Chemiluminescence from alkoxy-substituted acridinium dimethylphenyl ester labels

View Online / Journal Homepage / Table of Contents for this issue

Dynamic Article Links

Organic &

Biomolecular

Chemistry

Cite this: Org. Biomol. Chem., 2012, 10, 3432

www.rsc.org/obc

PAPER

Chemiluminescence from alkoxy-substituted acridinium dimethylphenyl ester

labels†

Anand Natrajan,* David Sharpe and David Wen

Received 4th January 2012, Accepted 23rd February 2012

DOI: 10.1039/c2ob00022a

Chemiluminescent acridinium dimethylphenyl ester labels are used in automated immunoassays for

clinical diagnostics. Light emission from these labels is triggered by alkaline peroxide in the presence of

the cationic surfactant cetyltrimethylammonium chloride (CTAC). The surfactant plays a critical role in

the chemiluminescence process of these labels by both accelerating their emission kinetics and increasing

total light output enabling high throughout and improved assay sensitivity in automated immunoassays.

Despite the surfactant’s crucial role in the chemiluminescent reaction, no study has investigated how

structural perturbations in the acridinium ring could impact the inﬂuence of the surfactant. We describe

herein the synthesis and properties of three new alkoxy-substituted, acridinium dimethylphenyl esters

where the nature of the alkoxy group in the acridinium ring was varied (hydrophobic or hydrophilic).

Chemiluminescence measurements of these alkoxy-substituted labels indicate that hydrophilic functional

groups in the acridinium ring, in particular sulfobetaine zwitterions, disrupt surfactant-mediated

compression of emission times but not enhancement of light yield. These results support the hypothesis

that surfactant-mediated effects require the binding of two different reaction intermediates to surfactant

aggregates and, that surfactants inﬂuence light emission from acridinium esters by two separate

mechanisms. Our studies also indicate that preservation of both surfactant effects on acridinium ester

chemiluminescence and low non-speciﬁc binding of the label can be achieved with a relatively

hydrophobic acridinium ring coupled to a hydrophilic phenolic ester leaving group.

treatment restores the electrophilic center at C-9 by rapidly con-

verting the pseudobase to the acridinium ester ii (Fig. 2). Sub-

Introduction

Chemiluminescent acridinium dimethylphenyl esters (9d and

10d, Fig. 1) are used as labels for clinical diagnostic immuno-

assays in automated instruments.¹These acridinium esters are

used to label proteins such as antibodies, antigens or small ana-

lytes and, the labeled reagents are used in conjunction with mag-

netic microparticles for sensitive analytical measurements of a

wide range of clinically important analytes. At the end of the

assay, light emission from the acridinium ester label is triggered

by the sequential addition of two reagents. An initial addition of

0.1 M nitric acid containing 0.5% hydrogen peroxide is followed

by the addition of 0.25 M sodium hydroxide containing the cat-

ionic surfactant cetyltrimethylammonium chloride (CTAC). At

neutral pH, most acridinium esters exist as the water-adduct i

(Fig. 2) commonly referred to as the pseudobase²which cannot

participate directly in the chemiluminescent reaction. The acid

sequent addition of base ionizes the hydrogen peroxide and

initiates the chemiluminescent process. Hydroperoxide ion

addition to C-9 forms the adduct iii which eventually leads to

the formation of the primary emitter, excited state acridone vi.

Dioxetanone v has been proposed as an intermediate in this

process³but detailed theoretical studies⁴have suggested that

direct formation of the excited state acridone vi from dioxetane

iv is energetically favored.

In a recent study, we reported that micelles of cationic surfac-

tants such as CTAC play a critical role in the chemiluminescence

of acridinium dimethylphenyl ester labels (containing unsubsti-

tuted N-sulfopropylacridinium rings) and their conjugates.^1dThe

surfactant not only compressed emission times from approxi-

mately one minute in the absence of surfactant to <5 seconds

but, was also observed to increase total light output by 3–4 fold.

