Oligosaccharide Sensing in Aqueous Media Using Porphyrin–Curdlan

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Oligosaccharide Sensing in Aqueous Media Using PorphyrinCurdlan Conjugates: An Allosteric Signal-Amplification System Mayuko Sasaki, Yuma Ryoson, Munenori Numata, and Gaku Fukuhara J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.9b00040 • Publication Date (Web): 28 Feb 2019 Downloaded from http://pubs.acs.org on March 8, 2019

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Oligosaccharide Sensing in Aqueous Media Using Porphyrin-Curdlan Conjugates: An Allosteric SignalAmplification System Mayuko Sasaki,† Yuma Ryoson,‡ Munenori Numata,§ and Gaku Fukuhara*,‡,¶ † Department

‡ Department

of Applied Chemistry, Osaka University, 2-1 Yamada-oka, Suita 565-0871, Japan

of Chemistry, Tokyo Institute of Technology, 2-12-1 Ookayama, Meguro-ku, Tokyo 152-8551, Japan

§ Department

of Biomolecular Chemistry, Graduate School of Life and Environmental Sciences,

Kyoto Prefectural University, Shimogamo, Sakyo-ku, Kyoto 606-8522, Japan ¶

JST, PRESTO, 4-1-8 Honcho, Kawaguchi, Saitama, 332-0012, Japan [email protected]

RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required according to the journal that you are submitting your paper to)

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TOC Graphic

oligosaccharide In aqueous media

Random coil

Globule curdlan-saccharide co-aggregate

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Abstract A series of porphyrin-curdlan conjugates 1-5 of varying degree of substitution (DS) were synthesized to examine their morphological features, chiroptical properties, and oligosaccharide sensing in aqueous media, particularly for tetrasaccharide acarbose which is a drug to treat type-2 diabetes. The random coil state of compounds 1-5 in DMSO becomes the globule curdlan-saccharide co-aggregate upon interaction of acarbose in aqueous DMSO solution to induce various circular dichroism (CD) responses. The high cooperativity and positive homotropic allosterism were observed in the acarbose recognition, enabling the allosteric signal-amplification sensing, for which the DS, stacking character, and microenvironmental polarity changes of the tethered porphyrin reporters are likely to be responsible.

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Introduction Supramolecular saccharide sensing in aqueous media has been a long-term scientifically significant topic but a highly challenging target in current chemistry.1-18 Of particular, a simultaneous achievement of highly accurate selectivity and sensitivity for a saccharide to be analyzed is indeed required in medical practice for rapid and precise diagnoses. Nevertheless the saccharide structural diversity, heavy hydration, and low physiological concentration condition (typically ≤ 5 mM) hamper the rapid progress of saccharide chemosensors and thus make creation of the chemosensors much more difficult.1-18 Most of prosperous approaches hitherto examined in mono- and disaccharide recognition are the use of boronate formation with saccharide's diols19-26 or of rigid supramolecular cages through noncovalent interactions.27-39 Unfortunately, these strategies are not very suitable for sensing oligosaccharides, higher carbohydrate homologues since a cyclic or an acyclic host molecule should be expanded for exactly fitting to structurally complicated higher oligosaccharides in size and shape. Learned by Nature's sophisticated technique,3,8,9,10,12,13,15,18 spreading a highly ordered multiple hydrogen-bonding and CH- interactions network in a supramolecular architecture seems quite promising. In order to construct such a "smart" oligosaccharide sensor, we focus on polysaccharide curdlan (Cur; Figure 1a) as an adjustable hydrogen-bonding recognition moiety for oligosaccharides.17,40-43 Cur, essentially a linear glucan composed of (1→3)-linked -D-glucose monomer units, forms a triple helix in an aqueous solution and denatures to a random coil in dimethyl sulfoxide (DMSO),44-46 shown in Figure 1e. An important feature of Cur, particularly for oligosaccharide sensing, is the reversible renaturing/denaturing process just by changing the solvent from DMSO to water. We have so far demonstrated that DABz-Cur (Figure 1b) functions as a selective oligosaccharide sensor for acarbose (Figure 1c) among 24 mono- to tetra-saccharides by reading out circular dichroism (CD) spectral changes originated from conformational changes of the tethered 4-dimethylaminobenzoate (DABz) reporter on the Cur backbone upon addition of saccharides.40 Acarbose, an inhibitor of -glucosidase releasing glucose from higher carbohydrates, is a world-famous drug to treat type-2 diabetes and obesity, and hence for preventing side effects, highly selective and sensitive sensing of acarbose in blood, i.e., an ACS Paragon Plus Environment

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aqueous medium, is one of the most significant landmarks in medical practice.47 Recently, we have constructed more sensitive acarbose Cur sensors by replacing the DABz reporter with porphyrin chromophores (Figure 1d; two of the top).43 The random coil of the modified Cur sensors in DMSO reversibly converts to a globule upon addition of water (Figure 1f, top), and therefore the acarbose molecule is included in the globule to form an expanded globule Cur-saccharide co-aggregation during the random coil-to-globule conversion (Figure 1f, bottom). Since conformational changes of the attached chromophores in the Cur globule transform into CD signals, we expected more sensitive readout of the complexation-induced structural changes through exciton couplings by using porphyrin chromophores possessing a characteristic Soret band, rather than the DABz reporter. Eventually, the limit of detection (LOD) was greatly improved from 10 mM for DABz-Cur, to 5 mM for H2Por-Cur5.2, and to 0.2 mM for AlPor-Cur3.3.40,43 The saccharide sensing results obtained for Por-Cur (Figure 1d; two of the top) are rather satisfactory, but we do not completely understand why and how such the excellent acarbose sensing is achievable by using porphyrin reporters. In the present study, we elaborated the effects of coordinated metals, aryl substituted groups at the meso position, and the degree of substitution (DS) of the porphyrin reporters, and comparatively studied morphological features, chiroptical properties, and acarbose sensing behaviors of the newly synthesized Por-Cur sensors (Figure 1d; five of the bottom; (1-5)) by comparing to those obtained from the previous sensors. The comparative studies can provide us with deeper mechanistic insights into the factors controlling the sensing outcomes of the Cur-based sensors which enable the oligosaccharide sensing in aqueous media.

