Letter pubs.acs.org/macroletters
Inhibition of Ice Recrystallization by Nylon‑3 Polymers Melissa J. MacDonald, Natasha R. Cornejo, and Samuel H. Gellman* Department of Chemistry, University of WisconsinMadison, Madison, Wisconsin 53706, United States S Supporting Information *
ABSTRACT: Nontoxic cryoprotectants are needed for storage of tissues and food preservation. Frozen tissue is particularly susceptible to damage caused by formation of large ice crystals during the thawing process. The current practice of using 5 wt % DMSO for cryopreservation does not produce 100% cell viability post-thaw, at least in part because of DMSO toxicity that is manifested during the freezing and thawing stages of the process. Recently, poly(vinyl alcohol) (PVA) has shown promise in inhibiting ice recrystallization, an activity that is critical for cryoprotection. Inspired by this discovery, we have evaluated nylon-3 polymers for ice recrystallization inhibition activity and for toxicity toward mammalian cells. A survey of homo- and heteropolymers, with side chains bearing variable functionality, has identified new nylon-3 materials that display excellent ice recrystallization inhibition activity and low toxicity.
M
effects: ice recrystallization inhibition, thermal hysteresis activity (TH), and dynamic ice shaping (DIS).8 Studies with synthetic AFP/AFGP mimics have shown that ice recrystallization inhibition activity correlates with cellular preservation.1 The use of AFPs or AFGPs for cryopreservation applications is hindered by the difficulty of accessing sufficient quantities of these natural materials.9 Efforts to address this challenge have led to studies of synthetic macromolecules that might mimic key chemical properties of AFPs and AFGPs.10 Although the ice-binding surfaces of many antifreeze peptides and proteins feature hydroxyl arrays,11 and hydroxyl groups are prominent components of AFGPs,11b it is clear that hydrophobic surfaces and ionic groups can be components of AFP ice-binding surfaces.11a The hydroxyl-rich nature of AFGPs inspired Inada et al. to examine poly(vinyl alcohol) as an ice recrystallization inhibitor.12 Poly(vinyl alcohol), which is commercially available and relatively nontoxic, displays potent ice recrystallization inhibition activity when the average chain length is above 20 subunits.13 As pointed out by Gibson in a very thorough review of the literature, the activity observed for atactic poly(vinyl alcohol) is noteworthy because of the lack of a specific hydroxyl arrangement within these polymer chains, which contrasts with the highly organized hydroxyl clustering observed for AGFPs.13 Poly(vinyl alcohol) can enhance the ability of DMSO to cryopreserve cells, but this polymer alone is not an effective cryopreservative.13 Furthermore, it may not be straightforward to tune poly(vinyl alcohol) properties via modification of the polymer functionality. Here we describe a preliminary evaluation of nylon-3 polymers as ice recrystallization inhibitors; the properties of nylon-3 materials can be widely
inimization of ice recrystallization during cryopreservation is a significant technical challenge for food preservation and cryo-medicine.1 Storing cells or tissues at −196 °C is an essential practice in biochemistry and medicine;2 however, this process can be damaging because of the formation of large ice crystals during the thawing process.3 Cells or tissues are also harmed during the freezing process from increased osmotic pressure caused by ice formation outside the cell.4 The availability of additives that protect cells or tissues from such damage without exerting toxic effects is essential for cryopreservation. Use of dimethyl sulfoxide (DMSO) or glycerol, at 5−10 wt %, is currently very common for cryopreservation. These additives are nonoptimal, however, because of their toxicity at room temperature and their ability to cross cell membranes.5 Ben et al. discovered that small molecules, such as glucose and galactose, could display ice recrystallization inhibition properties.6 These sugars are biocompatible and support cell viability even at high concentrations.7 However, hexoses do not approach the universal cryopreservation potency of DMSO or glycerol. Thus, there is need for more biocompatible additives for ice recrystallization inhibition. Many organisms that live in cold environments contain proteins and/or peptides that modulate ice formation, including glycopeptides (Figure 1). These antifreeze proteins (AFPs) or antifreeze glycopeptides (AFGPs) exert at least three relevant
