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. Author manuscript; available in PMC: 2010 May 1.
Published in final edited form as: J Org Chem. 2009 May 1;74(9):3544–3546. doi: 10.1021/jo9002008

Highly Water Soluble Monoboronic Acid Probes that Shows Optical Sensitivity to Glucose Based on 4-Sulfo-1,8-Naphthalic Anhydride

Zhi Cao 1, Premchendar Nandhikonda 1, Michael D Heagy 1,*
PMCID: PMC2736147  NIHMSID: NIHMS108647  PMID: 19351178

Abstract

graphic file with name nihms108647f4.jpg

Two highly water soluble monoboronic acid probes that display the more desirable OFF-ON fluorescence response were synthesized based on 4-Sulfo-1,8-naphthalic anhydride and a remarkable sensitivity for glucose rather than fructose and galactose was also observed.

With cases of diabetes reaching epidemic proportions, there continues to be a strong demand for methods of detecting saccharide concentration in blood for those patients who are suffering from this chronic disease.1–6 Since the ability of recognition of saccharides, boronic acids with diol moiety are widely investigated for their huge potential biomedical applications.1,3,7–9 Unfortunately, most of carbohydrate probes based on boronic acid moiety continue to have a water solubility and those based on enzymes exhibit poor stability or consumption of substrate during the detection procedure which also limit further biosensing applications.10–11 Because of their ability to bind to the diols of sugars, phenylboronic acid and its derivatives have been developed for saccharide sensing based on different measurements, such as fluorescence,12–13 UV-vis absorption14–15 and other methods.16–18 Some key challenges in this field that continue to limit the number of useful probes are: (1) the design of synthesis of reporters that have excitation and emission wavelength above 500 nm, (2) low molecular weight, (3) photostability and (4) perhaps most important water solubility.19–20

As a highly photostable and fluorescent probe, naphthalic anhydrides and their derivatives have been widely used for fluorescent tags and receptor antagonists.21–26 Of particular interest, when appropriately substituted at both the naphthalic and phenyl rings of N-aryl-1,8-naphthalimide, a clear dual-fluorescence was observed.26–27 For instance, by introducing a nitro group into the naphthalic anhydride ring, two emission bands(430nm/550nm, respectively) of the dye molecule were reported by our group.27

A bis-boronic acid based probe was first synthesized by Shinkai et. al.28 when 3-aminophenylboronic acid was added into a protoporphyrin system. At pH 10.5, in the presence of fructose, the fluorescence signal of this probe could be increased 100-fold. In addition, other studies23, 29 have shown that bis-boronic acid probe designs exhibit higher binding affinity specific for glucose than monoboronic acid probes by comparing Kd values of both sensors, 10−5−10−4 M for bis-boronic acid probe and 10−3−10−2 M for monoboronic acid probe, respectively. Unlike changing distances between the boronic acids through synthetic modifications of bis-boronic acid probes, recent investigations on simple monoboronic acid probes showed that fluorophores and substituents on fluorophores also could contribute to saccharide selectivity.23, 27, 29 Therefore, more complicated synthetic schemes of bis-boronic acid probes could be avoided by employing appropriate substituents of fluorophore, without losing saccharide binding efficiency.

In this work, we synthesized two different N-phenylnaphthalimide based monoboronic acid probes. Upon consideration of the structurally related Lucifer yellow dye and its high water solubility,30 we utilized the 4-sulfo potassium salt group of 1,8- naphthalic anhydride. By changing substituted –B(OH)2 positions on the phenyl ring, we investigated steric effect on saccharide binding of different probes and structural configuration during this procedure.

To explore fluorescent properties through sugar binding, we synthesized two probes 1–2, by using a common fluorophore 4-sulfo-1,8-naphthalic anhydride as starting material. 3-aminophenylboronic acid and 2-aminophenylboronic pinacol ester were selected for investigation of isomeric effects on different boronic acid binding pathways with sugar. Sensor 1 was prepared in single-step through reaction of the commercial potassium 4-sulfo-1,8-naphthalic anhydride and 3-aminophenylboronic acid hemisulfate. The other probe was prepared through a two steps reaction. After reaction between naphthalic anhydride and aminophenylboronic pinacol ester, the product was treated with 33% aqueous H2O2 for 1 h and probe 2 was obtained.31 A synthetic scheme for construction of those isomers is given in Scheme 1. High water solubility and significant emission signal change are observed in our experiments.

Scheme 1.