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Figure 1. Structures of acarbose and native and modified curdlans. (a) curdlan (Cur), (b) DABz-Cur, (c) acarbose, (d) Por-Cur, (e) reversible renaturing/denaturing of Cur, and (f) the random coil-toglobule conversion in the absence (top) and presence (bottom) of a saccharide.

Results and Discussion Design and syntheses for Por-Cur. For better understanding the acarbose sensing behavior in aqueous media by using the Por-Cur sensors, H2Por-Cur3.6 (1) and AlPor-Cur8.9 (2) (Figure 1d) were designed as the different porphyrin DS for the comparison reasons with the previous data43 obtained from H2Por-Cur5.2 and AlPor-Cur3.3. The synthetic routes for all the porphyrins reporters and Cur conjugates reported in the present study were summarized in Scheme 1. Since the water solubility of the H2Por reporter was relatively lower than that of the positively charged AlPor salt, the DS for H2Por ACS Paragon Plus Environment

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was set to the lower value (3.6) for the solubility problem, in contrast to the higher incorporation of the AlPor reporter (from 3.3 to 8.9). In order to further ascertain the effect of a coordination metal in the porphyrin chromophore, a zinc metal was chosen due to the strong stacking nature of the zinccoordinated porphyrin,48,49 and thus the ZnPor reporter was attached to the Cur backbone (ZnPorCur2.6 (3) in Figure 1d) for comparing with the moderate-stacking H2Por and the stacking-blocked AlPor. To finally investigate the effect of the oligoethylene chain at the aryl substituent, two DS's AlTPP-Cur2.8 (4) and AlTPP-Cur12.7 (5) without the hydrophilic side chain (Figure 1d) were planned, which led us to the deep insights into a hydrophobic effect of the reporters on the acarbose sensing. The newly designed five Por-Cur sensors 1-5 were thus synthesized in 33-98% yields with the reaction of the corresponding porphyrin reporter and native Cur, which was swollen in N-methyl-2-pyrrolidinone (NMP) prior to use, in the presence of N,N-dimethyl-4-aminopyridine (DMAP) and 1-ethyl-3-(3dimethylaminopropyl)carbodiimide hydrochloride (EDC) (see the Experimental Section).

Scheme 1. Synthetic Scheme for Porphyrin Reporters and Curdlan Conjugates

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Morphological features of Por-Cur. Since the previous Por-Cur conjugates, i.e., H2Por-Cur5.2 and AlPor-Cur3.3, are known to form not the triplex observed for native Cur44 but globules43 in 1:9 (v/v) DMSO-H2O, the newly synthesized five Por-Cur conjugates 1-5 were also subjected to the atomic force microscopy (AFM) observation in order to elucidate the direct morphological information of the five sensors. As shown in Figure 2, in keen contrast to the AFM image of helical fibers observed for native Cur, undoubtedly the images of Por-Cur 1-5 indicate the globule formation as was the case with the previous Por-Cur sensors (H2Por-Cur5.2 and AlPor-Cur3.3), and the average globule sizes are listed in Table 1 (middle column; see the line profiles of Figures S10-14 in the SI). These findings indicate that the random-coil state of Por-Cur 1-5 also converts to the globule aggregate just by switching the solvent from pure DMSO to aqueous DMSO solution, as shown in Figure 1f (top). Very interestingly, only the image of compound 3 showed a broad, dispersed shape, although the sensors 1-3 possess the same porphyrin structure unit with and without metals. This critical difference is reasonably accounted by the strong stacking nature of Zn-coordinated porphyrin, which unfortunately led the failure of the oligosaccharide sensing (vide infra). The oligoethylene side chain also plays a decisive role in the globule aggregation (more expanded diameters as 172-208 nm for compounds 4 and 5 than 152 nm for compound 2) and subsequent oligosaccharide sensing. The tighter globule formation of the Cur conjugates 1 and 2 is highly ascribable to a better solvation by the appended amphiphilic arms rather than the hydrophobic unsubstitued benzene at the meso position, however the latter of which enables us to sensitively detect the target acarbose (vide infra). Although this globule formation seems to be driven by the stacking feature which is characteristic to a porphyrin chromophore, we further investigated morphological properties of other chromophore-Cur conjugates, i.e., DABz-Cur40 (see Figure 1b) and Nap-Cur50 (6-O-(2-naphthoyl)Cur; see the structure in Figure S15 of the SI), both of which were speculated as triplex in aqueous media. The AFM images (Figure S15 in the SI) of DABzCur and Nap-Cur prepared from their aqueous DMSO solutions showed formation of somewhat irregular dots, eventually revealing that these Cur conjugates do not renature to triple helices but form ACS Paragon Plus Environment

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globules in aqueous DMSO solution. It should be therefore noted that the random coil-to-globule conversion caused by incorporating not only porphyrin reporters but also standard  chromophores with the Cur backbone is a quite general glucan's behavior.

(a) Cur

1.00 m

(d) ZnPor-Cur2.6 (3)

2.00 m

(b) H2Por-Cur3.6 (1)

2.00 m

(e) AlTPP-Cur2.8 (4)

2.00 m

(c) AlPor-Cur8.9 (2)

2.00 m

(f) AlTPP-Cur12.7 (5)

2.00 m

Figure 2. AFM images of (a) native Cur, (b) H2Por-Cur3.6 (1), (c) AlPor-Cur8.9 (2), (d) ZnPor-Cur2.6 (3), (e) AlTPP-Cur2.8 (4), and (f) AlTPP-Cur12.7 (5) prepared from 1:9 (v/v) DMSO-H2O solutions (compound 1: 431 M; compound 2: 112 M; compound 3: 214 M; compound 4: 369 M; compound 5: 360 M in monomer unit) on mica surface.

