Enhanced fructose, glucose and lactose transport promoted by a lipophilic 2-(aminomethyl)-phenylboronic acid

Article abstract of DOI:10.1016/j.tet.2008.05.052

A lipophilic 2-(aminomethyl)-phenylboronic acid (1a) was prepared and used to transport saccharides across a thin supported liquid membrane. The result was the highest recorded fructose flux promoted by a monoboronic acid under near-neutral conditions, and one of the highest fructose/glucose transport selectivities (12.9). The application of a pH gradient across the membrane further enhanced carrier-mediated fructose flux but fructose/glucose selectivity was decreased. The first effective transport of a disaccharide, lactose, was achieved using supported liquid membranes containing a high concentration of 1a (250 mM). A strong carrier concentration effect on lactose transport and a battery of mass spectrometry evidence point to the involvement of 2:1 boronate/lactose esters in this transport process.

Full text of DOI:10.1016/j.tet.2008.05.052

Tetrahedron 64 (2008) 7122–7126
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Enhanced fructose, glucose and lactose transport promoted by a lipophilic
2-(aminomethyl)-phenylboronic acid
,
b
,
c
c
c
d
Peter J. Duggan a , Todd A. Houston , Milton J. Kiefel , Stephan M. Levonis , Bradley D. Smith ,
*
Melanie L. Szydzik b
a CSIRO Molecular and Health Technologies, Bag 10, Clayton South, Victoria 3169, Australia
b School of Chemistry, Monash University, Clayton, Victoria 3800, Australia
c Institute for Glycomics, Gold Coast Campus, Griffith University, Queensland 4222, Australia
d Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 3 April 2008
Received in revised form 9 May 2008
Accepted 12 May 2008
Available online 15 May 2008
A lipophilic 2-(aminomethyl)-phenylboronic acid (1a) was prepared and used to transport saccharides
across a thin supported liquid membrane. The result was the highest recorded fructose flux promoted by
a monoboronic acid under near-neutral conditions, and one of the highest fructose/glucose transport
selectivities (12.9). The application of a pH gradient across the membrane further enhanced carrier-
mediated fructose flux but fructose/glucose selectivity was decreased. The first effective transport of
a disaccharide, lactose, was achieved using supported liquid membranes containing a high concentration
of 1a (250 mM). A strong carrier concentration effect on lactose transport and a battery of mass spec-
trometry evidence point to the involvement of 2:1 boronate/lactose esters in this transport process.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
OH
OH
Ar
B
+
sugar
HO
Q
X
sugar
HO
+
OH
+
OH
There is great potential for membrane processes to be used in
environmentally benign industrial sugar production. We have been
particularly interested in the use of thin supported liquid mem-
branes containing boronic acid carriers for use in this application,
and results obtained in this area have been extensively reviewed.1
The transport process under investigation involves the transient
formation of tetrahedral sugar/boronate esters (Fig. 1). The ad-
vantages of employing the tetrahedral form of the organoboron
species are several-fold. The negatively charged tetrahedral boro-
nate esters are generally more stable than the corresponding
neutral trigonal boronates2 and the formation of such tetrahedral
esters also allows the use of lipophilic quaternary ammonium salts
to improve initial extraction into the membrane. The rate of ester
formation at the departure phase–membrane interface is also likely
to be faster if the boronic acid is in the negatively charged tetra-
hedral form.3c The mechanism by which the sugars are thought to
pass through the membrane as tetrahedral boronates is shown in
Figure 1.
OH
OH
sugar
X
2H2O
+
X
2H2O
+
O
O
B
Q
OH
Ar
Aqueous Departure
Phase
Organic
Aqueous Receiving
Phase
Membrane
Figure 1. Accepted ‘tetrahedral’ mechanism by which arylboronic acids, in conjunction
with lipophilic ammonium salts (QþXꢀ), assist the passage of sugars through a lipo-
philic organic membrane.
