C-linked Antifreeze Glycoprotein Research Articles

065 Designing potent inhibitors of Ice recrystallization - from C-linked antifreeze glycoproteins (AFGPs) to small molecules

Cell-based therapeutics has emerged as a critical aspect of modern health care and has shaped new models of care in stem cell and regenerative therapy. Dimethyl sulfoxide and glycerol are the two most common cryoprotectants. Unfortunately, many of the current cryopreservation protocols are suboptimal resulting in poor cell recoveries and/or compromised cellular function post cryopreservation thus limiting applications. One of the most significant causes of decreased cell viability and impaired function during cryopreservation is direct mechanical damage from ice crystals. Preventing this damage using ice recrystallization inhibitors may greatly reduce cellular injury during cryopreservation and enhance the recovery of specialized cellular functions. Antifreeze glycoproteins (AFGPs) are peptide-based structures found in many deep-sea Teleost fish inhabiting sub-zero environments. These compounds prevent the seeding of ice crystals in vivo and ultimately protect these organisms against cryoinjury and death. For the past decade our laboratory has been actively pursuing the rational design and synthesis of functional carbon-linked (C-linked) AFGP analogues possessing custom-tailored antifreeze activity. These compounds are potent inhibitors of ice recrystallization but do not exhibit thermal hysteresis properties making them attractive candidates as cryoprotectants for the preservation of various biological materials. Structure-function studies of these C-linked glycoconjugates have indicated that the most active ice recrystallization inhibitors contain highly hydrated carbohydrate residues. This discovery has lead to the development of several classes of small molecule carbohydrate derivatives that exhibit potent ice recrystallization inhibition activity and may constitute a novel class of cryoprotectants. To date, several different classes of small molecule ice recrystallization inhibitors (IRIs) have been identified including amino acid-based organogelators, non-ionic carbohydrate-based amphiphiles (aryl and alkyl alditols) and O-linked alkyl and aryl glycoside derivatives. The rational design of these compounds, assessment and quantification of their IRI activity will be presented and the key structural features necessary for potent ice recrystallization inhibition activity will be discussed. Finally, the ability of both C-linked AFGP and the small molecule IRIs to function as cryoprotectants will be described. To properly assess this, several cell lines and primary cells have been examined. In addition, we have investigated the cryopreservation of oncolytic viruses (both encapsulated non-encapsulated). Source of funding: NSERC, CIHR, Canadian Blood Services, Green Centre Canada. Conflict of interest: None declared. rben@uottawa.ca

Developing highly active small molecule ice recrystallization inhibitors based upon C-linked antifreeze glycoprotein analogues

Ice recrystallization during cryopreservation results in a significant amount of cellular damage making compounds that exhibit ice recrystallization inhibition (IRI) activity desirable as a novel class of cryoprotectants. Herein, we report a systematic structure–function study on a previously identified IRI active C-linked antifreeze glycoprotein (C-AFGP) analogue (1). These studies indicate that while C-AFGPs containing three tripeptide repeats exhibit weak IRI activity 5.5 μM, a minimum number of four tripeptide repeats is required for potent IRI activity at this concentration. In addition, the galactosyl–ornithine building block 5 exhibited only moderate activity at 22 mM, but IRI activity was significantly increased upon addition of two glycine units to the C-terminal end of the amino acid bearing the C-linked galactopyranose residue. Finally, we report that conjugation of long alkyl chains (octyl, nonyl and decyl) to the C-linked galactosyl moiety of 1 can furnish IRI active small molecules. The “ideal” hydrocarbon chain length was 10 carbons for potent activity in this series of compounds. Longer hydrocarbon chain lengths dramatically decreased solubilities. The results of this study emphasize the importance of hydrophobic moieties for IRI activity and while consistent with previously reported small molecule carbohydrate-based and lysine-based ice recrystallization inhibitors, is the first example where a large IRI active glycoconjugate has been successfully truncated to small molecule IRI active components.

Synthesis of C-Linked Triazole-Containing AFGP Analogues and Their Ability to Inhibit Ice Recrystallization

C-Linked antifreeze glycoprotein (C-AFGP) analogues have been shown to have potent ice recrystallization inhibition (IRI) activity. However, the lengthy synthesis of these compounds is not amenable to large-scale preparation for the many commercial, industrial, and medical applications that exist. This paper describes the synthesis of triazole-containing AFGPs using a convergent solid-phase synthesis (SPS) approach in which multiple carbohydrate derivatives are coupled to a resin-bound synthetic peptide in a single step. Modified "Click" conditions using dry DMF as solvent with catalytic Cu(II), sodium ascorbate, and microwave radiation afforded the synthesis of AFGP analogues 9-12 in 16-54% isolated yield. Compound 9 demonstrated no IRI activity, while compounds 10, 11, and 12 were moderate inhibitors of ice recrystallization. These results suggest that, while the triazole group is a structural mimetic of an amide bond, the amide bond in C-AFGP analogue 3 is an essential structural feature necessary for potent IRI activity.

