Investigation of enzyme activity by SERRS using poly-functionalised benzotriazole derivatives as enzyme substrates

Article abstract of DOI:10.1039/b603439j

New methods of measuring biologically relevant concentrations of enzymes are necessary to allow greater understanding of biological systems. We have previously shown that aryl azo benzotriazolyl alkyl esters can act as enzyme substrates, with the progress

Full text of DOI:10.1039/b603439j

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Investigation of enzyme activity by SERRS using poly-functionalised
benzotriazole derivatives as enzyme substrates
Andrew M. Ingram, Kirsten Stirling, Karen Faulds, Barry D. Moore and Duncan Graham*
Received 7th March 2006, Accepted 16th June 2006
First published as an Advance Article on the web 3rd July 2006
DOI: 10.1039/b603439j
New methods of measuring biologically relevant concentrations of enzymes are necessary to allow
greater understanding of biological systems. We have previously shown that aryl azo benzotriazolyl
alkyl esters can act as enzyme substrates, with the progress of the reaction being monitored using
SERRS (see Nat. Biotechnol., 2004, 22, 1133, ref. 1). This is a wholly novel analytical application of
SERRS, and the low detection levels of the technique allow for an ultra-sensitive enzyme assay. Masked
enzyme substrates are used that are invisible to SERRS until enzymatic hydrolysis. Turnover of the
substrate by the enzyme leads to the release of the surface-seeking dye necessary for SERRS, and
intense signals are produced. Here we report an improved synthesis of 2H-benzotriazolyl alkyl esters
via nucleophilic substitution of a chloromethyl ester by benzotriazolyl azo dyes, giving up to a ten-fold
increase on previously reported yields. Introduction of electron-withdrawing groups to the
benzotriazole ring allows control over the SERRS properties of the compounds. This is of great
significance in expanding the synthetic flexibility and subsequently the fundamental use of these
compounds as ultra-sensitive and selective reporters of enzyme activity.
halogen displacement in alkyl halides,15 hydroxyl displacement
in alcohols or alkoxy displacement in acetals.16,17 Benzotriazole
Introduction
can also add across carbonyls in aldehydes,18 to imines19 and
also to enamines.20 For the N-alkylation of benzotriazole with
alkyl halides, sodium hydroxide in DMF,15 sodium alkoxide
bases,21,22 sodium hydride bases,23 micellar reactions24 and phase
transfer conditions25,26 have all been employed. A mixture of 1H,
2H, and 3H isomers can potentially be formed, although in a
symmetrically substituted benzotriazole, the 1H and 3H isomers
will be degenerate. This isomerisation can be affected, and to a
certain extent controlled, by a number of factors.25,27,10
Isomerisation has proven essential in dictating the extent of
observed masking of the SERRS. 1H alkyl esters have previously
shown considerably higher SERRS than the 2H isomers, as
illustrated in Fig. 1. This could be governed by a number of factors.
It may be a steric hindrance factor or it may be an electronic
factor. It is known28 that 2H isomers of benzotriazole are weaker
The uses of benzotriazole bestride a wide range of chemical
disciplines. Described as a ‘tame halogen’, it has been extensively
employed as a synthetic auxiliary, and used in a number of
elegant syntheses.2,3 Benzotriazole derivatives are also of use as
photostabilisers4 and anti-corrosion inhibitors.5 The latter is made
possible due to the strong affinity benzotriazole has for metal
surfaces, and it is this property that makes benzotriazole and
its derivatives excellent analytes for SERS and SERRS.6–9 The
surface-enhanced Raman sensitivity of a benzotriazole moiety is
reliant on the capacity of the benzotriazole to bind to a silver or
gold surface. Through N-substitution of the benzotriazole species
we can prevent, wholly or partially, this binding to the metal
surface and thus mask the SERRS activity of the compound.
