Robert N. Ben

Total Synthesis of Homogeneous Antifreeze Glycopeptides and Glycoproteins

Antifreeze glycoproteins (AFGPs) are a class of natural products found in deep sea teleost fish in Arctic and Antarctic waters. The physiological role of these biomolecules is to protect against cryoinjury in environments with subzero temperatures by preventing the growth of ice crystals in vivo. Structurally, AFGPs are polymeric, mucin-type glycoproteins that consist of a single glycotripeptide repeat (Ala-Thr-Ala/Pro) in which each secondary hydroxy group on threonine is linked to the disaccharide b-d-galactosyl-(1!3)a-N-acetyl-d-galactosamine (Scheme 1). AFGPs range in molecular weight from approximately 2.6 kDa (4 repeat units) to 33.7 kDa (50 repeat units).

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 ...

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

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.

Reaction of (R)-pantolactone esters of alpha-bromoacids with amines a remarkable synthesis of optically active alpha-amino esters

(R)-Pantolactone esters of racemic α-bromo acids react with amines to give α-amino esters having the (S)-configuration at the α-carbon in yields which are considerably greater than the 50% expected on the basis of a simple SN2 displacement reaction.

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.

Antifreeze Glycoproteins—Preventing the Growth of Ice

Biological antifreezes constitute a diverse class of proteins found in arctic and antarctic fish, as well as in amphibians, trees, plants, and insects. These compounds are unique in that they have the ability to inhibit the growth of ice and consequently are essential for the survival of organisms inhabiting environments where sub-zero temperatures are routinely encountered. This is an unusual ability attributed only to biological antifreezes. There are two types of biological antifreezes, the antifreeze proteins (AFPs) and the antifreeze glycoproteins (AFGPs). Antifreeze proteins are divided into four subtypes (types 1 ± 4) each possessing a very different primary, secondary, and tertiary structure. In contrast, AFGPs are subject to considerably less structural variation. A typical AFGP is composed of a repeating tripeptide unit (threonyl ± alanyl ± alanyl) in which the secondary hydroxy group of the threonine residue is glycosylated with the disaccharide b-D-galactosyl-(1,3)-a-D-N-acetylgalactosamine (Figure 1). Eight distinct AFGP subtypes exist ; glycoproteins of 20 ± 33 kDa are referred to as AFGPs 1 ± 4 and those of less than 20 kDa constitute AFGPs 5 ± 8. The lower molecular weight

Potent inhibition of ice recrystallization by low molecular weight carbohydrate-based surfactants and hydrogelators

Ice recrystallization inhibition (IRI) activity is a very desirable property for an effective cryoprotectant. This property was first observed in biological antifreezes (BAs), which cannot be utilized in cryopreservation due to their ability to bind to ice. To date, potent IRI active compounds have been limited to BAs or synthetic C-linked AFGP analogues (1 and 2), all of which are large peptide-based molecules. This paper describes the first example of low molecular weight carbohydrate-based derivatives that exhibit potent IRI activity. Non-ionic surfactant n-octyl-β-D-galactopyranoside (4) exhibited potent IRI activity at a concentration of 22 mM, whereas hydrogelator N-octyl-D-gluconamide (5) exhibited potent IRI activity at a low concentration of 0.5 mM. Thermal hysteresis measurements and solid-state NMR experiments indicated that these derivatives are not exhibiting IRI activity by binding to ice. For non-ionic surfactant derivatives (3 and 4), we demonstrated that carbohydrate hydration is important for IRI activity and that the formation of micelles in solution is not a prerequisite for IRI activity. Furthermore, using solid-state NMR and rheology we demonstrated that the ability of hydrogelators 5 and 6 to form a hydrogel is not relevant to IRI activity. Structure–function studies indicated that the amide bond in 5 is an essential structural feature required for potent IRI activity.

Antifreeze glycoproteins: structure, conformation, and biological applications.

Antifreeze glycoproteins (AFGPs) are a novel class of biologically significant compounds that possess the ability to inhibit the growth of ice both in vitro and in vivo. Any organic compound that possesses the ability to inhibit the growth of ice has many potential medical, industrial, and commercial applications. In an effort to elucidate the molecular mechanism of action, various spectroscopic and physical techniques have been used to investigate the solution conformations of these glycoproteins. This review examines the characterization of AFGPs and potential biological applications relating to stabilization of lipid membranes and vitrification adjuvants.

The promise of organ and tissue preservation to transform medicine

The ability to replace organs and tissues on demand could save or improve millions of lives each year globally and create public health benefits on par with curing cancer. Unmet needs for organ and tissue preservation place enormous logistical limitations on transplantation, regenerative medicine, drug discovery, and a variety of rapidly advancing areas spanning biomedicine. A growing coalition of researchers, clinicians, advocacy organizations, academic institutions, and other stakeholders has assembled to address the unmet need for preservation advances, outlining remaining challenges and identifying areas of underinvestment and untapped opportunities. Meanwhile, recent discoveries provide proofs of principle for breakthroughs in a family of research areas surrounding biopreservation. These developments indicate that a new paradigm, integrating multiple existing preservation approaches and new technologies that have flourished in the past 10 years, could transform preservation research. Capitalizing on these opportunities will require engagement across many research areas and stakeholder groups. A coordinated effort is needed to expedite preservation advances that can transform several areas of medicine and medical science.

C-Linked Antifreeze Glycoprotein (C-AFGP) Analogues as Novel Cryoprotectants

Significant cell damage occurs during cryopreservation resulting in a decreased number of viable and functional cells post-thawing. Recent studies have correlated the unsuccessful outcome of regenerative therapies with poor cell viability after cryopreservation. Cell damage from ice recrystallization during freeze-thawing is one cause of decreased viability after cryopreservation. We have assessed the ability of two C-AFGPs that are potent inhibitors of ice recrystallization to increase cell viability after cryopreservation. Our results indicate that a 1-1.5 mg/mL (0.5-0.8 mM) solution of C-AFGP 1 is an excellent alternative to a 2.5% DMSO solution for the cryopreservation of human embryonic liver cells.