We attributed the acceleration in emission kinetics to increased

local concentration of hydroperoxide ions at the surface of

CTAC aggregates which should facilitate formation of the per-

oxide adduct iii (Fig. 2). Similar enhancements in observed rates

have also been reported in other bimolecular reactions such as

ester hydrolysis by hydroperoxide ions in the presence of cetyl-

trimethylammonium micelles.⁵To explain the effect of the

Siemens Healthcare Diagnostics, Advanced Technology and Pre-

Development, 333 Coney Street, East Walpole, MA 02032, USA. E-mail:

anand.natrajan@siemens.com; Fax: +1-508-660-4591; Tel: +1-508-

660-4582

†Electronic supplementary information (ESI) available: See DOI:

10.1039/c2ob00022a

3432 | Org. Biomol. Chem., 2012, 10, 3432–3447

This journal is © The Royal Society of Chemistry 2012

View Online

Fig. 1 Structures of zwitterionic acridinium esters.

Fig. 2 Simpliﬁed reaction pathway of chemiluminescence from N-sulfopropyl acridinium esters.^1dCationic surfactants inﬂuence conversion of ii to

iii by increasing the local concentration of hydroperoxide ions. Surfactants also facilitate formation of dioxetane iv from iii by offering a lower polarity

medium for this intramolecular cyclization reaction that involves dispersal of negative charge in the transition state.

surfactant in signiﬁcantly increasing light output, we proposed

that the lower polarity of the micellar medium would be condu-

cive to the formation of the critical dioxetane intermediate iv

from intramolecular cyclization of iii. This reaction involves dis-

persal of negative charge in the transition state and classical

Hughes–Ingold theory⁶predicts that a lower polarity medium

would lower the activation energy of the reaction. In fact, our

proposal was based on analogous micellar effects that have been

reported in detailed studies of unimolecular reactions such as the

intramolecular cyclization reactions of ortho-haloalkyl-substi-

tuted phenoxides, decarboxylation of 6-nitrobenzisoxazole-3-

carboxylate and, 1,2-elimination reactions.^7,8All these reactions

involve dispersal of negative charge in their transition states and,

are catalyzed by aggregates of cationic and zwitterionic surfac-

tants because of reduced polarity (alcohol-like) at the micellar

phase.^7,8

This journal is © The Royal Society of Chemistry 2012

Org. Biomol. Chem., 2012, 10, 3432–3447 | 3433

View Online

Solute binding to ionic micelles such as those of cetyltri-

methylammonium salts results from a combination of hydro-

phobic and Coulombic interactions.⁹Given the critical role of

the surfactant in the chemiluminescence of acridinium dimethyl-

phenyl esters, any useful structural alteration of acridinium ester

must ensure that the surfactant’s inﬂuence is not disrupted.

Based on the chemiluminescence mechanism outlined in Fig. 2,

strong binding of the acridine ring in the acridinium ester ii and

the peroxide adduct iii, to surfactant aggregates is crucial to

observe the effect of the surfactant on the chemiluminescence

process i.e. acceleration of emission kinetics and enhanced light

output. However, these requirements pose an interesting

dilemma. In immunoassays that use microparticles and ﬂuor-

escent or chemiluminescent labels, high non-speciﬁc binding,

attributable to label hydrophobicity and/or charge often limits

assay sensitivity.^1aIs there a way to reconcile these seemingly

contradictory requirements in the design of new acridinium ester

labels so that the critical inﬂuence of the surfactant on the chemi-

luminescence process is retained without exacerbating the non-

speciﬁc binding of the labels? We report herein a study aimed to

answer this question as well as to outline some general principles

that, until now, have been lacking, for the design of acridinium

dimethylphenyl ester labels that not only exhibit fast light emis-

sion and high light output in the presence of surfactants but, also

display low non-speciﬁc binding to magnetic microparticles. Our

conclusions are based on the study of the chemiluminescence of

new, C-2 alkoxy-substituted, acridinium ester labels (Fig. 1),

where the nature of the alkoxy group was varied (hydrophobic or

hydrophilic). Our results on chemiluminescence measurements

of these labels also lend further support to the hypothesis that

surfactants inﬂuence light emission from acridinium dimethyl-

phenyl ester labels by two discrete mechanisms.

glycol (10c). To ensure aqueous solubility of all three, alkoxy-

substituted acridinium esters, a hydrophilic linker containing a

sulfobetaine zwitterion, disclosed previously,^1dwas incorporated

para to the phenolic ester leaving group as illustrated in Fig. 1.