Through judicious choice of this N-substituent, it is possible
to prepare a SERRS-inactive species that can be chemically, or
in our case enzymatically cleaved to generate a SERRS-active
species. Given the high sensitivity of SERRS,10 this technique
has shown the capacity to monitor enzyme turnover at very low
substrate concentrations. We have recently demonstrated that a
2-benzotriazol-2-yl alkyl ester azo dye can act as an enzyme
substrate, with the rate of enzymatic reaction being monitored
by SERRS.1
The synthetic organic utility of benzotriazole has been well
reported and there is considerable scope available for N-
functionalisation of the benzotriazole moiety. As a synthetic
auxiliary, it has been used for a number of elegant purposes; in
syntheses of ketones,11 esters,12 ethers13 and amides14 to name only
a few. N-Alkylated benzotriazole derivatives can be formed from
Centre for Molecular Nanometrology, WestCHEM, Department of Pure
and Applied Chemistry, University of Strathclyde, 295 Cathedral Street,
Glasgow, G1 1XL, UK; Tel: +44 0141 548 4701
Fig. 1 Superimposed SERRS spectra of dye 2, the 1H/3H isomers and
the unmasked dye.
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Brønsted bases than their 1H counterparts, and it would not be
unreasonable to suggest they are also weaker Lewis bases.
From our previous study, the synthesis of benzotriazolyl alkyl
esters, as per Katritzky et al.,29 yielded a very small amount of
the desired 2H isomer, with the 1H predominant. This method
involved acylation of the benzotriazole, then reaction of the N-acyl
benzotriazole with formaldehyde in the presence of base. We can
now report an improved synthetic route to these compounds, with
an up to ten-fold increase in yield of the 2H isomers. Furthermore,
we have now demonstrated that 1H-benzotriazolyl alkyl esters can
completely mask SERRS by introduction of electron-withdrawing
groups to the benzotriazole ring. This opens up the range of
substrates that can be used in this approach and provides further
understanding on the requirements for the masking of these dyes
in terms of SERRS activity.
The starting benzotriazolyl dyes for 2, 3 and 4 were prepared
according to literature procedures.1,32,33
The N-alkylation of the benzotriazolyl dyes was carried out
by reaction of the appropriate sodium benzotriazolate with
chloromethyl ester 1, as outlined in Scheme 2. Acetone was found
to be the most effective solvent in terms of overall yield, but
acetonitrile, DMF and DMSO were also used with success. NMR
analysis of the reactions indicated the presence of all 3 isomers,
with the ratio 1 : 2.5 for the 2H and combined 1H + 3H isomers
respectively. The 1H and 3H isomers were not isolated and their
structural isomerism not determined, but crude NMR data has
been previously obtained.1 Trace amounts of N-acyl benzotriazole
were also formed. The yield of benzotriazolyl alkyl ester formed
by this route is comparable to the yields reported in literature,29
but the yield of 2H isomer is higher. Outside of our own previous
work,1 2H-benzotriazolyl alkyl esters had only been reported as
a trace amount from 1H syntheses. Phase transfer conditions,
micellar reactions, or organic bases in acetone all led to a reduced
proportion of 2H isomer. A summary of the compounds prepared
and their yields is contained in Table 1.
Results and discussion
2H-Benzotriazolyl alkyl ester synthesis
For this study, benzotriazolyl alkyl esters were prepared by
nucleophilic substitution of chloromethyl ester 1, using a sodium
benzotriazolate as the nucleophile. A number of factors were found
to increase the relative yield of 2H isomer; the use of acetone as a
solvent, preformation of the benzotriazole anion and anhydrous
conditions. Chloromethyl ester 1 was prepared by alkylation of
the relevant carboxylic acid using chloromethyl chlorosulfate30,31
(CMCS), under phase transfer conditions, in yields consistently
higher than 90% (Scheme 1).
Scheme 2 Reagents and conditions: (i) Acetone.
SERRS studies. In the first instance, the masked compounds
were interrogated to ascertain that the compounds were indeed
SERRS-inactive.
