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

Aug 4, 2011 - Computational Biology and Chemistry 2018 73, 13-24 ... Ye Yuan , Ying Yang , Yuchen Tian , Jinkyu Park , Aihua Dai , R. Michael Roberts ...
10 downloads 9 Views 2MB Size
Download PDF
ARTICLE pubs.acs.org/bc

C-Linked Antifreeze Glycoprotein (C-AFGP) Analogues as Novel Cryoprotectants Mathieu Leclere,† Bonnie K. Kwok,† Luke K. Wu,‡ David S. Allan,‡ and Robert N. Ben*,† †

Department of Chemistry Department of Medicine, Regenerative Medicine Program University of Ottawa, Ontario, Canada, K1N 6N5

‡

bS Supporting Information ABSTRACT: 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 freezethawing 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 11.5 mg/mL (0.50.8 mM) solution of C-AFGP 1 is an excellent alternative to a 2.5% DMSO solution for the cryopreservation of human embryonic liver cells.

’ INTRODUCTION Cryopreservation is a simple and economic option for the long-term storage of biological materials. However, one of the main drawbacks of this method is that large percentages of cells do not survive the freezethawing cycle. There are many reasons for this decreased viability which include cell necrosis due to intracellular freezing, mechanical damage to the external cell membrane by either ice or osmotic flux, and cold-induced apoptosis.13 Cord blood banking has become routine in the past decade and this can be attributed to two reasons. The first is that stem cells from cord blood are now regarded as a practical alternative to autologous bone marrow stem cells,4 and second, the documented successes of new stem cell-based regenerative therapies has increased dramatically in the last ten years. However, decreased cell viability after thawing is still a significant problem, particularly since recent studies have linked the successful outcome of regenerative therapies with the proportion of viable cells prior to transplant.5,6 Consequently, improved cryopreservation protocols that increase cell viability are urgently required. Dimethyl sulfoxide (DMSO) is currently regarded as the “gold standard” for cryopreservation. While all cryoprotectants are potentially cytotoxic in vitro, DMSO has exhibited significant cytotoxic effects in the clinical setting.7,8 While various biopolymers9 have been explored as cryoprotectants, these fail to provide the high cell viabilities observed with DMSO or glycerol. Simple mono- and disaccharides have also been investigated as cryoprotectants; however, the structure of the carbohydrate, the freezing protocol, cell type, and reported cell viabilities vary dramatically between studies making it difficult to ascertain the true ability of these compounds to protect cells against cryoinjury. Native antifreeze glycoproteins (AFGPs) found in r 2011 American Chemical Society

Teleost fish are naturally occurring antifreezes that have the ability to inhibit the growth of ice crystals in vivo (Figure 1).10,11 Unfortunately, the thermal hysteresis (TH) properties of these proteins (the ability to selectively depress the freezing point of a solution relative to its melting point) actually exacerbates cell damage at temperatures outside of the TH gap during cryopreservation.12 Despite this undesirable effect, biological antifreezes have been explored as cryoprotectants. The majority of this work has been carried out using antifreeze proteins (AFPs) and reports of AFGPs as cryoprotectants have been limited to preservation of cells via vitrification.13 While AFGPs and AFPs also inhibit ice recrystallization in frozen samples, a property speculated to be beneficial in a cryoprotectant,14 they have not proven to be effective cryoprotectants due to the TH activity that damages cells at cryopreservation temperatures. This has been demonstrated using AFP type I for the cryopreservation of red blood cells (RBCs) where it was observed that micromolar concentrations of AFP I appeared to confer some protection against cell damage, but millimolar concentrations fostered increased damage to cells during freezethawing.14,15 It should be pointed out that these studies were performed using thawing rates that were nonoptimal, and thawing was performed slowly in order to increase the potential for ice recrystallization and subsequent cell damage. In typical cryopreservation protocols, thawing is done very quickly to mitigate cellular damage due to ice recrystallization. Given that an atypical thawing protocol was utilized, it is not clear whether inhibitors of ice recrystallization Received: April 11, 2011 Revised: July 15, 2011 Published: August 04, 2011 1804

dx.doi.org/10.1021/bc2001837 | Bioconjugate Chem. 2011, 22, 1804–1810

Bioconjugate Chemistry

ARTICLE

Figure 1. Structure of native AFGP 8 and C-linked analogues 1 and 2.

would actually increase cell viabilities under conditions in which thawing was performed quickly.

’ EXPERIMENTAL PROCEDURES The synthesis and characterization of precursors 8 and 9 as well as the general synthetic procedures details are described in the Supporting Information.

(S)-N-benzyloxycarbonyl-2,2-dimethyl-4-((E)-10 -deoxy20 ,30 ;40 ,60 -di-O-isopropylidene-R-D-galactopyranosyl)vinyl)oxazolidine (7). Sulfone 9 (4.50 g, 10.0 mmol, 1.5 equiv) was

