Mimicking the Properties of Antifreeze Glycoproteins: Synthesis and

J. Phys. Chem. B 2005, 109, 15849-15859

15849

Mimicking the Properties of Antifreeze Glycoproteins: Synthesis and Characterization of a Model System for Ice Nucleation and Antifreeze Studies Markus Hederos and Peter Konradsson DiVision of Chemistry, IFM, Linko¨ping UniVersity, SE-581 83 Linko¨ping, Sweden

Annika Borgh and Bo Liedberg* DiVision of Molecular Physics, IFM, Linko¨ping UniVersity, SE-581 83 Linko¨ping, Sweden ReceiVed: February 11, 2005; In Final Form: June 9, 2005

Synthesis of β-D-Gal-(1 f 3)-β-D-GalNAc coupled to HOC2H4NHCOC15H30SH is described. This compound was coadsorbed at various proportions with C2H5OC2H4NHCOC15H30SH to form statistically mixed selfassembled monolayers (SAMs) on gold in an attempt to mimic the properties of the active domain in antifreeze glycoproteins (AFGPs). The monolayers were characterized by null ellipsometry, contact angle goniometry, X-ray photoelectron spectroscopy, and infrared reflection-absorption spectroscopy. The disaccharide compound adsorbed preferentially, and SAMs prepared at a solution molar ratio >0.3 displayed total wetting. The mixed SAMs showed well-organized alkyl chains up to a disaccharide surface fraction of 0.8. The amount of gauche conformers in the alkyls increased rapidly above this point, and the monolayers became disordered and less densely packed. Furthermore, the generated mixed SAMs were subjected to water vapor at constant relative humidity and the subsequent ice crystallization on a cooled substrate was monitored via an optical microscope. Interestingly, rapid crystallization occurred within a narrow range of temperatures on mixed SAMs with a high disaccharide content, surface fraction >0.3. The reported crystallization temperatures and the ice layer topography were compared with results obtained for a much simpler reference system composed of -OH/CH3 terminated n-alkanethiols in order to account for changes in topography of the water/ice layer with surface energy. Although preliminary, the obtained results can be useful in the search for the molecular mechanism behind the antifreeze activity of AFGPs.

Introduction Biological antifreezes in fishes, insects, bacteria, fungi, and plants inhibit the growth of microscopic ice crystals at subzero temperatures and thereby allow the organisms to survive in subzero environments. Antifreeze proteins (AFPs) and glycoproteins (AFGPs) found in polar fishes are divided into several groups with significant differences in primary, secondary, and tertiary structure (see review 1-4). A typical AFGP is composed of a repeating tripeptide (Thr-Ala-Ala) unit, in which every hydroxyl of the threonine residue is glycosylated with the disaccharide β-D-galactosyl-(1 f 3)-R-D-N-acetyl-galactosamine (Figure 1, top).5 A lot of research efforts have been devoted to detailed studies of the structure-function relationships and potential applications of AFGPs. Unfortunately, the molecular mechanisms behind antifreeze activity are still not clear and remain a source of debate. The generally accepted adsorption inhibition model involves binding of AFPs and AFGPs to certain crystal planes of ice preventing further growth. Early studies suggested a mechanism dominated by hydrogen bond formation between hydrophilic side chains of the protein and the ice lattice. Hydrophobic side chains have also proven crucial for function of AFPs2,3,6 and they were recently found to influence antifreeze activity of synthesized AFGP analogues.7 Moreover, chemical and enzymatic modifications of native AFGPs have revealed that the oligosaccharide moiety is absolutely necessary for * Corresponding author. Phone: +46-13-281877. Fax: +46-13-288969. E-mail: [email protected].

obtaining antifreeze activity.8,9 Detailed structure-activity studies, however, suffer from substantial problems related to isolation and purification of the AFGPs from natural sources. Thus, instead of working with complex glycoproteins, we have designed a two-dimensional model system containing both the hydrophilic disaccharide and the hydrophobic methyl domains found in AFGPs. To our knowledge, this monolayer design is a new approach when studying the antifreeze properties of artificial AFGPs. Thin organic layers on solid supports have been used extensively in studies aiming at modeling interfacial phenomena and processes.10 One attractive approach involves the preparation of such layers by spontaneous adsorption of alkanethiols from solution onto gold surfaces, so-called self-assembled monolayer (SAM) formation. Long chain n-alkanethiols, for example, enable the formation of highly organized monolayer architectures on gold, which can be manipulated with respect to thickness, wettability, composition, and molecular conformation by combining thiols with different tail groups and chain lengths.11 The overall aim of this work is to describe the synthesis of a disaccharide terminated alkanethiol (1) and characterization of mixed SAMs of 1 and a methyl terminated alkanethiol (2). An illustration of the mixed SAM structure formed by 1 and 2 (bottom) to mimic the properties of the AFGP (top) is shown in Figure 1. The amide group between the alkyl chain and the tail group was introduced to enhance the organization/stability through intermolecular hydrogen bonding as well as to reduce the risk

10.1021/jp050752l CCC: $30.25 © 2005 American Chemical Society Published on Web 08/04/2005

15850 J. Phys. Chem. B, Vol. 109, No. 33, 2005

Figure 1. Top: chemical structure of an antifreeze glycoprotein (AFGP). Below: a schematic illustration of a mixed SAM of the disaccharide (1) and the methyl terminated alkanethiol (2).

for phase segregation. The amide group also serves as a firstorder model of the polypeptide backbone in APGPs. The fraction of disaccharide species in the SAM was controlled carefully by varying the ratio between 1 and 2 in the incubation solution. Initial observations of water freezing on statistically mixed SAMs of 1 and 2, using an optical microscope equipped with a cooled stage, are also presented. These observations are compared to those obtained using a much simpler reference system composed of a mixed SAM of -OH/-CH3-terminated n-alkanethiols to account for topographic changes in the water/ ice layer with wettability of the SAM surface. The long-term goal of this work is to improve understanding of the molecular mechanisms behind antifreeze activity by designing and developing a completely new class of artificial model systems. The present approach may also open for entirely new applications of these biomimetic SAMs/materials. Results and Discussion Synthesis. The synthetic route is depicted in Scheme 1, and synthetic data are presented in the Experimental Section. The strategy was to synthesize the β-D-Gal-(1 f 3)-β-D-GalNAc disaccharide prior to introducing the long alkanethiol chain. Moreover, a disaccharide donor having great synthetic flexibility, with respect to methods for activation, was used to facilitate the introduction of different spacers (the linkage

