at SciVerse ScienceDirect

Tetrahedron 69 (2013) 2758e2766

Contents lists available

Tetrahedron

journal homepage: www.elsevier .com/locate/ tet

Chemical and bacterial reduction of azo-probes: monitoringa conformational change using fluorescence spectroscopy

Nicholas J.W. Rattray a, Waleed A. Zalloum a, David Mansell a, Joe Latimer a,Mohammed Jaffar b, Elena V. Bichenkova a,*, Sally Freeman a,*

a School of Pharmacy & Pharmaceutical Sciences, University of, Manchester, Oxford Road, Manchester M13 9PT, UKbMorvus Technology Limited, Ty Myddfai, Llanarthne, Carmarthen SA32 8HZ, UK

a r t i c l e i n f o

Article history:Received 30 October 2012Received in revised form 12 January 2013Accepted 28 January 2013Available online 8 February 2013

Keywords:Fluorescent probeDimerConformational lockAzo-reductaseEscherichia coliAMBER

* Corresponding authors. Tel.: þ44 (0) 161 275 2366rattray@manchester.ac.uk (N.J.W. Rattray), elena.bi(E.V. Bichenkova), sally.freeman@manchester.ac.uk (

0040-4020/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.tet.2013.01.086

a b s t r a c t

Sterically constrained probes 2,4-O-bisdansyl-6,7-diazabicyclo[3.2.1]oct-6-ene (8) and 2,4-O-bispyr-enoyl-6,7-diazabicyclo[3.2.1]oct-6-ene (9) exhibit specific dimer fluorescent characteristics (lmax 555 nmand 511 nm, respectively), attributed to the 2,4-diaxial arrangement of the dansyl or pyrene groups.Reduction of the azo-conformational locking group in (8) and (9) yielded 1,3-bisdansyl-4,6-diaminocyclohexane (16) and 1,3-bispyrenoyl-4,6-diaminocyclohexane (17) in the tetra-equatorialchair conformation, thus minimising interaction of the bisdansyl or bispyrenoyl groups. This inducesa change in fluorescence from a cooperative green emission dimer band to a blue-shifted, monomer typefluorescence, with lmax 448 nm and 396 nm for the reduced forms (16) and (17), respectively. Theazo-bond conformational lock can either be reduced under biomimetic conditions (using sodiumdithionite) or with bacteria (Clostridium perfringens or Escherichia coli) utilising azo-reductase enzymes.These fluorescent probes have the potential to specifically detect azo-reductase expressing bacteria.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

The ability of fluorescent probes to identify biochemical eventsand abnormalities is an ever expanding area of research. From thedetection of DNA mismatches,1,2 through to the identification ofantigens expressed by specific cancer cells3 and hypoxic cellularconditions,4 the range of practical applications spans from themolecular to the multi-cellular level. The detection of bacteria hastraditionally relied upon direct culturing and subsequent micro-scopic analysis of cellular detail or colonial growth patterns, whichis a comparatively timely process. Research has previously shownhow specific bacteria can be identified using different methods,such as conjugationwith DNA,5 PCR based hybridisation6 and ELISAassays.7 Although great sensitivity is achieved by these strategies,they are expensive multistep procedures that require a high degreeof technical expertise.

Utilising bacterial enzyme expression as an indicative biomarkeris an alternative approach. A fluorescent probe can be designed asa substrate for the relevant enzyme and emits a fluorescent re-sponse upon contact. This confers a high degree of specificity to the

; e-mail addresses: nicholas.chenkova@manchester.ac.ukS. Freeman).

All rights reserved.

probe system, with the advantages of lower costs, flexibility andease of use. Through application of this type of probe to broth, plateculturing media or cellular suspensions, it is possible to detect thefluorogenic products of bacterial enzymatic action.8,9

The detection of Escherichia coli using glycosidic enzymes asa biomarker has been achieved using glycoside-coumarin probes.4-Methyl-umbelliferyl-b-D-glucuronide (1) is a substrate that uponhydrolysis of the ester linkage by b-glucuronidase, releases highlyfluorescent 4-methyl-coumarin (2) (Fig. 1).10 Altering the glycosideattached to 4-methyl-coumarin can give rise to a range of probesthat can detect other enzymes expressed by bacteria, such as b-galactosidase or b-glucosidase.10e12 However, the limitations of the4-methyl-coumarin fluorophore are well documented. The narrow

Fig. 1. b-Glucuronidase catalysed hydrolysis of 4-methyl-coumarin-b-D-glucuronideprobe (1) yields fluorescent product 4-methyl-coumarin (2).

Fig. 4. Fluorescent probes (8) and (9) held in a strained tetra-axial conformation via

N.J.W. Rattray et al. / Tetrahedron 69 (2013) 2758e2766 2759

working pH range due to the high pKa value of the fluorophore isknown to compromise reproducibility. These probes often requirea base work up at the end of the assay in order to increase ionisa-tion and release full fluorescence.10,13

In this research we have designed a fluorescent probe that in-teracts with a different bacterial enzyme system. E. coli and Clos-tridiumperfringens are known to express azo-reductase activity14e17

and it has been proposed that the monitoring of this enzyme usingfluorescence could lead to rapid clinical detection of such bacteria.18

Previous work has shown that the intramolecular heterodimerfluorescence-quenched probe 4-O-dabsyl-2-O-dansyl-myo-inosi-tol-1,3,5-orthoformate (3) can be reduced to (4) and decomposes to(5) to detect enzyme-based redox systems19 (Fig. 2). The fluores-cent dansyl and a quenching dabsyl group in (3) are located in closeproximity with electron transfer resulting in the static quenching ofthe dansyl fluorescence. Reduction of the azo bond within thedabsyl quenching groupwith either sodium dithionite (Na2S2O4) orosteosarcoma cell extracts (containing bioreductive enzymes)causes an increase in fluorescence, generating a measurable re-sponse to the presence of reductive conditions. Earlier work hasalso shown that the orthoformate trigger in (6) cleaves in thepresence of acid to give (7) with a ring flip of the myo-inositol ringresulting in fluorescence (Fig. 3).20

Fig. 2. Reduction of the azo bond in non-fluorescent 4-O-dabsyl-2-O-dansyl-myo-inositol-1,3,5-orthoformate (3) gives a fluorescent signal from 2-O-dansyl-myo-inosi-tol-1,3,5-orthoformate (5) via the decomposition of intermediate (4).

Fig. 3. Acid induced activation of static quenched non-fluorescent probe (6) generat-ing fluorescent probe (7).20

the azo locking group.

Fig. 5. Synthesis of key intermediate azo-diol (10). Reagents and conditions: (a)m-chloroperbenzoic acid, DCM; 31% yield (b) hydrazine, 2-methoxyethanol; 82% yield(c) H2O2, H2O, 69% yield.