The structures illustrated in Fig. 1 can be classiﬁed in simple

terms, while referring to the acridinium ring and the phenolic

ester with the sulfobetaine linker respectively as, hydrophobic–

hydrophilic (10a) and hydrophilic–hydrophilic (10b and 10c).

Compound 10d, with an unsubstituted acridinium ring was

described previously^1dand contains a relatively hydrophobic

acridinium ring along with a hydrophilic phenolic ester leaving

group.

The synthetic scheme for compounds 10a–10c is shown in

Fig. 3. Synthesis of the common intermediate 2-hydroxyacridine

ester 5 was accomplished in ﬁve steps starting with commer-

cially available isatin 1. N-Arylation of isatin 1 with 4-bromoani-

sole in the presence of copper(^I) iodide was followed by

rearrangement of the crude N-arylisatin derivative to 2-methoxy-

acridine-9-carboxylic acid 2 in reﬂuxing base.¹⁰Although the

overall yield of this two step process was modest (43%), the

reaction was amenable to scale up and the acridine carboxylic

acid 2 could be isolated without chromatography. Esteriﬁcation

of 2-methoxyacridine carboxylic acid 2 with methyl 4-hydroxy-

3,5-dimethylbenzoate 3 in the presence of p-toluenesulfonyl

chloride in pyridine afforded the C-2 methoxy-substituted acri-

dine ester 4 in 86% yield after puriﬁcation by chromatography

on silica. Cleavage of the methyl ether in 4 was carried out

using boron tribromide in dichloromethane which also cleaved

the methyl ester. However, during the process of quenching

excess boron tribromide with cold methanol, the methyl ester

was reinstalled to give compound 5 in 90% isolated yield follow-

ing puriﬁcation on silica.

With compound 5 in hand, we used the Mitsunobu reaction¹¹

to install the requisite aryl ether linkages in compounds 6a–6c.

Thus, treatment of compound 5 with 2-propanol, 3-dimethyl-

amino-1-propanol and methoxy(hexaethylene) glycol in the pres-

ence of triphenylphosphine and diisopropylazodicarboxylate

(DIAD) afforded the alkoxy-substituted acridine esters 6a, 6b

and 6c respectively. Isolated yields of 6a and 6b were high ≥

80% whereas 6c was isolated in modest (38%) yield. Conversion

of the alkoxy-substituted acridine esters 6a–6c to the N-sulfopro-

pyl acridinium esters was accomplished by N-alkylation of the

acridine nitrogen with 1,3-propane sultone in the ionic liquid 1-

butyl-3-methylimidazolium hexaﬂuorophosphate [BMIM][PF₆].

We reported previously that ionic liquids are excellent media for

the N-alkylation of the hindered nitrogen of acridine esters with

1,3-propane sultone¹²and, we now routinely use this protocol

for a variety of acridine ester substrates. In the case of substrate

6b, both the acridine nitrogen as well as the dimethylamino

group were N-alkylated in one step in excellent (∼80%) conver-

sion as judged by HPLC analysis. The N-sulfopropyl acridinium

methyl ester intermediates were directly hydrolyzed to the acridi-

nium carboxylic acids 7a–7c and subsequently puriﬁed by pre-

parative HPLC. Conversion to the ﬁnal targets 10a–10c ﬁrst

entailed addition of the zwitterionic sulfobetaine linker 8^1dto the

carboxylic acids in 7a–7c using N-hydroxysuccinimide (NHS)

ester chemistry for carboxylate activation. The amine-containing,

zwitterionic acridinium esters 9a–9c were converted to the

targets 10a–10c by ﬁrst condensing with glutaric anhydride

Results and discussion

Synthesis of C-2 alkoxy-substituted, zwitterionic acridinium

esters and protein conjugates

To study the effect of structural perturbation of the acridinium

ring on the chemiluminescence reaction both in the presence and

absence of surfactants, we elected to synthesize three different,

C-2 alkoxy-substituted acridinium dimethylphenyl ester labels

(10a–10c, Fig. 1). Acridinium esters with methoxy groups at

C-2 and/or C-7 (see Fig. 1 for numbering system) display

enhanced light output but poor chemiluminescence stability^1a

and, in the current study, we wanted to optimize the structure of

the alkoxy group not only for fast light emission and maximum

light output in the presence of surfactants but also for good

chemiluminescence stability and low non-speciﬁc binding.