dissolved in 20 mL of THF. The solution was cooled to 78 °C, and 10.0 mL of 1 M NaHMDS in THF (10 mmol, 1.5 equiv) was added. After 5 min of stirring, a solution of the crude aldehyde 8 (6.7 mmol) in 10 mL of THF was added via cannula. The stirring was continued for 1 h at 78 °C and the temperature slowly warmed to room temperature over 2 h. After an additional hour of stirring, the mixture was quenched with few drops of water and diluted with 100 mL of Et2O. The organic layer was washed with water and brine, dried over Na2SO4, concentrated, and the crude purified by flash chromatography (Pet. Ether/AcOEt 5/2) to give 2.2 g of the alkene 7 (65% from 14). 1H NMR (DMSO-d6, 500 MHz, 343 K) δ 7.36 (m, 5H), 5.82 (ddd, J = 16.2, 6.3, 1.9 Hz, 1H), 5.68 (dd, J = 16.2, 3.8 Hz, 2H), 5.06 (s, 2H), 4.78 (brt, J = 4.4 Hz, 1H), 4.49 (t, J = 6.2 Hz, 1H), 4.274.11 (br, 1H), 4.07 (dd, J = 9.0, 6.2 Hz, 1H), 4.04 (dd, J = 10.2, 5.81 Hz, 1H), 3.94 (d, J = 13.2 Hz, 1H), 3.75 (dd, J = 9.0, 2.0 Hz, 1H), 3.64 (d, J = 13.2 Hz, 1H), 3.41 (brs, 1H), 3.13 (brs, 1H), 1.54 (s, 3H), 1.47 (s, 6H), 1.38 (s, 6H), 1.33 (s, 6H) 1.29 (s, 3H), 1.27 (s, 3H). 13 C NMR (DMSO-d6, 125 MHz, 343 K) δ 152.2, 137.1, 133.1, 128.8, 128.2, 127.9, 124.9, 109.5, 97.9, 94.0, 74.3, 73.6, 70.9, 68.4, 67.3, 66.5, 63.9, 63.0, 58.5, 29.5, 27.0, 26.8, 23.4, 18.5. HRMS (EI) m/z calcd. for C27H37N1O8 [M]+ 503.2519 found 503.2521. IR (neat, cm1) 2985, 2935, 1701. (S)-3-amino-4-(10 deoxy-20 ,30 ;40 ,60 -di-O-isopropylidene-RD-galactopyranosyl)-butanol (17). H2 was bubbled for 5 min through a solution of the alkene 7 (2.10 g, 4.2 mmol) and Pd(OH)2/C (600 mg, 0.42 mmol, 0.1 equiv) in 20 mL of EtOH. Bubbling was stopped and stirring was continued for 1 h under H2 atmosphere. The flask was then purged with N2 and the mixture was filtered through Celite and the solvent removed in vacuo. After drying under high vacuum for several hours, 1.32 g of amino alcohol 17 was obtained (95%). 1H NMR (CDCl3, 400 MHz) δ 4.41 (brs, 1H), 4.32 (m, 1H), 4.25 (dd, J = 9.7, 5.4 Hz, 1H), 4.06 (dd, J = 12.9, 2.0 Hz, 1H), 3.85 (d, J = 12.9 Hz, 1H), 3.67 (dd, J = 9.9, 2.9 Hz, 1H), 3.59 (dd, J = 10.7, 3.3 Hz, 1H), 3.23 (brs, 1H), 3.203.00 (brs, 3H), 2.92 (brs, 1H), 1.61 (m, 3H), 1.44 (s, 3H), 1.43 (s, 3H), 1.42 (s, 3H), 1.38 (s, 3H), 1.36 (m, 1H). 13C (CDCl3, 100 MHz) δ 110.0, 98.6, 73.9, 70.8, 67.4, 65.6, 63.1, 52.8, 30.1, 29.2, 26.7, 26.5, 21.0, 18.4.

