Synthesis and characterisation of N-(3-dihydroxyborylphenyl)-5-mercapto-pentanamide: A novel self-assembling vicinal diol receptor

Article abstract of DOI:10.1039/a906542c

A novel self-assembling vicinal diol receptor, N-(3-dihydroxyborylphenyl)-5-mercaptopentanamide, has been synthesised via a three step procedure, characterised by NMR, mass spectroscopy and FTIR spectroscopy and shown to be functionally active by contact

Full text of DOI:10.1039/a906542c

Synthesis and characterisation of N-(3-dihydroxyborylphenyl)-5-mercapto-
pentanamide: a novel self-assembling vicinal diol receptor
Karl D. Pavey,a Cedric J. Olliff,*b the late Jim Bakerb and Frank Paula
a
Novel Methods Group, CASS, SmithKline Beecham Pharmaceuticals, NFSP (North), Third Avenue, Harlow, Essex,
UK CM19 5AW
b Department of Pharmacy and Biomolecular Science, Cockcroft Building, University of Brighton, Lewes Road,
Brighton, East Sussex, UK BN2 4GJ. E-mail: cj01@brighton.ac.uk
Received (in Oxford, UK) 9th August 1999, Accepted 27th September 1999
A novel self-assembling vicinal diol receptor, N-(3-di-
hydroxyborylphenyl)-5-mercaptopentanamide, has been
synthesised via a three step procedure, characterised by
NMR, mass spectoscopy and FTIR spectroscopy and shown
to be functionally active by contact angle analysis, surface
plasmon resonance and QCM.
Analysis of 4 by 1H NMR† spectroscopy showed that > 85%
of the sample was the desired thiol boronic acid. A small
amount of the 3-aminophenylboronic acid starting material was
also present. Further characterisation by LC-MS‡ and FTIR§
analysis gave molecular weight and structural information
supporting the NMR data (Table 1).
Table 1 FTIR data for 4
The recent explosion in biosensor technology has led to the need
for a greater variety of surface bound receptor systems. Thiol
monolayer self-assembly is a technique of growing importance
in many fields, particularly for the protection1 or modification2
of surfaces and the production of functionalised layers in sensor
systems.3–5. The chemistry is versatile and robust, with a variety
of headgroups attached to long-chain, thiol-terminated back-
bones. It has been suggested that the development of this
technology may be hampered by a lack of new receptor
systems.6 Introduction of novel headgroup chemistry would
continue to expand the uses of this technology. Here we report
the synthesis of a new self-assembling vicinal diol receptor,
based upon a thiol-modified boronic acid.
The interactions of sugars has been of interest to many with
an increase in the use of boronic acids as receptors, however
much of the work has taken place in free solution7 or at air–
water interfaces.8 Boronic acids have long been known to
interact with heavily hydroxylated species such as glycerol.9
The semi-specific attachment point for vicinal diol groups,
based upon the orientation of two adjacent hydroxy groups
within a molecule, is covered in an excellent review by James
et al.10 Many saccharides contain hydroxy groups in the correct
orientation and form stable cyclic esters when brought into
contact with boronic acids.11 Phenylboronic acids are known to
form five-membered rings with cis-1,2-diol groups and less
stable six-membered rings with the trans-isomer.7 The synthe-
sis of a self-assembling molecular layer capable of binding a
wide variety of diol-containing moieties may open a route to
producing a range of more specific multi-layer sensors.
Reaction of 3-aminophenylboronic acid 1 with 6-bromo-
pentanoyl chloride in ethanolic solution (Scheme 1) yielded
3-(5-bromopentanoylamino)phenylboronic acid 2. Further reac-
tion with thiourea in methanolic solution over four days gave
thioamidine 3, which when treated with base prior to acid-
ification was reduced to N-(3-dihydroxyborylphenyl)-5-mer-
captopentanamide 4.
Position/cm21
Transmittance (%) Assignment
3322
2934
2361
1663
1538
1344
4.20
41.7
79.0
3.20
B–OH
CH2
CH3 (Possible S–H)
meta substituted aromatic
CH2–NH
5.70
5.80
C–N
The self-assembly characteristics of 4 were investigated by
several techniques. Time resolved, sessile drop contact angle
measurements of 2 ml water droplets were recorded on 38 nm
gold films evaporated onto polished microscope slides. Initial
contact angles for water droplets of 64 ± 3° (N = 6) were noted.
Upon exposure to a 1 mM ethanolic solution of 4 in a clean
environment, the contact angle was observed to decrease with
time to 37 ± 2° (N = 6) after 2.5 h (Fig. 1). This is indicative of
the rapid self-assembly mechanisms observed in simple long
chain thiols on gold surfaces12 and strongly suggested that
surface modification had taken place via the widely accepted
thiol–gold self-adsorption route,13 the boronic acid headgroups
rendering the surface more hydrophilic.
Characterisation of the self-assembly in real time using a
Kretschmann configuration surface plasmon resonance (SPR)
instrument, also employing 38 nm gold films as the SPR
substrate, gave a 0.31º dual rate increase in SPR angle upon a
2.5 h exposure, at a concentration of the compound in ethanolic
solution equal to that used for the contact angle measurements.
Scheme
1 Reagents and conditions: i, 6-bromopentanoyl chloride,
NaHCO3, H2O–EtOH, 25 °C, 2 h, stirred; ii, thiourea, MeOH, 25 °C, 4 days,
stirred under N2; iii, NaOH, MeOH, 25 °C, 4 days, stirred under N2; iv,
HCl.
Fig. 1 Plot of sessile drop, water contact angle vs. time of immersion for a
38 nm gold coated microscope slide treated with a 1 mM ethanolic solution
of 4 (droplet size 2 ml, T = 25 °C, N = 6 ± standard deviation).
Chem. Commun., 1999, 2223–2224
This journal is © The Royal Society of Chemistry 1999
2223