Scheme 1

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Synthetic route of ortho- and meta- phenyl monoboronic acid probes for saccharides based on 4-sulfo-1,8-naphthalic anhydride

Beyond our expectations, both saccharide probes displayed a greater optical sensitivity to glucose than fructose. Numerous papers indicate that most of the monoboronic saccharide chemsensors favor fructose more than glucose. Some reports3, 21, 23, 29 indicated that the affinity of probe to fructose is approximately 100 times greater than glucose for monoboronic acid sensors. Probes 1–2 show large fluorescence intensity changes through chelation-enhanced fluorescence (CHEF) via three most abundant monosaccharides in human blood and receptors interactions, as (a, b and c) shown in Figure 1. Currently, we attribute the increased fluorescence in the presence of analyte to rigidification of the biaryl probe.32 Next, we examined the selectivity of these probes to common monosaccharides at pH = 7.4 condition. Figure 1d shows the relative fluorescence of 1 at 400 nm and 474 nm as a function of carbohydrate concentration. Photophysical properties of monoboronic acid probes 1–2 are listed in Table 1. The increase in fluorescence intensity ratios (I in the presence of saccharide, I0 in the absence of saccharide) for this series shows an increase of about 2–5 times, respectively. Probe 1 displays an increase in the ratio of intensities for three common monosaccharides, showing the largest increase in fluorescence for glucose. Similar observations were made by our group previously in which the largest quenching effect took place on glucose.23 The dissociation constant Kd for fructose was found to be 6.5 mM, while a higher Kd of 28.5 mM was obtained by calculation for glucose at pH 7.4. Though several bisboronic acid sensors have been synthesized to favor glucose over fructose among saccharide bindings, their limitations to complex glucose make them less efficient as glucose probes at high blood glucose levels. Consequently, probe 1 shows an advantage at pH 7.4 by displaying the largest fluorescence increase to glucose while maintaining an affinity within physiological limits, similar to our probe synthesized from 3-nitronaphthalic anhydride and 3-aminophenylboronic acid.21 The incorporation of the boronic acid group in the meta position does not lead to significant spectroscopic and photophysical changes in comparison with ortho derivative. A relatively smaller dissociation constant was observed for meta-derivative (probe 1) in the presence of saccharides, which could be explained by the effect of less steric hindrance.24 Quantum yields for all two probes were within approximately the same order of magnitude. In addition, the apparent dissociation constants for meta- (probe 1) and ortho- (probe 2) derivatives are listed in Table 2. Fluorescence spectra as well as graphical analyses to detail Kd values are also provided in supporting information.

Fig. 1.

Fig. 1

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Fluorescent spectral changes of boronic acid probe 1 upon addition of different saccharides in phosphate buffer (0.1 M) at pH 7.4: λex = 360 nm, λ1em = 400 nm, λ2em = 474 nm. (a) fructose; (b) glucose; (c) galactose; (d) plots of fluorescent intensity changes of 1 as a function of sugar concentration at 474 nm.

Table 1.

Photophysical Properties of 1–2 monoboronic acid probes.

Entry Sensor λex
(nm)		 λ1em
(nm)		 λ2em
(nm)		 ΦF
1 graphic file with name nihms108647t1.jpg 360 400 474 0.092
2 graphic file with name nihms108647t2.jpg 340 391 / 0.112
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Table 2.

Dissociation constants (Kd) of probes 1–2 in the presence of monosaccharides

Kd (mmol)
Probe D-fructose D-galactose D-glucose
1 6.5 ± 0.2 15.2 ± 0.5 28.5 ± 1.2
2 13.2 ± 0.4 22 ± 0.6 35.4 ± 1.5
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In conclusion, two highly water soluble monoboronic acid probes that exhibit large fluorescence increases in the presence of monosaccharides show remarkable sensitivity for glucose rather than fructose and galactose. To our knowledge, this is the first highly water soluble monoboronic acid probe to display the more desirable off-on fluorescence response. While both the monoboronic acid probes displayed a greater change in fluorescence intensity for glucose, it should be emphasized that their binding affinities are still greater for fructose. By changing position of the boronic acid group from ortho to -meta positions of the phenyl ring, there are no significant spectroscopic and photophysical changes in comparison with ortho derivative. Plans are currently underway to extend the absorption wavelength of theses dyes to longer wavelength.