In this research we have combined these approaches to designa probe system where reduction of the azo group leads to a con-formational change of a cyclohexane ring. Cleavage of a reduction-sensitive locking group by biomimetic chemical reduction or bac-terial azo-reductase enzymes should confer a notable fluorescentchange within the reporter groups. Tracking of this change viafluorescence-based spectroscopic methods could lead to the rapidand sensitive identification of certain bacterial strains.

Here we outline the design and synthesis of two novel fluores-cent molecular probes, 2,4-O-bisdansyl-6,7-diazabicyclo[3.2.1]oct-6-ene (8) and 2,4-O-bispyrenoyl-6,7-diazabicyclo[3.2.1]oct-6-ene(9) (Fig. 4). Fluorescence spectroscopy is used to characterise theprobes and follow their subsequent chemical/biological reductionsthat influence their conformation. This research is also supportedby molecular dynamic simulations that are implemented to de-termine themost likely conformation (axial or equatorial) followingreduction of the azo trigger. Preliminary results on (8) with C.perfringens have been reported in a communication.21

2. Results and discussion

2.1. Synthesis

Synthesis of probes (8) and (9) first required the synthesis of theazo-linked, tetra-axial precursor, 6,7-diazabicyclo[3.2.1]oct-6-ene-(2,4)-diol (10), achieved by adapting literature protocols22e24

(Fig. 5). Diol (10) was prepared from the bis-epoxidation of 1,4-cyclohexadiene (11). Ring-opening of the bis-epoxide (12) withhydrazine gave (13), followed by oxidation with H2O2 yielded (10).

The syntheses of probes (8) and (9), together with mono-dansyland mono-pyrene standards (14) and (15), are shown in Fig. 6. Duallabelled dansyl probe (8) was synthesised in a 55% yield via sulfo-nation at the 2- and 4-positions of (10) using dansyl chloride in thepresence of sodium hydride. Bis-pyrenoyl labelled probe (9) wasalso synthesised from precursor (10) by a Steglich type esterifica-tion25 using pyrene-1-carboxylic acid with the DCC coupling agentand DMAP catalyst, in a 21% yield.

Singly labelled dansyl standard (14) was synthesised in a 45%yield froma regiospecificmethodusingdiol (10) anddansyl chloridein the presence of silver oxide and potassium iodide. A coordinationcomplex formed at the 2- and 4-positions of the diol with the silverion, causes the deprotonationof one of thehydroxyl groups and thusexpediting the mono-sulfonyl esterification.26,27 Singly labelledpyrene standard (15) was adapted from the method used to syn-thesise probe (9). A careful addition of reagentswas carried outwithan equimolar equivalent of 1-pyrenecarboxylic acid being slowly

Fig. 6. Synthesis of azo-probes (8) and (9) and standards (14) and (15) from diol (10).Reagents and conditions: (a) dansyl chloride, sodium hydride, dry DMF; 55% yield (b)pyrene-1-carboxylic acid, DCC, DMAP, dry DCM; 21% yield for (9) and 28% yield for (15)(c) dansyl chloride, AgO, KI, dry DMF, 45% yield.

Fig. 7. Reduction of the azo-locking group within (8) and (9) results in the formationof the bis-ammonium tetra-axial conformations (16ax) and (17ax), respectively.A subsequent ring flip to the more stable tetra-equatorial conformations (16eq) and(17eq) then occurs.

N.J.W. Rattray et al. / Tetrahedron 69 (2013) 2758e27662760

added over an hour to a mixture of diol (10), DCC and DMAP at 0 �C,to give (15) in a 28% yield.

It was also desirable to synthesis the reduced forms of (8) and(9) as standards for fluorescence studies. However, due to technicaldifficulties, this was only achieved for probe (8), where (16) wasgenerated using a H-Cube� Continuous-flow Hydrogenation Re-actor. Using a Pd/C catalyst accompanied with H2 generation viaH2O electrolysis, (8) was converted into (16) in a 36% yield.

2.2. Probe characterisation

Novel azo-probes (8) and (9) alongside standards (14), (15) and(16) were characterised by a combination of 1H and 13C NMRspectroscopy, atmospheric pressure chemical ionisation massspectrometry (APCI-MS), infrared spectroscopy and elementalanalysis. The assignments of proton and carbon signals wereassisted by 1H COSY and DEPT techniques. The numbering of theatoms in each compound is shown in Fig. 6. Analysis of the 1H NMRchemical shifts indicated that a proportion of the aromatic protonsfrom bisdansylated probe (8) and bispyrene probe (9) participate inpep stacking interactions. Selected 1H NMR chemical shifts andcoupling constants for compounds (8), (9), (14) and (15) are shownin Table 1.

Table 1Chemical shifts (d, ppm) and coupling constants (J, Hz) for selected aromatic protonsfor compounds (8) and (9) alongside (14) and (15) to evaluate pep stackinginteractions

Compound Dan H-30 Dan H-50 Dan H-60

Bis-dansyl (8) d 8.02 (d), J¼8.0 d 7.51 (t), J¼8.5 d 8.26 (d), J¼8.5Mono-dansyl (14) d 8.22 (d), J¼8.6 d 7.58 (m) d 8.30 (d), J¼7.5

Compound Pyr H-10 Pyr H-50 Pyr H-90

Bis-pyrene (9) d 9.16 (d), J¼9.4 d 7.63 (d), J¼8.7 d 8.49 (d), J¼8.9Mono-pyrene (15) d 9.30 (d), J¼9.5 d 8.07 (d), J¼8.4 d 8.65 (d), J¼8.9

By comparison with mono-derivatised standards (14) and (15),up-field proton shifts are seen in the 1H NMR spectra of probes (8)and (9), attributed to the presence of stabilising interactions be-tween the aromatic rings in each bis-substituted structure. Whencomparing the pyrene protons H-10, H-50 and H-90, up-field shifts of0.14, 0.44 and 0.16 ppm, respectively, were observed, indicating

p-stacking interactions between closely located pyrenoyl groupswithin probe (9). For the dansyl probe (8) protons H-30, H-50 and H-60 experience up-field shifts of 0.20, 0.07 and 0.04 ppm, re-spectively, upon comparisonwith the spectrum of themono-dansylstandard (14). There is greater evidence of pep stacking in-teractions in the bis-pyrene probe (9) when comparedwith the bis-dansyl probe (8), which is rationalised by the closer proximity andincrease in size of the aromatic structures participating in thestacking. Probe (8) is functionalised by tetrahedral sulfonyleesterbonds and a steric repulsionwill occur between these large groups,thus reducing the pep overlap. In the case of probe (9), the pyrenegroups are linked via planar carbonyl ester bonds. A combined ef-fect of a smaller central ester atom and a planar structuremeans thepyrene groups can be located closer and afford a stronger overlap,when compared with the bis-dansyl derivative (8).

Reduction of the azo-locking group is expected to affordbis-ammonium intermediates (16ax) and (17ax) (Fig. 7). With thecyclohexane ring no longer retained in the tetra-axial conforma-tions, relaxation to the tetra-equatorial conformations (16eq)and (17eq) is anticipated, which is next studied using moleculardynamic simulations.