Reducing the bulk of the label and synthetic complexity were

other important objectives that we wanted to achieve. The pheno-

lic oxygen at C-2 on the acridine ring provided a convenient

handle to attach different functional groups using a common syn-

thetic precursor (compound 5 in Fig. 3). A C-2 isopropyloxy

group was selected as representative of a relatively hydrophobic

substituent (10a, Fig. 1). For hydrophilic functional groups, two

non-ionic alkoxy groups were selected, one containing a highly

polar sulfobetaine zwitterion (10b) and the other containing the

poly(ethylene) glycol (PEG) derivative, methoxy(hexaethylene)

3434 | Org. Biomol. Chem., 2012, 10, 3432–3447

This journal is © The Royal Society of Chemistry 2012

View Online

Fig. 3 Synthetic scheme for C-2, alkoxy-substituted, acridinium esters. Reagents: (a) sodium hydride, DMF; (b) 4-bromoanisole, copper(I) iodide,

DMF; (c) 10% potassium hydroxide; (d) p-toluenesulfonyl chloride, pyridine; (e) boron tribromide, dichloromethane; (f) methanol; (g) diisopropyl

azodicarboxylate, triphenylphosphine, THF; (h) 1,3-propane sultone, 2,6-di-tert-butylpyridine, 1-butyl-3-methylimidazolium hexaﬂuorophosphate; (i)

1 N HCl; ( j) N,N,N′,N′-tetramethyl-O-(N-succinimidyl)uronium tetraﬂuoroborate (TSTU), diisopropylethylamine, DMF; (k) 0.25 M sodium bicarbon-

ate, DMF (l) glutaric anhydride, diisopropylethylamine, methanol; (m) TSTU, diisopropylethylamine, DMF–water.

followed by conversion of the glutarate derivatives to the NHS

esters.

labels per antibody molecule using 7.5–10 equivalents input of

compounds 10a–10c in the labeling reactions.

HPLC analyses of all the intermediates and ﬁnal compounds

of Fig. 3 are shown in the ESI† (Fig. S1–S16†).

Protein conjugates of compounds 10a–10c were prepared

using a murine anti-TSH (thyroid stimulating hormone) mono-

clonal antibody as described in the Experimental section. Label

incorporation was measured by mass spectroscopy which indi-

cated a similar level of label incorporation of approximately 4

Emission spectra and pseudobase formation

Acridinium esters with electron-donating methoxy groups at C-2

and/or C-7 of the acridinium ring display bathochromic shifts in

their emission spectra compared to unsubstituted acridinium

This journal is © The Royal Society of Chemistry 2012

Org. Biomol. Chem., 2012, 10, 3432–3447 | 3435

View Online

Fig. 4 Emission spectra of acridinium esters. Compounds 10a, 10b,

and 10c with C-2 alkoxy groups exhibit a shift in the emission wave-

length maximum to 458 nm compared to 430 nm for the unsubstituted

acridinium ester 10d.

Fig. 5 pH Titrations of acridinium esters illustrating conversion of the

acridinium form ii to the pseudobase i. Compounds 10a, 10b, and 10c

with C-2 alkoxy groups exhibit a shift in the pK_ato 4.5 from 4.0 for the

unsubstituted acridinium ester 10d.

esters.^1aA single C-2 methoxy group was observed to shift the

emission maximum to 458 nm compared to 426 nm for an

unsubstituted acridinium ester.^1aEmission spectra (Fig. 4) of the

alkoxy-substituted acridinium esters 10a–10c were recorded

using a spectral camera to determine whether there are any

differences in the emission spectra of these compounds caused

by the different alkoxy groups. As illustrated in Fig. 4, all three

compounds 10a–10c emitted light with spectral maxima centered

at 458 nm. In comparison, compound 10d, with an unsubstituted

acridinium ring, exhibited an emission maximum centered at

430 nm. These results suggest that the electron donating ability

of the C-2, aryl ether oxygen in compounds 10a–10c is not

affected by structural differences in the alkoxy functional groups.

