Organic reactions promoted by mucin glycoproteins

Article abstract of DOI:10.1021/ja9040626

(Chemical Equation Presented) Mucins are heavily glycosilated proteins that exhibit a broad range of adhesive interactions with various hydrophobic organic materials. However, there has been no clear evidence that mucins are capable of promoting chemical

Full text of DOI:10.1021/ja9040626

Published on Web 08/12/2009
Organic Reactions Promoted by Mucin Glycoproteins
Natalie Shraga, Bogdan Belgorodsky, and Michael Gozin*
School of Chemistry, Raymond and BeVerly Sackler Faculty of Exact Sciences, Tel-AViV UniVersity, Ramat AViV,
Tel-AViV 69978, Israel
Received May 19, 2009; E-mail: cogozin@mgchem.tau.ac.il
Mucins are glycoproteins that constitute 80% of the organic
components of mucus,1 which coats many organs, including the
respiratory, digestive, and reproductive tracts and, in some amphibia,
the skin.2 It is believed that the main function of these glycoproteins
is to protect epithelial cells from infection and dehydration as well
as from physical and chemical injuries.1-3 Although mucins exhibit
a broad range of adhesive interactions with various hydrophobic
materials,1a,2,4 including polycyclic aromatic hydrocarbons,2c there
has been no clear evidence that mucins are capable of promoting
chemical reactions. Here we demonstrate for the first time that under
physiological conditions, two representative mucins, bovine sub-
maxillary mucin type I (BSM) and porcine gastric mucin type III
(PGM), accelerate the rate of fatty acid ester hydrolysis up to 337
times relative to the mucin-free reference reaction. Moreover, under
the same reaction conditions, a Diels-Alder (DA) reaction between
N-propylmaleimide and anthracene is promoted by these glyco-
proteins. The latter reaction does not occur in aqueous media
without mucins, and the rate was accelerated up 200 times in the
presence of a mucin relative to the rate of the reference process
performed in chloroform. Mucins consist of branched oligosaccha-
ride chains attached to a protein backbone.1,4a,5 This unique
structure was discovered to be critically important to the rate
acceleration, as various cyclic and noncyclic oligosaccharides were
far less efficient in promoting the same reactions.
Figure 1. (a) Hydrolysis reaction of pNP esters 1a-d. (b) Kinetic data
for hydrolysis of 1a-d by BSM and PGM vs the mucin-free reference
reaction.
γ-cyclodextrin,8 amylose,9 and arabic acid, were evaluated as
promoters of pNP octanoate and pNP dodecanoate ester hydrolysis.
We found that the smaller lactose and γ-cyclodextrin sugars had
practically no effect on the rate of hydrolysis of either pNP ester.
In contrast, a clearly detectable acceleration in the reaction rate
was observed when the larger amylose and arabic acid oligosac-
charides were present in the solution. More specifically, amylose
promoted hydrolysis of pNP octanoate and dodecanoate with 5-
and 24-fold rate increases, respectively, relative to the reference
oligosaccharide-free reactions, whereas arabic acid exhibited 9- and
21-fold accelerations, respectively. However, the rate accelerations
by amylose and arabic acid oligosaccharides were significantly less
than those observed with BSM and PGM glycoproteins (Figure S1
in the Supporting Information). Our findings strongly suggest that
the spatial arrangement of glycosidic moieties resulting from their
attachment to mucin protein backbone1,4a,5 plays an important role
in the hydrolytic activity of these glycoproteins. To support this
hypothesis, a deglycosilated BSM (dBSM) was prepared and
evaluated as a hydrolysis promoter. For the pNP octanoate and
dodecanoate ester hydrolyses, the dBSM gave 26-fold and 13-fold
slower reactions, respectively, than the corresponding parent BSM
(Figure S1). For comparison, a control mixture of dBSM and arabic
acid (mimicking the parent BSM) was capable of promoting the
hydrolysis of these esters at rates very similar to that for arabic
acid only.
The first reaction we investigated was the hydrolysis of car-
boxylic esters6 (Figure 1a). A series of p-nitrophenol (pNP) esters
of octanoic, decanoic, dodecanoic, and tetradecanoic acids were
used as substrates. The reaction was evaluated under physiological
conditions using a mucin concentration of 1 mg/mL. For all of the
evaluated substrates, both the BSM and PGM glycoproteins
dramatically increased the hydrolysis rate relative to that for the
reaction in the absence of mucin. The rates fitted typical pseudo-
first-order kinetic curves,7 with longer alkyl chain esters reacting
at lower rates (Figure 1b). The evaluated mucins were capable of
accelerating the rate of fatty acid ester hydrolysis up to 337 times
relative to the mucin-free reference reaction performed in sodium
phosphate buffer (Figure 1b). We found that in the case of both
BSM and PGM, complete conversion of pNP esters to the
corresponding products was achieved. Interestingly, PGM exhibited
a much greater difference in hydrolysis rates (kobs) than BSM for
pNP octanoate and pNP tetradecanoate (16-fold). Also, BSM
demonstrated less selectivity: there was only a 4-fold difference
between the highest and lowest rates for the evaluated esters. These
rate variations probably result from differences in the spatial
arrangements of these amorphous glycoproteins.
The second chemical transformation that we studied was a DA
cycloaddition. To demonstrate the remarkable and unprecedented
properties of mucins to promote the DA reaction in phosphate buffer
solution, highly hydrophobic unsubstituted anthracene and N-
propylmaleimide were selected as the diene and dienophile,
respectively (Figure 2a).10 Both the BSM and PGM glycoproteins
not only were capable of solubilizing these substrates2c (anthracene
was added to the reaction mixture in its solid form and not as a
nanosuspension) but also markedly promoted the DA process
To assess the influence of the glycosidic moieties on the
capability of mucins to promote hydrolytic reactions, we compared
the rate of ester hydrolysis in the presence of BSM and PGM
glycoproteins with those observed in the presence of various
oligosaccharides. A series of oligosaccharides, including lactose,
9
12074 J. AM. CHEM. SOC. 2009, 131, 12074–12075
10.1021/ja9040626 CCC: $40.75  2009 American Chemical Society