MS(ESI) m/z calcd. for C16H30N1O6 [M+H]+ 332.207 found 332.273. IR (neat, cm1) 3400, 2991, 2935. ((S)-3-(N-(9-fluorenylmethyloxycarbonyl)amino)-4-(10 deoxy-20 ,30 ;40 ,60 -di-O-isopropylidene-R-D-galactopyranosyl)butanol (18). To a solution of 17 (1.30 g, 3.9 mmol) and NaHCO3 (0.65 g, 7.8 mmol, 2 equiv.) in a mixture of 20 mL of 1,4-dioxane and 20 mL of water was added Fmoc-OSu (1.4 g, 4.2 mmol, 1.1 equiv) at 0 °C. The solution was stirred overnight at room temperature, then diluted with CH2Cl2, and the organic layer was washed with water, separated, dried over Na2SO4, and concentrated in vacuo. The crude was purified by flash chromatography (CH2Cl2/MeOH 98/2) to yield 1.60 g (75%) of the title compound 18. 1H NMR (C6D6, 500 MHz) δ 7.58 (dd, J = 6.5, 1.6 Hz, 2H), 7.51 (m, 2H), 7.22 (m, 4H), 4.87 (brs, 1H), 4.50 (dd, J = 10.0, 5.6 Hz, 1H), 4.45 (m, 2H), 4.27 (ddd, J = 9.2, 5.8, 2.8 Hz, 1H), 4.07 (m, 2H), 3.84 (dd, J = 12.8, 1.4 Hz, 1H), 3.65 (dd, J = 12.8, 2.4 Hz, 1H), 3.51 (dd, J = 10.0, 3.3 Hz, 1H), 3.44 (m, 1H), 3.37 (dd, J = 11.0, 5.1 Hz, 1H), 2.78 (s, 1H), 1.64 (m, 2H), 1.51 (m, 1H), 1.46 (s, 3H), 1.42 (s, 3H), 1.37 (s, 3H), 1.28 (m, 1H), 1.20 (s, 3H). 13C NMR (C6D6, 125 MHz): δ 157.0, 144.7, 144.6, 141.9, 127.4, 125.4, 125.3, 120.3, 109.8, 98.4, 76.8, 74.4, 71.4, 67.9, 66.6, 65.4, 63.8, 63.5, 53.7, 48.0, 29.4, 29.0, 27.1, 26.9, 21.5, 18.8. MS (ESI) m/z calcd. for C31H40NNaO8 [M+Na]+ 576.257 found 576.288. IR (neat, cm1) 2989, 2937, 1703. ((S)-3-(N-(9-fluorenylmethyloxycarbonyl)amino)-4-(10 -deoxy0 0 0 0 2 ,3 ;4 ,6 -di-O-isopropylidene-R-D-galactopyranosyl)-butanoic acid (3b). Alcohol 18 (1.50 g, 2.7 mmol) was dissolved in 20 mL of acetonitrile. NaHCO3 (0.68 g, 8.1 mmol, 3 equiv), TEMPO (42 mg, 0.27 mmol, 0.1 equiv), and PhI(OAc)2 (1.3 g, 4.0 mmol, 1.5 equiv) were added and the mixture was stirred vigorously. The reaction was monitored by TLC (CH2Cl2/ MeOH 98/2). Upon completion of the reaction (ca. 6 h), the mixture was diluted with CH2Cl2 and washed with water. The organic layer was separated, dried over Na2SO4, and the solvent evaporated. The crude aldehyde was redissolved in 20 mL of t-BuOH and 6 mL of 2-methyl-2-butene. The solution was then cooled to 0 °C and 14 mL of 1 M solution of aqueous KH2PO4 (14.0 mmol, 5.2 equiv) followed by 14 mL of 1 M solution of NaClO2 (14.0 mmol, 5.2 equiv). After stirring at 0 °C for 2 h, the mixture was partitioned with AcOEt (100 mL) and water (50 mL). The organic layer was separated; the aqueous phase was extracted with 100 mL of AcOEt. The combined organic layers were dried over Na2SO4 and the solvents evaporated. Purification of the crude by flash chromatography (CH2Cl2/ MeOH 96/4) yielded 1.22 g of the building block 3b (80%). 1H NMR (DMSO-d6, 500 MHz, 343 K) δ 7.85 (d, J = 7.6 Hz, 2H), 7.70 (dd, J = 7.8, 7.4 Hz, 2H), 7.41 (dd, J = 7.5, 7.5 Hz, 2H), 7.32 (ddd, J = 7.8, 7.5, 1.6 Hz, 2H), 4.39 (d, J = 3.0 Hz, 1H), 4.30 (dd, J = 10.0, 7.0 Hz, 1H), 4.27 (d, J = 6.8 Hz, 1H), 4.254.28 (m, 2H), 4.07 (m, 2H), 3.72 (dd, J = 10.0, 2.8 Hz, 1H), 3.66 (dd, 1805

dx.doi.org/10.1021/bc2001837 |Bioconjugate Chem. 2011, 22, 1804–1810

Bioconjugate Chemistry J = 12.8, 1.0 Hz, 1H), 3.28 (brs, 1H), 1.89 (m, 1H), 1.74 (m, 2H), 1.58 (m, 1H), 1.39 (s, 3H), 1.33 (s, 6H), 1.30 (s, 3H). 13C NMR (DMSO-d6, 125 MHz, 293 K) δ 174.1 (br), 156.3 (br), 149.1, 149.0, 145.9, 132.9, 132.3, 130.4, 125.4, 114.0, 102.6, 80.5, 78.4, 75.5, 72.0, 70.9, 67.8, 67.7, 51.8, 34.5, 31.9, 23.9. MS (ESI) m/z calcd. for C31H37N1O9Na [M+Na]+ 590.237 found 590.286. IR (neat, cm1) 2987, 2933, 1712, 1704. Glycoconjugate 2. A typical SPPS cycle consisted in the following steps: 300 mg (0.18 mmol) of Wang Fmoc-Gly-OH resin were swollen in DMF for 1 h. Fmoc deprotection was achieved by adding a mixture of piperidine/DBU/DMF 2/2/96 for 30 min. The resin was washed with DMF and treated with a premixed solution of Fmoc-Gly-Gly-OH and HCTU (6 equiv each) in DMF. DIPEA (12 equiv) was added and the resin was shaken gently for 3 h. After washing the resin with DMF, the Fmoc carbamate was cleaved as describe above. The resin was washed and treated with the premixed building block 3b and HCTU (1.5 equiv each) and DIPEA (3 equiv) for 24 h. The resin was then washed with DMF (3  10 mL) and the Fmoc carbamate cleaved as described above. This cycle was repeated 3 more times in order to achieve the desired peptide length. The resin was washed with DMF, CH2Cl2, and MeOH and dried under high vacuum for 6 h. Final cleavage using a mixture of TFA/i-Pr3SiH/H2O 92.5/5/2.5 for 3 h furnished the crude glycoconjugate. The resin was filtered and washed with TFA and the combined filtrates were concentrated in vacuo at 35 °C. Acetone was added to precipitate the crude glycopeptide which was further purified by SPE on a Discovery C18 cartridge, followed by preparative HPLC (C18) to give 55 mg of the glycoconjugate 2 (20%). 1H NMR (D2O, 500 MHz, presaturated on HOD frequency) δ 4.21 (brs, 1H), 4.003.68 (brm, 8H), 3.653.47 (brm, 4H), 1.951.41 (brm, 4H). 13C NMR (D2O, 125 MHz) δ 174.5, 171.8, 171.6, 170.4, 75.2, 74.7, 71.8, 71.6, 69.5, 69.0, 68.0, 67.9, 61.1, 54.0, 52.9, 46.6, 42.5, 42.2, 41.5, 40.4, 27.3, 27.2, 20.2, 19.3. HRMS (MALDI-TOF) m/z calcd. for C58H97N13NaO34 (M+Na)+ 1542.6159; found 1542.6965. Cryopreservation Assay. WRL 68 cells were cultured and trypsinized as described above. Cells were diluted with culture media and counted using a hematocytometer. Aliquots containing 1  106 cells were added to 2 mL Eppendorf vial and cells were pelleted by centrifugation for 10 min at 2000 rpm. The supernatant was removed and the cells were resuspended in 0.1 mL HTK supplemented with glycoconjugate 1 or 2 to a concentration of 1, 1.5, 2, and 5 mg/mL. HTK supplemented with DMSO to a concentration 2.5% or 5% served as the positive controls. Cell suspensions were transferred to 2 mL cryogenic vials (Corning) and placed in a “Mr. Frosty” freezing container (Nalgene). The container was placed in an ultracold freezer (80 °C) for 24 allowing a cooling rate of approximately 1 °C/min. Samples were then stored in a cryogenic tank in the vapor phase of liquid nitrogen (150 °C) for 6 days. Following storage, samples were rapidly thawed in a 37 °C water bath with gentle agitation. Thawed samples were transferred to 2 mL centrifuge tubes and slowly diluted to 1 mL by dropwise addition of cell culture medium. Cells were pelleted by centrifugation for 10 min at 2000 rpm. The supernatant was decanted and the pellets resuspended in 0.5 mL of Annexin V binding buffer (BD Pharmingen). Flow Cytometry. 100 μL of the suspension of cells in binding buffer was transferred to a flow cytometry tube. Subsequently, 5 μL of 7-Actinomycin D (7AAD) (BD Pharmingen) and 4 μL of Annexin V-PE (BD Pharmingen) were added and samples were