Another characteristic of acridinium esters that is a reﬂection

of the electronics of the acridinium ring is the pH of pseudobase

formation from the addition of water to C-9 (formation of i from

ii in Fig. 2). As reported previously by Bunting et al.,¹³acridi-

nium cations, at low pH, display a strong absorption band at

approximately 360 nm that disappears as the pH is raised due to

disruption of the acridinium ring chromophore caused by

addition of hydroxide ion to C-9. UV-Visible spectra of com-

pounds 10a–10d at low pH (pH = 2.8) and the corresponding

amines 9a–9d at high pH (pH = 9) are shown in Fig. S17–S20

(ESI†) and, they illustrate the absorption bands of the acridinium

forms of these compounds and their corresponding pseudobases.

(The amine derivatives 9a–9d were used at pH 9 to avoid NHS

ester hydrolysis in 10a–10d.) As can be noted from the spectra,

the acridinium forms of the compounds 10a–10c, containing C-2

alkoxy groups, displayed strong absorption bands centered at

383 nm whereas compound 10d with an unsubstituted acridi-

nium ring, displayed the same absorption band at 371 nm. At pH

9, this long wavelength absorption band was bleached for all

compounds because they exist predominantly as the pseudobase.

Titration of the acridinium chromophore as a function of pH is

thus a convenient way to measure the pK_aof the acridinium to

pseudobase transition of acridinium esters.

ring should reduce the electrophilic character of C-9 and shift

the pH of pseudobase formation to more basic pH. This predic-

tion is indeed borne out for all three alkoxy-substituted com-

pounds 10a–10c that exhibited a pK_aof 4.5 for conversion of the

acridinium forms to their corresponding pseudobases. Com-

pound 10d had a more acidic pK_aof 4.

Identical chemiluminescence emission spectra, UV-Visible

spectra and pK_aof pseudobase formation of compounds 10a–

10c indicate that the different alkoxy groups in these compounds

have very similar effects on the electronics of the acridinium

ring.

Chemiluminescence measurements

Chemiluminescence measurements of the alkoxy-substituted

labels 10a–10c as well their anti-TSH antibody conjugates were

carried out as described previously for compound 10d and its

conjugates^1dboth in absence and presence of surfactants. In a

typical experiment, 1–2 mg mL⁻¹solutions of HPLC-puriﬁed

labels 10a–10c and the corresponding protein conjugates of

these compounds were serially diluted in phosphate buffer to

approximately 0.5 nM (nm = nanomolar, 10⁻⁹M) for the free

labels and, approximately 0.2 nM for the protein conjugates as

described in the Experimental section. Chemiluminescence from

0.01 mL of each diluted sample was triggered by the addition of

0.3 mL each of 0.1 M nitric acid containing 0.5% (∼80 mM)

hydrogen peroxide followed by the addition of 0.25 M sodium

hydroxide. Light was collected for a total of two minutes, inte-

grated at 0.5 second intervals, using a luminometer. The light

collection time was sufﬁciently long for complete emission

under all conditions. In experiments involving surfactants, the

surfactant was included in the second reagent (0.25 M sodium

hydroxide) at ﬁve times its reported critical micelle concentration

(CMC) in water. The surfactants selected for the current study

were two cationic surfactants cetyltrimethylammonium chloride

(CTAC) and cetyltripropylammonium chloride (CTPAC), as well

as the sulfobetaine surfactant N,N-dimethyldodecylammonio-

Titration of the acridinium chromophore of compounds 10a–

10d as a function of pH (pH range 1–7) is illustrated in Fig. 5.

Electron-donating groups at C-2 and/or C-7 of the acridinium

3436 | Org. Biomol. Chem., 2012, 10, 3432–3447

This journal is © The Royal Society of Chemistry 2012

View Online

Table 1 Time (seconds^a) for emission of >90% total light of alkoxy-

substituted acridinium ester labels in the absence and presence of

surfactants

Compound

No surfactant

CTAC^b

CTPAC^b

DDAPS^b

10a

10b

10c

52.5

55

41.5

1.0

17.5

1.5

1.0

19.0

3.5

29.0

18.0

^aAverage of three replicates. ^bAbbreviations used: CTAC

cetyltrimethylammonium chloride (cationic), CTPAC

=

cetyltripropylammonium chloride (cationic), DDAPS

dimethyldodecylammonio-1,3-propane sulfonate (zwitterionic).

