial design step was the use of ringꢀopening metathesis
polymerization, ROMP, which introduces rigid alkene
backbones, whilst balancing the hydrophobiꢀ
city/hydrophilicity to maintain both solubility and the
Antifreeze glycoproteins, AFGPs, are found in the tisꢀ
sues and blood serum of extremophile fish species, and
act to modulate the growth of extracellular ice.1 A key
property of AFGPs is ice recrystallization inhibition,
IRI, which slows ice crystal growth (distinct from nucleꢀ
ation2).3 Ice recrystallization is a major cause of cell
death during the freezing of cells and tissue for transfuꢀ
sion, fundamental biomedicine and cell biology. Hence,
AFGPs (or their mimics) have many potential applicaꢀ
tions.4 Cryopreservation with AFGPs (and nonꢀ
glycosylated antifreeze proteins – AFPs)5 is limited,
however, by their secondary property of dynamic ice
shaping, whereby the AFGPs shape the ice into needleꢀ
like (spicular) morphologies, which can pierce cell
membranes.3 AFGPs are also challenging to synthesize,
by multistep procedures.6,7 Gibson and coworkers have
developed synthetic polymers8,9 based upon poly(vinyl
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presentation of hydrophobic faces. These have shown
The polymer library was assessed for IRI activity using
particular potency as potential antimicrobials.26
a ‘splat’ assay: where ice crystals are nucleated and their
growth after 30 minutes at –8 ºC is recorded. Activity is
expressed as the mean grain area (MGA) relative to a
PBS control, with smaller values representing more acꢀ
tivity. Polymers derived from M2 containing the ‘fulvo’
motif were significantly less soluble than those derived
from M3, containing the ‘oxo’ꢀether units. The solution
concentrations of these were therefore determined by
UVꢀVis spectroscopy (Supp. Info for BeerꢀLambert
plots) at saturation. In the case of poly(Fulvo), 1% v/v
DMSO was required and controls were adjusted to acꢀ
count for this.
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Considering the above, we designed and synthesized
locallyꢀrigid, facially amphipathic glycopolymers. A
combination of ice binding assays, modelling and smallꢀ
angle neutron scattering (SANS) provides compelling
evidence that local amphipathy is an essential motif for
introducing IRI activity, providing design rules for new
materials to mimic AFGP function.
Figure 1A shows the solution state structure of AFGP,7
with the disaccharide units spatially segregated from the
hydrophobic peptide backbone. Our approach was to use
ROMP polymerization to introduce local rigidity,27
compared to flexible backbones obtained from radical
polymerization. Four monomers were synthesized to
give a range of amphipathies: M1 was prepared by acetꢀ
ylation of a commercial norbornene diol. M2/3 were
synthesized by Koenigs–Knorr coupling of acetobromoꢀ
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Table 1 Polymer Characterization
Mn(THEO) Mn (SEC)
Đ
DP
Conv
(g.molꢀ1) (g.molꢀ1) (ꢀ)
(ꢀ)
%
[a]
[a]
[a]
[a]
[b]
2200 1.01 14
10,000 5300 1.01 34
8,400 1.02 54
poly(Diol) [c]
100
αꢀDꢀgalactose
with
exo,exoꢀ
[oxo/fulvene]norborneneimide. M4 was synthesized by
substitution of monomethoxyhexaethylene glycol
monotosylate, Fig 1B. The hydrophilic galactose and
hydrophobic fulvene motifs were selected in particular
due to their intrinsic rigidities, giving monomers with
structurally distinct domains of opposing polarity. These
monomers were polymerized using Grubbs 3rd generaꢀ
tion catalyst and acetate protecting groups removed by
treatment with sodium methoxide followed by ion exꢀ
change, Fig 1C. The panel of amphipathic polymers
25,000 10,300 1.21 28
10,000 7,300 1.18 22
10,000 35,900 1.38 133
100
100
100
poly(Fulvo)
poly(Oxo)
poly(FPEG)
5,000 10,700 1.12 14,35 94/97
10,000 16,800 1.10 22,54 100
poly(FulvoꢀcoꢀDiol)ꢀ11
poly(FulvoꢀcoꢀDiol)ꢀ17
poly(FulvoꢀcoꢀDiol)ꢀ35
poly(FulvoꢀcoꢀOxo)
25,000 34,600 1.26 47,112 100
10,000 7,700 1.35 11,11 100/96
10,000 55,600 1.47 76,58
71
poly(FulvoꢀcoꢀFPEG)
[a] Determined by SEC. [b] Determined by 1H NMR. [c] Single
species.
were characterized by SEC (ᴆ < 1.4), NMR and IR
(Supp. Info.), Table 1.