2.3. Molecular dynamic simulations

Upon reduction of the azo bond in (8) and (9) to give (16) and(17), it is possible for three different amine protonation states to beformed: neutral, monocation and dication (Fig. 8). It is important toconsider these states since they influence the ability of the cyclo-hexane ring to undergo the conformational ring flip from tetra-axial to tetra-equatorial. It is proposed that if the resultant neu-tral bis-amine was formed, the lone pairs of electrons would repeleach other in the diaxial conformation and aid in the conforma-tional change to diequatorial. A similar situation is expected in thebis-ammonium product (dication) where the positive chargeswould repel and promote the cyclohexane ring to relax to the tetra-equatorial conformation. This is the predicted protonation statesince the pKa of a primary amine group on a cyclohexyl ring isw9.8.28 The third possibility involves the mono-protonation of an

Fig. 8. Potential protonation states of the diamine product after reduction of the azogroups to give (16) and (17): ring flip from tetra-axial (ax) to tetra-equatorial(eq) conformations. (R¼dansyl or pyrenoyl).

N.J.W. Rattray et al. / Tetrahedron 69 (2013) 2758e2766 2761

amine group (monocation) within the probe. This is likely to createa hydrogen bond bridgewhere the lone pairs on both amine groupsparticipate in proton sharing, locking the ring in the tetra-axialconformation.

With the reduced amineproducts (16) and (17) proposed to adoptthe equatorial conformations, further molecular dynamic simula-tionswere carried out to determine fluorophore interactions (Fig. 8).The products of reduction, 1,3-bisdansyl-4,6-diaminocyclohexane(16ax) and 1,3-bispyrenoyl-4,6-diaminocyclohexane (17ax), re-spectively, are expected to ring-flip to the tetra-equatorial confor-mations, (16eq) and (17eq). Theoretically this will cause a spatialrelocation of each of the fluorescent moieties (>10 �A apart20,29)(Fig. 8), thus disabling the groups’ ability to form dimer complexes,with a concomitantfluorescence shiftwhen comparedwith the non-reduced probes (8) and (9). Insights into these stacking patterns andmost favourable geometries of the locked and reduced probes wereinvestigated theoretically. AMBER modelling software was used toascertain the theoretical lowest energy structural conformations ofazo-probes (8) and (9) and their reduced analogues (16) and (17),under aqueous solvation conditions. As can be seen from the struc-tures in Fig. 9, the fluorescent dansyl groups in (8) andpyrene groups

Fig. 9. Lowest energy conformations of (8) and (9) and their reduced amine analogues(16eq) and (17eq) as predicted by simulations on AMBER.

in (9) are in orientations that promotes pep stacking, an essentialprerequisite for dimer/excimer fluorescence emission.

From Fig. 9 and Table 2 the neutral and dication forms of (16)and (17) prefer to adopt the tetra-equatorial conformations. Incontrast, the lowest energy conformations of themonocation formsof (16) and (17) are in the tetra-axial conformation. Viewing theconformation (not shown), this is attributed to H-bonding betweenthe protonated and neutral amine groups across the ring. It must benoted that energetically the dication and neutral forms of (16) and(17) are in the tetra-equatorial forms and are significantly morestable than the monocation in the tetra-axial forms (Table 1).

2.4. Fluorescence studies

A comparison of the fluorescence spectra of probe (8) and itsreduced form (16), also shown in our communication,21 indicatesthat a change of fluorescence signal from dimer to monomer, re-spectively, is observed. This is attributed to the strained ringconformation in (8) no longer being present within the reducedform as the N]N bridge has been cleaved. Subsequently, the pepstacking interaction indicative of a dimer is minimised within(16) leading to the blue shifted signal. Probes (8) and (9), togetherwith the monodansyl or monopyrenoyl standards (14) and (15),were analysed for their fluorescence emission spectra, shown inFig. 10.

From the modelling data, it is proposed that cleavage of the azo-locking functionality will cause a spatial relocation of each of thefluorescent groups to equatorial positions (>10�A apart20,29) (Fig. 8),thus preventing the group’s ability to form dimer complexes.Spectra were recorded at a low concentration (5 mM) to eliminateany intermolecular aggregation effects that might alter the truefluorescent signal of each compound. The spectra of mono-substituted standards (14) and (15) showed locally excited stateemissions that are indicative of dansyl and pyrenoyl monomerswith emission lmax at 444 nm and 395 nm, respectively. In contrast,the emission spectra for probes (8) and (9) showed dimer andtypical green pyrene excimer emission bands at 555 nm and505 nm, respectively. These large shifts in fluorescence emissionprovide further evidence that the close proximity of the fluorescentgroups on the tetra-axial azo-locked cyclohexane ring conforma-tion in (8) and (9) gives rise to associated pep stacking of the ar-omatic groups.

In order to test the response of probes (8) and (9) to act assensors for biological reductive conditions, initial reduction underbiomimetic conditions was carried out. The fluorescence spectra of(8) (5 mM)were recorded over a 16 h period after the addition of thebiomimetic reducing agent Na2S2O4. Fig. 11 shows the initial fluo-rescence spectra of (8)dS0. Upon addition of Na2S2O4 and sub-sequent tracking over 16 h (S0eS16), a gradual disappearance of thedimer band at 555 nm is noted with the appearance of a blueshifted structure-less band at 448 nm. A slight red-shift of 4 nm(upon comparison to standard (14)) is noted, which is attributed todifferences in ionic strength from the Na2S2O4.

Probe (9) was also subjected to the same biomimetic conditionsused to reduce (8) and the individual time course fluorescencespectra is shown in Fig. 12. Initial S0 excitation of probe (9) at351 nmyields a strong green coloured excimer fluorescence band atlmax 511 nm. Again, this value is shifted slightly in comparison tothe monomer standard due to environmental effects attributed toNa2S2O4. Upon comparison of the consecutive fluorescence spectra(S0eS12), the excimer band is shown to decrease in intensity whilsta typical structured pyrene monomer band appeared at 396 nm.This shift is attributed to the disappearance of pep stackinginteractions of the two pyrenoyl moieties in the ground state of (9),initiated by the reduction and subsequent ring flip of the cyclo-hexane ring from (17ax) to (17eq).

Fig. 10. A) Emission spectra of 2,4-O-bisdansyl-6,7-diazabicyclo[3.2.1]oct-6-ene(8, ———) and its monodansylated standard, 2-dansyl-6,7-diazabicyclo[3.2.1]oct-6-ene-4-ol (14,▬). Excited at 335 nm. B) 2,4-O-Bispyrenoyl-6,7-diazabicyclo[3.2.1]oct-6-ene (9, ———) and its monopyrene standard 2-pyrenoyl-6,7-diazabicyclo[3.2.1]oct-6-ene-4-ol (15, ▬). Excited at 351 nm. Spectra were recorded at 5 mM in DMSO/100 mM pH 7.4 phosphate buffer (70/30 v/v) at 20 �C.