Successful approaches have included the attachment of multiple
boronic acid functionalities to a semi-rigid scaffold4 and the in-
corporation of a quaternary ammonium group within the carrier.5
There has been much interest for over a decade in 2-(amino-
methyl)-arylboronic acids 1 (Fig. 2) due to their application in
sensor research.3 These compounds were first investigated in detail
by Wulff for chromatographic applications,6 but it was later found
that when the amine is attached to a fluorophore, they provide
a means of sensing the presence of sugars. This is because the
fluorescence that is initially quenched by intramolecular photo-
electron transfer from the amine is enhanced when the boronic
Although significant progress has been made towards an in-
dustrially feasible process, further improvements are required. One
issue that has received much of our attention is flux enhancement.
* Corresponding author. Tel.: þ61 3 9545 2560; fax: þ61 3 9545 2446.
E-mail address: peter.duggan@csiro.au (P.J. Duggan).
0040-4020/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.05.052

P.J. Duggan et al. / Tetrahedron 64 (2008) 7122–7126
7123
R
universally disappointing, presumably due to the more hydrophilic
nature of the disaccharide cf. monosaccharides, and its low
association constant with boronic acids.2 The binding constant of
lactose to phenylboronic acid is only slightly stronger than that
of sucrose,2 but the greater transport efficiency expected for 2-
(aminomethyl)-arylboronic acids gave us hope that lactose trans-
port with these compounds might be viable. Herein we report
the preparation and superior fructose, glucose and lactose trans-
port properties of the lipophilic 2-(aminomethyl)-phenylboronic
acid 1a.
R
N
B
OH
OH
1
a R =n-octyl
Figure 2. General structure of 2-(aminomethyl)-phenylboronic acids.
acid binds a sugar.3 The amine also improves the stability of bor-
onate esters7 and lowers the pKa of the boronic acid (w6.5 cf. 8.8 for
phenylboronic acid), so that tetrahedral forms are present at neu-
tral pH. Until recently the nature of the interaction between the
nitrogen and boron in these boronate esters was thought to involve
exclusively a dative B–N bond (3, Scheme 1). This was called into
question by Wang and Franzen8 and thorough investigations re-
cently published by Anslyn and co-workers9 have shown that in the
presence of protic solvents at neutral pH, the boronate ester exists
in equilibrium between 3 and a ‘solvent-inserted’ form (4 when the
solvent is water), as illustrated in Scheme 1. In aqueous solvent the
zwitterionic 4 tends to dominate.
2. Results
2.1. Synthesis of 1a
The 2-(aminomethyl)-phenylboronic acid 1a used in this study
was prepared from o-formylphenylboronic acid 6 and dioctylamine
as shown in Scheme 2. This involved the initial formation of the
imine 7.10 This reaction could be significantly accelerated by heat-
ing at reflux for 1 h in the presence of molecular sieves and catalytic
acetic acid. Careful reduction of 7 at 0 ꢁC with sodium borohydride
provided 1a in 64% isolated yield.
In the past, our best membrane transport results have been
obtained using a basic departure phase to ensure that the tetrahe-
dral transport mechanism was in operation. We were encouraged to
investigate the sugar transport properties of 2-(aminomethyl)-
arylboronic acids because their lower pKa may allow good transport
to occur at near-neutral pH and might also negate the need for
a quaternary ammonium salt in the membrane.
Our quest for a membrane-based technology for the production
of high fructose syrup has focused most of our energies on im-
proving fructose flux and selectivity over glucose. There are, how-
ever, other potential applications of boronic acid-containing
membranes. Lactose, for example, is used in the food, feed, phar-
maceutical and cosmetics industries. A major source of lactose is
cheese whey, but existing lactose manufacturing processes are time
consuming and complex, resulting in low lactose yield and/or poor
product purity and consistency. There is a need to develop new
technologies for the isolation and purification of lactose from feed
streams, particularly from dairy protein whey. Membrane tech-
nologies may have an impact here and we were interested to learn
if boronic acid-mediated transport through thin organic mem-
branes could be feasible with lactose. Sucrose is the only other di-
saccharide to be tested extensively in boronic acid assisted
membrane transport,1c and results with this sugar have been
2.2. Transport studies
The competitive transport of fructose and glucose promoted by
1a was first examined. The membrane consisted of 2-nitrophenyl
octyl ether (NPOE) supported on a thin sheet of porous poly-
propylene Accurel, as previously described in detail.11 The results
are shown in Table 1 and compared to that previously obtained
with a reference monoboronic acid 8 (Fig. 3),11 which has a similar
molecular weight to 1a.