ARTICLE

Figure 2. Retrosynthetic strategy to prepare building block 3a.

incubated for 20 min in the dark. Following incubation, samples were diluted to 500 μL with 1 Annexin-V binding buffer. Flow cytometry analysis on 10 000 cells was carried out on a BD LSR flow cytometer using CellQuest (BD). Annexin V-PE was measured on FL-2 while 7-AAD was measured on FL-3. Complete viability was denoted as Annexin V-/7AAD- cells, early apoptotic as Annexin V+ /7AAD- cells, late apoptotic as Annexin V+ /7AAD+ cells, and Annexin V- /7AAD+ as necrotic cells. Compensation was set from single stain experiments with 7-AAD and Annexin-V. Aliquots from noncryopreserved samples labeled with equivalent fluorochromes were used to set the control gate excluding 99% 7AAD+ cells. Time between thawing and analysis by flow cytometry was 2 h. All trials were performed in duplicate.

’ RESULTS AND DISCUSSION Our laboratory has successfully designed and synthesized a number of C-linked analogues of antifreeze glycoproteins (1 and 2, Figure 1) that are potent inhibitors of ice recrystallization but do not exhibit thermal hysteresis.1621 These compounds also possess increased chemical and biological stability relative to native AFGP. The two major structural differences between these C-linked AFGPs and native AFGP are that the disaccharide has been replaced with a C-linked galactosyl moiety and the alanine residues have been replaced with glycine residues. These structural changes were initially incorporated for ease of synthesis and to avoid C-terminal racemization during solid-phase synthesis. More recently, truncated versions of these structures bearing fluorine atoms have been prepared but these compounds failed to demonstrate the ability to inhibit ice recrystallization.22 The “custom-tailored” antifreeze activity observed in 1 and 2 makes these compounds ideally suited for cryopreservative applications. In addition, 1 and 2 exhibit lower in vitro cytotoxicity and offer increased chemical stability relative to native AFGPs.23 Unfortunately, the previously reported synthesis20 of 2 was not amenable to the scale-up required for in vitro studies. The key step in our original synthesis of building block 3a required for the solidphase synthesis of 2 employed a catalytic asymmetric hydrogenation of the galactosyl enamide 4 as shown in Figure 2. This reaction proceeded with only moderate diastereoselectivity (69% de) yielding a complex mixture that was difficult to purify by HPLC making this approach unattractive for the preparation of 2. A relatively small number of syntheses of C-galactosyl serines have been reported. In these examples, the key step has employed a Wittig olefination,24 condensation of silyl enol-ether25 or enolate26 or a catalytic asymmetric hydrogenation27 to incorporate the stereocenter at the R-carbon. In order to assess the ability of 2 as a cryoprotectant, a more efficient highly stereoselective synthesis affording a single diastereoisomer was desirable. Recently, Chen et al. reported the synthesis of C-galactosyl ceramides using the Julia-Kocienski-Lythgoe (JKL) olefination reaction.28 We envisioned a similar approach in which a one-pot 1806