Hederos et al. between the glyco moiety and the n-alkanethiol). The use of n-pentenyl glycosides (NPGs) as glycosyl donors has been known since the late 80’s, and there are various methods to activate these.12 For this reason, a n-pentenyl disaccharide, which could be selectively activated in glycosylations or elongated via chemical manipulation of the pentenyl double bond,13 was synthesized as a promising candidate. The glycosyl portion, β-DGal-(1 f 3)-β-D-GalNAc, was coupled to the hexadecyl chain via an ethanolamine linkage. The synthesis started with the preparation of a NPG acceptor following a protocol described by Madsen et al. for corresponding glucosamine compound.14 The phthalimido tetra acetate 315 was treated with 4-penten-1-ol and stannic chloride in CH2Cl2 to introduce the n-pentenyl group. Deacetylation with sodium methoxide in MeOH and subsequent conversion into the 4,6O-benzylidene derivative by standard conditions produced acceptor 4 in 50% overall yield. The glycosylation to the disaccharide proved to be the most crucial step in the synthetic route. Several glycosyl donors were tested before finding the successful condensation of 4 with 1-O-(2,3,4,6-tetra-O-benzoylR-galactose)-trichloroacetimidate 516, using trimethylsilyl triflate as promoter in toluene, to give the desired β-(1 f 3) disaccharide 6 in 70% yield. To introduce the ethanolamine spacer, we activated the NPGdonor with N-iodosuccinimide/silvertriflate and linked it to benzyl N-(2-hydroxyethyl) carbamate to produce 7 in 75% yield. Removal of the phthalimido group was performed using ethylenediamine in a mixture of n-butanol and ethanol at 90 °C for 24 h. Under these conditions, a complete cleavage of all of the benzoyl groups was observed. Subsequent acetylation gave N-acetamido derivative 8 in 85% yield. Prior to introducing the alkanethiol chain, the benzylcarbamate- and benzylidene groups were removed by hydrogenolysis of 8 using palladium hydroxide as catalyst in ethanol/acetic acid. Acylation of the 2-aminoethyl spacer using N-hydroxy-succinimide ester 917 introduced the S-acetyl protected 16-mercapto-hexadecanoyl chain. Disaccharide-terminated alkanethiol 10 was obtained in 75% yield over two steps. Deacetylation with methanolic sodium methylate and presence of dithiothreitol to prevent disulfide formation, furnished thiol 1 in 90% yield. Composition, Coverage, and Phase Characteristics of Mixed SAMs. Ellipsometry. The ellipsometric thicknesses for the single-component and mixed SAMs versus the molar fraction of 1 in solution (χsol1) are shown in Figure 2a. The thickness of the single-component SAM of 2, ∼25 Å, is in good agreement with the value obtained from molecular modeling. The thicknesses of the mixed monolayers increase to ∼31 Å at χsol1 ∼0.3. The rapid increase is followed by a gradual decrease to ∼28 Å for the single-component SAM of 1, a thickness that does not coincide with the expected value from molecular modeling, ∼32 Å. We expected a monotonic increase in monolayer thickness from the terminal point of 2 (25 Å) to the corresponding point of 1 (32 Å) with an increasing fraction of 1 in solution. The observed decrease in thickness is most likely due to the complex nature (size) of the saccharide tail leading to a less ordered structure for these high χsol1 monolayers, see below for further discussion. Riepl et al.18 obtained a similar result upon formation of mixed biotin/oligo(ethylene glycol) SAMs on gold. Contact Angle Goniometry. The contact angle measurements with water for the mixed SAMs are plotted against χsol1 in Figure 2b. The contact angle measurements show total wetting (contact angles with water < 10°) for monolayers of χsol1 g 0.3. Thus, the disaccharide-containing species appear to adsorb preferen-

Mimicking Antifreeze Glycoproteins

J. Phys. Chem. B, Vol. 109, No. 33, 2005 15851

SCHEME 1: Reagents and Conditions for the Synthesis of Disaccharide Terminated Alkanethiol 1a

a (i) 4-penten-1-ol, SnCl4, CH2Cl2; (ii) NaOMe, MeOH; (iii) p-TsOH, R,R-dimethoxy-toluene, acetonitrile; (iv) TMSOTf, 4-Å molecular sieves, toluene, 0 °C; (v) AgOTf, NIS, 4-Å molecular sieves, benzyl N-(2-hydroxyethyl) carbamate, CH2Cl2; (vi) ethylenediamine, n-BuOH/EtOH 1:1, 90 °C; (vii) Ac2O, pyridine; (viii) Pd(OH)2/C, H2, EtOH/AcOH 1:1; (ix) DIPEA, DMF; (x) NaOMe, dithiothreitol, MeOH.

Figure 2. The ellipsometric thickness (a) and the contact angles with water (b) for the mixed SAMs as a function of the molar fraction of 1 in solution (χsol1). The advancing and receding contact angles are represented by filled and open circles, respectively.

tially. Consequently χsurf1 > χsol1, and a nonlinear relationship between the surface composition and the solution molar ratio is evident. A much smaller hysteresis is observed for monolayers consisting only of 2 compared to the mixed SAMs. This effect is most likely due to an increased roughness and heterogeneity of the mixtures. Infrared Reflection-Absorption Spectroscopy (IRAS). Infrared spectra of monolayers produced from incubation solutions with 0 < χsol1 < 1 and the single-component SAMs as terminal points

suggest that the two thiols coadsorb to form statistically mixed monolayers, Figure 3. The spectrum of the mixed monolayer at the lowest concentration of 1 in the incubation solution, χsol1 ) 0.09, resembles the spectrum of pure 2, although there are differences. When the concentration of compound 1 increases, the peaks shift in intensity and position toward the spectrum of pure 1. In the CH stretching region, the νasCH2 and νsCH2 peaks appear at 2918 and 2850 cm-1, respectively, for a SAM of 2. The same peaks broaden and move to higher frequencies when


Hederos et al.

Figure 3. Infrared RA spectra of mixed SAMs of 1 and 2 in the methylene stretching region (a) and the fingerprint region (b). The spectra represent, from the top, SAMs formed from solutions of 1 mixed with 2 with the following molar ratios: χsol1 ) 1.0, 0.75, 0.50, 0.25, 0.14, 0.09, 0.01, and 0.0. The negative peaks marked by * in b originate from the deuterated reference sample, and the hatched line in b displays the region used to integrate the C-O peaks, 1195-1005 cm-1.