Transport experiments were also performed with lactose pres-
ent in the departure phase (0.3 M). Initially, conditions that gave
the highest fluxes with the monosaccharides (condition (c) in Table
1) were used. With a carrier concentration of 50 mM in the
membrane, negligible lactose transport was detected even after
two days. This is similar to what has previously been observed
with sucrose.5 Increasing the carrier concentration in the mem-
brane to 250 mM, however, produced strong lactose transport
with 1a (89.4ꢂ10ꢀ8 mol mꢀ2 sꢀ1) and was found to be >20 times
the flux measured with the reference monoboronic acid 811
(4.1ꢂ10ꢀ8 mol mꢀ2 sꢀ1).
The nature of the boronate esters formed between 1a and lac-
tose was studied using electrospray ionisation mass spectrometry.
R
N
R
R
R
R
R
H
H
N
N
R
R
O
OH
OH
H2O
N
OH
O
OH
O
B
O
O
B
O
B
O
B
O
2
3
4
5
Scheme 1. The various protonation states of 2-(aminomethyl)-phenylboronate sugar esters.
O
N(n-C8H17 2
)
N(n-C8H17 2
)
Di-n-octylamine
H
MeOH reflux
NaBH4
H
cat. HOAc
mol. sieves
0 °C, 64%
B(OH)2
B(OH)2
B(OH)2
6
7
1a
Scheme 2. Synthesis of lipophilic 2-(aminomethyl)-phenylboronic acid sugar transporter (1a).

7124
P.J. Duggan et al. / Tetrahedron 64 (2008) 7122–7126
Table 1
repeated with 1a and fructose, a strong signal for a 1:1 boronate
ester was observed and only a very weak signal for a 2:1 boronate
ester. Interestingly, evidence for strong association between the
lactose boronate esters (e.g., 10) and chloride could be observed in
the low resolution negative ion spectrum (Fig. 4). Association of the
fructose monoester with chloride was also observed in its negative
ion spectrum. No strong evidence for the formation of ‘solvent-
inserted’ boronate esters similar to 4 was observed in any mass
spectra recorded with 1a.
Fructose and glucose fluxes through supported liquid membrane
b
Boronic acid
Conditionsa
Flux (10ꢀ8 mol mꢀ2 sꢀ1
)
Ratio of fluxes
Fructose
Glucose
1a
1a
1a
8c
a
b
c
c
3.2
14.2
84.1
42.3
0.54
1.10
18.1
10.6
7.0
12.9
4.7
4.0
a
[Boronic acid] in membrane¼50–52 mM, [sugars] in departure phase¼0.30 M
each, T¼298 K. Other conditions: (a) pH departure and receiving phases¼7.4 (0.1 M
sodium phosphate buffer); (b) same as (a) with addition of 1 equiv Aliquat 336 to
membrane; (c) same as (b) but with pH departure phase¼11.3 (0.1 M sodium
carbonate) and pH receiving phase¼6.0 (0.1 M sodium phosphate).
3. Discussion
b
Fluxes are the averages of 2–3 independent runs and uncertainties are estimated
The monosaccharide fluxes promoted by 1a alone when the
departure and receiving phases were set to pH 7.4 are unremark-
able and similar to those previously observed for simple mono-
boronic acids under similar conditions.12 The addition of Aliquat
336 (mainly trioctylmethylammonium chloride), however, in-
creased the fructose flux by more than four-fold, giving the highest
reported fructose flux promoted by a boronic acid under near-
neutral conditions. This is a significant result because when simple
boronic acid carriers are used, the addition of Aliquat at near-
neutral conditions has very little effect on saccharide fluxes.12 The
addition of Aliquat to the membrane had a weaker effect on glucose
flux, leading to one of the best fructose/glucose transport selec-
tivities (12.9) observed thus far.
to be ꢃ10%.
c
Data from Ref. 11.