From Fig. 1 it can be seen that the 2H isomer of dye 2 gives better
masking of SERRS. All spectra recorded were performed using
single-scan measurements, 1 second spectral acquisition times with
514.5 nm excitation.
Dye 2 was tested against a range of lipases, which were all
succesful in hydrolysing the substrate, but with varying intensity
of SERRS response. The results are illustrated in Fig. 2.
It can be seen from Fig. 2 that the Candida-derived enzymes
seemingly hydrolyse the substrate more rapidly than those from
Pseudomonas. When dye 3 was tested against the lipase from
Scheme 1 Reagents and conditions: (i) CMCS, NaHCO3, Bu4NHSO4,
DCM–H2O.
Table 1 Summary and yield of 2H-benzotriazolyl alkyl esters. Reported yield of 1H + 3H taken from crude NMR only
Compound
R1
R2
H
Yield of 2H (%)
Yield of 1H + 3H (%)
2
21
65
3
NH2
23
65
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Scheme 3 Reagents and conditions: (i) (S)-iodomethyl 3-phenylbutanoate,
K2CO3, DMF, rt, 16% (65% conversion to 1H/3H).
Fig. 2 SERRS spectra of 2 before and after treatment with a selected
range of lipases. All experiments were carried out in situ in silver colloidal
solution, with the substrate present at 1 × 10−7 M and the enzyme at
0.002 mg ml−1. The spectrum was recorded at 1200 seconds after lipase
addition. For ease of interpretation the enzymes are listed in decreasing
order of size of SERRS response. a) Candida aspergillus, b) Candida
cylindracea, c) Candida rugosa, d) Pseudomonas stutzeri, e) Pseudomonas
cepacia, f) Pseudomonas fluorescens, g) no enzyme added.
Fig. 3 Superimposed SERRS spectra of 1H isomers of 2 and 4. The
electron-deficient benzotriazole 4 gives greater blocking of SERRS.
Pseudomonas cepacia, no appreciable turnover could be observed
by SERRS. It is suggested that by moving the bulky naphthylazo
group from the 5 to the 4 position of the benzotriazole, the ester
functionality is hindered from accessing the active site of the
enzyme.
cepacia, i.e. addition of enzyme causes the substrate to become
SERRS-active. The parent or “unmasked” dyes of 2 and 4 give
similar SERRS spectra, as can be seen in Fig. 4, with the main
difference being that a single peak is observed at 1125 cm−1 for dye
4, whereas dye 2 has two bands at 1110 and 1137 cm−1.
Electron-deficient benzotriazolyl alkyl esters
The occurrence of SERRS is dependent on binding to an
appropriate metal surface, in this case colloidal silver. As part of
the same study, an electron-deficient 3H-benzotriazolyl alkyl ester
4 was prepared, to investigate the effect the withdrawing group
would have on the binding to silver.34
This approach was taken in an attempt to produce masked
1H/3H substrates that were as well masked to SERRS as the
2H compounds. This would allow for simpler chemistry towards
our targets, as more synthetic chemistry is known with regards to
1H compounds. Accordingly, a nitro group was introduced to the
aromatic ring. However, this caused a decrease in nucleophilicity
of the benzotriazole anion, and subsequently reaction with the
chloromethyl ester as described in Scheme 1 did not occur. Fur-
thermore, the anion was no longer soluble in acetone. A modified
alkylation procedure was therefore used, with the chloromethyl
ester being converted to the iodomethyl analogue, and DMF used
as the solvent. No 2H isomer was observed using these conditions,
with the 3H isomer being isolated in 20% yield, the structure of
which was confirmed by X-ray crystallography. The 1H isomer
was observed by NMR analysis of the crude reaction mixture, in
∼20% conversion, but was not isolated to a suitably high degree
of purity for characterisation. The synthetic route and yield of the
compound are illustrated in Scheme 3.
Fig. 4 Superimposed spectra of the ‘unmasked’ dyes from 2 and 4.