dx.doi.org/10.1021/bc2001837 |Bioconjugate Chem. 2011, 22, 1804–1810

Bioconjugate Chemistry JKL olefination29,30 using carbohydrate derivative 8 and sulfone 9 as outlined in Figure 3 would furnish 7. Reduction of the carboncarbon double bond in 7 with concomitant cleavage of the Cbz group was expected to furnish the amino alcohol, which could be converted to amino acid derivative 3b necessary for the synthesis of 2. In this approach, we chose to employ 5-phenyl-tetrazolyl (PT) sulfone 9 (prepared following a modified procedure reported by Albrecht et al.31), as it has been reported to be less prone to self-condensation than the benzothiazolyl sulfone.32 The synthesis of 3b is described in Scheme 1. Aldehyde 8 was prepared from R-C-allyl galactose derivative 11 which in turn was prepared by radical-mediated allylation of galactosyl bromide 10 using allyltributylstannane and Et3B/air.33 This afforded the R-C-allyl derivative 11 in 70% yield with a trace amount of 12 (6%) resulting from in situ acyl migration.34 The terminal alkene was isomerized to 2-propenyl derivative 13 using (Ph2MeP)2Ir(cod)PF6 catalyst.35 Initial attempts to isomerize olefin 13 followed by ozonolysis failed to produce the desired aldehyde. Hence, isopropylidenes were installed in 75% yield and ozonolysis of 14 followed by reduction with PPh3 yielded aldehyde 8 in 84% yield. Due to the propensity of this compound to epimerize and undergo elimination, it was not purified but used directly in the JKL olefination. Sulfone 9

Figure 3. New retrosynthetic strategy to prepare building block 3b.

ARTICLE

was prepared from orthogonally protected L-serine methyl ester 15 via Mitsunobu reaction with 5-phenyl-tetrazolyl thiol to furnish 16 in 82% yield. Subsequent reduction with LiBH4 followed by N,O-isopropylidene formation and oxidation using Sharpless conditions furnished the sulfone 9 in 65% yield. Sulfone 9 was then metalated at 78 °C with NaHMDS and followed by dropwise addition of aldehyde 8. The reaction was then warmed to room temperature over 2 h. After workup, olefin 7 was obtained in 65% isolated yield. The geometry of the double bond was found to be exclusively trans by 1H NMR spectroscopy. Reduction of the carboncarbon double bond and removal of the Cbz carbamate using wet Pd(OH)2/C in ethanol was accompanied by concomitant cleavage of the N,O-isopropylidene moiety. The resulting amino-alcohol 17 was protected as the Fmoc carbamate (18) and oxidized furnishing carboxylic acid 3b in 80% yield. Synthesis of glycoconjugate 2 using building block 3b was carried out using standard Fmoc-based solid-phase synthesis with Wang resin. Cleavage of the glycoconjugate from the resin and simultaneous deprotection by treatment with TFA furnished the crude glycopeptide which was purified by solidphase extraction on a C-18 SPE cartridge followed by reversedphase HPLC to afford glycoconjugate 2 in 21% overall yield. C-AFGP analogues 1 and 2 were then assessed for cryoprotective abilities using human embryonic liver cells (WRL-68) as a model. This specific cell model was chosen because liver cells have been identified as a potential source of stem cells for various regenerative therapies.36 Stem cells have changed the course of treatment for many malignant and nonmalignant conditions in adults and pediatric patients.37,38 For instance, mesenchymal stem cells (MSCs) have been featured prominently as they have the ability to differentiate into many different cell types and have been successfully utilized to treat cartilage lesions,39 spinal cord injury,40 bone defects,41 and cardiac disease.42 Unfortunately, recent studies have correlated poor in vivo differentiation of these

Scheme 1. Synthesis of Building Block 3b

1807

dx.doi.org/10.1021/bc2001837 |Bioconjugate Chem. 2011, 22, 1804–1810

Bioconjugate Chemistry

Figure 4. WRL-68 cell viability 2 h post thaw. Error bars indicate SEM.

cells with low survival rates after cryopreservation, thus underscoring the need for improved cryopreservation protocols.43 It was hypothesized that, since these C-AFGPs were potent inhibitors of ice recrystallization and that this phenomenon has been implicated as a significant source of cell injury during cryopreservation,15,44 addition of 1 and 2 to cell medium during freezing would enhance cell viabilities after thawing. In order to assess the cryoprotective potential of C-AFGP analogues 1 and 2, a cryopreservation assay was developed, based on currently utilized cryopreservation protocols. Standard preservation conditions designed to minimize cell damage due to ice recrystallization were used. In this assay, C-AFGP analogue 1 or 2 was added to a suspension of 1  106 human embryonic liver cells (WRL68) in a 100 μL histidine-tryptophane-ketoglutarate (HTK) custodiol solution. Custodiol HTK solution was a logical choice as it is routinely used in organ transplantation. The cryoprotection ability of 1 and 2 was directly compared to 2.5% and 5% DMSO solutions. This is a reasonable control, as cells are routinely frozen with DMSO in the medium.45 Cell suspensions were transferred into 2 mL cryovials and placed into a Mister Frosty inside a 78 °C freezer to achieve rate controlled freezing of 1 °C/min. At a temperature of 80 °C, the cryovials were transferred into a liquid N2 storage container. After 6 days, the samples were thawed quickly (to minimize ice recrystallization and subsequent cell damage) and resuspended in fresh medium. An aliquot was withdrawn and total cell viability was determined 2 h after thawing. A combination of 7-amino-actinomycin (7AAD, an indicator of cell membrane integrity) and Annexin-V PE (a marker of apoptosis) was used to distinguish the viable cells from the apoptotic and necrotic ones. Cell viabilities after freezing WRL 68 cells in the presence of C-AFGP analogues 1 and 2 using appropriate controls (DMSO and HTK solution) is presented in Figure 4. Experiments were performed using 1, 1.5, 2, and 5 mg/mL concentrations of 1 and 2. WRL 68 cells cryopreserved using a 1 mg/mL solution of 1 resulted in total cell viability comparable to a 2.5% DMSO solution. This 1 mg/mL solution of 1 is 30% more effective than HTK solution alone but is approximately 10% less effective then the 5% DMSO solution. Similar results were obtained when a 1.5 mg/mL solution was utilized; however, 2 and 5 mg/mL concentrations of 1 proved detrimental, resulting in a marked decrease in total cell viability. While intercellular concentrations of 2 and 5 mg/mL are in the millimolar range, similar concentrations of AFP type I have been reported to result in significant cell damage during freezing and poor viabilities after thawing.13 Analogue 2 gave similar results with maximum cryoprotective