Received: May 29, 2017 Accepted: June 14, 2017
Figure 1. Core structure of an antifreeze glycopeptide. © XXXX American Chemical Society
695
DOI: 10.1021/acsmacrolett.7b00396 ACS Macro Lett. 2017, 6, 695−699
Letter
ACS Macro Letters
average chain lengths near the expected values (Table 1). For HS, a degree of polymerization (DP) of 16 was estimated for the side-chain-protected sample via the multiangle lightscattering (MALS) detector interfaced with the gel permeation chromatograph (GPC). After side chain deprotection, 1H NMR analysis in water suggested a DP of 16 for the HS polymer (details in Supporting Information). For DH homopolymer synthesized with 5 mol % p-t-butylbenzoyl chloride, GPCMALS suggested a DP of 21 prior to side-chain deprotection, and 1H NMR in DMSO after deprotection suggested a DP of 23. Use of smaller proportions of the acid chloride delivered polymer samples with longer chains for both HS (protected DP of 45 by GPC-MALS; deprotected DP of 37 by 1H NMR) and DH (protected DP of 46 by GPC-MALS). Copolymerization of a 1:1 mixture of HSβ and DHβ with 5 mol % acid chloride provided a sample with ∼60% HS subunits and ∼40% DH subunits, according to 1H NMR analysis (protected DP of 27 by GPC-MALS; deprotected DP of 30 by 1H NMR). The nylon-3 polymers were tested for ice recrystallization inhibition via a modified splat-cooling assay.16 This method involves measuring the percent mean grain size (% MGS) in an ice sample formed in the presence of the candidate inhibitor relative to an ice sample formed from an aqueous PBS solution (Figure 3). A thin ice wafer is formed by dropping a 10 μL sample onto a cooled aluminum block. This wafer is transferred to a cooled cryostage microscope with a camera. A picture of the ice crystals is taken immediately to obtain a visual reference, and another picture is taken after the wafer has annealed at −8 °C for 30 min. The area of the largest 10 ice crystals, manually analyzed on ImageJ, observed in the 30 min data set, is averaged to calculate the % MGS value. Lower % MGS values indicate more potent ice recrystallization inhibitors. The currently preferred cryoprotectant for most cell types, 3−5 wt % DMSO, has been reported to display a % MGS value of ∼40.7 We measured a similar value for 3 mol wt % DMSO, as a standard in our experiments (Figure 4). It is important to note that the mechanism of action of DMSO is quite different from that of AFPs and their synthetic mimics. DMSO is a penetrating cryoprotectant, as it readily crosses the cell memebrane, while most synthetic polymers reduce the size of ice crystals formed outside the cell. We compare our polymers to DMSO because the latter is widely used for cryoprotection and despite the fact that the DMSO mechanism of action is not limited to ice recrystallization inhibition. Additional standards, for comparison with the new polymers, were 22 mM glucose (63% MGS) and 6 mg/mL of poly(vinyl alcohol) with a molecular weight range 31 000−50 000 (14% MGS). Our measurements with these two standards are consistent with published ice recrystallization inhibition data.7,12 Evaluation of ice recrystallization inhibition activity for new substances is typically conducted with samples in the 4−10 mg/mL concentration range in PBS.1 The HS and DH samples with shorter average chain lengths showed good ice recrystallization inhibition activity (Figure 4), with 14 and 6% MGS, respectively, at 6 mg/mL. These values compare favorably with our results for the benchmark poly(vinyl alcohol), with DP of ∼250, at the same concentration. The nylon-3 results surpass those for DMSO or glucose (Figure 4). Lengthening the HS or DH chains caused a substantial erosion of ice recrystallization inhibition activity; the origin of this length effect is unclear. A nylon-3
varied because a broad range of building blocks (β-lactams) is available.14,15 We hypothesized that nylon-3 polymers with hydroxylbearing side chains would be able to inhibit ice recrystallization and that these materials would not be toxic. Nylon-3 materials are synthesized via anionic ring-opening polymerization of βlactams; known β-lactams HSβ and DHβ have peripheral moieties that can be deprotected to reveal hydroxyl groups after polymerization (Figure 2).14
Figure 2. (A) β-Lactams used in this work. (B) Synthesis of a representative nylon-3 polymer, HS.