As has been previously stated, the occurrence of SERRS is
dependent on binding to an appropriate metal surface. In the 2H
compounds discussed above, this metal binding is prevented to a
greater extent than with the 1H counterparts, thus lower SERRS
is obtained. It is proposed that the reduced SERRS from electron-
poor benzotriazol-1-yl alkyl esters is because of the inductive effect
of the nitro group. The basicity of the triazole ring is reduced,
which prevents the binding to the silver surface, and subsequently
prevents SERRS to a greater extent than the de-nitro analogue.
Conclusions
A new synthesis of 2H-benzotriazolyl alkyl esters has been
reported, simplifying the preparation of such compounds and
expanding the scope of compounds that can be prepared. The
introduction of an electron-withdrawing group to the phenyl
ring, in this case a nitro group, has reduced the affinity of 1H-
benzotriazolyl alkyl esters for the silver surface, and subsequently
SERRS studies. Upon interrogation of dye 4, the SERRS
effect is completely masked. The contrast can be seen when the
same experiment is carried out with a mixture of 1H and 3H
isomers of 2, the de-nitro analogue (Fig. 3). Dye 4 gives a similar
response to 2 upon treatment with the lipase from Pseudomonas
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the compounds appear to be SERRS-invisible for the first time.
The nitro group seemingly does not affect enzyme selectivity. With
all 3 isomers from the reaction now being suitable precursors for
the SERRS analyses, the yield and efficiency of the reaction is
significantly increased.
(1H, d, J 6.9, CH), 2.71 (1H, d, J 6.9, CH), 3.31 (1H, sept, J 6.9,
CH), 5.64 (1H, d, J 6.8, CH), 5.68 (1H, d, J 6.8, CH), 7.20–7.33
(5H, m, ArH); dC (100 MHz; CDCl3) 21.9, 36.5, 68.7, 126.9, 128.8,
145.3, 170.5; m/z 230.0945 ([M + NH4]+ C11H17ClNO2 requires
230.0942); [a]2D0 +62.2 (c 1, MeCN)
3 - (S) - Phenylbutyric acid 5 - (4 - dimethylaminonaphthalen - 1-
ylazo)benzotriazol-2-ylmethyl ester 2. [4-(3H-Benzotriazol-5-
ylazo)naphthalen-1-yl]dimethylamine (0.250 g, 0.8 mmol) was
treated with 1.038 M NaOH solution (0.77 ml, 0.8 mmol). The
water was removed at reduced pressure and the residue dissolved in
anhydrous acetone (10 ml). (S)-Chloromethyl 3-phenylbutanoate
(0.170 g, 0.8 mmol) was then added, and the reaction mixture
left to stir for 2 h at room temperature. The solvent was then
removed at reduced pressure and the residue purified by column
chromatography, eluting with 20% ethyl acetate in hexane. The
2H isomer elutes more rapidly than the other isomers. The
title compound was furnished as a red oil (0.12 g, 21%), and
had spectroscopic data consistent with that already published.1
(Found: C, 70.92; H, 5.75; N, 17.01;.C29H28N6O2 requires C, 70.71;
H, 5.73; N, 17.06); dH (400 MHz; CDCl3) 1.30 (3H, d, J 7.0, CH3),
2.69 (1H, d, J 6.7, CH), 2.76 (1H, d, J 6.7, CH), 3.09 (6H, s, CH3),
3.31 (1H, sept, J 7.3, CH), 6.53 (1H, d, J 9.9, CH), 6.59 (1H, d, J
9.9, CH), 6.99 (1H, dd, J 7.6, ArH), 7.23–7.11 (6H, m, 6 × ArH),
7.61 (1H, dd, J 7.0, ArH), 7.68 (1H, dd, J 6.9, ArH), 7.98 (2H,
d, J 9.0, ArH), 8.25 (1H, d, J 7.6, ArH), 8.51 (1H, s, ArH), 9.04
(1H, d, J 7.6, ArH); m/z 493.2344 ([M + H]+. C29H29N6O2 requires
493.2347); [a]2D0 +60.2 (c 1, MeCN).