the fraction of 1 increases in the monolayer. Moreover, the progression bands at 1215-1350 cm-1 decrease as the fraction of 1 increases, and this will be discussed more thoroughly below. The SAM of compound 1 absorbed strongly in the disaccharide C-O stretching region (1195-1005 cm-1), whereas the absorption was negligible for the SAM of 2, Figure 3b. The peaks were integrated and normalized to the value obtained from single-component SAM of 1. As can be seen in Figure 4a, the surface fractions of the disaccharide derivative (χsurf1) are higher in the SAMs than the molar fraction in the incubation solutions. For example, even at low molar fraction (χsol1 ≈ 0.3) the surface fraction is as high as 80-90%, in agreement with the contact angle measurements, Figure 2b. We believe that this preferential adsorption of 1 can be explained in part by the significantly poorer solubility of 1 in ethanol. X-ray Photoelectron Spectroscopy (XPS). To test the validity of the compositions obtained from integrating the disaccharide C-O stretching region in IRAS, we performed XPS measurements. The oxygen spectra from 1 display a broad peak around 532 eV attributed to the O1s orbital. The corresponding nitrogen spectra showed a peak at 401 eV, which originates from the N1s orbital of the acetamide and amide links. The most interesting peaks are those arising from differently functionalized carbons.19 These C1s peaks are resolved at 288.8, 287.2, and 285.3 eV, respectively. The peak with highest binding energy originates from carbonyl groups, and a similar peak can be seen in the carbon C1s spectra of 2, but with reduced intensity. The peak at 287.2 eV is due to the C1s orbital representing singlebonded carbon to oxygen (C1sC-O), whereas the peak around 285 eV with highest intensity originates from aliphatic carbons. The 287.2 eV peak was used to calculate the amount of 1 in the mixed SAMs because it is the only C1s peak that uniquely represents the disaccharide tail. The fitted peaks were integrated and normalized to the intensity observed for the SAM prepared at χsol1 ) 1.0. The resulting plot, Figure 4b, agrees with that obtained from the IRAS integration method. The fraction of the disaccharide derivative in the SAM increases rapidly in the χsol1 ) 0.0-0.5 region. For higher ratios, the curve levels out to reach a maximum value at χsol1 ) 1.0. This convinced us that the IRAS integration method is trustworthy. In the following section, when discussing the surface fraction (χsurf1), the reported values originate from the IRAS integration method. An analogous plot of the C1s peak at 285 eV reveals that the intensity of aliphatic carbons decreases gradually as χsol1

increases, and the decrease is particularly apparent at high χsol1 values (data not shown). This can be due to shielding of the alkyl C1s photoelectrons by the disaccharide top layer and/or to a decrease in the absolute number (coverage) of alkanethiols (1 + 2) on the gold surface with increasing χsol1. Shielding is a phenomenon that always occurs when photoelectrons penetrate a surface layer and it will certainly contribute to the overall lowering of the aliphatic C1s intensity. However, if peak intensities from elements localized within the topmost portion of the SAMs are compared, then we expect shielding to play a less significant role. For example, the O1s and the C1sC-O photoelectrons (both originate from elements in the topmost part of the SAMs) are expected to follow essentially the same trend as long as the total number of alkanethiol molecules in the SAM is the same for all mixtures. This appears, however, not to be the case. The O1s intensity increases in a similar manner as the C1sC-O intensity, Figure 4b, up to χsol1 ) 0.8, above which it starts to decrease to a level that is approximately 85% of that at χsol1 ) 0.8. Thus, our O1s intensity data strongly suggests that the total number of oxygens decreases at the surface of the SAM at high χsol1 values, whereas the analyses of the C1sC-O intensity, Figure 4b, as well as the integrated IR peak intensity in the C-O stretching region, Figure 4a, clearly point out that the number of disaccharide species remains at a high and constant level. The observed reduction of the O1s intensity at χsol1 ) 1.0 is attributed to a decrease in the coverage of alkanethiols (1 + 2) at high χsol1 values. We therefore propose that the additional contribution to the increased O1s intensity in the SAM prepared at χsol1 ) 0.8, as compared to χsol1 ) 1.0, Figure 4b, originates from the carbonyl oxygen present in the filling compound 2. Note that a reduced coverage, as revealed by the decreasing O1s intensity at high χsol1 values, is consistent with the concomitant lowering of the aliphatic C1s peak intensity at 285 eV (data not shown). The observed lowering of the coverage at high χsol1 values may also influence the organization and packing properties of the alkyls and this issue will be addressed in more detail in the coming sections. Phase Characteristics and Cassie’s Law. Avoiding phase separation is crucial for the current approach. An amide group was therefore introduced in both compounds to reduce the risk for phase separation. Besides reducing the risk for phase separation, it also improves the organization and lateral stabilization of the monolayer by forming an in-plane hydrogenbonding network.20 When a surface is composed of two different



Figure 4. The calculated surface fraction (χsurf1) of 1 as a function of the molar fraction of 1 in solution (χsol1) using (a) IRAS integration in the C-O stretching region (1195-1005 cm-1) and (b) integrated XPS intensities of the C1sC-O (b). Also displayed is the O1s (O) peak intensity vs χsol1. (c) Advancing contact angles plotted against χsurf1 (determined from IRAS data). (d) Effective surface fraction χsurf1 obtained from Cassie’s law as a function of χsol1.

species, the observed contact angle (θA) can be estimated using Cassie’s law,21 cos θA ) f1 cos θ1 + f2 cos θ2, where θ1 and θ2 are the contact angles on homogeneous surfaces of 1 and 2, and f1 and f2 (f1 + f2 )1) are their surface fractions χsurf1 and 1-χsurf1, respectively. This equation is applicable only if the two components act independently. Consequently, a graph of cos θA versus χsurf1 is expected to be linear. The relationship in Figure 4c is linear all the way up to χsurf1 ) 0.9 (obtained by using the infrared data, Figure 4a), suggesting that phase separation into macroscopic domains does not occur. Thus, Cassie’s law suggests that statistical mixing occurs between 1 and 2. It also can be used to estimate the effective surface fraction, χsurf1, and the plot of χsurf1 versus molar ratio in solution is shown in Figure 4d. This plot agrees well with those obtained from IRAS and XPS, Figure 4a and b. Taken together, the ellipsometric, contact angle, and spectroscopic data obtained so far clearly suggest that the disaccharide compound adsorb preferentially on the gold surface. We are also confident about the compositional profiles because the three methods used provide almost identical plots. Our data also suggest that the coverage varies with composition and that the bulkiness of the disaccharide entity reduces the coverage at high χsol1 values. This will influence the conformation and packing of the SAMs, an issue to be discussed below. Conformational Properties of Mixed SAMs. When discussing the conformational properties of the mixed monolayers,

TABLE 1: Observed Frequencies (cm-1) in the Infrared Transmission (TR) and Reflection Absorption (RA) Spectra of Compounds 1 and 2 (in KBr) and the Corresponding Single-Component SAMs (on Gold)a TR 1

RA 1

TR 2

RA 2

tentative assignments

2922

2922

2975 2916

2850 1644 1557 1467 1376 1159 1120 1075 1038

2852 1652 1562 1467 1375 1158 1116 1084 1049

2974 2918 2879 2850

νasCH3 νasCH2 νsCH3 νsCH2 amide I amide II δsCH2 δsCH3 νC-O and νasC-O-C νC-O νC-O νC-O

a

2849 1639 1543 1471 1378

1559 1466 1378

Also shown are the tentative mode sssignments.