wash were made. Fig. 2 shows strong binding to the NAD
treated slides, with a residual SPR angle increase of 47 ± 3° (N
= 3) and very little evidence of desorption. No overall change
in SPR angle was observed for injection of LDH if the NAD was
not present. This would support the binding of NAD via its diol
groups to the surface, leaving the binding regions, at the adenine
and nicotinamide units, available for LDH and a range of other
dehydrogenases.15
In summary, this report describes a novel self-assembling
boronic acid derivative with the potential to specifically interact
with vicinal diol groups found within a range of nucleotides,
saccharides, antibiotics and other systems. It has been shown
that with a clean environment, formation of a correctly
orientated layer on the surface is achieved and that vicinal diol
containing species attach strongly only to the modified surface.
Further, enzyme–cofactor binding has been demonstrated as
part of a multilayer system, potentially opening new avenues for
enzyme studies, via a relatively simple attachment route,
applicable to a range of novel analysis tools such as SPR and
QCM.
Fig. 2 SPR angle change for the binding of NAD (1 mg m21) to a gold
substrate previously treated with 4 followed by lactate dehydrogenase (1 3
1026 M) (flow rate = 4 ml min21, T = 30 °C, pH 7.2 Sorensons PBS).
Increases in SPR angle equate to material attachment to the
substrate surface. Non-linear regression using a four parameter,
double exponential rise relationship to the SPR data gave R =
0.99 and rate constants, K1 = 1.1 3 1023 s21 and K2 = 6.91
3 1023 s21.The value of the measured K1 is of the order found
by Debono et al.,14 (4.3 3 1023 s21) with SPR for the
adsorption of dodecanethiol from ethanolic solution at an equal
concentration. These results follow the distinct two step
adsorption process found by DeBono et al.14 and Bain et al.12
The results show a very fast initial step lasting a few minutes
giving rise to an 80–90% monolayer coverage, followed by a
slower step lasting between minutes and hours depending upon
the structure of the thiol, in which time the monolayer
reorganises and completes its formation.
A similar two stage adsorption profile was observed upon the
exposure of a 10 MHz lapped quartz crystal, thickness shear
mode sensor with gold electrodes to a 1 mM ethanolic solution
of the compound. A total frequency decrease of 126 Hz was
recorded over an 8 h period for a single electrode in contact with
the solution. The increased time required for the adsorption
process to reach equilibrium is thought to be due to surface
roughness; 2–3 mm troughs are found in the lapped crystal
surface compared to the sub-micron polished surface used for
SPR analysis.
Surface functionality of the adsorbed boronic acid SAM was
further confirmed by contact angle and SPR experiments. Gold
coated slides, previously exposed to the monolayer forming
solution, with contact angles measuring 37 ± 2° (N = 6), were
placed in a 1 mg ml21 solution of nicotinamide adenine
dinucleotide (NAD) in Sorenson’s buffer, pH 7.2. Following a
15 min incubation at 30 °C the slides were removed, washed
with further buffer, dried and the water contact angle measured
as described previously. A further decrease in angle was
recorded [27 ± 1.0° (N = 6)], suggesting that binding of the
NAD to the surface may have occurred.
Real time analysis of NAD binding (Fig. 2) was carried out
using SPR. NAD (1 mg ml21) in a buffer stream was passed
over a boronic acid treated gold slide (4 ml s21). A residual SPR
angle increase of 40 ± 2 millidegrees, (N = 3) verified that
NAD had bound to the boronic acid layer. A similar profile was
observed for lactose and maltose with SPR angle increases of 32
± 3 (N = 3) and 24 ± 2° (N = 3) respectively. No residual
change in SPR angle was observed when untreated gold
surfaces were similarly challenged.
The authors wish to dedicate this paper to the memory of Dr
Jim Baker who passed away prior to submission. We thank the
EPSRC and SmithKline Beecham for financial support under
the CASE award scheme, Award #G77.
Notes and references
†
Selected data for 4: dH[250 MHz, CDCl3–DMSO-d6 (1+1)]1.3
(CH3CO2H), 2.1, 2.3, 2.5 (CH2), 3.0 (H2O), 2.6, 3.4 (CH2S), 7.1, 7.4, 7.6
(dd, CH aromatic), 9.0 (NH), 9.1 (CH3CO2H).
‡ LC/MS (Finnigan LCQ) yielded a single species of m/z (H+) 254.300 at a
retention time of 15.61 min comparable to the calculated mass of 254.101
(C11H16BNO3S).
§ FTIR analysis on a Perkin-Elmer 1720 spectrophotometer, resolution 4
cm21, 20 scans.
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After treatment of the SPR slides with NAD, injections of
lactate dehydrogenase (1 3 1026M, LDH), followed by a buffer
Communication 9/06542C
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Chem. Commun., 1999, 2223–2224