Experimental
Preparation of solutions for SERRS analysis
We prepared 0.001% wt/vol poly(L-lysine) in distilled water for
use as an aggregating agent. Enzyme solutions were prepared at
0.1 mg ml−1 in water. Stock solutions of the substrate were prepared
in acetonitrile at a concentration of 10−3 M, and subsequent
dilutions were made using distilled water to a concentration of
1 × 10−6 M.
Raman instrumentation
A Renishaw Mark III probe system with a Spectra Physics 362C,
15 mW, argon ion laser was used for the collection of spectra using
an excitation wavelength of 514.5 nm.
Reaction monitoring
All reactions were carried out in disposable plastic cuvettes (300–
800 nm transmission range) and analysis was done in situ. Silver
colloid (500 ll), de-ionised water (500 ll) and poly(L-lysine) (20 ll
of 0.001%) were added to a cuvette and allowed to aggregate for
15 min. Enzyme solution (100 ll) and substrate solution (100 ll)
were then added to the aggregated colloid, the contents were mixed
and the spectra accumulated. A spectral acquisition time of 1 s was
used. Further details on experimental conditions can be obtained
from our previous publication.1
3-(S)-Phenylbutyric
acid
5-amino-4-(naphthalen-1-ylazo)-
benzotriazol-2-ylmethyl ester 3. 5-Amino-4-(naphthalen-1-
ylazo)benzotriazole (0.100 g, 0.34 mmol) was treated with
1.038 M NaOH solution (0.33 ml, 0.34 mmol). The water
was removed at reduced pressure and the residue dissolved in
anhydrous acetone (10 ml). (S)-Chloromethyl 3-phenylbutanoate
(0.070 g, 0.34 mmol) was then added, and the reaction mixture
left to stir for 2 h at room temperature. The solvent was then
removed at reduced pressure and the residue purified by column
chromatography, eluting with 20% ethyl acetate in hexane. The
2H isomer elutes more rapidly than the other isomers. The title
compound was furnished as a red oil (0.04 g, 23%). (Found:
C, 68.88; H, 5.39;.C27H24N6O2 requires C, 69.81; H, 5.21); dH
(400 MHz; CDCl3) 1.30 (3H, d, J 7.7, CH3), 2.67 (1H, d, J 7.2,
CH), 2.72 (1H, d, J 7.2, CH), 3.30 (1H, sept, J 7.2, CH), 6.50
(1H, d, J 10.1, CH), 6.56 (1H, d, J 10.1, CH), 7.00 (1H, d, J 7.6,
ArH), 7.23–7.11 (6H, m, ArH), 7.59 (1H, dd, J 7.4, ArH), 7.64
(1H, dd, J 7.4, ArH), 7.77 (1H, d, J 9.20, ArH), 7.90–7.95 (3H,
m, ArH), 8.70 (1H, d, J 7.8, ArH); dC (100 MHz; acetone) 22.0,
37.0, 42.7, 75.4, 112.0, 123.6, 124.2, 124.4, 126.7, 127.0, 127.4,
128.8, 129.1, 130.0, 131.3, 135.4, 141.4, 144.0, 146.3, 149.7, 170.9;
m/z 465.2027 ([M + H]+. C27H25N6O2 requires 465.2034); [a]D20
+61.0 (c 1, MeCN).
Chemical synthesis
Unless otherwise stated, all chemical precursors were purchased
from Sigma. Chloromethyl chlorosulfate was purchased from
Acros. The precursor dyes for 2, 3 and 4 were prepared in
accordance with previously published work.1,32,33
1H NMR and 13C NMR were recorded on a Bruker DPX
400 MHZ spectrometer with the appropriate solvent peak as a
reference. J values are quoted in Hertz. Elemental analyses were
performed as by the University service using a Perkin–Elmer 240
elemental analyser. Mass spectrometry data was provided by the
EPSRC Mass Spectrometry Service Centre, Swansea.