ARTICLE

Figure 5. Viability of WRL-68 cells after 6 days cryopreservation using 1.5 mg/mL of analogue 1 with 1 and 2 h preincubation (P.-I.) at 37 °C. Error bars indicate SEM.

ability at 1 mg/mL. However, total viability was slightly lower than with analogue 1. Cellular concentrations 1 and 2 greater than 2 mg/mL failed to increase post-thaw viabilities. It has recently been reported that various cryoprotectants require preincubation with cells prior to freezing in order to ensure internalization46 to confer cellular protection against intracellular ice formation.1 This is an important property associated with cell penetrating cryoprotectants. Our laboratory has previously demonstrated that C-AFGP analogues 1 and 2 are readily internalized into liver cells within 10 min23 and that this internalization is a receptor-mediated process likely involving the asialoglycoprotein receptor (ASGPr).47,48 Encouraged by the fact that 1 and 2 where readily internalized, we investigated whether preincubation of cells with C-AFPGs would increase cell viabilities post-thaw. For this experiment, only C-AFGP analogue 1 was examined as it resulted in slightly better viabilities postthaw than C-AFGP analogue 2 (Figure 4) at 1 and 1.5 mg/mL. To ensure adequate time for internalization and thus maximize any cryoprotective effect, C-AFGP analogue 1 was preincubated with WRL 68 cells at 37 °C for 1 and 2 h prior to cryopreservation (Figure 5). No significant improvements in total cell viability were observed. This result suggests that adequate internalization occurs in a short amount of time and is consistent with our earlier studies.22 Cell viability dramatically decreased when preincubation times were increased to 2 h. The cytotoxic nature of the 2 h preincubation is currently under investigation in our laboratory.

’ CONCLUSIONS We have developed a new and highly selective synthesis of C-AFGP analogue 2. Both C-AFGP analogues 1 and 2 are potent inhibitors of ice recrystallization but do not exhibit thermal hysteresis activity and can protect embryonic liver cells from cryo-injury at millimolar concentrations. C-AFGP analogue 1 at 11.5 mg/mL (0.50.8 mM) in cell culture is an effective cryoprotectant which appears to be as good as a 2.5% DMSO. It is particularly noteworthy that such a large increase in cell viability is observed under “ideal” thawing conditions that are designed to mitigate but cannot abrogate cell damage from ice recrystallization. This result further validates the hypothesis that inhibiting ice recrystallization during cryopreservation is important for improving cell viability. To the best of our knowledge, this is the first example where an analogue of a biological antifreeze possessing custom-tailored antifreeze activity has been 1808

dx.doi.org/10.1021/bc2001837 |Bioconjugate Chem. 2011, 22, 1804–1810

Bioconjugate Chemistry shown to be an effective cryoprotectant. While the mechanisms by which cryoprotectants protect cells against cryoinjury are complex, the results presented in this paper indicate that C-AFGP analogues 1 and 2 are effective cryoprotectants for human embryonic liver cells and that inhibition of ice recrystallization during cryopreservation is an important function for a cryoprotectant. Novel cryoprotectants such as these that are specifically designed to inhibit ice recrystallization may be instrumental in overcoming the current limitations associated with existing cryoprotectants, thus facilitating the efficient preservation of not just cells but tissues and even organs. C-AFGP glycoconjuages 1 and 2 are currently being investigated as cryoprotectants with other cell types, and the results of these studies will be reported in due course.

’ ASSOCIATED CONTENT

bS

Supporting Information. Experimental procedures and spectroscopic data for all new compounds. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*[email protected].

’ ACKNOWLEDGMENT This work was supported with grants from the Natural Sciences and Engineering Research Council (NSERC) of Canada and Canadian Blood Services (CBS). ’ REFERENCES (1) Mazur, P. (1984) Freezing of living cells: mechanism and implications. Am. J. Physiol.: Cell Physiol. 247, C125–C142. (2) Karlsson, J. O. M., and Toner, M. (1996) Long-term storage of tissues by cryopreservation: critical issues. Biomaterials 17, 243–256. (3) Farrant, J., Walter, C. A., Lee, H., and McGann, L. E. (1977) Use of two-step cooling procedures to examine factors influencing cell survival following freezing and thawing. Cryobiology 14, 273–286. (4) Broxmeyer, H. E. (2010) Will iPS cells enhance therapeutic applicability of cord blood cells and banking? Cell Stem Cell 6, 21–24. (5) Allan, D. S., Keeney, M., Howson-Jan, K., Popma, J., K Weir, K., Bhatia, M., Sutherland, D. R., and Chin-Yee, I. H. (2002) Number of viable CD34 cells reinfused predicts engraftment in autologous hematopoietic stem cell transplantation. Bone Marrow Transplant. 29, 967–972. (6) Abrahamsen, J. F., Wentzel-Larsen, T., and Bruserud, Ø. (2004) Autologous transplantation: the viable transplanted CD34+ cell dose measured post-thaw does not predict engraftment kinetics better than the total CD34+ cell dose measured pre-freeze in patients that receive more than 2  106 CD34+ cells/kg. Cytotherapy 6, 356–362. (7) Liseth, K., Abrahamsen, J. F., Bjørsvik, S., Grøttebø, K., and Bruserud, Ø. (2005) The viability of cryopreserved PBPC depends on the DMSO concentration and the concentration of nucleated cells in the graft. Cytotherapy 7, 328–333. (8) Konuma, T., Ooi, J., Takahashi, S., Tomonori, A., Tsukada, T., Kobayashi, T., Sato, S., Kato, S., S., K., Ebihara, Y., Nagamura-Inoue, T., Tsuji, K., Tojo, A., and Asano, S. (2008) Cardiovascular toxicity of cryopreserved cord blood cell infusion. Bone Marrow Transplant. 41, 861–845. (9) Matsumura, K., and Hyon, S.-H. (2009) Polyampholytes as low toxic efficient cryoprotective agents with antifreeze protein properties. Biomaterials 30, 4842–4849.