IRAS measurements are very useful. The characteristic IR frequencies of the two alkanethiols are summarized in Table 1. One characteristic spectral region for alkanethiols is the CH stretching region, Figure 3a. For the SAM of 2, the peak frequencies of the asymmetric (νasCH2) and symmetric (νsCH2) CH2 stretch modes appear at 2918 and 2850 cm-1, respectively. These values are characteristic for densely packed alkyl chains in an extended all-trans conformation. The corresponding peaks


Hederos et al.

Figure 5. (a) The asymmetric CH2 stretching (νasCH2) frequency and (b) the ellipsometric thickness as a function of χsurf1.

for the SAM of 1 are broader and appear at 2922 and 2852 cm-1, respectively, indicating that the alkyls contain a substantial amount of gauche defects. Moreover, both molecules contain a methyl group and the asymmetric and symmetric CH3 stretching modes are clearly visible in the spectra of 2 at 2974 and 2879 cm-1, respectively. The neighboring carbonyl group in the acetamide influences (shifts) the intensity of the methyl stretching modes,22 and no CH3 stretching peaks are discernible in the spectra of 1. Further, the C-O stretch and the C-O-C asymmetric stretch from the disaccharide in 1 resulted in several broad and intense peaks between 1005 and 1195 cm-1, Figure 3b. The amide linkage between the alkyl chain and the terminal ethyl group in 2 absorbs strongly in the RA and TR spectra at ∼1560 cm-1 due to C-N-H in-plane bending and C-N stretching (amide II). The CdO stretching (amide I) is absent in the RA spectrum, but appears as a strong peak in the TR spectrum at 1639 cm-1. This intensity difference is due to an orientation effect of the amide link.20 The surface dipole selection rule in IRAS suggests that the amide I vibration (polarized parallel to the CdO axis) and the amide II vibration (polarized parallel to the C-N axis), and thereby the CdO bond and C-N bonds, are aligned parallel and perpendicular to the plane of the gold surface, respectively. In the RA spectrum of 1, both amide I and II are present. It is not that straightforward to determine the orientation of the amide linkage in this particular case because of interfering absorption from the acetamide group. Nevertheless, the intensity ratio of the amide I/amide II peaks in the TR spectrum is close to 2/1, whereas the same ratio in the RA spectrum equals 1/2. Thus, a substantial part of the CdO bonds in the SAM of 1 seems to be aligned parallel to the surface. Both 1 and 2 display a pronounced peak belonging to the CH3 symmetric deformation (δsCH3) at 1375 and 1378 cm-1, respectively, revealing the presence of a methyl group in 1 (acetamide) even though no methyl groups are discernible in the CH stretching region, Figure 3a. A series of distinct peaks are observed between 1215 and 1350 cm-1 for 2 but not for 1 (Figure 3b). They are referred to as the progressions peaks and are due to coupled CH2 wag modes in trans (ttt) CCC sequences of the alkyl chain, and they appear as sharp features only when the alkyl chain adopts an extended all-trans conformation.23 Thus, the absence of progression peaks in the RA spectrum of 1 suggests that the alkyl chain contains imperfections, gauche defects. The gauche defects appear most likely because of a mismatch between the cross-sectional area of the disaccharide

moiety and the optimal pinning distance alkanethiols on gold (∼5 Å for a (x3 × x3) R30° over layer structure on Au(111)). The appearance of disordered, gauche-rich, domains in the mixed SAMs of 1 and 2 can also be monitored by following the exact frequencies of the methylene stretching vibrations, in particular the asymmetric CH2 stretching mode. Figure 5a plots the νasCH2 as a function of χsurf1, and it is evident that it remains at 2918 cm-1 for χsurf1 e 0.8. Hence, the monolayer packing and the extended all-trans conformation remain intact as long as the bulky disaccharide thiol is mixed with a substantial amount of the shorter thiol 2. Thus, the shorter (filling) thiols seem to stabilize the all-trans structure. The νasCH2 peaks move rapidly to higher frequencies for χsurf1 > 0.8 and reach 2922 cm-1 for a single-component SAM of 1. Thus, the degree of disorder within the alkyl chains increases rapidly at these high χsurf1 values. The SAMs become obviously less densely packed and disordered at high fractions of 1 on the surface. This conformational flexibility of the alkyl chains can only be obtained if the coverage is less than 1, in full agreement with our O1s data obtained from XPS, Figure 4b. An incomplete coverage is also in line with the ellipsometric data in Figure 2a. The thicknesses can be replotted in a similar way as the νasCH2, Figure 5b, and it is evident that the thickness of the mixed SAMs increases linearly up to a breakpoint, χsurf1 ≈ 0.8, above which the thickness abruptly decreases to reach a minimum value of ∼2628 Å at χsurf1. This breakpoint coincides exactly with the disruption of the alkyl chain organization, that is, the SAMs appear thinner and less densely packed at high surface fractions of 1. In summary, our experimental data clearly suggest that the two derivatives form statistically mixed and conformational well-defined SAMs up to χsurf1 ≈ 0.8. The conformation and packing properties of the SAMs start to deteriorate above χsurf1 ≈ 0.8, a phenomenon that is attributed to the size and complex nature of the disaccharide tail. The critical range for the applications of these SAMs in antifreeze experiments is, however, around χsurf1 ≈ 0.33 (one disaccharide on every third threonine, Figure 1), that is, in the range where the SAMs possess ideal mixing and conformational properties. A first set on ice crystallization experiments are briefly described below. Ice Crystallization Studies. Initial assessment of our AFGP model was performed by studying water crystallization from humid atmosphere on mixed SAMs of 1 and 2 using a standard optical microscope equipped with a cooled stage. The substrate temperature was lowered continuously and the water vapor, kept



Figure 6. (a) Ice crystallization temperature (Tc) on the SAMs as a function of the surface free energy, cos(θA). The filled circles (b) and the unfilled circles (O) represent the mixed SAM of 1 and 2 and the reference SAMs of TOH and TCH3, respectively. The lines should be regarded as guides to the eye. Parts b and c show images obtained after crystallization on SAMs with a contact angle of 45° (cos θA ) 0.7), (b) mixed SAM of TOH and TCH3, (c) mixed SAM of 1 and 2. Insets in b and c show water on SAMs formed from χsolTOH ) 1 and χsol1 ) 0.5, respectively.