O
N(n-C8H17)3 Br
NO2
O
O
OH
OH
B
B
OH
OH
8
9
Figure 3. Structure of reference boronic acid (8) used in this study and the boronic
acid–quaternary ammonium conjugate (9) used in a previous study.5
The ability of fructose and lactose boronate esters of 1a to
readily associate with chloride is evident from the negative ion
mass spectrometry experiments. This suggests that the source of
the enhanced fluxes observed when the departure phase has a pH
of 7.4 and Aliquat is added to the membrane results from an en-
hanced extraction of the sugar into the membrane due to an as-
sociation of sugar boronate esters similar to 3 with the chloride of
Aliquat.
The application of a pH gradient (departure phase pH 11.3; re-
ceiving phase 6.0) caused the fructose/glucose selectivity to drop to
the level normally seen for simple monoboronic acids under these
conditions, but the overall saccharide flux was still significantly
The positive ion high resolution spectrum of a methanol solution of
1a and lactose, made slightly basic by the addition of a trace of
aqueous ammonia, showed two strong peaks (m/z¼682.4329,
1021.7522), which together with their isotopic distribution are
characteristic of protonated 1:1 and 2:1 boronate esters formed
between 1a and lactose (see Supplementary data). A similar ex-
periment with phenylboronic acid in the place of 1a showed only
a weak signal for the lactose/PBA monoester and no evidence for
diester formation. When the mass spectrometry experiment was
10 – H+
680.4
OH
HO
HO
OH
O
O
O
10 + Cl-
O
HO
HO
716.4
O
B
N(n-oct)2
10
600
700
800
900
1000
Figure. 4. Low resolution negative ion electrospray mass spectrum of a methanol solution of 1aþlactose with suggested chemical structure of the detected ions.

P.J. Duggan et al. / Tetrahedron 64 (2008) 7122–7126
7125
enhanced. The fructose flux observed in this system is second only
5.2. Synthesis of 2-((N,N-di-n-octyl)aminomethyl)-
to that recorded with the boronic acid-quaternary ammonium
conjugate 9 (5.2ꢂ10ꢀ6 mol mꢀ2 sꢀ1).5 In the present case, where
the departure phase is quite basic, the extracted and transported
species is most likely similar to 5, associated with the lipophilic
ammonium ion of Aliquat. Here the superior fluxes most likely
result from the greater stability of the tetrahedral boronate esters
as well as a stronger association with the cation of Aliquat.
The effect of the ortho-aminomethyl group on boronic acid
promoted transport was even more dramatic with lactose. With
disaccharides, fluxes are typically very low, but increasing the
membrane concentration of the carrier and Aliquat to 250 mM
provided strong lactose fluxes. The positive ion mass spectrometry
results show facile formation of 2:1 boronate esters between 1a
and lactose, which explains the strong enhancement of lactose flux
with rising membrane carrier concentration. As carrier concentra-
tion is increased in the membrane, the equilibrium is shifted in
favour of the highly lipophilic boronate diesters, and hence ex-
traction into membrane is promoted. It is most likely that the in-
dividual lactose boronate esters are formed at 1,2-position of the
glucosyl moiety and either 3,4- or 4,6-position of the galactosyl
moiety;3b a preference for trigonal boronate ester formation at 4,6-
position of galactosides having been previously demonstrated in
organic solvents.13
phenylboronic acid (1a)
2-Formylphenylboronic acid (250 mg, 1.67 mmol) was com-
bined with dioctylamine (500 L, 400 mg, 1.66 mmol) in anhydrous
m
methanol (5 mL) containing one drop acetic acid catalyst. This was
stirred at reflux along with 3 Å molecular sieves under argon for
1 h. The reaction mixture was then brought to 0 ꢁC before the ad-
dition of NaBH4 (120 mg, 3.2 mmol) portionwise over half an hour.