C O M M U N I C A T I O N S
rides, dBSM, and their mixtures, we propose that mucins perform
the task of dissolving the hydrophobic compound (with subsequent
promotion of organic reactions) by folding of their amorphous
oligomeric structure in order to create local hydrophobic environ-
ments in which such reactions can take place. Furthermore, our
results may lead to a better understanding of how various highly
reactive therapeutic agents undergo metabolic processes in a mucus
layer. Studies of mucin chemical reactivity are also relevant to
development of biocompatible materials for implantable devices,
such as birth control devices, orthodontic devices, digestive-tract-
implantable devices, and contact lenses, all of which have surfaces
that come into direct, long-term interaction with mucus.1a,4c,5,11,12
In this report, we have demonstrated mucin-based acceleration
of carboxylic ester hydrolysis and Diels-Alder carbon-carbon
bond-forming reactions. Further studies will determine whether
these glycoproteins accelerate other organic transformations in
aqueous solution, such as hetero-DA reactions and dipolar
cycloadditions.
Figure 2. (a) DA reaction for the preparation of compound 3 catalyzed by
mucins. (b) Kinetic data for DA reactions catalyzed by BSM, PGM, and
arabic acid vs the reference reaction performed in chloroform.
between them; in contrast, without the presence of mucins, no
product 3 could be detected, eliminating possibility of an
“on-water”-type10d,h chemical process involving these reactants. For
kinetic studies, solutions of either BSM or PGM were preincubated
with solid anthracene for 48 h. Next, mucin-bound anthracene
complexes were separated from nonbound solid anthracene by
filtration and reacted with excess N-propylmaleimide. After workup,
the amount of extracted product 3 was determined by HPLC using
separately synthesized 3 as a reference standard. We found that in
case of BSM, a typical conversion of anthracene to 3 was 65%,
whereas for PGM, 47% conversion was observed. Under the tested
conditions, the reaction was accelerated 39- and 24-fold by BSM
and PGM, respectively, relative to the rate of the same reaction in
chloroform (Figure 2b).
To date, several investigators have reported a rate enhancement
for certain DA reactions performed in aqueous solutions.10 This
phenomenon has generally been explained by the hydrophobic “on-
water” effect. However, in those cases, starting materials with better
water solubility were used, such as anthracene-9-carbinol and
N-ethylmaleimide.10e It should be stressed that numerous thermal
DA reactions between unsubstituted anthracene and various dieno-
philes are known.10f All of these thermal reactions require organic
solvents and high temperatures. In contrast, our mucin-promoted
DA process was successfully performed in phosphate buffer at 37
°C.
The importance of the mucin glycosidic moieties was evaluated
by analysis of the same cycloaddition reaction in the presence of
lactose, γ-cyclodextrin, amylose, or arabic acid oligosaccharides.
Only arabic acid accelerated the rate of the DA reaction (Figure
2b). However, in the presence of the latter oligosaccharide, the
measured acceleration in the rate was an order of magnitude less
than in the presence of either the BSM or PGM glycoprotein. We
hypothesize that the lack of activity of lactose, γ-cyclodextrin, and
amylose is due to the inability of these oligosaccharides to solubilize
anthracene. When the aforementioned dBSM protein was tested as
a promoter of the DA reaction, we found that for the evaluated
substrates the DA reaction was 6-fold slower than in the presence
of the parent BSM, while the dBSM-arabic acid mixture performed
at the level of arabic acid only (Figure 2b).
Acknowledgment. The authors thank Tel Aviv University for
providing financial support.