(S)-Chloromethyl 3-phenylbutanoate 1. (S)-3-Phenylbutyric
acid (0.335 g, 2 mmol), sodium bicarbonate (0.840 g, 10 mmol)
and tetra-n-butylammonium hydrogen sulfate were dissolved in
water (20 ml). Dichloromethane (20 ml) was added and the
mixture left to stir vigorously at 0 ◦C for 15 min, at which
point chloromethylchlorosulfate (0.5 g, 3 mmol) was added, with
continuous overnight stirring at room temperature. The organic
phase was separated, washed with brine, dried over sodium sulfate
before filtration and removal of the solvent by evaporation. The
residue was purified by column chromatography, eluting with 20%
ethyl acetate in hexane to afford the title compound as a colourless
oil (0.4 g, 94%). (Found: C, 61.88; H, 6.15; C11H13ClO2 requires C,
62.12; H, 6.16); dH (400 MHz; CDCl3) 1.33 (3H, d, J 6.9, CH3), 2.61
3-(S)-Phenylbutyric acid 6-(4-dimethylamino-naphthalen-1-
ylazo)-5-nitrobenzotriazol-2-ylmethyl ester 4. (S)-Chloromethyl
3-phenylbutanoate (0.070 g, 0.33 mmol) was dissolved in
anhydrous acetone (5 ml). Sodium iodide (0.05 g, 0.33 mmol)
was added and the reaction mixture left to stir for 15 min, after
which time a precipitate of sodium chloride was seen to form. The
solution was filtered and the filtrate collected, with the solvent
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being removed at reduced pressure. The residue was dissolved in
DMF (5 ml), and dimethyl [5-(6-nitro-3H-benzotriazolylazo)-
naphthalen-1-yl]amine (0.12 g, 0.33 mmol) and potassium
carbonate (0.045 g, 0.33 mmol) were added. The reaction mixture
was left to stir overnight at room temperature and the solvent was
then removed at reduced pressure. The residue was partitioned
between diethyl ether (50 ml) and water (50 ml). The organic phase
was separated and dried over sodium sulfate before filtration and
removal of the solvent by evaporation. The residue was purified
by column chromatography, eluting with 0–20% ethyl acetate in
hexane to afford the title compound as a dark red solid (0.050 g,
19%). (Found: C, 64.38; H, 5.05;.C29H27N7O4 requires C, 64.79;
H, 5.05); dH (400 MHz; CDCl3) 1.23 (3H, d, J 7.0, CH3), 2.71 (2H,
m, CH2), 3.16 (6H, s, CH3), 3.31 (1H, sept, J 7.4, CH), 6.53 (1H,
d, J 11.3, CH), 6.58 (1H, d, J 11.4, CH), 7.10–6.99 (5H, m, 5 ×
ArH), 7.10 (1H, d, J 8.0, ArH), 7.61 (1H, dd, J 7.3, ArH), 7.71
(1H, dd, J 7.3, ArH), 7.96 (1H, s, ArH), 8.00 (1H, d, J 8.6, ArH),
8.24 (1H, d, J 8.5, ArH), 8.55 (1H, d, J 7.1, ArH), 9.03 (1H, dd,
J 8.5, ArH); dC (100 MHz; acetone) 22.4, 37.3, 42.4, 44.8, 68.6,
68.8, 113.7, 116.5, 124.2, 125.9, 126.2, 126.9, 127.1, 128.3, 128.5,
128.9, 132.6, 134.2, 134.4, 134.7, 142.7, 143.8, 144.5, 145.6, 146.2,
146.7, 147.0, 148.8, 157.1, 157.5, 171.9; m/z 538.2199 ([M + H]+.
C29H29N7O4 requires 538.2197); [a]2D0 +61.5 (c 1, MeCN).
8 G. McAnally, C. McLaughlin, R. Brown, D. C. Robson, K. Faulds,
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