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Table 1. Average Sizes for Globule and Globule Curdlan-Saccharide Co-Aggregate Confirmed by AFM sensor

globule

globule curdlan-saccharide co-aggregate

diameter/nm

height/nm

diameter/nm

height/nm

1

114 ± 19

5.0 ± 2.5

119 ± 62

2

152 ± 21

3.7 ± 0.7

227 ± 57

3

185 ± 51

6.6 ± 2.5

285 ± 44

4.0 ± 1.1

4

172 ± 18

4.4 ± 1.8

195 ± 37

5.5 ± 2.1

5

208 ± 32

5.9 ± 2.0

270 ± 82

2.4 ± 1.0

2.9 ± 0.9 17 ± 8.8

Chiroptical properties for Por-Cur. The chiroptical properties of Por-Cur 1-5 were subsequently examined spectroscopically in DMSO and 1:9 DMSO-H2O, where the Cur conjugates form random coil and globule, respectively. As can be seen from the UV/vis spectra in Figure 3, the strong and sharp Soret bands around 420 nm of Por-Cur 1-5 in DMSO (top panel, black line) are broadened with large hypochromic effect in 1:9 DMSO-H2O (top panel, red line), indicating that the porphyrin reporters stacked to each other in the globule structure. The CD spectra (bottom panel) of compound 1 in both solvents showed negative exciton couplets, in which a couplet amplitude (A) was slightly increased from 12.6 (black line) to 14.5 (red line) M-1 cm-1; all the A values are summarized in Table 2. This result suggests that the H2Por reporter aligned in a left-handed helical manner on average, according to the exciton chirality theory.51 Although the couplet changes pattern from negative to negative is similar to those for the previous H2Por-Cur5.2 sensor (see Figure S16a in the SI), the enhancement (1.9) of the A values is much smaller than that (6.8) obtained from H2Por-Cur5.2 due to the lower DS. On the other hand, the CD spectrum of compound 2 in DMSO exhibited an opposite positive couplet, i.e., righthanded alignment, with larger A value of 83.3 M-1 cm-1 than that (10.2 M-1 cm-1) for AlPor-Cur3.3 due to the higher DS (see Figure S16b (black line) in the SI). This CD couplet inversion of the random coil in DMSO for H2Por-Cur vs AlPor-Cur regardless of DS is likely to be caused by the different stacking feature for standard H2Por or charge-repulsive AlPor reporter. More significantly, the exciton coupling ACS Paragon Plus Environment

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of compound 2 in 1:9 DMSO-H2O further inverts to a negative couplet with the smaller A value of 13.1 M-1 cm-1, the helicity of which is same as H2Por-Cur, although the trisignate Cotton effect was observed from AlPor-Cur3.3 (see Figure S16b (red line) in the SI). The small negative couplet may be originated from forcibly regular alignment of the repulsive AlPor reporter crowded by the relatively high DS. These different chiroptical properties of the different DS's H2Por-Cur or AlPor-Cur particularly in 1:9 DMSO-H2O play critical roles in the oligosaccharide sensing. Intriguingly, the CD spectra of compound 3 gave a right-handed positive couplet in DMSO with A as 18.6 M-1 cm-1 and a trisignate Cotton effect (negative-positive-negative) of relatively large A as 71.3 M-1 cm-1 in 1:9 DMSOH2O, the latter of which is responsible for an overlap of two opposite exciton couplings. Effects of the amphiphilic side chain can be examined by comparing the chiroptical properties of AlPor-Cur3.3, compounds 2, 4, and 5. The CD spectra of compound 4 showed both positive couplets from A as 13.8 M-1 cm-1 in DMSO to A as 12.6 M-1 cm-1 in 1:9 DMSO-H2O, indicating that the AlTPP reporter may weakly interact each other in the larger globule structure (estimated by the AFM results) probably due to the less-favored solvation of the unsubstituted aryl group at the meso position. The CD spectral changes of compound 5 in the two solvents (from black line to red line) were very similar to those obtained from compound 2 but the differential A = 37.3 (A in DMSO - A in 1:9 DMSO-H2O) for compound 5 is much smaller than that (70.2) for compound 2. This fact further indicates that the phenyl groups with and without the oligoethylene chain significantly affect on the chiroptical properties, globule volume, and subsequent sensing behaviors.

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Figure 3. UV/vis (top panel) and CD (bottom panel) spectra of (a) H2Por-Cur3.6 (1), (b) AlPor-Cur8.9 (2), (c) ZnPor-Cur2.6 (3), (d) AlTPP-Cur2.8 (4), and (e) AlTPP-Cur12.7 (5) in DMSO (black) and 1:9 (v/v) DMSO-H2O (red), measured in a 1 cm cell at 25 °C.

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Table 2. Exciton Coupling Amplitude (A) of the Por-Cur Conjugates in DMSO and 1:9 (v/v) DMSO-H2O and Their Helical Pattern sensor

A in DMSO/M-1 cm-1 (helicitya)

A in 1:9 (v/v) DMSO-H2O/M-1 cm-1 (helicitya)

1

12.6 (left-handed)

14.5 (left-handed)

H2Por-Cur5.2b

14.2 (left-handed)

21.0 (left-handed)

AlPor-Cur3.3b

10.2 (right-handed)

32.5 (overlapc)

2

83.3 (right-handed)

13.1 (left-handed)

3

18.6 (right-handed)

71.3 (overlapc)

4

13.8 (right-handed)

12.6 (right-handed)

5

54.9 (right-handed)

17.6 (left-handed)

a

According to the exciton chirality theory. b ref. 43. c trisignate.

Oligosaccharide sensing by Por-Cur 1-5. We first estimated the oligosaccharide sensing behavior of compound 3 in 1:9 DMSO-H2O by means of CD spectroscopy and AFM. As can been seen from Figure 4, even the addition of a large amount of 30 mM acarbose can only induce an extremely slight CD change, nevertheless the globule shape of compound 3 became a larger and more dispersed sheetlike structure upon addition of acarbose by the AFM images (see Table 1; right column), indicating the formation of globule curdlan-saccharide co-aggregate shown in Figure 1f (bottom): the globule volume of all the sensors 1-5 expanded upon addition of acarbose and hence the co-aggregation behavior can be applied to Por-Cur 1-5 (see all the AFM images and line profiles of Por-Cur 1-5 with acarbose in Figures S17-21 of the SI). This inappropriate sensing ability of compound 3 can be accounted in terms that strongly stacked ZnPor reporters no longer move in the Cur conformational changes or expansion caused by co-aggregation with acarbose analyte.

