Methods of using ice-controlling molecules

Details Methods of using ice-controlling molecules Methods of using ice-controlling molecules

1997-10-03

2004-08-10

Saucier, Sandra E. (Department: 1651)

Chemistry: molecular biology and microbiology

Differentiated tissue or organ other than blood, per se, or...

Including freezing; composition therefor

C244S13400A, C037S197000, C252S070000

Reexamination Certificate

active

06773877

ABSTRACT:

BACKGROUND OF THE INVENTION
Ice formation is damaging to living systems and food products and may be a nuisance and a hazard to human beings who must cope with snow and ice in their environment. The field of the present invention is the provision of processes for the preparation of specific chemical agents, referred to herein as ice interface dopants (IID), that will effectively reduce ice formation and make ice that does form innocuous to living systems and foodstuffs and less troublesome and hazardous to humans and machinery in the environment.
Referring to
FIGS. 1A-1B
, ice crystallizes in the shape of a hexagonal plate
10
. A plane defined by the a axis
12
and the b axis
14
(which is crystallographically identical to the a axis) and perpendicular to the c axis
16
defines a hexagonal cross section called the basal plane
18
. The six faces of the hexagon are called prism faces
20
. Crystallographically, the basal plane
18
is referred to as the 0001 surface, and the prism face is referred to as the 1{overscore (1)}00 surface or the 1{overscore (1)}20 surface depending on the orientation.
FIGS. 2A-2D
show that the units of the crystal that give rise to this macroscopic structure are also hexagonal. In
FIG. 2A
, following common usage, only the oxygen atoms are represented. Stippling indicates oxygens projecting out of the plane of the sheet. The hydrogen atoms lie along the straight lines shown bonding each oxygen atom to its four nearest neighbors.
FIG. 2A
shows the basal plane 0001 surface as seen from above. Within each hexagon, three vertices project upward (or forward), and the three intervening vertices project downward (or backward). The upward vertices are separated by 4.5 ű0.02 Å and are located at a 60° angle with respect to each other. Their fourth bonds extend perpendicularly out of the page toward the viewer. Another spacing at 7.36 Å separates alternate bilayers
21
of oxygen atoms in the lattice, or, viewed differently, separates each oxygen-defined hexagon from an identical hexagon located immediately adjacent to it.
FIG. 2B
shows a perspective view of the prism face. Crystallographically, the prism face may be represented as the c1{overscore (1)}00 (
FIG. 2C
) or d11{overscore (2)}0 (
FIG. 2D
) prism faces, depending on the angle of the viewer.
Several natural molecules exist that alter the behavior of ice and of water. Antifreeze glycoproteins (AFGPs) and antifreeze proteins or antifreeze peptides (AFPs) produced by several species of fish are believed to adsorb preferentially to the prism face
20
of ice and thus to inhibit ice crystal growth perpendicular to the prism face, i.e, in the direction extending along the basal plane
18
and along the a and b axes
12
and
14
.
This capability is sufficient to permit certain fish to live their entire lives at a body temperature about 1° C. below the thermodynamic freezing point of the fishes' body fluids. These fish can ingest and contact ice crystals that might otherwise provide crystal nucleation sites without being invaded by the growth of ice through their supercooled tissues because the AFGPs present in their tissues and body fluids block ice growth despite the presence of supercooling. Insect antifreeze or “thermal hysteresis” proteins (THPs) are even more effective, being active at supercooling levels of 2° C. or more below the thermodynamic freezing point.
The natural “antifreeze” or “thermal hysteresis” proteins found in polar fish and certain terrestrial insects are believed to adsorb to ice by lattice matching (Davies and Hew,
FASEB J.,
4; 2460-2468, 1990) or by dipolar interactions along certain axes (Yang, Sax, Chakrabartty and Hew,
Nature,
333:232-237, 1988).
AFGPs and AFPs found in certain organisms provide natural “proofs of principle” for the concept of novel man-made IIDs. However, natural ice interface doping proteins are not sufficiently active or abundant for most practical applications of interest. Furthermore, a disadvantage of growth inhibition on the prism faces is that, when supercooling becomes sufficient to overcome ice crystal growth inhibition, growth occurs, by default, predominantly in the direction of the c axis
16
, perpendicular to the basal plane
18
. This results in the formation of spicular or needle-shaped ice crystals (
FIG. 1B
) that are more damaging to living cells than normal ice, apparently for mechanical reasons. (In
FIG. 1B
, AFGPs are represented as blobs blocking the prism faces. Other layers are similarly blocked, but the details are omitted for simplicity.)
Natural IIDs are commercially available only in a very limited quantity and variety. Furthermore, they must have fairly high relative molecular masses (typically at least about 4,000 daltons) to be effective. This tends to make them expensive, and they often require complex interactions with other hard-to-acquire proteins and often require carbohydrate moieties for full effectiveness. Insect antifreeze proteins, recently shown to be extremely effective compared to fish antifreeze proteins, still have relative molecular masses of around 8,400 daltons. (Graham, Liou, Walker and Davies,
Nature,
388:727-728, 1997).
Furthermore, addition of natural fish AFGP to a concentrated solution of cryoprotectant (30-40% v/v DMSO) had minimal effect on ice crystal growth rates below −20 to −40° C. (Fahy, G. M., in
Biological Ice Nucleation and its Applications
, chapter 18, pp. 315-336, 1995), thus making questionable its effectiveness for use in organ vitrification for cryopreservation.
Another problem with natural antifreeze proteins is that continuing confusion over their precise mechanisms of action hampers the development of recombinant variants that could be more effective. Recently, Warren and colleagues reported some progress in this direction (U.S. Pat. No. 5,118,792).
Caple et al. (
Cryo
-
Letters,
4:51-58, 1983) made several apparently arbitrary synthetic polymers and showed that some of them were able to prevent nucleation of water by silver iodide crystals. They suggested that these polymers adsorbed either to the silver iodide or to ice crystal nuclei, but they did not suggest any specific interactions, and their polymers were made without regard to any consideration of the structure of ice or of AgI. Further, except for noting that a 2 to 1 ratio of hydrophobic to hydrophillic groups on their polymers gave maximum inhibition of nucleation, they provided no guidance or general principles as to how one could approach the synthesis of ice-binding polymers on a systematic theoretical or empirical basis or maximize the ice-binding effectiveness of such polymers. They also taught that higher concentrations of their polymers nucleated their solutions, and failed to teach that their polymers would slow ice crystal growth rates or have other than academic uses. Caple et al. (
Cryo
-
Letters,
4: 59-64, 1983) also reported detecting unidentified, uncharacterized, and unpurified nucleation-inhibiting substances from natural sources, but again suggested no applications.
The concept of designing specific artificial chemical agents whose purpose is to control the physics of ice was first mentioned by Fahy in
Low Temperature Biotechnology
, McGrath and Diller, eds., ASME, pp.113-146, 1988. The sole mention of this idea was the single statement that “insight into the mechanism of AFP action . . . opens the possibility of designing molecules which may be able to inhibit ice crystal growth in complementary ways, e.g., along different crystallographic planes.” However, no method of preparing such molecules was suggested.
Kuo-Chen Chou (“Energy-optimized structure of antifreeze protein and its binding mechanism”,
J. Mol. Biol.,
223:509-517, 1992) mentions an intention to specifically design ice crystal growth inhibitors. However, it is confined to minor modifications of existing antifreeze molecules, and does not envision the present radically different approach of preparing synthetic IIDs de novo.
Based on these observations, it is advantageous to d

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