The availability of β-lactams DMβ, CAβ, and C3β allowed us to survey copolymers in which hydroxyl functionality was combined with cationic, anionic, or hydrophobic functionality, respectively. This effort began with evaluation of homopolymers in which each subunit bears one hydroxyl group (HS) or two hydroxyl groups (DH). All of the β-lactams employed in this study, including HSβ and DHβ, were racemic; therefore, all polymer chains were heterochiral. p-t-Butylbenzoyl chloride was used for in situ generation of an N-acyl-β-lactam coinitiator, which allows initiation of all polymer chains at the start of the reaction and minimizes dispersity of the product.14 Choice of this co-initiator precursor places a p-t-benzoyl unit at the N-terminus of each polymer chain. The molar proportion of co-initiator relative to β-lactam(s) should control average chain length; thus, for example, use of 5 mol % p-t-butylbenzoyl chloride is predicted to result in an average polymer chain length of 20 subunits. Analysis of HS and DH homopolymers generated with 5 mol % p-t-butylbenzoyl chloride revealed 696
DOI: 10.1021/acsmacrolett.7b00396 ACS Macro Lett. 2017, 6, 695−699
Letter
ACS Macro Letters Table 1. Characterization of Nylon-3 Polymers GPC characterizatione polymer
β-lactam feed ratio
expected length
HS DH HS (Long) DH (Long) HS:DH DM DM:HS DM:DH CA CA:HS CA:DH 3C:HS 3C:DH
50:50 50:50 50:50 50:50 50:50 20:80 20:80
20 20 40 60 20 20 20 20 20 20 20 20 20
a
ĐGPC
b
1.2 1.2 1.3 1.4 1.3 1.2 1.3 1.2 1.2 1.4 1.3 1.2 1.3
c
MnGPC
4760 4010 17058 8515 5900 6762 3405 7859 5950 5150 5903 4944 3158
NMR characterizationf d
DpGPC 16 21 45 46 27 29 12 37 33 19 32 21 20
d
DpNMR
observed subunit ratio
16 23 37 25 30 25 23 28 25 20 27 24 20
60:40 40:60 64:36 10:90 11:89 25:75 20:80
a
Predicted based on the monomer:coinitiator ratio. bDispersity index based on GPC. cMn: number-average molecular weight. dAverage degree of polymerization. eGPC characterization in THF, performed prior to deprotection. fNMR characterization in D2O performed after deprotection (DMSO-d6 was used for NMR analysis of DH (Long)).
and other ice-modifying activities but that hydrophobic surface elements can be favorable for synthetic polymers.13 However, ionic groups are tolerated in the ice-binding surfaces of natural AFPs.11a In addition, synthetic polyampholytes can display potent ice recrystallization inhibition activity, which provides further evidence that ionic species are compatible with this activity.17 The bifunctional copolymers were prepared with 5 mol % pt-butylbenzoyl chloride, i.e., targeting an average 20-mer length, based on our findings with hydroxylated homopolymers. βLactam DMβ has previously been used to incorporate basic side chains that are expected to be cationic in PBS,15 and β-lactam CAβ has previously been used to incorporate acidic (anionic) side chains.15e C3β is a new compound that introduces small hydrophobic moieties (n-propyl side chains) into nylon-3 chains. Copolymerization of a 1:1 mixture of HSβ and DMβ led to material containing similar levels of HS and DM subunits, but a comparable reaction with 1:1 DHβ:DMβ provided material with a 2:1 preference for DM incorporation (Table 1). Copolymerization of 1:1 mixtures of either HSβ or DHβ with CAβ produced hydroxyl-rich polymers containing only ∼10% of the acidic CA subunit. For copolymerizations involving C3β, the polar component, HSβ or DHβ, was used in excess (4:1 ratio), and the resulting polymer samples displayed the expected subunit proportions. All bifunctional polymers displayed lower ice recrystallization inhibition activity relative to the hydroxylated homopolymers with comparable average chain length. The activity decline for cationic DM-containing copolymers and for anionic CAcontaining copolymers is consistent with observations of Gibson;13 however, the apparent favorability of hydrophobic surface components in AFGs11 had led us to predict that the C3-containing copolymers would match or surpass the HS and DH homopolymers. Cationic and anionic nylon-3 homopolymers (DM and CA, respectively) displayed even poorer ice recrystallization inhibition activity than did bifunctional copolymers containing both ionic and hydroxylated subunits, which strengthens the conclusion that the side chain hydroxyl groups are crucial for ice recrystallization inhibition activity of nylon-3 materials. Our general interest in exploring nylon-3 polymers as functional mimics of surface-active natural polypeptides has
Figure 3. Examples of ice crystal size (10×magnification).