Experimental Section

N-(1,8-Naphthaloyl)-3-aminophenyl Boronic Acid (1). 3-Aminophenylboronic acid hemisulfate (0.100g, 0.54mmol), 4-sulfo-1,8-naphthalic anhydride, potassium salt (0.143g, 0.45mmol), 10ml pure water were placed in a 25mL round-bottom flask equipped with a Dean-Stark receiver and condenser. The reaction mixture was allowed to reflux for 24 h. Water was removed by drying for overnight, and over amount of 3-aminophenylboronic acid was removed by crystallization in hot ethanol. The reaction offered a pink-white powder (0.162g, 81%), mp 290–2930C: 1H NMR (D2O) δ 9.04 (d, J = 9 Hz, 1H), 8.54 (d, J = 6 Hz, 2H), 8.30 (d, J = 7.7 Hz, 1H), 7.93 (t, J = 6.6 Hz, 1H), 7.65 (m, J = 8.8 Hz, 2H), 7.50 (m, 2H); 13C NMR (DMSO) δ 164.5, 164.0, 150.5, 135.0, 134.8, 131.1, 131.0, 129.8, 128.3, 127.4, 125.6, 123.9, 123.0; IR 1217, 1366, 1730, 2970 cm−1; HRMS for C18H9KSBNO7: Expected m/z, 396.0349. Found: m/z, 396.0351.

N-(1,8-Naphthaloyl)-2-aminophenyl Boronic Acid (2). 2-Aminophenylboronic acid pinacol ester (0.165g, 0.75mmol), 4-sulfo-1,8-naphthalic anhydride, potassium salt (0.143g, 0.45mmol), 10ml pure water were placed in a 25mL round-bottom flask equipped with a Dean-Stark receiver and condenser. The reaction mixture was allowed to reflux for 24 h. The mixture was allowed to reflux by adding 2ml 30% aqueous hydrogen peroxide at room temperature for 1 hour. Water was removed by drying for overnight, and over amount of 3-aminophenylboronic acid was removed by crystallization in hot ethanol. The reaction offered a orange-yellow powder (0.174g, 87%), mp 355–3580C: 1H NMR (D2O) δ 8.98 (d, J = 8.2 Hz, 2H), 8.50 (d, J = 6.6 Hz, 2H), 8.27 (d, J = 7.3 Hz, 1H), 7.89 (t, J = 7.2 Hz, 1H), 7.61 (d, J = 8.1 Hz, 1H), 7.35 (m, J = 7.2 Hz, 2H); 13C NMR (DMSO) δ 164.6, 164.0, 150.7, 135.1, 131.0, 130.8, 129.0, 128.3, 127.2, 125.4, 124.1, 123.1, 123.0; IR 1209, 1368, 1732, 3011 cm−1. HRMS for C18H9KSBNO7: Expected m/z, 396.0349. Found: m/z, 396.0348.

Supplementary Material

1_si_001

Fig. 2.

Fig. 2

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Prediction of apparent dissociation constants of probe 1 among different saccharides.

Acknowledgment

This work was supported by a grant from the National Institutes of Health (GM R15-57855-03 the M.D.H wishes to thank Ms. Sherrel of Dept. of Chemistry and Chemical Biology, University of New Mexico for HRMS analysis).

Footnotes

Supporting Information Available: Fluorescent spectral study and apparent dissociation constants in the presence of monosaccharides of sensor 2. This material is available free of charge via the Internet at http://pubs.acs.org.