0

50

100

150

200

250

300

350

360 410 460 510 560 610 660

Inte

nsi

ty (a

.u)

Wavelength (nm)

S0

S16

Fig. 11. Fluorescent time course spectra of the real time reduction of 2,4-O-bisdansyl-6,7-diazabicyclo[3.2.1]oct-6-ene (8) to generate its reduced form (16eq). The probe(5 mM) was treated with the biomimetic reducing agent sodium dithionite (23 mMNa2S2O4 in 100 mM phosphate buffer). Excitation at 335 nm initially showed a dimertype emission at 555 nm. Subsequent tracking of fluorescence over a 16 h periodshowed the appearance of a monomer band at 448 nm. Spectra at 2 h intervals areshown.

0

50

100

150

200

250

300

350

365 415 465 515 565 615

Inte

nsi

ty (a

.u)

Wavelength (nm)

S0

S12

Fig. 12. Fluorescent time course spectra of the real time reduction of 2,4-O-bispyr-enoyl-6,7-diazabicyclo[3.2.1]oct-6-ene (9) to generate its reduced form (17eq). At 5 mMconcentration the probe was reacted with the biomimetic reducing agent sodiumdithionite (23 mM Na2S2O4 in phosphate buffer). Excitation at 351 nm initially showedan excimer type emission at ca. 511 nm. Subsequent tracking of fluorescence overa 12 h period showed the appearance of a monomer band at ca. 396 nm. Spectra at 2 hintervals are shown.

Table 2Theoretical geometry based energy minimisation calculations for the reduction products (16) or (17) in the three possible protonation states indicated in Fig. 8 (EEL¼Energy ofElectrostatic interactions, EGB¼Energy of Generalised Born)

DE (kcal/mol) 16 Neutral 16 Monocation 16 Dication 17 Neutral 17 Monocation 17 Dication

Bond stretching 3.4 3.5 3.3 5.7 5.6 5.9Angular bending 11.2 16.4 7.5 9.5 8.9 10.1Torsional 45.0 84.6 43.5 14.3 11.2 15.6Van der Waals �15.7 �13.6 �8.7 �13.6 �16.2 �12.5EEL 155.7 23.5 106.9 44.2 �16.6 66.4EGB �202.0 �66.1 �184.8 �148.8 �69.0 �170.51e4 Van der Waals 22.9 23.9 24.1 38.3 37.9 38.71e4 EEL �195.1 �178.0 �182.7 10.1 13.8 �2.1Total �174.6 �105.9 �190.9 �40.3 �24.5 �48.7Axial or Equatorial Eq Ax Eq Eq Ax Eq

N.J.W. Rattray et al. / Tetrahedron 69 (2013) 2758e27662762

2.5. Fluorescence based microbiological assay

The initial biomimetic reduction studies of (8) and (9) showedthat a dissociative state (monomer) type emission was observedupon reduction of the azo locking group to give (16eq) and (17eq),thus exhibiting a suitably large fluorescence shift, that is, distin-guishable and quantifiable when compared to the original non-reduced probe. The probes were then subsequently tested fortheir suitability for detection of azo-reductase enzymes expressedby E. coli and C. perfringens. Due to poor aqueous solubility, probe(9) was excluded from the microbiological studies. Initial bacterialcell cultures were tested for evidence of azo-reductase activityusing a standard amaranth dye absorbance assay (Fig. 13).30

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0 30

Abs

525

nm

Time (min)

C. perfringens

0

0.1

0.2

0.3

0.4

0.5

0.6

0 30

Abs

525

nm

Time (min)

E. coli

Fig. 13. UV absorbance based amaranth enzyme viability assay indicating that the azobond within the amaranth dye has been reduced causing a decrease in absorbance at525 nm. This indicates azo-reductase activity in the E. coli and C. perfringens cell culturesupernatants.

N.J.W. Rattray et al. / Tetrahedron 69 (2013) 2758e2766 2763

Probe (8) was tested using E. coli cell culture supernatant andthe reduction was monitored over 12 h using fluorescence (Fig. 14,A/B). These results showed a modest decrease in dimer fluores-cence (18%), together with an increase in monomer type fluores-cence, indicating that some reduction was taking place, attributedto azoreductive enzymes contained within the E. coli supernatant.Probe (8) was also tested with C. perfringens supernatant, whichexhibited a substantial shift from dimer to monomer fluorescenceover 12 h (Fig. 14, C/D). The dimer emission dropped by 89%accompanied by a symmetrical increase in monomer fluorescencefor 16eq.

Fig. 14. Fluorescent spectra and kinetic plots of the reduction of 2,4-O-bisdansyl-6,7-diazabicyclo[3.2.1]oct-6-ene (8) to generate its reduced form (16eq). At 10 mMconcentration, the probe was treated with E. coli (A,B) or C. perfringens (C,D) cellculture supernatants known to contain azoreductive enzymes. Excitation at 335 nminitially showed a dimer type emission at ca. 552 nm (blue line). Subsequent trackingof fluorescence over a 12 h period (S0eS12) showed the appearance of a monomer bandat ca. 451 nm (red line). In both cases kinetic profiles (B) and (D) indicate a decrease indimer emission for (8) mirrored by an increase in monomer fluorescence for (16eq).

2.6. Limit of detection

The limit of bacterial detection by probe (8) was also deduced(Fig. 15). An overall concentration value of 300 nM of probe (8) wasdetermined as the lowest limit of detection bywhich a fluorescencesignal could be determined above baseline noise (being determinedas three times over this noise baseline level).31 A series of bacterial

Fig. 15. Fluorescence spectra of 300 nM (8) upon exposure to cell supernatant from1�105 C. perfringens colony forming units containing azo-reductase enzymes. The limitof detection for this probe system against azo-reductase expression by C. perfringens is300 nM of (8) versus 1�105 colony forming units/mL after a 14 h time period. (Greenline (T0)¼0 h red line (T14)¼14 h).

broth dilutions were then carried out to deduce the lowest numberof colony forming units (C.F.U) capable of eliciting a detectablefluorescent response from (8). Results indicated that after a 14 htime period the cell extract from 1�105 C.F.U of C. perfringens wascapable of reducing 300 nM (8) and displaying the monomerfluorescence signal indicative of its reduced form (16).