The reaction mixture was further stirred while being allowed to
warm to room temperature over half an hour. After concentration
of the reaction mixture under vacuum, the residue was dissolved in
dichloromethane and filtered to produce a dark oil (670 mg) that
was shown by 1H NMR to contain both the desired product and
some unreacted dioctylamine. This oil was dissolved in hexanes
(50 mL) and shaken vigorously with 0.04 M HCl (50 mL). At the
phase boundary a white solid formed. The hexane layer was col-
lected and to this was added potassium carbonate (200 mg) and the
mixture was agitated before filtering. The filtrate was finally con-
centrated under vacuum to produce 1a (395 mg, 64%) as a colour-
less oil.
1H NMR (300 MHz, MeOD)
d
0.89 (t, 6H, J¼6.9 Hz, 2ꢂCH3), 1.30
(m, 20H, 10ꢂCH2), 1.65 (m, 4H, 2ꢂCH2CH2N), 2.92 (t, 4H, J¼8.4 Hz,
2ꢂCH2CH2N), 4.19 (s, 2H, CH2–Ar), 7.13 (d, 1H, J¼4.2 Hz, ArH), 7.24
(m, 2H, 2ꢂArH), 7.67 (d, 1H, J¼6.9 Hz, ArH) ppm. 13C NMR (75 MHz,
MeOD) d 14.5, 23.7, 24.4, 27.8, 30.2, 30.4, 32.9, 51.7, 61.0, 127.3, 127.4,
4. Conclusions
128.8,131.6,136.1 ppm. m/z (þve ESI): (33% aq MeCN/NH4OH) 376.2
(MþH)þ, 715.7 (dimeric anhydrideþH)þ, 1073.3 (trimeric
anhydrideþH)þ. HRMS (þve FTMS): calcd for C23H43BNO2,
375.3418; found, 375.3413.
A
lipophilic 2-(aminomethyl)-phenylboronic acid (1a) was
prepared and used to transport saccharides across a thin supported
liquid membrane consisting of 2-nitrophenyl octyl ether. Under
near-neutral conditions, and in the absence of any other additives,
observed transport fluxes were similar to those obtained with other
monoboronic acids. However, when Aliquat 336 was added to the
membrane, the highest reported fructose flux promoted by
a monoboronic acid under near-neutral conditions and one of the
highest fructose/glucose transport selectivities (12.9), were ob-
served. The results of mass spectrometry experiments suggest that
the flux enhancement produced by Aliquat reflects an association
between the zwitterionic saccharide ester and chloride. The ap-
plication of a pH gradient across the membrane resulted in one of
the highest fructose fluxes ever recorded. The superior transport
properties of 1a were further demonstrated by transport experi-
ments conducted with the disaccharide, lactose. A strong concen-
tration effect on lactose transport and further mass spectrometry
evidence point to the involvement of 2:1 boronate/lactose esters in
this transport process. This suggests that bis(aminoboronate)
compounds of proper spacing may be highly effective lactose
transporters.
5.3. Transport experiments
Apparatus and methods used in the transport of fructose and
glucose have previously been described in detail.11 Lactose con-
centrations in the receiving phase were measured using a similar
enzymatic method to that used for measuring glucose concentra-
tion, with the addition of b-galactosidase to catalyse the hydrolysis
of lactose to glucose and galactose.
Acknowledgements
The Australian Research Council is acknowledged for the award
to P.J.D. and B.D.S. a Linkage Grant, which funded this work. Regine
Stockmann, Food Sciences Australia, Werribee, Vic. is acknowl-
edged for alerting us to the importance of lactose in dairy whey.
Carl Braybrook and Jo Cosgriff, CSIRO Molecular and Health Tech-
nologies, and Hoan The Vu, Griffith University, are thanked for their
assistance with mass spectrometry.