ARTICLE

(10) Harding, M. M., Anderberg, P. I., and Haymet, A. D. J. (2003) ‘Antifreeze’ glycoproteins from polar fish. Eur. J. Biochem. 270, 1381–1392. (11) Feeney, R. E., Burcham, T. S., and Yeh, Y. (1986) Antifreeze glycoproteins from polar fish blood. Annu. Rev. Biophys. Biophys. Chem. 15, 59–78. (12) Wang, T., Zhu, Q., Yang, X., Layne, J. R., and Devries, A. L. (1994) Antifreeze glycoproteins from antarctic notothenioid fishes fail to protect the rat cardiac explant during hypothermic and freezing preservation. Cryobiology 31, 185–192. (13) Rubinsky, B., Arav, A., and Devries, A. L. (1992) The cryoprotective effect of antifreeze glycopeptides from antarctic fishes. Cryobiology 29, 69–79. (14) Carpenter, J. F., and Hansen, T. N. (1992) Antifreeze protein modulates cell survival during cryopreservation: mediation through influence on ice crystal growth. Proc. Natl. Acad. Sci. U.S.A. 89, 8953–8957. (15) Chao, H., Davies, P. L., and Carpenter, J. F. (1996) Effects of antifreeze proteins on red blood cell survival during cryopreservation. J. Exp. Biol. 199, 2071–2076. (16) Tam, R. Y., Rowley, C. N., Petrov, I., Zhang, T., Afagh, N. A., Woo, T. K., and Ben, R. N. (2009) Solution conformation of C-linked antifreeze glycoprotein analogues and modulation of ice recrystallization. J. Am. Chem. Soc. 131, 15745–15753. (17) Eniade, A., Purushotham, M., Ben, R., N., Wang, J. B., and Horwath, K. (2003) A Serendipitous discovery of antifreeze proteinspecific activity in c-linked antifreeze glycoprotein analogs. Cell Biochem. Biophys. 38, 115–124. (18) Ben, R. N., Eniade, A., and Hauer, L. (1999) Synthesis of a Clinked antifreeze glycoprotein (AFGP) mimic: probes for investigating the mechanism of action. Org. Lett. 1, 1759–1762. (19) Eniade, A., and Ben, R. N. (2001) Fully convergent solid phase synthesis of antifreeze glycoprotein analogues. Biomacromolecules 2, 557–561. (20) Liu, S., and Ben, R. N. (2005) C-linked galactosyl serine AFGP analogues as potent recrystallization inhibitors. Org. Lett. 7, 2385–2388. (21) Czechura, P., Tam, R. Y., Dimitrijevic, E., Murphy, A. V., and Ben, R. N. (2008) The importance of hydration for inhibiting ice recrystallization with C-linked antifreeze glycoproteins. J. Am. Chem. Soc. 130, 2928–2929. (22) Chaytor, J. L., and Ben, R. N. (2010) Assessing the ability of a short fluorinated antifreeze glycopeptide and a fluorinated carbohydrate derivative to inhibit ice recrystallization. Bioorg. Med. Chem. Lett. 20, 5251–5254. (23) Liu, S., Wang, W., Von Moos, E., Jackman, J., Mealing, G., Monette, R., and Ben, R. N. (2007) In vitro studies of antifreeze glycoprotein (AFGP) and a C-linked AFGP analogue. Biomacromolecules 8, 1456–1462. (24) Bertozzi, C. R., Hoeprich, P. D. J., and Bednarski, M. D. (1992) Synthesis of carbon-linked glycopeptides as stable glycopeptide models. J. Org. Chem. 57, 6092–6094. (25) Dondoni, A., Marra, A., and Massi, A. (1999) Design and use of an oxazolidine silyl enol ether as a new homoalanine carbanion equivalent for the synthesis of carbon-linked isosteres of O-glycosyl serine and N-glycosyl asparagine. J. Org. Chem. 64, 933–944. (26) Nolen, E. G., Watts, M. M., and Fowler, D. J. (2002) Synthesis of C-linked glycopyranosyl serines via a chiral glycine enolate equivalent. Org. Lett. 4, 3963–3965. (27) Debenham, S. D., Debenham, J. S., Burk, M. J., and Toone, E. J. (1997) Synthesis of carbon-linked glycopeptides through catalytic asymmetric hydrogenation. J. Am. Chem. Soc. 119, 9897–9898. (28) Chen, G., Chien, M., Tsuji, M., and Franck, R. W. (2006) E and Z R-C-galactosylceramides by JuliaLythgoeKocienski chemistry: a test of the receptor-binding model for glycolipid immunostimulant. ChemBioChem 7, 1017–1022. (29) Baudin, J. B., Hareau, G., Julia, S. A., and Ruel, O. (1993) A direct synthesis of olefins by reaction of carbonyl compounds with lithio derivatives of 2-[alkyl or (20 -alkenyl)- or benzyl-sulfonyl]-benzothiazoles. Tetrahedron Lett. 32, 1175–1178. 1809