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(a)

(b)

(c)

4

 / mdeg

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0

-4

400

500

600

Wavelength / nm

700

2.00 m

2.00 m

Figure 4. (a) CD spectra of compound 3 (214 M in monomer unit) in the absence (black) and presence of 30 mM acarbose (red) in 1:9 (v/v) DMSO-H2O, measured in a 1 cm cell at 25 °C. AFM images of compound 3 in the absence (b; the same image as the one in Figure 2d) and presence of acarbose prepared from 1:9 (v/v) DMSO-H2O solution (compound 3 of 214 M in monomer unit with 30 mM acarbose) on mica surface.

The oligosaccharide sensing of compound 1 in aqueous DMSO solution was next performed by reading out CD changes. As shown in Figure 5a and b, the CD spectral changes or the net change of coupling amplitude (A) upon addition of acarbose showed a typical sigmoidal curve to reach a plateau at 60-90 mM, in sharp contrast to the steady increase observed from H2Por-Cur5.2 (see Figure S22a in the SI). Thus, this binding isotherm was subjected to the Hill analysis (Figure 5c) to afford the Hill coefficient and the apparent binding constant (n = 10.2 and Ka = 27 M-1). The cooperative binding (n > 1), i.e., sigmoidal binding isotherm, is a quite general behavior observed in a system where positive homotropic allosterism is operative.52-55 The Hill coefficient (n) thus observed in this positive allosteric system is relatively large compared with hemoblobin (n = ca. 3),56 general allosteric host molecules (n = 2~8.4),57-66 and allosteric supramolecular self-assembly (n = 51).67 Since the cooperative binding isotherm in the positive allosteric case is very characteristic, at the sudden jump region (30-45 mM; blue dotted regression line in Figure 5b), the signal response obtained can be amplified in the narrow guest concentration range, which is so-called "supramolecular allosteric signal-amplification sensing" ACS Paragon Plus Environment

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proposed by us.17,68 Hence, the sensitivity factor calculated from the slope of the blue dotted regression line in Figure 5b was rather amplified as 0.50 mdeg mM-1 which was enhanced by a factor of 3.1 compared to that obtained from H2Por-Cur5.2. This positive allosterism may be accounted in terms that the initial acarbose-binding is difficult to chirally move the H2Por reporter due to the low DS but further acarbose-incorporation facilitates one by one co-aggregation to induce the amplified response originated from the remarkable chiral movement of the H2Por reporter. It is therefore to note that the positive homotropic allosterism-based signal amplification of oligosaccharide sensing in aqueous media can be attainable by the exquisite valance of a medium-stacking ability and relatively low DS of the H2Por reporter.

Figure 5. (a) CD spectra of compound 1 (431 M in monomer unit) in the absence (black) and presence of acarbose (15-90 mM; colored lines) in 1:9 (v/v) DMSO-H2O, measured in a 1 cm cell at 25 °C. (b) The net changes of coupling amplitude (A) against the acarbose concentration (c), the sudden jump region of which (c = 30-45 mM) is fitted to a regression line (blue dotted line; r = 0.987, A = 0.50c). (c) A Hill plot for acarbose sensing (r = 0.969, Y represents the fractional saturation).

Since the previous AlPor-Cur3.3 sensor sensitively respond to acarbose with the sensitivity factor of 2.17 mdeg mM-1 (see Figure S22b in the SI), a positively charged aluminum porphyrin serves as a ACS Paragon Plus Environment

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promising reporter and thus the oligosaccharide sensing of compound 2 with higher DS was examined in aqueous DMSO solution. As shown in Figure 6a, the coupling amplitude of compound 2 was gradually augmented with increasing acarbose concentration to reach a maximum at 2 mM and then further acarbose addition (5-10 mM) caused the partial precipitation of 2•acarbose co-aggregation formed in 1:9 DMSO-H2O. From the linear response region (0-2 mM) of the A value, the sensitivity factor was determined as 0.58 mdeg mM-1 which was unfortunately reduced down approximately by a one-quater sensitivity of AlPor-Cur3.3. According to the chiroptical property analysis, this lower sensitivity could be ascribed to a more inert-moving of the forcibly stacking AlPor reporter by the high DS. Since the effect of the amphiphilic side chain on the oligosaccharide sensing has not been figured out, the sensing ability of AlTPP-Cur2.8 4 without the oligoethylene chain was examined. In Figure 6b, the exciton coupling gradually decreased upon addition of acarbose, but obviously the sensitivity factor becomes lower as 0.25 mdeg mM-1. This result can be reasonably explained by the low DS and related smaller volume change from globule (172 nm) to co-aggregation expansion (195 nm) confirmed by the AFM results (see Table 1). To overcome the problem and further construct a sensitive sensor, AlTPPCur12.7 5 with higher DS was designed and its oligosaccharide sensing was investigated. As shown in Figure 6c, the couplet amplitude gradually increased with increasing acarbose concentration, and surprisingly the highest sensitivity factor was observed as 6.13 mdeg mM-1, which is 2.8-fold higher than that for AlPor-Cur3.3. The LOD was also attainable as low as 100 M, which is the lowest value ever reported for reporter-curdlan conjugate acarbose sensors. The AFM morphological and CD chiroptical property analyses jointly revealed that more hydrophobic AlTPP reporter could be twisted much chirally surrounded by the hydrophilic oligosaccharide, i.e., acarbose, in the larger co-aggregation (270 nm). It is therefore deduced that not only the relative (chiral) orientation but also the microenvironmental polarity of porphyrin reporters in the Cur globule should be a critical factor controlling CD responses upon co-aggregation of an oligosaccharide.

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Figure 6. Upper panels: CD spectral changes of (a) compound 2 (112 M in monomer unit), (b) compound 4 (369 M in monomer unit), and (c) compound 5 (103 M in monomer unit) in the absence (black) and presence of acarbose (colored lines) in 1:9 (v/v) DMSO-H2O, measured in a 1 cm cell at 25 °C. Lower panels: The net changes of A against the acarbose concentration (c); (a) r = 0.860, A = 0.58c, (b) r = 0.964, A = -0.25c, and (c) r = 0.931, A = 6.13c.