Poly(Oxo) was found to inhibit ice crystal growth by
approximately 50% MGA at concentrations above 5
mg.mLꢀ1, Fig 2A, which is more active than many previꢀ
ously reported IRI active polymers.28,29 The poly(Fulvo)
derivative featuring the hydrophobic face, however, was
considerably more activity, inhibiting by ~ 50% MGA at
just 0.5 mg.mLꢀ1 (solubility limit), supporting the facialꢀ
ly amphipathic hypothesis for IRI. Molecular models
corroborate this (Fig 2B), and illustrate the relative inꢀ
crease in hydrophobicity across the poly(Oxo) and
poly(Fulvo) homopolymers. To improve the solubility, a
1:1 statistical copolymer of M2/M3, poly(Fulvoꢀcoꢀ
Oxo), was prepared. This coꢀpolymer had significantly
improved solubility and comparable overall IRI activity
to poly(Fulvo) showing that some coꢀmonomer incorpoꢀ
ration is tolerated, unlike PVA,30 and example ice waꢀ
fers are shown in Fig 2C. However the nonꢀideal copolꢀ
ymerization kinetics of the oxo (M2) and fulvo (M3) coꢀ
monomers will have led to a blocky, rather than statistiꢀ
cal copolymerization.31,32 Infrared analysis confirmed
incomplete acetate removal (in contrast to the homo polꢀ
ymers), suggesting an internalized domain structure
and/or aggregation, with some (hydrophobic) surfaces
being solvent inaccessible and hence limiting the total
activity poly(FulvoꢀcoꢀOxo). The monomers alone also
had no activity (Supp. Info), confirming that a macromoꢀ
lecular architecture is essential.
Fig 1 A) Concept of facially amphipathic ROMP polymers to
mimic AFGP. Figure partially reproduced from Tachibana et al.
(2004)7 B/C) Monomers and polymers synthesized here; hydroꢀ
philic groups are indicated in blue, and hydrophobic in red.
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Fig 3 IRI activities of the poly(FulvoꢀcoꢀDiol) molecular weight
series
AF(G)Ps bind to specific ice crystal faces,35,36 leading to
dynamic ice shaping (unwanted in cryopreservation3).
Control ice crystals (Fig 4A) showed no dynamic ice
shaping, but addition of AFGPs (Fig 4B) produced disꢀ
tinctive spicular (needleꢀlike) crystals. Poly(Fulvoꢀcoꢀ
Diol) (Fig 4C) did not lead to ice shaping, ruling out
strong and specific ice face recognition and showing that
these effects can be separated by macromolecular deꢀ
sign.
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Fig 2 A) IRI activities of the poly(Fulvo), (Oxo), and copolymer
series. B) Hydrophobic surface map of poly(Fulvo) and (Oxo)
C/D) Ice crystal wafer of PBS and poly(FulvoꢀcoꢀOxo).
To improve solubility, a norbornene diol monomer, M1,
with a nonꢀhydrophilic bridgehead was investigated. Deꢀ
acetylated homopolymers of M1, poly(Diol), were found
to have surprisingly low solubility and no activity at
their solubility limit of 0.5 mg.mLꢀ1. However, when M1
was incorporated as a coꢀmonomer with the (IRI active)
‘fulvo’ monomer M2 to give poly(FulvoꢀcoꢀDiol), an
overall increase in solubility was achieved. Poly(Fulvoꢀ
coꢀDiol)ꢀ17 had remarkable IRI activity; 40% MGA at
just 1.3 mg.mLꢀ1, Fig 3. This polymer showed some moꢀ
lecular weight dependence on activity, with 17 kDa havꢀ
ing more activity than 11 kDa (and far more than the
monomer, indicating the need for a macromolecular arꢀ
chitecture). Increasing the molecular weight further to
35 kDa lowered the solubility of the copolymer, and
hence activity, highlighting a ‘sweet spot’. Work underꢀ
Fig 4 Ice morphology analysis. A) Water (ꢀ6 °C); B) AFGPꢀ8 (ꢀ5
°C); C) poly(FulvoꢀcoꢀDiol)ꢀ17 [0.72 mg.mLꢀ1] (ꢀ8 °C).