at a constant relative humidity (RH), was allowed to condense, nucleate, and crystallize into ice. The temperature of ice crystallization (Tc) was determined by observing the crystallization process in the microscope. A well-characterized set of mixed monolayers composed of HS(CH2)16OH (TOH) and HS(CH2)15CH3 (TCH3) was used as a reference system.24,25 This particular system was chosen to account for the change in the heat conductance between the SAM surface and the water/ice layer because of the variation in droplet size, shape, and thus the effective surface coverage with composition (surface energy) of the SAMs. Figure 6a displays the observed crystallization temperatures (Tc) for the mixed SAMs of 1 and 2 and the TOH/TCH3 reference SAMs as a function of surface free energy (cos θA). The crystallization temperature (note: this temperature is not related to the antifreeze temperature reported in the literature for AFGPs) for the reference TOH/TCH3 SAMs (unfilled circles) displays a gradual decrease with decreasing surface free energy. This trend is expected for a water layer that changes topography from a 2D “sandwich-like”- into a 3D “dropletlike”- appearance upon lowering the surface energy (increasing contact angle). The effective contact area between the water droplets and the substrate decreases and thus also the heat conductance (dissipation) with decreasing surface energy. The same trend is seen for the antifreeze model (filled circles) but

with a deviation in the range cos θA > 0.45 (corresponding to a surface composition χsurf1 > 0.3). Interestingly, this plateau region contains SAMs with a high disaccharide content including those mimicking the active domain, that is, the repeating tripeptide unit, of AFGPs. Thus, it seems that SAMs with high disaccharide content, χsurf1 > 0.3, behave similarly and act as nucleation initiators for ice crystallization. This observation is consistent with the accepted ice binding (inhibition) model in which the AFPs as well as AFGPs are known to bind to specific prism planes of microscopic ice crystals formed at subzero temperatures.4 This specific interaction will inhibit nucleation of water on the prism faces (the growth planes) and thereby macroscopic ice formation. Thus, the native AFGPs appear to attach to specific ice planes, whereas the water molecules in the present set of experiments seem to organize and nucleate on SAM surfaces with high disaccharide contents, χsurf1 > 0.3. Crystallization is induced within a narrow range of substrate temperatures (-5 to -6 °C) on these SAMs. It is also very interesting to observe that the knee in the curve for the AFGP model appears at cos θA ) 0.45, that is, at a surface energy that corresponds to a surface composition of χsurf1 ) 0.3. Note again that the mixing ratio given by the repeating unit in AFGPs equals 0.33. We have also investigated the topography of ice layers by following the crystallization process under microscope. Figure

15856 J. Phys. Chem. B, Vol. 109, No. 33, 2005 6b and c shows images obtained at crystallization of water at mixed SAMs of TOH and TCH3 and 1 and 2, respectively. The two SAMs have a contact angle of ∼45°, represented here by the data points at cos θA ≈ 0.7 in a. The appearance of these two images is very different. The image of the TOH/TCH3 SAM consists of spherical ice droplets, Figure 6b. Such topography is expected for a surface with a fairly high contact angle with water considering the shape of the water droplets formed. The images of the TOH/TCH3 SAMs look qualitatively the same except at the single-component SAM (χsurfTOH )1) on which larger “2D-like” structures (droplets) are formed (see inset, Figure 6b). The images of the ice layer on the mixed SAMs of 1 and 2 with cos θA e 0.45 look more or less identical to those obtained for the TOH/TCH3 model system, Figure 6b. However, the appearance of the images changes dramatically at cos θA ) 0.7, Figure 6c, where the spherical droplets are replaced by wormlike features. These features change gradually into sheets with a typical size of about 125 µm (see inset, Figure 6c) and ultimately into a single and homogeneous ice sheet at χsurf1 ) 1.0 that covers the entire SAM surface (image not shown). Thus, the constant ice crystallization temperatures for cos θA > 0.45 in Figure 6a do not seem to correlate exactly with the 2D-like ice topography at cos θA ) 0.45 and above. Taken together, our crystallization and microscopic data clearly reveal that the two model systems behave very differently. It is, however, far too early to make a direct comparison between the antifreeze activity for the native AFGPs and the ice crystallization data for the model AFGP system examined here. It is interesting though to observe that the mixed SAMs of 1 and 2 seem to act as a template for ice nucleation and crystallization, a behavior consistent with the adsorption inhibition model for AFGPs as well as AFPs. Experimental Section Synthesis, General. Organic extracts were dried over MgSO4, filtered, and concentrated in vacuo below 40 °C. NMR spectra were recorded on a Varian Mercury 300 (1H 300 MHz and 13C 75.4 MHz) instrument at 25 °C in CDCl3 with TMS as the internal standard (δ ) 0.00 ppm) or in MeOH-d4 using CHD2OD as the indirect standard (δ ) 3.31 ppm) at 25 °C unless otherwise stated. Signals were assigned by using DEPT and 2D (COSY and HMQC) experiments. TLC was performed on Silica Gel F254 (E. Merck) plates with detection by UV light (254 nm) and/or by charring with ethanol/sulfuric acid/p-anisaldehyde/ acetic acid 90:3:2:1 followed by heating at ∼250 °C. Silica gel MERCK 60 (0.040-0.063 mm) was used for Flash Chromatography (FC) or, for reversed-phase separations, Silica gel Fluka 100 C18 RP (0.040-0.063 mm). Preparative HPLC was performed using a reversed-phase (C18) column at 230 nm. Optical measurements were recorded at room temperature with a PerkinElmer 141 polarimeter. Melting points were recorded with an uncorrected Gallenkamp melting point apparatus. MALDI-TOF mass spectra were recorded with a Voyager-DE STR Biospectrometry Workstation, in positive mode, using an R-cyano-4hydroxycinnamic acid matrix and the dimer (m/z 379.0930) as the internal standard. IR spectra were recorded (KBr pellets) on a Perkin-Elmer SPECTRUM 1000 FT-IR Spectrometer. Pent-4-enyl 4,6-O-benzylidene-2-deoxy-2-N-phthalimidoβ-D-galactopyranoside (4). Tin(IV) chloride (1.25 mL, 10.64 mmol) was added at 0 °C to a solution of 1,3,4,6-tetra-O-acetyl2-deoxy-2-N-phthalimido-β-D-galactopyranoside 315 (2.0 g, 4.19 mmol) and 4-penten-1-ol (1.08 mL, 10.59 mmol) in CH2Cl2 (40 mL). The reaction mixture was allowed to reach room temperature during 2 h, diluted with CH2Cl2, washed with water (3 × 50 mL), dried, filtered, and concentrated. The crude syrup