dx.doi.org/10.1021/bc2001837 |Bioconjugate Chem. 2011, 22, 1804–1810

Bioconjugate Chemistry

ARTICLE

(30) Bellingham, R., Jarowicki, K., Kocienski, P. J., and Martin, V. (1996) Synthetic approaches to Rapamycin: synthesis of a C10-C26 fragment via a one-pot Julia olefination reaction. Synthesis 285–296. (31) Albrecht, B. K., and Williams, R. M. (2004) A concise, total synthesis of the TMC-95A/B proteasome inhibitors. Proc. Natl. Acad. Sci. U.S.A. 101, 11949–11954. (32) Kocienski, P. J., Bell, A., and Blakemore, P. R. (2000) 1-tertButyl-1H-tetrazol-5-yl sulfones in the modified Julia olefination. Synlett 365–366. (33) Guindon, Y., Lavallee, J.-F., Boisvert, L., Chabot, C., Delorme, D., Yoakim, C., Hall, D., Lemieux, R., and Simoneau, B. (1991) Stereoselective radical-mediated reduction and alkylation of R-halo esters. Tetrahedron Lett. 32, 27–30. (34) Giese, B., Gr€oninger, K. S., Witzel, T., Korth, H.-G., and Sustmann, R. (1987) Synthese von 2-desoxyzuckern. Angew. Chem. 99, 246–247. (35) Patman, R., Juarez-Ruiz, J. M., and Roy, R. (2006) Subtle stereochemical and electronic effects in iridium-catalyzed isomerization of C-allyl glycosides. Org. Lett. 8, 2961–2964. (36) Petrenko, Y. A., Jones, D. R. E., and Petrenko, A. Y. (2008) Cryopreservation of human fetal liver hematopoietic stem/progenitor cells using sucrose as an additive to the cryoprotective medium. Cryobiology 57, 195–200. (37) Barker, J. N., and Wagner, J. E. (2002) Umbilical cord blood transplantation: current state of the art. Curr. Opin. Oncol. 14, 160–164. (38) Wagner, J. E., Broxmeyer, H. E., Byrd, R. L., Zehnbauer, B., Schmeckpeper, B., Shah, N., Griffin, C., Emanuel, P. E., Zuckerman, K. S., Cooper, S., Carow, C., Bias, W., and Santos, G. W. (1992) Transplantation of umbilical cord blood after myeloablative therapy: analysis of engraftment. Blood 79, 1874–1881. (39) Can, A., and Karahuseyinoglu, S. (2007) Human umbilical cord stroma with regard to the source of fetus-derived stem cells. Stem Cells 25, 2886–2895. (40) Sykova, E., Jendelova, P., Urdzíkova, L., Lesny , P., and Hejcl, A. (2006) Bone marrow stem cells and polymer hydrogels—two strategies for spinal cord injury repair. Cell. Mol. Neurobiol. 26, 1113–1129. (41) Kraus, K. H., and Kirker-Head, C. (2006) Mesenchymal stem cells and bone regeneration. Veterinary Surgery 35, 232–242. (42) Stamm, C., Kleine, H.-D., Westphal, B., Petzsch, M., Kittner, C., Nienaber, C. A., Freund, M., and Steinhoff, G. (2004) CABG and bone marrow stem cell transplantation after myocardial infarction. Thorac. Cardiovasc. Surgeon 152–158. (43) Quevedo, H. C., Hatzistergos, K. E., Oskouei, B. N., Feigenbaum, G. S., Rodriguez, J. E., Valdes, D., Pattany, P. M., Zambrano, J. P., Hu, Q., McNiece, I., Heldman, A. W., and Hare, J. M. (2009) Allogeneic mesenchymal stem cells restore cardiac function in chronic ischemic cardiomyopathy via trilineage differentiating capacity. Proc. Natl. Acad. Sci. U.S.A. 106, 14022–14027. (44) Fowler, A., and Toner, M. (2006) Cryo-injury and biopreservation. Ann. N.Y. Acad. Sci. 1066, 119–135. (45) Morris, C. B. (2007) Cryopreservation of Animal and Human Cell Lines, in Cryopreservation and Freeze-Drying Protocols, 2nd ed. (Day, J. G., and Stacey, G. N., Eds.) pp 227236, Humana Press, Totowa, NJ, USA. (46) Mase, J., Mizuno, H., Okada, K., Sakai, K., Mizuno, D., Usami, K., Kagami, H., and Ueda, M. (2006) Cryopreservation of cultured periosteum: Effect of different cryoprotectants and pre-incubation protocols on cell viability and osteogenic potential. Cryobiology 52, 182–192. (47) Stockert, R. J. (1995) The asialoglycoprotein receptor: relationships between structure, function, and expression. Physiol. Rev. 75, 591–609. (48) Meier, M., Bider, M. D., Malashkevich, V. N., Spiess, M., and Burkhard, P. (2000) Crystal structure of the carbohydrate recognition domain of the h1 subunit of the asialoglycoprotein receptor. J. Mol. Biol. 300, 857–865.

1810

dx.doi.org/10.1021/bc2001837 |Bioconjugate Chem. 2011, 22, 1804–1810