Figure 4. Ice recrystallization inhibition activities of nylon-3 polymers (6 mg/mL or ∼2 mM) in PBS, pH 7.4 (n = 3). Glucose (22 mM) and 6 mg/mL (∼0.2 mM) of PVA (molecular weight 31 000−50 000) were used as standards. Three wt % DMSO is 500 mM. PBS = phosphate buffered saline, PVA = poly(vinyl alcohol).
copolymer containing both hydroxylated subunits was less active than either of the shorter hydroxylated homopolymers. For the most potent HS and DH polymers, we evaluated the effect of lowering polymer concentration on ice recrystallization inhibition activity. Both polymers maintained good activity at 2 mg/mL. However, further dilution to 1 mg/mL led to a substantial decline in ice recrystallization inhibition efficacy (see Supporting Information, Figure S1). We took advantage of the accessibility of binary nylon-3 copolymers to survey the effects on ice recrystallization inhibition activity of combining hydroxyl functionality with ammonium, carboxylate, or hydrophobic functionality. Based on a careful evaluation of literature on hydroxyl-rich natural AFGP mimics, Gibson has concluded that introduction of charged groups is unfavorable for ice recrystallization inhibition 697
DOI: 10.1021/acsmacrolett.7b00396 ACS Macro Lett. 2017, 6, 695−699
ACS Macro Letters
■
been motivated by the hypothesis that these materials will tend to display low toxicity because of their protein-like backbone (the nylon-3 subunits are derived from β-amino acids, which are similar to the α-amino acid subunits of proteins). We tested this hypothesis by evaluating nylon-3 toxicity toward HEK 293FT cells at 2 mg/mL in Dulbecco’s Modified Eagle’s medium (DMEM). Among the two nylon-3 samples with the best ice recrystallization inhibition activity, the short HS and DH homopolymers, HS was less toxic (Figure 5). This nylon-3
Letter
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Melissa J. MacDonald: 0000-0001-6161-395X Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS This work was supported in part by DARPA Cooperative Agreement N66001-15-2-4023. NRC thanks NSF for support (CHE-1565810). We thank Peter Kitin for technical suggestions and the Newcomb Imaging Center, Department of Botany, UW-Madison for the use of their microscopy instrumentation. We thank UW-Madison soft materials lab for use of their Differential Scanning Calorimeter (DSC) Q100 and Anna V. Kiyanova for access to the instrument. Marlies V. Hager (UW-Madison) is thanked for assistance with cell culture.
Figure 5. Cell viability assay of nylon-3 polymers (2 mg/mL or ∼1 mM) in DMEM (n = 3). Glucose (22 mM) and 2 mg/mL (∼0.01 mM) of PVA (MW 31 000 to 50 000) were used as standards.