3. Conclusion

Fluorescence spectroscopy has been used to examine the bio-mimetic and biological reduction of probes (8) and (9), whichcontain an azo bond trigger in a strained tetra-axial cyclohexanechair conformation. Molecular modelling studies predict the closeproximity of the fluorescent partners in probes (8) and (9), withparticipation in pep stacking interactions to account for the for-mation of dimer/excimer type fluorescent emission. Upon re-duction of the azo-bond trigger, changes in the fluorescence spectraand NMR chemical shift data support that the tetra-axial confor-mations of (8) and (9) undergo a ring flip to the tetra-equatorialconformations to give (16) and (17). Both probes indicatedchanges from dimer/excimer fluorescence to a monomer emissionupon biomimetic and bacterial azo-reductase reduction to (16eq)and (17eq) in the tetra-equatorial conformations, with molecularmodelling studies backing up these conformational changes. Hav-ing tested azo-reductase expression in two bacterial strains wehave shown that, at a proof-of-principle level, how these types ofnovel molecular probes may find application in the detection ofenzymatic biomarkers for the identification of bacteria.

4. Experimental

4.1. Materials and instrumentation

Chemicals were purchased from Aldrich Chemical Co., Gilling-ham, UK. Syntheses were monitored by thin layer chromatographyon pre-coated 60 F254 silica gel aluminium backed plates (Merck,Darmstadt). Visualisation of spots for thin layer chromatographywas performed using a UV GL-58 Mineral-Light lamp. Flash columngrade 40e63 mm silica gel (Apollo Scientific, Stockport, UK) wasused in preparative scale column chromatography. Melting pointswere determined using a Gallenkamp Melting Point apparatusmicroscope (UK). IR spectra were recorded using a Jasco FT/IR 4100spectrometer, resolution 1 cm�1. NMR spectra were recorded usinga Bruker Avance spectrometer equipped with a 5 mm single-axis Z-gradient quattro nucleus probe, operating at 300 MHz for 1H and at75 MHz for 13C unless otherwise stated. The spectrometers wererunning TOPSPIN NMR system software (Version 2.1). Chemicalshifts (d) are reported in parts per million (ppm), peak positionsrelative to Me4Si (0.00 ppm) for 1H NMR and 13C NMR spectra.Abbreviations used for splitting patterns are: s, singlet; d, doublet;t, triplet; m, unresolved multiplet. Mass spectra were recorded atthe Swansea University EPRSC National Mass Spectrometry ServiceCentre using a Thermo Scientific LTQ Orbitrap XL Hybrid FourierTransform Mass Spectrometer. Elemental analyses were recordedat the School of Chemistry, University of Manchester.

4.2. Synthesis

4.2.1. 6,7-Diazabicyclo[3.2.1]oct-6-ene-2,4-diol (10). The synthesesof (10) and its precursors were adapted from previous meth-ods.22e24 A solution of 6,7-diazabicyclo[3.2.1]octane-2,4-diol (13)(500 mg, 3.5 mM) was dissolved in water (5 mL) and 35% hydrogenperoxide (1.1 mL, 4 M equiv) was stirred in the dark for 72 h. Aftercompletion of the reaction aqueous Raney Nickel slurry (0.1 mL)was added to quench excess peroxide. The solutionwas filtered andconcentrated under reduced pressure to yield a crystalline solid.

N.J.W. Rattray et al. / Tetrahedron 69 (2013) 2758e27662764

Purification using flash column chromatography (acetone/CH2Cl2,1:2) was carried out to yield 339 mg (69%) of (10) as a colourlesssolid. Mp 142e145 �C. Lit. 140e142 �C.24 IR nmax/cm�1: 1089 (CeOstr), 1298 (OH str), 1716 (N]N cis str), 2935 (CH2 str), 3312 (OH str).1H NMR (CDCl3) dH 4.95 (2H, dd, 3J2e1/3eq 3.5 Hz, 3J4e3eq/5 5.4 Hz, H-2/4), 3.75 (2H, dt, 3J1e2/6eq 3.5 Hz, 3J5e4/6eq 5.5 Hz, H-1/5), 2.48 (1H,d, 2J6axe6eq 12.3 Hz, H-6ax), 1.67 (1H, dt, 3J3eq-2/4 6.5 Hz, 2J3eqe3ax15.4 Hz, H-3eq), 1.42 (1H, dt, 3J3axe2/4 3.5 Hz, 2J3axe3eq 15.4 Hz, H-3ax), 1.32 (1H, dt, 3J6eqe1/5 3.5 Hz, 2J6eqe6ax 12.3 Hz, H-6eq). 13C NMRdC 85.4 (C-2/4), 61.2 (C-1/5), 37.2 (C-3), 23.7 (C-6). MS (APCI) m/z[MþNH4]þ 160.1.

4.2.2. 2,4-O-Bisdansyl-6,7-diazabicyclo[3.2.1]oct-6-ene (8). Sodiumhydride (60%, 60 mg, 1.4 mM) was suspended in dry DMF (20 mL)and the mixture was stirred at room temperature for 10 min undera nitrogen atmosphere. Diol (10) (100 mg, 0.7 mM) was added andthe mixture was stirred for 10 min. Dansyl chloride (450 mg,1.7 mM) was added and the mixture was stirred under a nitrogenatmosphere for 12 h. Completion of the reaction was monitored byTLC (ethyl acetate/hexane, 2:1) Rf 0.38. The reaction was quenchedby the addition of water (20 mL) and the mixture was diluted withethyl acetate (100 mL). The layers were separated and the organiclayer was washed with water (6�25 mL), followed by 10% aqueousNaHCO3 (2�20 mL). The organic layer was dried (MgSO4), filteredand the filtrate concentrated under reduced pressure. The residuewas purified by flash column chromatography (ethyl acetate/hex-ane 2:1) to yield 233mg (55%) of a yellow solid (8). Mp 134e138 �C.IR nmax/cm�1: 1172, 1358 (S]O str), 1572 (N]N str cis). 1H NMR(400MHz, CDCl3) dH 8.64 (2H, d, 3J10e20 8.0 Hz, H-10), 8.27e8.22 (4H,m, H-30, H-60), 7.61e7.52 (4H, m, H-20, H-50), 7.26 (2H, d, 3J40e508.5 Hz, H-40), 5.02 (2H, dt, H-2/4), 4.54 (2H, dt, 3J1e2/8eq, 5e4/8eq5.0 Hz, H-1/5), 2.90 (12H, s, H-70), 2.48 (1H, d, 2J3axe3eq 13.8 Hz,H-3ax), 1.67 (1H, dt, 2J8axe8eq 17.3 Hz, 3J8axe1/5 4.7 Hz, H-8ax), 1.36(1H, d, 2J8eqe8ax 17.3 Hz, H-8eq), 1.20 (1H, dt, 2J3eqe3ax 13.8 Hz,3J3eqe2/4 5.0 Hz, H-3eq). 13C NMR dC 132.1, 131.3, 130.4, 129.8, 129.6,129.3, 123.0, 119.1, 115.8, 81.6 (napth), 68.8 (C-2/4), 45.4 (C-10/100),41.0 (C-1/5), 30.6 (C-3), 24.0 (C-6). MS (APCI) m/z [MþNH4]þ 608.2.Elemental analysis calcd for C30H32N4O6S2: C, 59.19; H, 5.30; N,9.20. Found: C, 58.94; H, 5.02; N, 9.14%.