Conclusion We have demonstrated the oligosaccharide sensing in aqueous media using the newly synthesized porphyrin-curdlan conjugates 1-5. Only by changing the DS, the neutral free-base porphyrin system showed the large cooperativity and positive homotropic allosterism, which was attainable for the first time in acarbose sensing. The amphiphilic oligoethylene side chain at the meso position in the positively charged aluminum porphyrin systems plays pivotal roles in the globule and globule curdlan-saccharide ACS Paragon Plus Environment

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co-aggregate formation, chiroptical properties, and oligosaccharide sensing. The most excellent sensor 5 exhibited the highest sensitivity factor as 6.13 mdeg mM-1 and the lowest LOD as 100 M. Eventually, it can be emphasized that the allosteric signal-amplification sensing thus observed may be expanded to other oligosaccharides and guests that are difficult to sense in water. Experimental Section Instruments. Melting points were measured with a BÜCHI B-545 apparatus. FAB-MS spectra was obtained by using a JEOL JMS-700 instrument. 1H NMR (400 MHz) and

13C

NMR (100 MHz, 150

MHz) spectra were recorded in CDCl3, CD3OD or DMSO-d6 on JNM-AL400, JNM ECS400, or Varian INOVA-600; the 1H and 13C NMR peaks of the newly synthesized porphyrin derivatives and porphyrincurdlan conjugates were assigned by comparing to those observed in analogous H2Por-COOMe43 and DABz-Cur40 reported previously. UV/Vis spectra were measured in a quartz cell by using JASCO V650, V-670 or V-560 and CD spectra with a J-820YH spectrometer, all equipped with an ETCS-761 temperature controller. AFM images were obtained with a Shimadzu SPM-9600 microscope. IR spectra were recorded on a JASCO FT/IR-6200 spectrometer. Materials. Fluorescence-free grade water (Milli-Q), DMSO, and acarbose were used as received. Native Cur, purchase from Wako, was dried at 80 °C under high vacuum prior to use and its number average molecular weight (Mn) and polydispersity (PDI) were determined as 1.3 x 106 and 1.5, respectively. Synthesis of ZnPor. H2Por43 (263 mg, 0.230 mmol) was dissolved in CHCl3 (10 mL). Methanol (10 mL) and Zn(OAc)2 (422 mg, 2.29 mmol) were added to the CHCl3 solution. After stirring for 22 h at room temperature under N2 atmosphere, the mixture was extracted with CH2Cl2. The organic layer was washed with water (three times) and dried over anhydrous MgSO4. After the solvent was evaporated, twice flash silica gel column chromatography (30:1 - 10:1 CH2Cl2/MeOH) of the purple residue gave the desired product as purple solid (156 mg, 0.129 mmol) in 56% yield. mp 127 °C; 1H NMR (400 MHz, CDCl3) δH 8.99-8.97 (m, 6H, Ha), 8.89 (d, 2H, J = 4.7 Hz, Hb), 8.49 (d, 2H, J = 8.1 Hz, Hc), 8.34 (d, 2H, J = 8.2 Hz, Hd), 8.12-8.09 (m, 6H, He), 7.30-7.27 (m, 6H, Hf), 4.44-4.40 (m, 6H, Hg), 4.06-4.03 ACS Paragon Plus Environment

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(m, 6H, Hi), 3.88-3.85 (m, 6H, Hj), 3.79-3.75 (m, 6H, Hk), 3.74-3.69 (m, 6H, Hl), 3.61-3.57 (m, 6H, Hm), 3.42 (s, 9H, Hn); 13C NMR (100 MHz, CDCl3) C 171.0 (CC=O), 151.2, 151.0, 152.9, 149.9, 149.2, 135.8 (Ce), 135.1 (Cd), 132.8, 132.6, 132.5, 131.8, 128.9 (Cc), 121.8, 121.5, 119.6, 113.2 (Cf), 72.3 (Cm), 71.3, 71.2, 71.1, 71.0, 70.9, 70.8, 70.9, 70.3 (Ci), 68.0 (Cg), 59.4 (Cn); HR-MS (FAB, sector type): m/z: [M]+ Calcd for C66H70N4O14Zn 1206.4180; Found 1206.4149. Synthesis of ZnPor-COOMe. H2Por-COOMe43 (568 mg, 0.490 mmol) was dissolved in CHCl3 (10 mL). Methanol (10 mL) and Zn(OAc)2 (899 mg, 4.90 mmol) were added to the CHCl3 solution. After stirring for 22 h at room temperature under N2 atmosphere, the mixture was extracted with CH2Cl2. The organic layer was washed with water (three times) and dried over anhydrous MgSO4. After the solvent was evaporated, flash basic alumina column chromatography (170:1 dichloromethane/methanol) of the purple residue gave the desired product as purple solid (537 mg, 0.437 mmol) in 90% yield. mp 120 °C; 1H

NMR (400 MHz, CDCl3) H 8.83 (d, 6H, J = 4.1 Hz, Ha), 8.87 (d, 2H, J = 4.6 Hz, Hb), 8.39 (d, 2H, J

= 8.6 Hz, Hc), 8.30 (d, 2H, J = 8.5 Hz, Hd), 8.11-8.09 (m, 6H, He), 7.26-7.23 (m, 6H, Hf), 4.31-4.29 (m, 6H, Hg), 4.06 (s, 3H, Hh), 3.92-3.89 (m, 6H, Hi), 3.72-3.70 (m, 6H, Hj), 3.63-3.59 (m, 6H, Hk), 3.563.53 (m, 6H, Hl), 3.43-3.40 (m, 6H, Hm), 3.25 (s, 9H, Hn);