Hederos et al. was dissolved in MeOH (20 mL), and NaOMe (100 mg, 1.85 mmol) was added. The solution was stirred for 2 h, then neutralized with Dowex-H+, filtered, and evaporated. The obtained syrup was treated with R,R-dimethoxytoluene (3.0 mL, 19.99 mmol) and p-toluenesulfonic acid (0.080 g, 0.42 mmol) in acetonitrile (25 mL) for 4 h and then concentrated. FC (toluene/EtOAc 5:1 f 1:1) gave 4 (0.975 g, 2.10 mmol, 50%) as a white solid. Rf 0.52 (toluene/EtOAc 1:1) mp (from MeOH) 153-155 °C; [R]D + 17 (c 0.8, CHCl3); IR Vmax cm-1 3066, 3035, 2916, 2873, 1773, 1712, 1640, 1612, 1468, 1452, 1391, 1367, 1171, 1113, 1089, 1044, 995, 757, 722, 707, 700; NMR 1H (300 MHz, CDCl ): δ 1.47-1.60 (2H, m, CH CH O), 1.803 2 2 1.92 (2H, m, CH2-CHd), 3.45 (1H, dt, J ) 9.6, 6.3 Hz, OCHaHb), 3.62 (1H, m, H-5), 3.88 (1H, dt, J ) 9.6 6.3 Hz, OCHbHa), 4.12 (1H, dd, J ) 1.8 12.4 Hz, H-6a), 4.27 (1H, dd, J ) 1.1 3.6 Hz, H-4), 4.40 (1H, dd, J ) 1.4 12.4 Hz, H-6b) 4.43 (1H, dd, J ) 8.0 11.0 Hz, H-2), 4.49 (1H, dd, J ) 3.6 11.0 Hz, H-3), 4.70-4.74 (2H, m, CH2dCH), 5.25 (1H, d, J ) 8.0 Hz, H-1), 5.54-5.63 (2H, m, CHPh, CHdCH2), 7.36-7.42 (3H, m, Ph), 7.52-7.57 (2H, m, Ph), 7.67-7.73 (2H, m, Ph), 7.78-7.86 (2H, m, Ar). 13C (75 MHz, CDCl3): δ 28.5 (CH2CH2-O), 29.9 (CH2-CHd), 54.8 (C-2), 66.7 (C-5), 67.9 (C3), 68.6 (OCH2), 69.2 (C-6), 75.1 (C-4), 98.3 (C-1), 101.5 (CHPh), 114.6 (CH2dCH), 123.0 (Ph), 123.5 (Ph), 126.5 (Ph), 128.3 (Ph), 129.3 (Ph), 131.8 (Ph), 137.4 (Ph), 137.9 (CHd CH2), 168.3 (CON), 168.8 (CON); MALDI-TOF Calcd for C26H27NO7: [M + Na]+ 488.2. Found: [M + Na]+ 488.2. Pent-4-enyl (2,3,4,6-tetra-O-benzoyl-β-D-galactopyranosyl)-(1 f 3)-4,6-O-benzylidene-2-deoxy-2-N-phthalimidoβ-D-galactopyranoside (6). A mixture of 4 (740 mg, 1.590 mmol), 516 (1.410 g, 1.908 mmol), and 4-Å molecular sieves in dry toluene (30 mL) was stirred at 0 °C under argon for 30 min. Trimethylsilyltriflate (14 µL, 0.072 mmol) was added, and after 1.5 h the reaction mixture was quenched with triethylamine, filtered, and concentrated. FC (toluene/EtOAc, 9:1) gave 6 (1.170 g, 1.113 mmol, 70%) as a white solid. Rf 0.42 (toluene/ EtOAc 4:1) [R]D +104 (c 0.5, CHCl3); IR Vmax cm-1 3065, 3034, 2978, 2939, 1777, 1730, 1715, 1603, 1452, 1392, 1369, 1316, 1267, 1179, 1110, 1095, 1069, 1028, 749, 709; NMR 1H (300 MHz,CDCl3): δ 1.36-1.50 (2H, m, CH2CH2O), 1.67-1.80 (2H, m, CH2CHd), 3.35 (2H, m, H-5, OCHaHb), 3.78 (1H, dd, J ) 2.2 12.0 Hz, H-6b), 3.81 (1H, dt, J ) 9.6 6.3 Hz, OCHbHa), 4.24 (1H, d, J ) 12.0 Hz, H-6a), 4.27-4.34 (2H, m, H-5′, 6a′), 4.44 (1H, d, J ) 3.3 Hz, H-4), 4.55 (1H, dd, J ) 8.8 13.2 Hz, H-6b′), 4.59-4.67 (2H, m, CH2dCH), 4.71 (1H, dd, J ) 8.5 11.3 Hz, H-2), 4.89 (1H, dd, J ) 3.3 11.3 Hz, H-3), 5.05 (1H, d, J ) 8.5 Hz, H-1), 5.07 (1H, d, J ) 8.0 Hz, H-1′), 5.45 (1H, s, CHPh), 5.50 (1H, dd, J ) 3.3 10.2 Hz, H-3′), 5.52 (1H, m, CHdCH2), 5.75 (1H, dd, J ) 8.0 10.2 Hz, H-2′), 5.90 (1H, d, J ) 3.3 Hz, H-4′), 7.03-7.68 (25 H, m, Ph), 7.98-8.03 (4H, m, Ph). 13C (75 MHz, CDCl3): δ 28.4 (CH2-CH2-O), 29.7 (CH2-CHd), 51.8 (C-2), 62.4 (C-6′), 66.7 (C-5), 68.1 (C-4′), 68.4 (OCH2), 69.0 (C-6), 69.6 (C-2′), 71.5 (C-5′), 71.9 (C-3′), 75.3 (C-3), 75.5 (C-4), 98.5 (C-1), 101.0 (CHPh), 101.8 (C-1′), 114.5 (CH2dCH), 122.6 (Ph), 123.3 (Ph), 126.3 (Ph), 128.2130.0 (Ph), 131.1 (Ph), 132.9 (Ph), 133.3 (Ph), 133.5 (Ph), 133.7 (Ph), 134.0 (Ph), 137.7 (CHdCH2), 164.7 (COPh), 165.5 (COPh), 165.6 (COPh), 165.9 (COPh), 167.2 (CON), 169.2 (CON); MALDI-TOF Calcd for C60H53NO16: [M + Na]+ 1066.3. Found: [M + Na]+ 1066.3. N-(benzyloxycarbonyl)-2-aminoethyl-(2,3,4,6-tetra-O-benzoyl-β-D-galactopyranosyl)-(1 f 3)-4,6-O-benzylidene-2deoxy-2-N-phthalimido-β-D-galactopyranoside (7). A mixture of 6 (950 mg, 0.91 mmol), benzyl N-(2-hydroxyethyl)