4.2.3. 2,4-O-Bispyrenoyl-6,7-diazabicyclo[3.2.1]oct-6-ene (9). Diol(10) (230, 1.6 mM), 1-pyrenecarboxylic acid (1.009 g, 4.1 mM) andDMAP (19.5 mg, 0.16 mM) were dissolved in dry DCM (20 mL)under a nitrogen atmosphere. The mixture was then cooled to 0 �Cbefore DCC (700 mg, 3.4 mM) was added, which was stirred for5 min at 0 �C and 24 h at 20 �C. Completion of the reaction wasmonitored by TLC (ethyl acetate/hexane, 1:1) Rf 0.28. Filtration ofthe reaction removed any precipitated dicyclohexylurea, then thefiltrate was evaporated in vacuo. The residue was dissolved in DCM,washed twice with 0.5 N HCl and saturated NaHCO3, then driedover MgSO4. The organic layer was subsequently concentratedunder vacuum and the residue was purified via flash columnchromatography (ethyl acetate/hexane 1:3) to yield (9) 202 mg(21%) as a brown solid. Mp 178e180 �C. IR nmax/cm�1: 1249 (CeOstr), 1595 (N]N cis str), 1701 (C]O str). 1H NMR (CDCl3) dH 9.16(2H, d, 3J10e20 9.4 Hz, H-10), 8.49 (2H, d, 3J90e80 8.9 Hz, H-90), 8.19 (2H,d, 3J20e10 9.4 Hz, H-20), 8.10 (2H, d, 3J80e90 8.9 Hz, H-80), 7.95e7.88 (6H,m, H-30, 40, 7), 7.82 (2H, d, 3J60e50 8.7 Hz, H-60), 7.63 (2H, d, 3J50e608.7 Hz, H-50), 5.73 (2H, t, 3J2e1/3eq, 4e3eq/5 5.5 Hz, H-2/4), 5.64 (2H, t,3J1e2/8eq, 5e4/8eq 6.0 Hz, H-1/5), 2.89 (1H, d, 2J8axe8eq 12.6 Hz, H-8ax),2.31 (1H, d, 2J3eqe3ax 17.1 Hz, H-3eq), 2.01 (1H, dt, 3J3axe2/4 5.5 Hz,2J3axe3eq 17.1 Hz, H-3ax), 1.67 (1H, dt, 3J8eqe1/5 6.0 Hz, 2J8eqe8ax12.6 Hz, H-8eq). 13C NMR dC 166.4 (C]O), 134.4, 131.3, 130.9, 130.1,129.9, 129.8, 128.4, 126.9, 126.5, 126.3, 126.3, 124.7, 124.4, 123.9,123.8, 122.6 (PyreC), 81.6 (C-2/4), 63.4 (C-1/5), 33.9 (C-3), 24.9(C-6). MS (APCI) m/z [MþNH4]þ 598.2. Elemental analysis calcd for

C40H26N2O4: C, 80.25; H, 4.38; N, 4.68. Found: C, 79.97; H, 4.14; N,4.42%.

4.2.4. 2-Dansyl-6,7-diazabicyclo[3.2.1]oct-6-ene-4-ol (14). This pro-cedure describing the mono-esterification of a diol has beenadapted from Bouzide et al.27 6,7-Diazabicyclo[3.2.1]oct-6-ene-2,4-diol (10) (78 mg, 0.55 mM) was dissolved in dry DMF (10 mL). Sil-ver oxide (180 mg, 0.8 mM) was added and the mixture was stirredfor 10 min at room temperature. Dansyl chloride (162 mg, 0.6 mM)and potassium iodide (10 mg, 0.06 mM) were added and the re-actionwas stirred at room temperature for 90min. The mixture wasthen passed through a sintered filter and the filtrate was dilutedwith ethyl acetate (50 mL). The organic layer was washed with6�5 mL of water, dried over MgSO4 and reduced in vacuo. The re-sultant crude solid was crystallised from chloroform to yield 93 mg(45%) of (14). Mp 78e84 �C. IR nmax/cm�1 1171, 1351 (S]O str), 1572(N]N str cis), 3388 (OH str). 1H NMR (CDCl3) dH 8.62 (1H, d, 3J10e208.6 Hz, H-10), 8.30 (1H, d, 3J60e50 7.5 Hz, H-60), 8.22 (1H, d, 3J30e208.6 Hz, H-30), 7.57e7.48 (2H, m, H-20/50), 7.20 (1H, d, 3J40e50 7.5 Hz,H-40), 5.16 (1H, t, 3J4e3eq/5 4.8 Hz, H-4), 4.95 (1H, t, 3J2e1/3eq 4.9 Hz,H-2), 4.61e4.53 (1H, m, H-5), 3.98e3.88 (1H, m, H-1), 2.90 (6H, s,H-10), 2.51 (1H, d, 2J8axe8eq 12.8 Hz, H-8ax), 1.48e1.43 (2H, m, H-3ax/3eq), 1.28e1.16 (1H, m, H-8eq). 13C NMR dC 152.0, 132.1, 131.3, 130.7,129.9, 129.7, 129.0, 123.2, 119.0, 115.7 (naptheC), 84.2 (C-4), 81.4(C-2), 70.7 (C-5), 59.9 (C-1), 45.4 (NMe2), 33.2 (C-3), 23.7 (C-6). MS(APCI) m/z [MþNH4]þ 376.13. Elemental analysis calcd forC18H21N3O4S: C, 57.58; H, 5.64; N, 11.19. Found: C, 57.19; H, 5.32, N,10.98%.

4.2.5. 2-Pyrenoyl-6,7-diazabicyclo[3.2.1]oct-6-ene-4-ol (15). Diol(10) (50 mg, 0.35 mM), DCC (77 mg, 0.37 mM) and a catalyticamount of DMAP (2 mg, 0.016 mM) was dissolved in dry DCM(10 mL). This mixture was then cooled to 0 �C. Separately,1-pyrenecarboxylic acid (111 mg, 0.45 mM) was dissolved dry DCM(10 mL). Both mixtures were kept under an inert nitrogen atmo-sphere and left to stir for 10 min. 1 mL of the pyrene solution wasadded every 5 min to the diol solution. After 45 min, the additionwas complete and the mixture was left to stir in the dark for 24 h.Completion of the reaction was monitored by TLC (ethyl acetate/hexane, 1:1) Rf 0.15. Filtration of the reaction mass was carried outto remove any precipitated dicyclohexylurea, then the filtrate wasevaporated in vacuo. The residue was dissolved in DCM, washedtwice with 0.5 N HCl and saturated NaHCO3, and then dried overMgSO4. Evaporation of solvent and flash column chromatography(ethyl acetate/hexane, 1:1) yielded 32 mg (28%) of a brown solid(15). Mp 102e106 �C. IR nmax/cm�1: 1593 (N]N str cis), 1711 (C]Ostr), 3270 (OH str). 1H NMR (CDCl3) dH 9.30 (1H, d, 3J10e20 9.5 Hz,H-10), 8.65 (1H, d, 3J90e80 8.9 Hz, H-90), 8.16e8.09 (6H, m, H-20/30/40/60/70/80), 8.07 (1H, d, 3J50e60 8.4 Hz, H-50), 5.54e5.47 (2H, m, H-2/4),5.35e5.31 (1H, m, H-1), 4.27e4.25 (1H, m, H-5), 2,76 (1H, d,2J3eqe3ax 12.55 Hz, H-3eq), 2.00 (1H, br s, HeOH), 1.93e1.83 (1H, m,H-8ax/8eq), 1.57e1.51 (1H, m, H-3ax). 13C NMR dC 166.8 (C]O), 134.7,131.4, 131.0, 130.4, 129.9, 129.8, 128.4, 127.2, 126.5, 126.5, 126.4,124.9, 124.7, 124.2, 124.1, 122.4 (PyreC), 84.4 (C-4), 81.3 (C-2), 63.8(C-1), 60.6 (C-5), 33.2 (C-3), 24.4 (C-6). MS (APCI) m/z [MþNH4]þ