13C

NMR (100 MHz, CDCl3) C 167.6

(CC=O), 158.6, 150.8, 150.7, 150.6, 149.7, 149.2, 135.5 (Ce), 134.7 (Cd), 132.3, 131.5, 129.4, 127.9 (Cc), 121.4, 121.1, 119.4, 112.9 (Cf), 71.9 (Cm), 70.9 (Cj), 70.8 (Ck), 70.7 (Ci), 67.8 (Cg), 59.1 (Cn), 52.5 (Ch); HR-MS (FAB, sector type): m/z: [M]+ Calcd for C67H72N4O14Zn 1220.4347; Found 1220.4343. Synthesis of AlTPP-COOMe. To a 30 mL three-necked round-bottomed flask containing TPPCOOMe69 (100 mg, 0.149 mmol) dissolved in dehydrated CH2Cl2 (15 mL) was slowly added 1 M diethylaluminum chloride in hexane (0.5 mL, 0.5 mmol) from a dropping funnel at 0 °C and then the mixture was stirred for 5 h at room temperature under N2 atmosphere. The reaction solution was quenched by adding methanol and water at 0 °C. After the solvent was evaporated, flash basic alumina column chromatography (100:1 dichloromethane/methanol) of the residue gave the desired product as green solid (81 mg, 0.110 mmol) in 74% yield. mp 267-268 °C; 1H NMR (400 MHz, CDCl3) δH 9.05 (s, ACS Paragon Plus Environment

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6H, -pyrrole H), 8.76 (s, 2H, -pyrrole H), 8.44 (d, 2H, J = 7.8 Hz, 5 Ar-o-H), 8.19 (s, 8H, Ph-H) 7.73 (m, 9H, Ph-H), 4.11 (s, 3H, -PhCO2CH3); 13C NMR (100 MHz, CDCl3) C 167.1, 148.7, 141.2, 134.1, 132.3, 132.0, 128.0, 127.8, 126.8, 52.3; HR-MS (FAB, sector type): m/z: [M-Cl]+ Calcd for C46H30N4O2Al 697.2184; Found 697.2192. Synthesis of AlTPP. AlTPP-COOMe (120 mg, 0.164 mmol) was dissolved in THF (10 mL) and MeOH (10 mL). After adding an aqueous KOH solution (2.5 mmol KOH in 10 mL H2O), the mixture was refluxed for 2 h under N2 atmosphere. After the organic solvent was evaporated completely, the aqueous solution was acidified by 1 M HCl. The purple precipitation was washed with 1:4 diethylether/n-hexane to afford the desired product as purple solid (89.1 mg, 0.124 mmol) in 76% yield. mp > 300 °C; 1H NMR (400 MHz, CD3OD) δH 9.13 (s, 8H, -pyrrole H), 8.44 (d, 2H, J = 4.6 Hz, PhH), 8.29-8.22 (m, 8H, Ph-H), 7.83 (m, 9H, Ph-H);

13C

NMR (100 MHz, CD3OD) C 149.6, 149.5,

149.4, 148.9, 142.9, 135.7, 133.9, 133.7, 133.3, 129.7, 128.4; HR-MS (FAB, sector type): m/z: [MCl]+ Calcd for C45H28N4O2Al 683.2028; Found 683.2046. Synthesis of H2Por-Cur3.6 (1). In a 30 mL round-bottomed flask, curdlan (95.5 mg, 0.589 mmol in glucose unit) was added to dry N-methyl-2-pyrrolidinone (NMP) (10 mL) and the resulting solution was heated to 100 °C and stirred for 12 h under N2 atmosphere at that temperature. After cooling down to room temperature, H2Por43 (100 mg, 0.087 mmol) was dissolved in the NMP solution. Then EDC (167 mg, 0.873 mmol) and DMAP (107 mg, 0.873 mmol) were added to the NMP solution and the resulting solution was stirred for 3 d. In order to promote the reaction, further EDC (167 mg, 0.873 mmol) and DMAP (107 mg, 0.873 mmol) were added every 24 h (three times in total). After 3 d, the reaction mixture was slowly poured onto MeOH (50 mL) to give a purple precipitate, which was collected, triturated and washed with MeOH (300 mL) and then dried under high vacuum to afford a crude product (129 mg). The product thus obtained was dissolved in DMSO (24 mL) and heated to 80 °C for 12 h. Then the solution cooled down to room temperature was slowly poured onto MeOH (250 mL) to give a purple precipitate, which was collected, triturated and washed with MeOH (300 mL) and then dried ACS Paragon Plus Environment

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under high vacuum to afford a crude product (104 mg). The product thus obtained was dissolved in DMSO (10 mL) and heated to 80 °C for 12 h again. Then the solution cooled down to room temperature was slowly poured onto MeOH (100 mL) to give a purple precipitate, which was collected, triturated and washed with MeOH (300 mL) and then dried under high vacuum to afford pure H2Por-Cur3.6 in 33% yield (39.6 mg, 0.195 mmol in monomer unit) as purple solid. 1H NMR (400 MHz, DMSO-d6, 80 °C) δH 8.79 (s, 8H, Ha, Hb), 8.41 (s, 2H, Hc), 8.30 (s, 2H, Hd), 8.05 (s, 6H, He), 7.35 (s, 6H, Hf), 4.50 (H1,1’), 4.37 (H6a’), 3.91 (H6b’), 3.70 (H6a), 3.50 (H6b,3,3’), 3.24 (H2, H2’, H4, H4’, H5, H5’); 13C NMR (150 MHz, rt) C 149-134 (CAr), 102.9 (C1, C1’), 86.4 (C3, C3’), 76.4 (C5, C5’), 72.8 (C2, C2’), 68.6 (C4, C4’), 60.9 (C6, C6’); Anal. Calcd for C8.376H12.52N0.144O5.468: C, 49.63; H, 6.23; N, 0.99. Found: C, 47.09; H, 5.89; N, 1.37; IR  3452, 2995, 2911, 1658, 1438, 1404, 1318, 1054, 952, 699, 665 cm-1. The DS (x) of H2Por-Cur was determined by UV/vis spectroscopy using a DMSO solution of H2Por-COOMe as a reference compound. Thus, the DS (x) of H2Por-Cur was determined as 0.036 from the equation: Abs (H2Por-Cur) =  (H2Por-COOMe) × c (concentration of H2Por-Cur in chromophore unit) × l (passlength). Synthesis of AlPor-Cur8.9 (2). In a 10 mL round-bottomed flask, curdlan (50 mg, 0.308 mmol in glucose unit) was added to dry NMP (5 mL) and the resulting solution was heated to 100 °C and stirred for 12 h under N2 atmosphere at that temperature. After cooling down to room temperature, AlPor43 (148.3 mg, 0.123 mmol) was dissolved in the NMP solution. Then EDC (211 mg, 1.23 mmol) and DMAP (150 mg, 1.23 mmol) were added to the NMP solution and the resulting solution was stirred for 3 d. In order to promote the reaction, further EDC (211 mg, 1.23 mmol) and DMAP (150 mg, 1.23 mmol) were added every 24 h (three times in total). After 3 d, the reaction mixture was slowly poured onto MeOH (120 mL) to give a purple precipitate, which was collected, triturated and washed with MeOH (300 mL) and then dried under high vacuum to afford pure AlPor-Cur8.9 in 69% yield (56.5 mg, 0.211 mmol in monomer unit) as purple solid. 1H NMR (600 MHz, DMSO-d6, rt) δH 8.97 (s, 8H, Ha, Hb), 8.44 (s, 2H, Hc), 8.28 (s, 2H, Hd), 8.04 (s, 6H, He), 7.37 (s, 6H, Hf), 4.58 (H1,1’), 4.36 (H6a’), 3.89 (H6b’), 3.66 (H6a), 3.44 (H6b, H3, H3’), 3.23 (H2, H2’, H4, H4’, H5, H5’);