=

N,N-

Table 2 Time (seconds^a) for emission of >90% total light of anti-TSH

antibody conjugates of alkoxy-substituted acridinium ester labels in the

absence and presence of surfactants

Conjugate

No surfactant

CTAC

CTPAC

DDAPS

Fig. 7 Chemiluminescence proﬁles of alkoxy-substituted acridinium

esters in the presence of the cationic surfactant CTAC.

Anti-TSH-10a

Anti-TSH-10b

Anti-TSH-10c

40.5

47.5

37.5

1.0

14.5

1.0

14.5

2.0

4.5

27.5

7.5

^aAverage of three replicates.

Fig. 8 Chemiluminescence proﬁles of alkoxy-substituted acridinium

esters in the presence of the cationic surfactant CTPAC.

Fig. 6 Chemiluminescence proﬁles of alkoxy-substituted acridinium

esters in the absence of surfactant.

1,3-propane sulfonate (DDAPS). In our previous study^1dthese

surfactants afforded the greatest impact on the chemilumines-

cence of unsubstituted acridinium esters and their conjugates.

The effect of the three surfactants on the emission times of the

alkoxy-substituted acridinium ester labels 10a–10c and their cor-

responding anti-TSH antibody conjugates are illustrated in

Tables 1 and 2. These emission times reﬂect the ability of the

surfactant in accelerating light emission. The emission proﬁles of

the labels 10a–10c are shown in Fig. 6–9 and those of the anti-

TSH antibody conjugates, which were similar, are shown in

Fig. S21–S24 (ESI†).

Fig. 9 Chemiluminescence proﬁles of alkoxy-substituted acridinium

As can be noted from Tables 1 and 2, in the absence surfactant

light emission was observed to be quite slow for both the free

labels and the protein conjugates. Although the protein conju-

gates showed marginally faster light emission, yet emission

times for the emission of >90% of total light required >35

esters in the presence of the zwitterionic surfactant DDAPS.

seconds. These results are similar to what we observed pre-

viously for unsubstituted acridinium esters.^1dIn the presence of

the two cationic surfactants CTAC and CTPAC, light emission

This journal is © The Royal Society of Chemistry 2012

Org. Biomol. Chem., 2012, 10, 3432–3447 | 3437

View Online

Table 3 Speciﬁc activity (RLUs^aper mole × 10⁻¹⁹) of alkoxy-

substituted acridinium ester labels at a measurement time of 2 min.

Values in parentheses reﬂect the enhancement in light output in the

presence of surfactant

The results in Tables 1–4 prompts the following question:

why are surfactants equally effective in enhancing light output of

acridinium ester labels with hydrophobic (10a) and hydrophilic

acridinium rings (10b and 10c) but, are signiﬁcantly less effec-

tive in accelerating emission kinetics of the latter (especially

10b)?

Compound

No surfactant

CTAC

CTPAC

DDAPS

10a

10b

10c

2.0

1.8

2.4

8.4 (4.2)

6.1 (3.4)

7.7 (3.2)

10.3 (5.0)

6.9 (3.8)

9.3 (3.9)

10.0 (5.0)

5.4 (3.0)

6.6 (2.8)

Micellar catalysis of bimolecular reactions results from

increased local concentrations of the reactants in a small volume

of the micellar phase.¹⁴Cationic micelles in particular can bind

negatively charged ions such as hydroperoxide ions and, provide

a high local concentration of these reactive ions to micelle-

bound substrates. For zwitterionic micelles, this ion binding is

weaker.¹⁵Micelles also provide a low polarity (alcohol-like)

environment that facilitates unimolecular reactions whose tran-

sition states involve charge dispersal.^7,8For these unimolecular

reactions, the magnitude of the catalysis has been observed to be

greater in less polar micelles derived from CTPAC and

DDAPS.^7,8Substrate binding to ionic micelles is through a com-

bination of hydrophobic and charge interactions. In an elegant

study, Bunton and Sepulveda^9aexamined the binding of a series

of phenol derivatives of increasing hydrophobicity to cetyltri-

methylammonium bromide (CTAB) micelles at both low and

high pH. At low pH, where the phenols were uncharged, the

magnitude of the binding to CTAB micelles increased with

increasing hydrophobicity of the phenol. At high pH, where the

phenols were ionized to the phenoxides, binding for each phenol

analog was signiﬁcantly stronger compared to its unionized

counterpart.