■
polymer matched poly(vinyl alcohol) in terms of minimal deleterious effects on cell viability. Increasing HS length led to a moderate decline in cell viability, as did use of DH instead of HS. Among the copolymers, incorporation of acidic (CA) or hydrophobic (C3) subunits did not enhance toxicity; in contrast, incorporation of the basic subunit (DM) resulted in lower cell viability. The acidic and basic homopolymers behaved quite differently from one another, with the DM homopolymer displaying very high toxicity, but only minimal toxicicity from the CA homopolymer. The DM homopolymer has previously been shown to be a potent inducer of red blood cell lysis.18 It is noteworthy that nearly all of the nylon-3 materials are superior to 5 wt % DMSO in terms of maintenance of HEK 293FT cell viability. We have shown that hydroxyl-rich nylon-3 polymers based on the HS subunit can exhibit potent inhibition of ice recrystallization without cytotoxicity. Relatively short HS and DH homopolymers surpass established cryoprotectants DMSO and glucose at commonly used concentrations, and these new polymers are comparable to poly(vinyl alcohol). The potent ice recrystallization inhibition demonstrated here for certain nylon3 polymers is promising from an application perspective because the properties of materials in this class are readily tailored via subunit choice. Our results highlight this feature of nylon-3 chemistry via the preparation and evaluation of bifunctional nylon-3 polymers in which hydroxyl groups have been combined with acidic, basic, or hydrophobic groups.
■
REFERENCES
(1) (a) Balcerzak, A. K.; Capicciotti, C. J.; Briard, J. G.; Ben, R. N. Designing Ice Recrystallization Inhibitors: from Antifreeze (glyco) Proteins to Small Molecules. RSC Adv. 2014, 4, 42682. (b) Gibson, M. I. Slowing the growth of ice with synthetic macromolecules: Beyond antifreeze (glyco) proteins. Polym. Chem. 2010, 1, 1141. (c) Ustun, N. S.; Turhan, S. J. AntiFreeze Proteins: Characteristics, Function, Mechanism of Action, Sources and Application to Foods. J. Food Process. Preserv. 2015, 39, 3189. (2) Fowler, A. Cryo-Injury and Biopreservation. Ann. N. Y. Acad. Sci. 2005, 1066, 119. (3) Mazur, P. Freezing of Living Cells: Mechanisms and Implications. Am. J. Physiol. 1984, 247, 125. (4) Karlsson, J. O.; Cravalho, E. G.; Borel Rinkes, I. H.; Tompkins, R. G.; Yarmush, M. L.; Toner, M. Nucleation and Growth of Ice Crystals Inside Cultured Hepatocytes During Freezing in the Presence of Dimethyl Sulfoxide. Biophys. J. 1993, 65, 2524. (5) (a) Trubiani, O.; Salvolini, E.; Staffolani, R.; Di Primio, R.; Mazzanti, L. DMSO Modifies Structural and Functional Properties of RPMI-8402 Cells by Promoting Programmed Cell Death. Int. J. Immunopathol. Pharmacol. 