371.23. Elemental analysis calcd for C23H18N2O3: C, 74.58; H, 4.90;N, 7.56. Found: C, 74.43; H, 4.61; N, 7.23%.

4.2.6. 1,3-Bisdansyl-4,6-diaminocyclohexane (16). Azo-linked probe(8) (50 mg) was dissolved in dry DMF and cycled within a Tha-lesNanoH-Cube� continuousflowhydrogenation reactor for 30minwhilst passing through a Pd/C catalyst cartridge. The internalhydrogen source was obtained from H2O electrolysis and the pres-surewithin the systemwasmaintained at 60 bar alongside a systemtemperature of 60 �C. Upon completion, concentration underreduced pressure yielded a brown residue that when purified by

N.J.W. Rattray et al. / Tetrahedron 69 (2013) 2758e2766 2765

flash column chromatography (ethyl acetate/hexane, 2:1) gave19 mg (36%) of a yellow solid (16). 1H NMR for reduced probe (16)(400 MHz, DMSO-d6) dH 8.65 (2H, d, 3J10e20 8.2 Hz, H-10), 8.04 (2H, d,3J60e50 7.9 Hz, H-60), 7.86 (2H, d, 3J30e20 8.0 Hz, H-30), 7.70e7.60 (2H,m,3J20e10/30, H-20), 7.64e7.55 (2H, m, 3J50e40/60, H-50), 7.33 (2H, d, 3J40e50

8.0 Hz, H-40), 4.55e4.51 (2H, m, H-2/4), 3.94e3.88 (2H, m, H-1/5),2.80 (12H, s, H-70), 2.31 (1H, d, 2J3axe3eq 14.2 Hz, H-3ax), 2.20e2.12(1H, m, H-8ax), 1.70e1.62 (1H, m, H-8eq), 1.58e1.50 (1H, m, H-3eq)m/z [MþNH4]þ 612.2.

4.3. UV and fluorescence studies

UVevisible spectra were recorded in 1.4 mL Hellma QG Far-UVquartz cuvettes using a Cary 4000 UV-visible spectrophotometerequipped with a Peltier-thermostated cuvette holder. pH wasmeasured using a Hanna-instruments HI 9321 microprocessor pHmeter, calibrated with standard buffers (Sigma) at 20 �C. Fluores-cence emission and excitation spectra were recorded in 1.4 mLHellma QS Fluorescence quartz thermostated cuvettes usinga temperature controlled Cary-Eclipse fluorescence spectropho-tometer. Emission and excitation spectra were recorded in 0.1 Mphosphate buffer pH 7.4. The excitation wavelength used for com-pound (8) was 335 nm and for compound (9) was 351 nm (bothvalues were determined by the absorption bands on UV analysis).Slit-widths were 10 nm. Photo bleaching of the sample was mini-mised by using the automatic shutter on function.

To monitor the biomimetic reduction of (8) and (9) in real-time(with subsequent decomposition to (16) and (17)), 4.2 mg ofsodium dithionite (23.0 mM) was added to the quartz cuvettecontaining (8) or (9) (at 5 mM) dissolved in 1 mL of DMSO/pH 7.4P.B.S mixture (50/50 v/v). The reaction mixture was stirred at 20 �Cwith the first fluorescence spectrum being recorded immediatelyafter the addition of sodium dithionite.

The UV amaranth dye assay confirmed the ability of E. coli and C.perfringens to produce azo-reductase enzymes. An aliquot (1 mL) ofcultured cell media was placed in a cuvette alongside 10 mg ofamaranth dye (10 mL of a 1 mg/mL stock). The absorbance maximaat 525 nm was then noted. Readings were then taken after 30 minto deduce the extent of amaranth dye reduction.

The reduction of probe (8) using E. coli (4.6�108 colony formingunits per mL) and C. perfringens (1.8�107 colony forming units permL) cell culture supernatants was also tracked by fluorescence. A10 mM concentration of the probe was observed over a 12 h periodin a bacterial thioglycollate broth supernatanteDMSO mix (50/50v/v at pH 7.4). Spectra were recorded every hour and correctedagainst background interference.

4.4. Microbiology

For the initial reduction profile of (8) going to (16), stock culturesof E. coli (4.6�108 colony forming units per mL) and C. perfringens(1.8�107 colony forming units per mL) were maintained on brainheart infusion agar (Oxoid, UK). Single colonies were used toinoculate 5 mL brain heart infusion/thioglycollate broth (Oxoid,UK), which was incubated for 24 h. Cultures were then incubatedfor a further 30 min. All of the above steps were carried out understrictly anaerobic conditions in a MkIII workstation (Don Whitley,UK) at 37 �C. Cell culture aliquots (1 mL) were then centrifuged at13,000 rpm for 4 min and the culture supernatant was passedthrough a 0.22 mm syringe filter. For the limit of detection study, C.perfringens was grown in a thioglycollate broth and a cell count of1.8�107 colony forming units per mL was noted. Initially, a DMSO/broth supernatant mixture (50/50 v/vd9�106 colony units/mL)was applied to a 300 nM sample over 15 h. This gave the initial highconcentration value which was reduced by serial dilution to givea reduced bacterial cell unit count of 3 � 105/mL.

