1570
A. Ingram et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1569–1571
3000
2500
N
N
(i)
N
2000
N
3
1
1500
N
1000
N
OH
O
500
PO3H2
0
1000 1100
1200 1300 1400 1500
Raman Shift (cm -1
1500
CN
N
)
(ii)(iii)
CN
N
N
Figure 2. Superimposed SERRS spectra for unmasked dyes 1 and 3. The peaks used
N
for discrimination, at 1267 cmÀ1 and 1340 cmÀ1 are highlighted.
OH
N
N
O
O
OH
HO
OH
OH
2000
Scheme 1. Reagents and conditions: (i) POCl3, 75% (ii) tetraacetyl
topyranose, Cs2CO3, acetone, reflux, 14 h, 21% (iii) MeOH, 5% NaOMe, rt, 2 h, 80%.
a-bromogalac-
1500
c
1000
500
0
b
a
of using them as reporters of enzymatic activity for alkaline phos-
phatase and b-galactosidase respectively. 1 and 3 were prepared as
previously reported,18 phosphate 2 was prepared by treatment of 1
with phosphorous oxychloride, and the product isolated by reverse
phase silica gel chromatography. A number of conditions for glyco-
syl transfer to 3 were explored, but apparent low nucleophilicity of
both the phenol and its conjugate phenolate anion hindered reac-
1000
1200
1400
1600
Raman Shift (cm-1
)
Figure 3. SERRS spectra obtained from a mixture of 2 and 4. (a) without enzyme
added (b) with alkaline phosphatase added (c) with b-galactosidase added.
tion with tetraacetyl
a-bromogalactopyranose. Two possible rea-
sons for low reactivity of the phenolate anion are proposed:
firstly a resonance effect via the electron-withdrawing azo group
and secondly base-catalysed conversion to hydrazo species. Addi-
tion of base will shift the hydrazo-azo tautomerisation towards
the hydrazo species, which will minimise availability of phenolate
at room temperature for 30 min. A clearly distinguishable increase
in SERRS response was found (Fig. 3), and due to the emergence of
a peak at 1267 cmÀ1, and lack of signal at 1340 cmÀ1, this increase
in SERRS can be attributed to the emergence of dye 3 only. Thus,
the b-galactosidase had only hydrolysed 4 and masked substrate
2 remained intact. In each case a blank was also run, where water
was added in place of the relevant enzyme, to investigate whether
non-enzymatic hydrolysis was occurring. The reverse experiment
was conducted, with alkaline phosphatase replaced with b-galac-
tosidase. This time the peak at 1340 cmÀ1 was emergent, but no
trace of the peak at 1267 cmÀ1 was observed. Thus the alkaline
phosphatase has hydrolysed 2 selectively (Fig. 3).
In conclusion, we have demonstrated that 8-hydroxy quinolinyl
azo dye derivatives can act as SERRS reporters of both alkaline
phosphatase and b-galactosidase. Furthermore we have shown
that it is possible to simultaneously detect the presence of either
of these enzymes using a mixture of the substrates. To our knowl-
edge, this is the first time a simultaneous assay for the detection of
alkaline phosphatase or galactosidase using SERRS has been dem-
onstrated. By demonstrating simultaneous enzyme detection in
this manner, the technique allows for one-pot detection of multi-
ple analytes, offering more expeditious analysis times, plus the
prospect of multiplexing ELISA’s.
anion for reaction. In the first instance tetraacetyl a-bromogalacto-
pyranose with silver oxide as a heavy metal promoter was at-
tempted but did not afford any alkylated product. Phase transfer
conditions were attempted,19,20 but again no alkylation was ob-
served. Finally galactose derivative 4 was prepared by reaction of
tetraacetyl
a-bromogalactopyranose with phenolic dye 2 in the
presence of cesium carbonate in acetone at reflux. Cesium carbon-
ate has been reported as exhibiting modified reactivity in pheno-
late alkylations in comparison to carbonate salts of sodium and
potassium, Ouyang et al. found that reaction rate and nucleophile
regioselectivity could be manipulated by variation of alkali me-
tal.21 It has been suggested that O-alkylations using Cs2CO3 in
non-aqueous solvents occur via the naked phenolate anion, which
will be a stronger nucleophile and more reactive, which may ex-
plain the modified reactivity seen when using cesium carbonate.22
A subsequent Zemplen de-acetylation procedure23 afforded the
unprotected galactopyranoside 4 in good yield. Dyes 1 and 3, the
unmasked analogues of 2 and 4 respectively, can be easily discrim-
inated using SERRS, as can be seen from the superimposition of
their spectra (Fig. 2). Dye 3 has a peak at 1267 cmÀ1 which is not
present in 1, dye 1 has a peak at 1340 cmÀ1 which is not present
in 3. These bands have been previously assigned to in-plane C–H
bending, and N@N-aryl stretching respectively.18
To facilitate simultaneous reactions of multiple enzymes, a
mutually compatible medium for satisfactory activity of all en-
zyme components was sought. It has been reported that alkaline
phosphatase and b-galactosidase have an optimum reaction pH
of 8–924 and 6–8,25 respectively. Accordingly, all reactions were
buffered at pH 8, in Tris buffer. To maintain consistency the SERRS
spectra of 1 and 3 were also recorded in the same buffer. A mixture
of 10À6 M galactosidase substrate 4 and 10À6 M phosphatase sub-
strate 2 was treated solely with b-galactosidase and left to react
Supplementary data
Supplementary data (solvents and reagents) associated with
this article can be found, in the online version, at doi:10.1016/
References and notes
1. Michael, K. L.; Taylor, L. C.; Schultz, S. L.; Walt, D. R. Anal. Chem. 1998, 70, 1242.
2. Gibbons, I. Drug Discov. Today: HTS Supplement 2000, 1, 33.
3. Vieytes, M. R.; Fontal, O. I.; Leira, F.; Baptista de Sousa, J. M. V.; Botana, L. M.
Anal. Biochem. 1996, 240, 258.
4. Berger, C. N.; Tan, S. S.; Sturm, K. S. Cytometry 1994, 17, 216.
5. Lin, S.; Yang, S.; Hopkins, N. Dev. Biol. 1994, 161, 77.
Ingram, Andrew