2003, 16, 253. (b) Tjernberg, A.; Markova, N.; Griffiths, W. J.; Hallén, D. DMSO-Related Effects in Protein Characterization. J. Biomol. Screening 2006, 11, 131. (6) (a) Czechura, P.; Tam, R. Y.; Dimitrijevic, E.; Murphy, A. V.; Ben, R. N. The Importance of Hydration for Inhibiting Ice Recrystallization with C-linked Antifreeze Glycoproteins. J. Am. Chem. Soc. 2008, 130, 2928. (b) Tam, R.; Czechura, P.; Ferreria, S. S.; Chaytor, J.; Ben, R. N. Hydration Index. A Better Parameter for Explaining Small Molecule Hydration in Inhibition of Ice Recrystallization. J. Am. Chem. Soc. 2008, 130, 17494. (c) Capicciotti, C. J.; Kurach, J. D. R.; Turner, T. R.; Mancini, R. S.; Acker, J. P.; Ben, R. N. Small Molecule Ice Recrystallization Inhibitors Enable Freezing of Human Red Blood Cells with Reduced Glycerol Concentrations. Sci. Rep. 2015, 5, 9692. (7) Chaytor, J. L.; Tokarew, J. M.; Wu, L. K.; Leclere, M.; Tam, R. Y.; Capicciotti, C. J.; Guolla, L.; von Moss, E.; Findlay, C. S.; Allan, D. S.; Ben, R. N. Inhibiting Ice Recrystallization and Optimization of Cell Viability After Cryopreservation. Glycobiology 2012, 22, 123. (8) (a) Bar-Dolev, M.; Celik, Y.; Wettlaufer, J. S.; Davies, P. L.; Braslavsky, I. New Insights into Ice Growth and Melting Modifications by AntiFreeze Proteins. J. R. Soc., Interface 2012, 9, 3249. (b) Harding,
ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.7b00396. NMR/GPC of all polymers and images of ice recrystallization (PDF) 698
DOI: 10.1021/acsmacrolett.7b00396 ACS Macro Lett. 2017, 6, 695−699
Letter
ACS Macro Letters M. M.; Anderberg, P. I.; Haymet, A. D. J. ’Antifreeze’ Glycoproteins from Polar Fish. Eur. J. Biochem. 2003, 270, 1381. (9) Liu, S.; Wang, W.; von Moos, E.; Jackman, J.; Mealing, G.; Monette, R.; Ben, R. N. In VitroStudies of Antifreeze Glycoprotein (AFGP) and a C-Linked AFGP Analogue. Biomacromolecules 2007, 8, 1456. (10) (a) Deller, R. C.; Pessin, J. E.; Vatish, M.; Mitchell, D. A.; Gibson, M. I. Enhanced Non-Vitreous Cryopreservation of Immortalized and Primary Cells by Ice-Growth Inhibiting Polymers. Biomater. Sci. 2016, 4, 1079. (b) Deller, R. C.; Vatish, M.; Mitchell, D. A.; Gibson, M. I. Synthetic Polymers Enable Non-Vitreous Cellular Cryopreservation by Reducing Ice Crystal Growth During Thawing. Nat. Commun. 2014, 5, 3244. (c) Mancini, R. J.; Lee, J.; Maynard, H. D. Trehalose Glycopolymers for Stabilization of Protein Conjugates to Environmental Stressors. J. Am. Chem. Soc. 2012, 134, 8474. (d) Stidham, S. E.; Chin, S. L.; Dane, E. L.; Grinstaff, M. W. Carboxylated Glucuronic Poly-amido-saccharides as Protein Stabilizing Agents. J. Am. Chem. Soc. 2014, 136, 9544. (11) (a) Dolev, M. B.; Braslavsky, I.; Davies, P. L. Ice-Binding Proteins and Their Function. Annu. Rev. Biochem. 2016, 85, 515. (b) Urbańczyk, M.; Góra, J.; Latajka, R.; Sewald, N. Antifreeze Glycopeptides: From Structure and Activity Studies to Current Approaches in Chemical Synthesis. Amino Acids 2017, 49, 209. (c) Deller, R. C.; Congdon, T.; Sahid, M. A.; Morgan, M.; Vatish, M.; Mitchell, D. A.; Notman, R.; Gibson, M. I. Ice Recrystallisation Inhibition by Polyols: Comparison of Molecular and Macromolecular Inhibitors and Role of Hydrophobic Units. Biomater. Sci. 2013, 1, 478. (12) (a) Inada, T.; Lu, S.-S. Inhibition of Recrystallization of Ice Grains by Adsorption of Poly(Vinyl Alcohol) onto Ice Surfaces. Cryst. Growth Des. 2003, 3, 747. (b) Inada, T.; Lu, S.