Mimicking Antifreeze Glycoproteins carbamate (467 mg, 2.39 mmol), and 4-Å molecular sieves in dry CH2Cl2 (15 mL) was stirred for 20 min. Silvertriflate (760 mg, 2.96 mmol) and N-iodosuccinimide (733 mg, 3.26 mmol) were added to the solution. After 2 h, the mixture was diluted with CH2Cl2 and filtered and washed with saturated aqueous Na2S2O3 and water. The organic phase was dried, filtered, and concentrated to give a colorless glue, which was purified by FC (toluene/EtOAc 2:1) to give 7 (788 mg, 0.683 mmol, 75%) as a white solid. Rf 0.36 (toluene/EtOAc 2:1) [R]D +78 (c 0.5, CHCl3); IR Vmax cm-1 3064, 3033, 2967, 2876, 1779, 1728, 1715, 1602, 1541, 1452, 1391, 1370, 1317, 1265, 1177, 1109, 1097, 1070, 1051, 1027, 749, 710; NMR 1H (300 MHz, CDCl3): δ 3.20 (2H, m, CH2NH), 3.39 (1H, m, H-5), 3.56 (1H, dt, J ) 10.4 4.9 Hz, OCHaHb), 3.74-3.77 (2H, m, H-6a, OCHbHa), 4.20 (1H, d, J ) 11.8 Hz, H-6b), 4.28-4.32 (2H, m, H-5′, 6a′), 4.45 (1H, d, J ) 3.3 Hz, H-4), 4.55 (1H, dd, J ) 9.1 13.2 Hz, H-6b′), 4.70 (1H, dd, J ) 8.5 11.3 Hz, H-2), 4.86 (1H, d, J ) 12.0 Hz, CHaHbPh), 4.89 (1H, dd, J ) 3.3 11.3 Hz, H-3), 4.97 (1H, d, J ) 12.0 Hz, CHbHaPh), 5.00 (1H, bs, NH), 5.06 (1H, d, J ) 8.5 Hz, H-1), 5.08 (1H, d, J ) 7.7 Hz, H-1′), 5.43 (1H, s, CHPh), 5.47 (1H, dd, J ) 3.3 10.2 Hz, H-3′), 5.72 (1H, dd, J ) 7.7 10.2 Hz, H-2′), 5.90 (1H, d, J ) 3.3 Hz, H-4′), 7.05-7.67 (30H, m, Ph), 7.98-8.07 (4H, m, Ph). 13C (75 MHz, CDCl3): δ 40.8 (CH2NH), 51.5 (C-2), 62.4 (C-6′), 66.4 (CH2Ph), 66.9 (C-5), 68.1 (C-4′), 68.3 (OCH2), 68.9 (C-6), 69.6 (C-2′), 71.5 (C-5′), 71.9 (C-3′), 75.1 (C-3), 75.4 (C-4), 98.6 (C-1), 100.9 (CHPh), 101.7 (C-1′), 122.7 (Ph), 123.4 (Ph), 125.3 (Ph), 126.3 (Ph), 127.8 (Ph), 127.9 (Ph), 128.1130.0 (Ph), 131.0 (Ph), 132.8 (Ph), 133.2 (Ph), 133.5 (Ph), 133.6 (Ph), 133.7 (Ph), 133.9 (Ph), 136.6 (Ph) 137.7 (Ph), 137.8 (Ph), 156.2 (NHCOO), 164.5 (COPh), 165.4 (COPh), 165.5 (COPh), 165.9 (COPh), 167.2 (CON), 169.2 (CON); MALDI-TOF Calcd for C65H56N2O18: [M + Na]+ 1175.3. Found: [M + Na]+ 1175.3. N-(benzyloxycarbonyl)-2-aminoethyl-(2,3,4,6-tetra-O-acetylβ-D-galactopyranosyl)-(1 f 3)-4,6-O-benzylidene-2-deoxy2-N-acetamido-β-D-galactopyranoside (8). Ethylenediamine (3 mL, 44.95 mmol) was added to a solution of 7 (590 mg, 0.51 mmol) in n-butanol and ethanol (1:1, 50 mL). The mixture was heated to 90 °C for 24 h and then coevaporated with toluene and xylene. The obtained white solid was treated with pyridine and acetic anhydride (3:2, 40 mL) for 8 h at room temperature before coevaporation with toluene. FC (EtOAc/MeOH 9:1) gave 8 (355 mg, 0.435 mmol, 85%) as a white solid. Rf 0.54 (EtOAc/ MeOH 9:1) [R]D +13 (c 0.5, MeOH); IR Vmax cm-1 3088, 3067, 3036, 2978, 2937, 2883, 1751, 1653, 1541, 1457, 1371, 1232, 1138, 1080, 1060, 822, 739, 701, 603; NMR 1H (300 MHz, MeOH-d4): δ 1.92 (3H, s, CH3COO), 1.94 (3H, s, CH3CON), 2.02 (6H, s, 2 x CH3COO), 2.12 (3H, s, CH3COO), 3.28-3.36 (2H, m, CH2-NH), 3.53 (1H, m, H-5), 3.65 (1H, dt, J ) 10.4 5.5 Hz, OCHaHb), 3.85 (1H, dt, J ) 10.4 5.5 Hz, OCHbHa), 4.02-4.09 (3H, m, H-2,3,6a), 4.13-4.23 (4H, m, H-5′,6a′,6b′,6b), 4.34 (1H, d, J ) 1.9 Hz, H-4), 4.60 (1H, d, J ) 8.2 Hz, H-1), 4.79 (1H, d, J ) 7.7 Hz, H-1′), 5.06-5.11 (4H, m, CH2Ph, H-3′, 2′), 5.37 (1H, dd, J ) 1.1 3.0 Hz, H-4′), 5.60 (1H, s, CHPh), 7.27-7.34 (8H, m, Ph), 7.51-7.54 (2H, m, Ph); 13C (75 MHz, MeOH-d4): δ 20.5 (CH3COO, 2 C), 20.7 (CH3COO), 20.9 (CH3COO), 23.3 (CH3CON), 41.9 (CH2NH), 52.7 (C-2), 62.7 (C-6′), 67.5 (CH2Ph), 68.0 (C-5), 68.9 (C-4′), 69.5 (OCH2), 70.2 (C-6), 70.3 (C-3′), 72.1 (C-5′), 72.5 (C-2′), 76.7 (C-4), 78.8 (C-3), 102.1 (CHPh), 102.5 (C-1), 103.1 (C-1′), 127.5 (Ph), 128.9 (Ph), 129.0 (Ph), 129.5 (Ph), 129.8 (Ph), 139.6 (Ph), 158.7 (NHCOO), 171.3 (CH3COO), 171.5 (CH3COO), 172.0 (CH3COO,