SANS was employed to evaluate the solution conforꢀ
mation and rigidity of the poly(FulvoꢀcoꢀDiol) series,
Fig 5 (and Supp. Info). The persistence lengths, bt,
were estimated from the position of the characteristic
crossover between the scattering profile typical for
taken by Inada et. al. described the molecular weight
33
dependence on IRI of PVA.
Similarly, a previous
fractal aggregates (qꢀ3.5) to that of rigid rods (qꢀ1) (see
study by Deswal et. al reported on the IRI activity of
proteins extracted from the leaves of the freeze tolerant
plant Seabuckthorn, of which superior antifreeze activity
was observed only for polypeptides of elevated molecuꢀ
lar weights.34 Replacing the glycan with a short oliꢀ
go(ethylene glycol), PEG, chain to give both
poly(FulvoꢀcoꢀFPEG) and poly(FPEG), decreased activiꢀ
ty as the (flexible) PEG can access numerous conforꢀ
mations, reducing the overall amphipathy. See ESI. Hyꢀ
drogenation of the alkene backbone to increase flexibilꢀ
ity resulted in a wholly insoluble polymer. See ESI.
These observations demonstrate that precise macromoꢀ
lecular engineering is essential to achieve a potent IRI
mimetic.
37,38
the ESI).
The estimated bt values for poly(Fulvoꢀ
coꢀDiol)ꢀ11 and poly(Fulvoꢀco-Diol)ꢀ17 are 38.9 and
44.4 Å, respectively. It should be noted that the overlap
may actually occur at a lower q region, but is masked by
aggregate scattering. Thus, these values should be taken
as the minimum persistence lengths for each polymer.
Nevertheless, each bt is much larger than the monomer
length (~10 Å) which suggests that the chain backbones
are locally stiff.38 Furthermore, given the approximate
contour length, L, of both polymer chains (490 Å and
760 Å for poly(FulvoꢀcoꢀDiol)ꢀ11 and poly(Fulvoꢀcoꢀ
Diol)ꢀ17, respectively), the large bt suggests rigid rather
than highly flexible aggregates, of potentially rodꢀlike
structures. This rigidity coupled with the intrinsic amꢀ
phipathy of the polymers is aligned with the hypotheꢀ
3
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erated at the Heinz MaierꢀLeibnitz (MLZ), Garching, Gerꢀ
many.
sized semiꢀrigid (and generally amphipathic) ice binding
faces of AFPs,14,39 and the flexible hydrophilic ‘glycan
face’ of AFGPs, providing evidence that facial amꢀ
phipathy is a key motif for introducing IRI activity into a
diverse range of polymers.
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ASSOCIATED CONTENT
Supporting Information.
Full
experimental
details,
including
syntheꢀ
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Mancini, R. S.; Acker, J. P.; Ben, R. N. Sci. Rep.
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sis/characterization, additional IRI data and SANS analysis
is in the Supporting Information. This material is available
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Mitchell, D. E.; Clarkson, G.; Fox, D. J.; Vipond,
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AUTHOR INFORMATION
Corresponding Author
Prof. Matthew I. Gibson, m.i.gibson@warwick.ac.uk.
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Notes
No competing financial interests have been declared.
ACKNOWLEDGMENT
MIG holds an ERC Starter Grant (CRYOMAT 638661).
Thanks to A. L. DeVries for supplying the antifreeze glyꢀ
coprotein AFGPꢀ8. The SANS portion of this work is based
upon experiments performed at the KWSꢀ2 instrument opꢀ
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