13C

NMR (150 MHz,

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DMSO-d6, rt) C 149.8, 135.5 (CAr), 103.1 (C1, C1’), 86.5 (C3, C3’), 76.8 (C5, C5’), 72.8 (C2, C2’), 68.5 (C4, C4’), 60.5 (C6, C6’); IR  3470, 2992, 2912, 1599, 1442, 1408, 1312, 1058, 951, 703, 659 cm-1. The DS (x) of AlPor-Cur was determined by UV/vis spectroscopy using a DMSO solution of AlPor-COOMe43 as a reference compound. Thus, the DS (x) of AlPor-Cur was determined as 0.089 from the equation: Abs (AlPor-Cur) =  (AlPor-COOMe) × c (concentration of AlPor-Cur in chromophore unit) × l (passlength). Synthesis of ZnPor-Cur2.6 (3). In a 30 mL round-bottomed flask, curdlan (89.5 mg, 0.552 mmol in glucose unit) was added to dry NMP (10 mL) and the resulting solution was heated to 100 °C and stirred for 12 h under N2 atmosphere at that temperature. After cooling down to room temperature, ZnPor (100 mg, 0.083 mmol) was dissolved in the NMP solution. Then EDC (159 mg, 0.83 mmol) and DMAP (101 mg, 0.83 mmol) were added to the NMP solution and the resulting solution was stirred for 3 d. In order to promote the reaction, further EDC (159 mg, 0.83 mmol) and DMAP (101 mg, 0.83 mmol) were added every 24 h (three times in total). After 3 d, the reaction mixture was slowly poured onto MeOH (50 mL) to give a purple precipitate, which was collected, triturated and washed with MeOH (300 mL) and then dried under high vacuum to afford a crude product (143 mg). The product thus obtained was dissolved in DMSO (10 mL) and heated to 80 °C for 12 h. Then the solution cooled down to room temperature was slowly poured onto MeOH (100 mL) to give a purple precipitate, which was collected, triturated and washed with MeOH (300 mL) and then dried under high vacuum to afford pure ZnPorCur2.6 in 95% yield (101 mg, 0.523 mmol in monomer unit) as purple solid. 1H NMR (400 MHz, DMSO-d6, 80 °C) δH 8.79 (s, 8H, Ha, Hb), 8.41 (s, 2H, Hc), 8.30 (s, 2H, Hd), 8.05 (s, 6H, He), 7.35 (s, 6H, Hf), 4.50 (H1,1’), 4.37 (H6a’), 3.91 (H6b’), 3.70 (H6a), 3.50 (H6b,3,3’), 3.24 (H2, H2’, H4, H4’, H5, H5’); 13C NMR (150 MHz, rt) C 149-134 (CAr), 102.9 (C1, C1’), 86.4 (C3, C3’), 76.4 (C5, C5’), 72.8 (C2, C2’), 68.6 (C4, C4’), 60.9 (C6, C6’); Anal. Calcd for C7.716H11.768N0.104O5.338Zn0.024: C, 48.03; H, 6.15; N, 0.75. Found: C, 52.00; H, 6.10; N, 1.94; IR  3469, 2995, 2911, 2588, 2323, 2267, 2222, 2098, 1996, 1664, 1438, 1409, 1308, 1138, 1037, 952, 693, 665 cm-1. The DS (x) of ZnPor-Cur was determined by UV/vis spectroscopy using a DMSO solution of ZnPor-COOMe as a reference ACS Paragon Plus Environment

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compound. Thus, the DS (x) of ZnPor-Cur was determined as 0.026 from the equation: Abs (ZnPorCur) =  (ZnPor-COOMe) × c (concentration of ZnPor-Cur in chromophore unit) × l (passlength). Synthesis of AlTPP-Cur2.8 (4). In a 10 mL round-bottomed flask, curdlan (15 mg, 0.0925 mmol in glucose unit) was added to dry NMP (2 mL) and the resulting solution was heated to 100 °C and stirred for 12 h under N2 atmosphere at that temperature. After cooling down to room temperature, AlTPP (9.98 mg, 0.014 mmol) was dissolved in the NMP solution. Then EDC (26.5 mg, 0.14 mmol) and DMAP (16.9 mg, 0.14 mmol) were added to the NMP solution and the resulting solution was stirred for 3 d. In order to promote the reaction, further EDC (26.5 mg, 0.14 mmol) and DMAP (16.9 mg, 0.14 mmol) were added every 24 h (three times in total). After 3 d, the reaction mixture was slowly poured onto MeOH (50 mL) to give a purple precipitate, which was collected, triturated and washed with MeOH (300 mL) and then dried under high vacuum to afford pure AlTPP-Cur2.8 in 98% yield (16.4 mg, 0.0902 mmol in monomer unit) as purple solid. 1H NMR (600 MHz, DMSO-d6, 25 °C) δH 8.98 (s, 8H, Ha, Hb), 8.84 (s, 2H, Hc), 8.48 (s, 2H, Hd), 8.18 (s, 6H, He), 7.84 (s, 6H, Hf), 4.50 (H1,1’), 4.37 (H6a’), 3.91 (H6b’), 3.70 (H6a), 3.50 (H6b,3,3’), 3.24 (H2, H2’, H4, H4’, H5, H5’);