5. Experimental
5.1. General
Supplementary data
Exact positive ion mass spectra of lactose boronates formed
with 1a are provided. Supplementary data associated with this
article can be found in the online version, at doi:10.1016/j.tet.
2008.05.052.
Commercial starting materials (Aldrich) and reagents were used
without further purification. 1H and 13C NMR spectra were recorded
on a Bruker Avance 300 MHz spectrometer. Methanol was distilled
and stored over 3 Å molecular sieves prior to use. Chemical shifts
References and notes
are expressed as parts per million (ppm,
d) relative to MeOD
(3.3 ppm in 1H and 49.0 ppm in 13C). Low resolution positive and
negative ion electrospray mass spectra were acquired with a VG
Platform mass spectrometer using a cone voltage of 50 V. The
source was maintained at 80 ꢁC and a solvent flow rate of 0.04 ml/
min was used. High resolution positive ion mass spectra was ac-
quired on a Bruker Daltonics Apex III 4.7e Fourier Transform Mass
Spectrometer fitted with an Apollo API source.
1. (a) Smith, B. D. Supramol. Chem. 1996, 7, 55; (b) Smith, B. D.; Gardiner, S. J. Adv.
Supramol. Chem. 1999, 5, 157; (c) Duggan, P. J. Aust. J. Chem. 2004, 57, 291.
2. Springsteen, G.; Wang, B. Tetrahedron 2002, 58, 5291.
3. (a) James, T. D.; Sandanayake, K. R. A. S.; Shinkai, S. J. Chem. Soc., Chem. Commun.
1994, 447; (b) James, T. D.; Shinkai, S. Top. Curr. Chem. 2002, 218, 160; (c) James,
T. D.; Phillips, M. D.; Shinkai, S. Boronic Acids in Saccharide Recognition; RSC:
Cambridge, 2006; (d) Fang, H.; Kaur, G.; Wang, B. J. Fluoresc. 2004, 14, 481.
4. Altamore, T. M.; Barrett, E. S.; Duggan, P. J.; Sherburn, M. S.; Szydzik, M. L. Org.
Lett. 2002, 4, 3489.

7126
P.J. Duggan et al. / Tetrahedron 64 (2008) 7122–7126
5. Gardiner, S. J.; Smith, B. D.; Duggan, P. J.; Karpa, M. J.; Griffin, G. J. Tetrahedron
1999, 55, 2857.
9. Zhu, L.; Shabbir, S. H.; Gray, M.; Lynch, V. M.; Sorey, S.; Anslyn, E. V. J. Am. Chem.
Soc. 2006, 128, 1222.
6. Wulff, G. Pure Appl. Chem. 1982, 54, 2093.
10. (a) Gray, C. W., Jr.; Houston, T. A. J. Org. Chem. 2002, 67, 5426; (b) Gray, C. W., Jr.;
Walker, B. T.; Foley, R. A.; Houston, T. A. Tetrahedron Lett. 2003, 44, 3309.
11. Duggan, P. J.; Szydzik, M. L. Aust. J. Chem. 2003, 56, 17.
12. Paugam, M.-F.; Riggs, J. A.; Smith, B. D. Chem. Commun. 1996, 2539.
13. Morin, G. T.; Paugam, M.-F.; Hughes, M. P.; Smith, B. D. J. Org. Chem.1994, 59, 2724.
7. (a) Lauer, M.; Wulff, G. J. Chem. Soc., Perkin Trans. 2 1987, 745; (b) Smith, B. M.;
Owens, J. L.; Bowman, C. N.; Todd, P. Carbohydr. Res. 1998, 308, 173.
8. (a) Franzen, S.; Ni, W.; Wang, B. J. Phys. Chem. B 2003, 107, 12942; (b) Ni, W.;
Kaur, G.; Springsteen, G.; Wang, B.; Franzen, S. Bioorg. Chem. 2004, 32, 571.