REFERENCES

  • 1.James TD, Shinkai S. Top. Curr. Chem. 2002;218:159–200. [Google Scholar]
  • 2.Fang H, Kaur G, Wang B. J. Fluoresc. 2004;14:481–489. doi: 10.1023/b:jofl.0000039336.51399.3b. [DOI] [PubMed] [Google Scholar]
  • 3.Cao H, Heagy MD. J. Fluoresc. 2004;14:569–584. doi: 10.1023/b:jofl.0000039344.34642.4c. [DOI] [PubMed] [Google Scholar]
  • 4.Finney SJ, Zekveld C, Elia A, Evans TW. J. Am. Med. Assoc. 2003;290:2041–2047. doi: 10.1001/jama.290.15.2041. [DOI] [PubMed] [Google Scholar]
  • 5.Wentholt IM, Vollebregt MA, Hart AA, Hoekstra JB, DcVrics JH. Dibetes Care. 2005;28:2871–2876. doi: 10.2337/diacare.28.12.2871. [DOI] [PubMed] [Google Scholar]
  • 6.Pickup JC, Hussain F, Evans ND, Rolinski OJ, Birch DJS. Biosens. Bioelectron. 2005;20:2555–2565. doi: 10.1016/j.bios.2004.10.002. [DOI] [PubMed] [Google Scholar]
  • 7.James TD, Sandanayake KRAS, Shinkai S. Nature. 1995;374:345–357. [Google Scholar]
  • 8.Wang W, Gao X, Wang B. Curr. Org. Chem. 2002;6:1285–1317. [Google Scholar]
  • 9.Striegler S. Curr. Org. Chem. 2003;7:81–102. [Google Scholar]
  • 10.Yan J, Fang H, Wang B. Med. Res. Rev. 2005;25:490–520. doi: 10.1002/med.20038. [DOI] [PubMed] [Google Scholar]
  • 11.Heller A. Annu, Rev. Biomed. Eng. 1999;1:153–175. doi: 10.1146/annurev.bioeng.1.1.153. [DOI] [PubMed] [Google Scholar]
  • 12.Yoon JY, Czarnik AW. J. Am. Chem. Soc. 1992;114:5874–5875. [Google Scholar]
  • 13.James TD, Linnane P, Shinkai S. Chem. Commun. 1996:281–288. [Google Scholar]
  • 14.Yamamoto H, Ori A, Ueda K, Dusemund C, Shinkai S. Chem. Commun. 1996:407–408. [Google Scholar]
  • 15.Shinmori H, Takeuchi M, Shinkai S. Tetrahedron. 1995;51:1893–1902. [Google Scholar]
  • 16.Gabai R, Sallacan N, Chegel V, Bourenko T, Katz E, Willner I. J. Phys. Chem. B. 2001;105:8196–8202. [Google Scholar]
  • 17.Lee M, Kim T, Kim KH, Kim JH, Choi MS, Choi HJ, Koh K. Anal. Biochem. 2002;310:163–170. doi: 10.1016/s0003-2697(02)00305-6. [DOI] [PubMed] [Google Scholar]
  • 18.Shoji E, Freund MS. J. Am. Chem. Soc. 2002;124:12486–12493. doi: 10.1021/ja0267371. [DOI] [PubMed] [Google Scholar]
  • 19.Norrild JC, Eggert H. J. Am. Chem. Soc. 1995;117:1479–1484. [Google Scholar]
  • 20.Eggert H, Frederiksen J, Morin C, Norrild JC. J. Org. Chem. 1999;64:3846–3852. [Google Scholar]
  • 21.Mader HS, Wolfbeis OS. Microchim Acta. 2008;162:1–34. [Google Scholar]
  • 22.DiCesare N, Pinto MR, Schanze KS, Lakowicz JR. Langmuir. 2002;18:7785–7787. doi: 10.1021/la0264926. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Cao H, Diaz DI, DiCesare NJ, Lakowicz R, Heagy MD. Org. Lett. 2002;4:1503–1505. doi: 10.1021/ol025723x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.DiCesare N, Adhikari DP, Heynekamp JJ, Heagy MD, Lakowicz JR. J. Fluoresc. 2002;12:147–154. [PMC free article] [PubMed] [Google Scholar]
  • 25.Adhikiri DP, Heagy MD. Tetrahedron Lett. 1999;40:7893–7896. [Google Scholar]
  • 26.Cao H, Chang V, Hernandez R, Heagy MD. J. Org. Chem. 2005;70:4929–4934. doi: 10.1021/jo050157f. [DOI] [PubMed] [Google Scholar]
  • 27.Cao H, McGill T, Heagy MD. J. Org. Chem. 2004;69:2959–2966. doi: 10.1021/jo035760h. [DOI] [PubMed] [Google Scholar]
  • 28.Murakami H, Nagasaki T, Hamachi I, Shinkai S. Tetrahedron Lett. 1993;34:6273–6276. [Google Scholar]
  • 29.DiCesare N, Lakowicz JR. Org. Lett. 2001;3:3891–3893. doi: 10.1021/ol016813p. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Coskun A, Akkaya EU. Org. Lett. 2004;6:3107–3109. doi: 10.1021/ol0488744. [DOI] [PubMed] [Google Scholar]
  • 31.Gypser A, Michel D, Nirschl DS, Sharpless KB. J. Org. Chem. 1998;63:7322–7327. doi: 10.1021/jo980850l. [DOI] [PubMed] [Google Scholar]
  • 32.Takeuchi M, Mizuno T, Shinmori H, Nakashima M, Shinkai S. Tetrahedron. 1996;52:1195–1204. [Google Scholar]

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Supplementary Materials

1_si_001
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