J. Phys. Chem. B, Vol. 109, No. 33, 2005 15857 2 C), 173.6 (CH3CON); MALDI-TOF Calcd for C39H48N2O17: [M + Na]+ 839.3. Found: [M + Na]+ 839.3. (S-acetyl)-N-(16-mercapto-palmitoyl)-2-aminoethyl-(2,3,4,6tetra-O-acetyl-β-D-galactopyranosyl)-(1 f 3)-2-deoxy-2-Nacetamido-β-D-galactopyranoside (10). A mixture of 8 (345 mg, 0.423 mmol) and palladium hydroxide on carbon (30 mg, 20% Pd) in EtOH and acetic acid (1:1, 15 mL) was stirred under H2 (1 atm) for 24 h, filtered, and concentrated. The obtained syrup, 917 (199 mg, 0.465 mmol), and N,N-diisopropylethylamine (81 µL, 0.465 mmol) were stirred in dry DMF (15 mL) for 3 h at room temperature and co-concentrated with toluene. The crude title compound was purified sequentially with FC (EtOAc/MeOH 9:1) and reversed-phase FC (MeOH/H2O 9:1) to give 10 (287 mg, 0.318 mmol, 75%) as a white solid. Rf 0.68 (EtOAc/MeOH/H2O 9:2:1) [R]D -6 (c 0.5, MeOH); IR Vmax cm-1 2923, 2852, 1752, 1699, 1653, 1558, 1372, 1232, 1171, 1135, 1079, 955, 913, 628, 602; NMR 1H (300 MHz, MeOH-d4, 30 °C): δ 1.29-1.36 (22H, m, CH2), 1.53-1.64 (4H, m, CH2), 1.93 (3H, s, CH3COO), 1.98 (3H, s, CH3CON), 2.03 (3H, s, CH3COO), 2.06 (3H, s, CH3COO), 2.13 (3H, s, CH3COO), 2.18 (2H, t, J ) 7.5 Hz, CH2CON), 2.30 (3H, s, CH3COS), 2.86 (2H, t, J ) 7.3 Hz, CH2S), 3.28 (2H, m, CH2NH), 3.51 (1H, t, J ) 6.1 Hz, H-5), 3.64 (1H, dt, J ) 10.4 5.8 Hz, OCHaHb), 3.77-3.88 (4H, m, OCHbHa, H-4,6), 4.04 (1H, dd, J ) 8.5 10.9 Hz, H-2), 4.08-4.23 (4H, m, H-3,5′,6′), 4.44 (1H, d, J ) 8.5 Hz, H-1), 4.78 (1H, d, J ) 8.0 Hz, H-1′), 5.08-5.18 (2H, m, H-2′,3′), 5.38 (1H, d, J ) 2.2 Hz, H-4′); 13C (75 MHz, MeOH-d4): δ 20.5 (CH3COO, 2C), 20.6 (CH3COO), 20.9 (CH3COO), 23.3 (CH3CON), 27.0 (CH2), 29.8 (CH2S), 29.9 (CH2), 30.2 (CH2), 30.4-30.7 (CH2 and CH3COS, several peaks), 37.1 (CH2CON), 40.6 (CH2NH), 52.6 (C-2), 62.4 (C-6), 62.6 (C-6′), 68.8 (C-4′), 69.1 (OCH2), 69.2 (C-3), 70.3 (C-2′), 72.0 (C-5′), 72.3 (C-3′), 76.4 (C-5), 81.4 (C-4), 102.9 (C-1), 103.1 (C-1′), 171.4 (CH3COO), 171.5 (CH3COO), 171.9 (CH3COO), 172.1 (CH3COO), 173.4 (CH3CON), 176.4 (CONH), 197.6 (CH3COS); MALDI-TOF Calcd for C42H70N2O17S: [M + Na]+ 929.4. Found: [M + Na]+ 929.4. N-(16-mercapto-palmitoyl)-2-aminoethyl-β-D-galactopyranosyl-(1 f 3)-2-deoxy-2-N-acetamido-β-D-galactopyranoside (1). Methanolic NaOMe (830 µL, 0.415 mmol, 0.5 M) was added to a solution of 10 (75 mg, 0.083 mmol) in MeOH (4 mL) while purging argon into the reaction solution. After 15 min, dithiothreitol (38 mg, 0.249 mmol) was added. The solution was stirred for 3 h before being neutralized with Dowex-H+, filtered, and concentrated. The obtained solid was purified by HPLC (MeOH/H2O 9:1) to give 1 (52 mg, 0.075 mmol, 90%) as a white solid. Rf 0.24 (EtOAc/MeOH/H2O 9:2:1) [R]D -8 (c 0.5, MeOH); IR Vmax cm-1 2922, 2850, 1644, 1557, 1467, 1376, 1307, 1159, 1120, 1075, 897, 781, 720, 707; NMR 1H (300 MHz, MeOH-d4/CDCl3 2:1, 30 °C): δ 1.28-1.39 (22H, m, CH2), 1.56-1.63 (4H, m, CH2), 1.98 (3H, s, CH3CON), 2.20 (2H, t, J ) 7.7 Hz, CH2CON), 2.51 (2H, t, J ) 7.1 Hz, CH2SH), 3.30-3.38 (3H, m, CH2NH, OCHaHb), 3.46-3.59 (4H, m, OCHbHa,H-2′,5′,5), 3.62-3.85 (7H, m, H-3,4,6,3′,6′) 3.96 (1H, dd, J ) 8.2 10.7 Hz, H-2), 4.11 (1H, m, H-4′), 4.32 (1H, d, J ) 7.4 Hz, H-1′), 4.53 (1H, d, J ) 8.2 Hz, H-1); 13C (75 MHz, MeOH-d4/CDCl3 2:1, 30 °C): δ 23.2 (CH3CON), 24.8 (CH2SH), 26.6 (CH2), 29.0 (CH2), 30.0-30.3 (CH2, several peaks), 34.7 (CH2), 36.9 (CH2CON), 40.2 (CH2NH), 52.8 (C2), 62.1 (CH2), 62.3 (CH2), 68.8 (CH), 69.0 (CH), 69.8 (CH), 72.1 (CH), 74.1 (CH2O), 75.7 (CH), 76.2 (CH), 81.1 (CH), 102.2 (C-1), 106.0 (C-1′), 174.0 (CH3CON), 175.9 (CON); MALDITOF Calcd for C32H60N2O12S: [M + Na]+ 719.4. Found: [M

15858 J. Phys. Chem. B, Vol. 109, No. 33, 2005 + Na]+ 719.4. Anal. Calcd for C32H60N2O12S: C, 55.15; H, 8.68; N, 4.02. Found: C, 54.93 H, 8.57 N, 3.89. Gold Films. Si(100) wafers were cut in appropriate sizes and cleaned. The organic contaminants were removed with a solution (TL1) consisting of 5 parts H2O (MilliQ), 1 part H2O2 (30%), and 1 part NH3 (25%) at 80 °C for 10 min, and then the inorganic contaminants and particles were removed in a solution (TL 2) consisting of 6 parts H2O (MilliQ), 1 part H2O2 (30%), and 1 part HCl (37%) at 80 °C for 10 min. The quality of the MilliQ water was 18.2 MΩcm, 0.22 µm filter, and

Mimicking the Properties of Antifreeze Glycoproteins: Synthesis and

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