-S. Thermal Hysteresis Caused by Non-Equilibrium Antifreeze Activity of Poly(Vinyl Alcohol). Chem. Phys. Lett. 2004, 394, 361. (13) (a) Gibson, M. I. Slowing The Growth of Ice with Synthetic Macromolecules: Beyond Antifreeze(Glyco) Proteins. Polym. Chem. 2010, 1, 1141. (b) Congdon, T.; Notman, R.; Gibson, M. I. Antifreeze (Glyco)protein Mimetic Behavior of Poly(Vinyl Alcohol): Detailed Structure Ice Recrystallization Inhibition Activity Study. Biomacromolecules 2013, 14, 1578. (14) Hashimoto, K. Ring-Opening Polymerization of Lactams. Living Anionic Polymerization and Its Applications. Prog. Polym. Sci. 2000, 25, 1411. (15) (a) Mowery, B. P.; Lindner, A. H.; Weisblum, B.; Stahl, S. S.; Gellman, S. H. Structure−activity Relationships among Random Nylon-3 Copolymers That Mimic Antibacterial Host-Defense Peptides. J. Am. Chem. Soc. 2009, 131, 9735. (b) Chakraborty, S.; Liu, R.; Hayouka, Z.; Chen, X.; Ehrhardt, J.; Lu, Q.; Burke, E.; Yang, Y.; Weisblum, B.; Wong, G. C. L.; Masters, K. S.; Gellman, S. H. Ternary Nylon-3 Copolymers as Host-Defense Peptide Mimics: Beyond Hydrophobic and Cationic Subunits. J. Am. Chem. Soc. 2014, 136, 14530. (c) Liu, R.; Chen, X.; Chakraborty, S.; Lemke, J. J.; Hayouka, Z.; Chow, C.; Welch, R. A.; Weisblum, B.; Masters, K. S.; Gellman, S. H. Tuning the Biological Activity Profile of Antibacterial Polymers via Subunit Substitution Pattern. J. Am. Chem. Soc. 2014, 136, 4410. (d) Liu, R.; Chen, X.; Hayouka, Z.; Chakraborty, S.; Falk, S. P.; Weisblum, B.; Masters, K. S.; Gellman, S. H. Nylon-3 Polymers with Selective Antifungal Activity. J. Am. Chem. Soc. 2013, 135, 5270. (e) Zhang, J.; Markiewicz, M. J.; Weisblum, B.; Stahl, S. S.; Gellman, S. H. Functionally Diverse Nylon-3 Copolymers from Readily Accessible β-Lactams. ACS Macro Lett. 2012, 1, 714. (16) (a) Knight, C. A.; Wen, D.; Laursen, R. A. Nonequilibrium Antifreeze Peptides and the Recrystallization of Ice. Cryobiology 1995, 32, 23. (b) Kitin, P.; Voelker, S. L.; Meinzer, F. C.; Beeckman, H.; Strauss, S. H.; Lachenbruch, B. Tyloses and Phenolic Deposits in Xylem Vessels Impede Water Transport in Low-Lignin Transgenic Poplars: A Study by Cryo-Fluorescence Microscopy. Plant Physiol. 2010, 154, 887. (c) Nakaba, S.; Kitin, P.; Yamagishi, Y.; Begum, S.; Kudo, K.; Nigroho, W. D.; Funada, R. Three-Dimensional Imaging of Cambium and Secondary Xylem Cells by Confocal Laser Scanning Microscopy. In Plant Microtechniques and Protocols; Yeung, E. C. T.,
Stasolla, C., Sumner, M. J., Huang, B. Q., Eds.; Springer: Switzerland, 2015. (17) (a) Mitchell, D. E.; Cameron, N. R.; Gibson, M. I. Rational, yet Simple, Design and Synthesis of an Antifreeze-Protein Inspired Polymer for Cellular Cryopreservation. Chem. Commun. 2015, 51, 12977. (b) Matsumura, K.; Hyon, S.-H. Polyampholytes as Low Toxic Efficient Cryoprotective Agents with Antifreeze Protein Properties. Biomaterials 2009, 30, 4842. (18) Mowery, B. P.; Lindner, A. H.; Weisblum, B.; Stahl, S. S.; Gellman, S. H. Structure−activity Relationships among Random Nylon-3 Copolymers That Mimic Antibacterial Host-Defense Peptides. J. Am. Chem. Soc. 2009, 131, 9735.
699
DOI: 10.1021/acsmacrolett.7b00396 ACS Macro Lett. 2017, 6, 695−699