Supporting Information Available: Detailed procedures for kinetic
analysis of hydrolysis and Diels-Alder reactions. This material is
available free of charge via the Internet at http://pubs.acs.org.
References
(1) For general references, see: (a) Bansil, R.; Turner, B. S. Curr. Opin. Colloid
Interface Sci. 2006, 11, 164. (b) Perez-Vilar, J.; Mabolo, R. Histol.
Histopathol. 2007, 22, 455. (c) Strous, G. J.; Dekker, J. Crit. ReV. Biochem.
Mol. Biol. 1992, 27, 57. (d) Lichtenberger, L. M. Annu. ReV. Physiol. 1995,
57, 565.
(2) (a) Perez-Vilar, J.; Robert, L. H. J. Biol. Chem. 1999, 274, 31751. (b)
Roussel, P.; Delmotte, P. Curr. Org. Chem. 2004, 8, 413. (c) Belgorodsky,
B.; Drug, E.; Fadeev, L.; Gozin, M. Submitted.
(3) Rose, M. C. Am. J. Physiol. 1992, 263, 413.
(4) For additional references, see: (a) Harding, S. E. Biochem. Soc. Trans. 2003,
31, 1036. (b) Holzer, P. Curr. Opin. Gastroenterol. 2000, 16, 469. (c)
Nanotechnology in Therapeutics; Huang, Y., Peppas, N. A., Eds.; Horizon
Bioscience: Wymondham, U.K., 2007; p 109.
(5) (a) Fendler, E. J.; Fendler, J. H. AdV. Phys. Org. Chem. 1970, 8, 271. (b)
Ma, Z.; Taylor, J. S. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 11159. (c)
Hydrolysis in Drug and Prodrug Metabolism; Testa, B. J., Mayer, M., Eds.;
Wiley-VCH: Weinheim, Germany, 2003.
(6) For reports on pNP ester hydrolysis, see: (a) Rodulfo, T.; Cordes, E. H. J.
Org. Chem. 1974, 39, 2281. (b) Broo, K. S.; Brive, L.; Ahlberg, P.; Baltzer,
L. J. Am. Chem. Soc. 1997, 119, 11362. (c) Sakurai, Y.; Ma, S.; Watanabe,
H.; Yamaotsu, N.; Hirono, S.; Kurono, Y.; Kragh-Hansen, U.; Otagiri, M.
Pharm. Res. 2004, 21, 285. (d) Varma, M. N.; Madras, G. Appl. Biochem.
Biotechnol. 2008, 144, 213. (e) Kokubo, T.; Uchida, T.; Tanimoto, S.;
Okano, M.; Sugimoto, T. Tetrahedron Lett. 1982, 23, 1593. (f) Hollfelder,
F.; Kirby, A. J.; Tawfik, D. S. Nature 1996, 383, 60.
(7) For references reporting pseudo-first-order kinetic curves for hydrolysis of
pNP esters, see: (a) Lawin, L. R.; Fife, W. K.; Tian, C. X. Langmuir 2000,
16, 3583. (b) Baltzer, L.; Broo, K. S.; Nilsson, H.; Nilsson, J. Bioorg. Med.
Chem. 1999, 7, 83. (c) Zuev, Y. F.; Mirgorodskaya, A. B.; Kudryavtseva,
L. A.; Idiyatullin, B. Z.; Khamidullin, R. N. Russ. J. Gen. Chem. 2004,
74, 1051.
(8) For a general review of the reactions catalyzed by cyclodextrins in water,
see: Breslow, R.; Dong, S. D. Chem. ReV. 1998, 98, 1997, and references
within.
(9) For an example of pNP ester hydrolysis bound by amylose, see: Hui, Y.;
Wang, S.; Jiang, K. J. Am. Chem. Soc. 1982, 104, 347.
(10) For examples of similar Diels-Alder reactions in water, see: (a) Seelig,
B.; Jaschke, A. Chem. Biol. 1999, 66, 167. (b) Seelig, B.; Keiper, S.;
Stuhlmann, F.; Jaschke, A. Angew. Chem., Int. Ed. 2000, 39, 4576. (c)
Yoshizawa, M.; Tamura, M.; Fujita, M. Science 2006, 312, 251. (d)
Narayan, S.; Muldoon, J.; Finn, M. G.; Fokin, V. V.; Kolb, H. C.; Sharpless,
K. B. Angew. Chem., Int. Ed. 2005, 44, 3275. (e) Breslow, R.; Rideout,
D. C. J. Am. Chem. Soc. 1980, 102, 7816. (f) Atherton, J. C. C.; Jones, S.
Tetrahedron 2003, 59, 9039. (g) Kumar, A.; Pawar, S. S. Tetrahedron 2002,
58, 1745. (h) Chanda, A.; Fokin, V. V. Chem. ReV. 2009, 109, 725. (i) Li,
C. Chem. ReV. 2005, 105, 3095.
The discovered property of mucins to accelerate organic chemical
reactions provides a new and unique example of natural nonenzy-
matic proteins capable of promoting reactions of hydrophobic
materials in aqueous solution. As heavily glycosylated mucins
showed unique properties in comparison with various oligosaccha-
(11) (a) Sandberg, T.; Carlsson, J.; Ott, M. K. Microsc. Res. Tech. 2007, 70,
864. (b) Shi, L. Trends Glycosci. Glycotechnol. 2000, 12, 229.
(12) Harding, S. E. Trends Food Sci. Technol. 2006, 17, 255.
JA9040626
9
J. AM. CHEM. SOC. VOL. 131, NO. 34, 2009 12075