Solution Conformation of C-Linked Antifreeze Glycoprotein Analogues and Modulation of Ice Recrystallization

Antifreeze glycoproteins (AFGPs) are a unique class of proteins that are found in many organisms inhabiting subzero environments and ensure their survival by preventing ice growth in vivo. During the last several years, our laboratory has synthesized functional C-linked AFGP analogues (3 and 5) that possess custom-tailored antifreeze activity suitable for medical, commercial, and industrial applications. These compounds are potent inhibitors of ice recrystallization and do not exhibit thermal hysteresis. The current study explores how changes in the length of the amide-containing side chain between the carbohydrate moiety and the polypeptide backbone in 5 influences ice recrystallization inhibition (IRI) activity. Analogue 5 (n = 3, where n is the number of carbons in the side chain) was a potent inhibitor of ice recrystallization, while 4, 6, and 7 (n = 4, 2, and 1, respectively) exhibited no IRI activity. The solution conformation of the polypeptide backbone in C-linked AFGP analogues 4-7 was examined using circular dichroism (CD) spectroscopy. The results suggested that all of the analogues exhibit a random coil conformation in solution and that the dramatic increase in IRI activity observed with 5 is not due to a change in long-range solution conformation. Variable-temperature (1)H NMR studies on truncated analogues 26-28 failed to elucidate the presence of persistent intramolecular bonds between the amide in the side chain and the peptide backbone. Molecular dynamics simulations performed on these analogues also failed to show persistent intramolecular hydrogen bonds. However, the simulations did indicate that the side chain of IRI-active analogue 26 (n = 3) adopts a unique short-range solution conformation in which it is folded back onto the peptide backbone, orienting the more hydrophilic face of the carbohydrate moiety away from the bulk solvent. In contrast, the solution conformation of IRI-inactive analogues 25, 27, and 28 had fully extended side chains, with the carbohydrate moiety being exposed to bulk solvent. These results illustrate how subtle changes in conformation and carbohydrate orientation dramatically influence IRI activity in C-linked AFGP analogues.

The Importance of Hydration for Inhibiting Ice Recrystallization with C-Linked Antifreeze Glycoproteins

The role of hydration in modulating solution conformation, molecular recognition, and biological activity of oligosaccharides, proteins, and nucleotides is widely recognized but is often neglected when investigating many biological processes such as the mechanism by which biological antifreezes inhibit the growth of ice. We have investigated the relationship between carbohydrate configuration and recrystallization-inhibition (RI) activity in functional C-linked antifreeze glycoprotein (AFGP) analogues using a series of analogues 1−4. While analogues 1−4 did not show any thermal hysteresis (TH) activity, 1 did exhibit weak dynamic ice shaping indicating that this compound had the ability to interact with the ice lattice. The d-mannose and d-talose analogues (3 and 4, respectively) exhibited very weak RI activity with mean largest grain size values similar to phosphate buffered saline, the negative control. d-Glucose analogue 2 exhibited moderate RI activity while d-galactose analogue 1 was the most potent ...

C-Linked Galactosyl Serine AFGP Analogues as Potent Recrystallization Inhibitors

[reaction: see text] A series of C-linked antifreeze glycoprotein analogues have been prepared to evaluate antifreeze activity as a function of distance between the carbohydrate moiety and polypeptide backbone. The building blocks for these analogues were prepared using either an olefin cross-metathesis or catalytic asymmetric hydrogenation. Analysis of antifreeze protein-specific activity revealed that only analogue 2a (n = 1) was a potent recrystallization inhibitor and thus has potential medical and industrial applications.

A general synthesis of structurally diverse building blocks for preparing analogues of C-linked antifreeze glycoproteins.

A synthetic methodology to afford unusual glycoconjugate building blocks useful for the solid-phase synthesis of C-linked antifreeze glycoprotein (AFGP) analogues is described. Such compounds are urgently required in order to elucidate the molecular mechanism by which AFGPs function. All reactions are general in nature and accommodate structural variation in the carbohydrate moiety, polypeptide backbone, and amino acid side chain.

Synthesis of a C-linked Antifreeze Glycoprotein (AFGP) Mimic: Probes for Investigating the Mechanism of Action

A general convergent synthesis has been developed to afford a low molecular weight C-linked antifreeze glycoprotein (AFGP) mimic (9). Structural mimics of AFGPs have tremendous potential as probes to better understand how native AFGPs inhibit ice crystal growth in organisms that inhabit subzero environments.