4.5. Molecular dynamic simulations

The structures of the molecular probes (8, 9), and their reducedforms (16, 17) in the neutral, monocation and dication states werebuilt using SYBYL software 8.0. Atomic point charges were derivedfrom the AM1-BCC chargemodel with ANTECHAMBER in AMBER10.Molecular dynamics (MD) simulations for all molecular probeswere carried out using AMBER10 with a GAFF force field32 under animplicit water system applying the Generalised Born continuumsolvent mode.33 The system was minimised using the steepestdescent and then conjugate gradient algorithms for 500 cycles toremove any strains. Then, the systemwas heated to 300 K for 15 pswith a gradual removal of the restraints on (8) and (9). Finally, the10 ns molecular dynamics simulation was performed without anyrestraints at a constant temperature of 300 K and a constantpressure of 1 atm. The SHAKE algorithm of the hydrogen atomsbond length was used. Cut-off distance for the calculation of non-bonded forces was set to 16 Ǻ. The integration time step was setat 2 fs and the energies and atomic coordinates were saved every1 ps.

4.6. Molecular dynamics and energy minimisation studies

The 15 ns of each MD simulation was clustered by Kclust script,where a fixed cluster radius (based on Cartesian coordinates RMSD)was enabled. Structures (8, 9, 16, 17 and charge study variants)obtained from the implicit water MD simulation were clusteredwith a fixed radius of 3.0 Ǻ and in the case of (9) a 2 Ǻ distance wasused. These returned many clusters with only one highly popu-lated. The structurewith the least RMSD to the ‘centroid’ of themostpopulated cluster was selected as a representative structure of eachmolecule (Fig. 9). These representative structures were againenergy-minimised using SYBYL to determine the energy of lowestenergy conformation (Table 2).

Acknowledgements

We thank Dr. R. Bryce for the use of computational facilities andDr. A. McBain for helpful discussions regarding the microbiologywork. We thank the EPSRC National Mass Spectrometry Centre inSwansea for MS analysis. NR thanks Morvus Technologies and theBBSRC for a CASE studentship.

References and notes

1. Bichenkova, E. V.; Gbaj, A.; Walsh, L.; Savage, H. E.; Rogert, C.; Sardarian, A. R.;Etchells, L. L.; Douglas, K. T. Org. Biomol. Chem. 2007, 7, 1039e1051.

2. Bichenkova, E. V.; Yu, X.; Bhadra, P.; Heissigerova, H.; Pope, S. J. A.; Coe, B. J.;Faulkner, S.; Douglas, K. T. Inorg. Chem. 2005, 44, 4112e4114.

3. Liu, T.; Wu, L. Y.; Hopkins, M. R.; Choi, J. K.; Berkman, C. E. Bioorg. Med. Chem.Lett. 2010, 20, 7124e7126.

4. Komatsu, H.; Harada, H.; Tanabe, K.; Hiraoka, M.; Nishimoto, S. Med. Chem.Commun. 2010, 1, 50e53.

5. Wang, Z. P.; Xu, H. A.; Wu, J.; Ye, J.; Yang, Z. Food Chem. 2010, 125, 779e784.6. Yu, G. F.; Niu, J. J.; Shen, M. S.; Shao, H. J.; Chen, L. Clin. Chim. Acta 2006, 366,

281e286.7. Paffard, S. M.; Miles, R. J.; Clark, C. R.; Price, R. G. J. Immunol. Methods 1996, 192,

133e136.8. Zaytsev, A. V.; Anderson, R. J.; Bedernjak, A.; Groundwater, P. W.; Huang, Y.;

Perry, J. D.; Orenga, S.; Roger-Dalbert, C.; James, A. Org. Biomol. Chem. 2008, 6,682e692.

9. Feng, P. C.; Hartman, P. A. Appl. Environ. Microbiol. 1982, 43, 1320e1329.10. Manafi, M.; Kneifel, W.; Bascomb, S. Microbiol. Rev. 1991, 55, 335e348.11. Dealler, S. F. Rev. Med. Microbiol. 1993, 4, 198e206.12. Perry, J. D.; James, A. L.; Morris, K. A.; Oliver, M.; Chilvers, K. F.; Reed, R. H.;

Gould, F. K. J. Appl. Microbiol. 2006, 101, 977e985.13. Gee, K. R.; Sun, W. C.; Bhalgat, M. K.; Upson, R. H.; Klaubert, D. H.; Latham, K. A.;

Haugland, R. P. Anal. Biochem. 1999, 273, 41e48.14. Chen, H.; Hopper, S. L.; Cerniglia, C. E. Microbiology 2005, 151, 1433e1441.15. McBain, A. J.; Macfarlane, G. T. J. Med. Microbiol. 1998, 47, 407e416.16. Stingley, R. L.; Zou, W.; Heinze, T. M.; Chen, H. Z.; Cerniglia, C. E. J. Med.

Microbiol. 2010, 59, 108e114.

N.J.W. Rattray et al. / Tetrahedron 69 (2013) 2758e27662766

17. McBain, A. J.; MacFarlane, G. T. J. Med. Microbiol. 2001, 50, 833e842.18. James, A. L.; Perry, J. D.; Jay, C.; Monget, D.; Rasburn, J. W.; Gould, F. K. Lett. Appl.

Microbiol. 2001, 33, 403e408.19. Kadirvel, M.; Rattray, N. J. W.; Rajendran, R.; Zalloum, W. A.; Gbaj, A.;

Demonacos, C.; Bichenkova, E. V.; Freeman, S. New. J. Chem. 2011, 35, 701e708.20. Kadirvel, M.; Gbaj, A.; Mansell, D.; Miles, S. M.; Arsic, B.; Bichenkova, E. V.;

Freeman, S. Tetrahedron 2008, 64, 5598e5603.21. Rattray, N. J. W.; Zalloum, W. A.; Mansell, D.; Latimer, J.; Schwalbe, C. H.; Blake,

A. J.; Bichenkova, E. V.; Freeman, S. Chem. Commun. 2012, 6393e6395.22. Chenevert, R.; Jacques, F. Tetrahedron: Asymmetry 2006, 17, 1017e1021.23. Kavadias, G.; Velkof, S.; Belleau, B. Can. J. Chem. 1978, 56, 404e409.24. Saumi, T. J. J. Org. Chem. 1975, 40, 456e461.

25. Neises, B.; Steglich, W. Angew. Chem., Int. Ed. Engl. 1978, 17, 522e524.26. Sureshan, K. M.; Shashidhar, M. S.; Praveen, T.; Gonnade, R. G.; Bhadbhade, M.

M. Carbohydr. Res. 2002, 337, 2399e2410.27. Bouzide, A.; Sauve, G. Org. Lett. 2002, 4, 2329e2332.28. Brown, H. C. Determination of Organic Structures by Physical Methods; Academic:

New York, NY, 1971.29. Turro, N. J. Modern Molecular Photochemistry of Organic Molecules; University

Science Books: 2010.30. Rau, J.; Knackmuss, H.-J.; Stolz, A. Environ. Sci. Technol. 2002, 36, 1497e1504.31. Crummett, W. Anal. Chem. 1980, 52, 2242e2249.32. Tsui, V.; Case, D. A. Biopolymers 2000, 56, 275e291.33. Cieplak, P.; Caldwell, J.; Kollman, P. J. Comput. Chem. 2001, 22, 1048e1057.