13C

NMR (150

MHz, rt) C 103.2 (C1, C1’), 86.3 (C3, C3’), 76.5 (C5, C5’), 73.1 (C2, C2’), 68.6 (C4, C4’), 61.1 (C6, C6’); IR  3455, 2992, 2911, 1440, 1407, 1313, 1057, 953, 693, 665 cm-1. The DS (x) of AlTPP-Cur was determined by UV/vis spectroscopy using a DMSO solution of AlTPP-COOMe as a reference compound. Thus, the DS (x) of AlTPP-Cur was determined as 0.028 from the equation: Abs (AlTPPCur) =  (AlTPP-COOMe) × c (concentration of AlTPP-Cur in chromophore unit) × l (passlength). Synthesis of AlTPP-Cur12.7 (5). In a 10 mL round-bottomed flask, curdlan (30 mg, 0.185 mmol in glucose unit) was added to dry NMP (4 mL) and the resulting solution was heated to 100 °C and stirred for 12 h under N2 atmosphere at that temperature. After cooling down to room temperature, AlTPP (60 mg, 0.083 mmol) was dissolved in the NMP solution. Then EDC (159.8 mg, 0.83 mmol) and DMAP (101.9 mg, 0.83 mmol) were added to the NMP solution and the resulting solution was stirred for 3 d. In order to promote the reaction, further EDC (159.8 mg, 0.84 mmol) and DMAP (159.8 mg, 0.84 mmol) were added every 24 h (three times in total). After 3 d, the reaction mixture was slowly poured onto ACS Paragon Plus Environment

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MeOH (100 mL) to give a purple precipitate, which was collected, triturated and washed with MeOH (300 mL) and then dried under high vacuum to give a purple product (51.7 mg). The product thus obtained was dissolved in DMSO (10 mL) and heated to 80 °C for 12 h. Then the solution cooled down to room temperature was slowly poured onto MeOH (100 mL) but no precipitation. Thus, the solvent was completely evaporated to afford AlTPP-Cur12.7 in 79% yield (37 mg, 0.147 mmol in monomer unit) as purple solid. 1H NMR (600 MHz, DMSO-d6, 25 °C) δH 8.84 (s, 8H, Ha, Hb), 8.47 (s, 2H, Hc), 8.23 (s, 2H, Hd), 8.14 (s, 6H, He), 7.81 (s, 6H, Hf), 4.66 (H1,1’), 4.49 (H6a’), 3.91 (H6b’), 3.70 (H6a), 3.50 (H6b,3,3’), 3.24 (H2, H2’, H4, H4’, H5, H5’); 13C NMR (150 MHz, rt) C 103.1 (C1, C1’), 86.3 (C3, C3’), 76.4 (C5, C5’), 73.7 (C2, C2’), 68.4 (C4, C4’), 60.9 (C6, C6’); IR  3471, 2989, 2912, 1594, 1434, 1410, 1307, 1056, 947, 929, 698 cm-1. The DS (x) of AlTPP-Cur was determined by UV/vis spectroscopy using a DMSO solution of AlTPP-COOMe as a reference compound. Thus, the DS (x) of AlTPP-Cur was determined as 0.127 from the equation: Abs (AlTPP-Cur) =  (AlTPP-COOMe) × c (concentration of AlTPP-Cur in chromophore unit) × l (passlength). Associated Content Supporting Information NMR Spectra of porphyrin reporters and curdlan conjugates, UV/vis and CD spectra of H2Por-Cur5.2 and AlPor-Cur3.3, and AFM images of 1-5, DABz-Cur, and Nap-Cur. This material is available free of charge via the Internet at http://pubs.acs.org. Author Information Corresponding Author *E-mail: [email protected] Notes The authors declare no competing financial interest.

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Acknowledgments We are grateful to Prof. Yoshihisa Inoue at Osaka University for valuable suggestions. G.F. appreciates the generous supports by a Grant-in-Aid for Young Scientists (A) (No. JP16H06041) from Japan Society for the Promotion of Science (JSPS), Japan Science Technology Agency (JST), PRESTO (No. JPMJPR17PA), and also Nakatani Foundation and Challenging Research Award of Tokyo Institute of Technology. References (1) Aoyama, Y. In Comprehensive Supramolecular Chemistry; Atwood, J. L., Davies, J. E. D., MacNicol, D. D., Vögtle, F., Lehn, J.-M., Eds.; Pergamon Press: Oxford, U.K., 1996; vol. 2, pp 279307. (2) James, T. D.; Sandanayake, K. R. A. S.; Shinkai, S. Saccharide Sensing with Molecular Receptors Based on Boronic Acid. Angew. Chem. Int. Ed. Engl. 1996, 35, 1910-1922. (3) Davis, A. P.; Wareham, R. S. Carbohydrate Recognition through Noncovalent Interactions: A Challenge for Biomimetic and Supramolecular Chemistry. Angew. Chem. Int. Ed. 1999, 38, 2978-2996. (4) Lützen, A. In Highlights in Bioorganic Chemistry: Methods and Applications; Schmuck, C., Wennemers, H., Eds.; Wiley-VCH: Weinheim, Germany, 2004; p 109. (5) Aslan, K.; Zhang, J.; Lakowicz, J. R.; Geddes, C. D. Saccharide Sensing Using Gold and Silver Nanoparticles-A Review. J. Fluorescence 2004, 14, 391-400. (6) Arnold, M. A.; Small, G. W. Noninvasive Glucose Sensing. Anal. Chem. 2005, 77, 5429-5439. (7) Borisov, S. M.; Wolfbeis, O. S. Optical Biosensors. Chem. Rev. 2008, 108, 423-461. (8) Kubik, S. Synthetic Lectins. Angew. Chem. Int. Ed. 2009, 48, 1722-1725. (9) Davis, A. P. Synthetic lectins. Org. Biomol. Chem. 2009, 7, 3629-3638.

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