Based on the chemiluminescence reaction pathway of acridi-

nium esters outlined in Fig. 2, acceleration of emission kinetics

requires efﬁcient binding of the acridinium esters ii to surfactant

aggregates whereas increase in light output requires binding of

the peroxide adduct iii. An examination of the structural features

of the acridine rings of ii and iii indicate some crucial differences

that can affect their binding to micelles. The acridinium esters ii

contain no net charge and consequently binding to micelles is

expected to be primarily through hydrophobic interactions of the

acridinium ring with the surfactant. On the other hand, the acri-

dine ring of the peroxide adduct iii contains two negative

charges. Binding of this reaction intermediate to cationic and

zwitterionic micelles is expected to be much stronger due to

both hydrophobic and charge interactions. The relatively weaker

interaction of the acridinium ring of ii with surfactants is

expected to be more easily disrupted by the introduction of

hydrophilic functional groups in the acridinium ring. Our results

with label 10b which, has a very polar sulfobetaine zwitterion in

the acridinium ring, indicates that this is indeed the case. This

label showed relatively slow light emission even in the presence

of cationic surfactants. The label 10c also contains a hydrophilic

acridinium ring but with a different functional group PEG, that

structurally is very similar to the non-ionic surfactant triton

X-100. The latter surfactant exhibits ideal mixing with cationic

surfactants,¹⁶which suggests that PEG introduction in the acridi-

nium ring is not likely to be as disruptive to binding to cationic

micelles. Chemiluminescence measurements of label 10c are

consistent with this expectation. Finally, because the acridine

ring of peroxide adduct iii (Fig. 2) of all three labels 10a–10c is

negatively charged, augmentation of hydrophobic binding with

signiﬁcant charge interactions should result in minimal

^aRLU = relative light unit.

Table 4 Speciﬁc activity (RLUs per mole × 10⁻¹⁹) of anti-TSH

antibody conjugates of alkoxy-substituted acridinium ester labels at a

measurement time of two minutes. Values in parenthesis reﬂect the

enhancement in light output in the presence of surfactant

Conjugate

No surfactant

CTAC

CTPAC

DDAPS

Anti-TSH-10a

Anti-TSH-10b

Anti-TSH-10c

1.7

1.2

2.2

7.1 (4.2)

4.1 (3.4)

7.4 (3.4)

8.1 (4.8)

4.3 (3.6)

7.9 (3.6)

8.0 (4.7)

3.6 (3.0)

6.1 (2.8)

from 10a and 10c and their antibody conjugates was signiﬁ-

cantly faster and the emission times were compressed to <5

seconds. On the other hand, the label 10b and its protein conju-

gate, showed a less dramatic acceleration of emission kinetics

even in the presence of these cationic surfactants. Acceleration

of emission kinetics was also attenuated in micelles of the zwit-

terionic surfactant DDAPS for all labels as well as their protein

conjugates. However, emission kinetics of the acridinium label

10a with a more hydrophobic ring was less severely affected

compared to 10b and 10c which have more hydrophilic acridi-

nium rings.

The effect of the three surfactants on total light output of the

alkoxy-substituted acridinium ester labels 10a–10c and their cor-

responding anti-TSH antibody conjugates are illustrated in

Tables 3 and 4. In Tables 3 and 4, light output for each label is

presented as speciﬁc activity in units of RLUs per mole (RLU =

relative light unit). The speciﬁc activity values in Tables 3 and 4

reﬂect the ability of the three surfactants in increasing total light

yield from the acridinium ester labels and their protein

conjugates.

In the absence of surfactant, the three labels 10a–10c showed

depressed light output but the speciﬁc activity was similar for all

three labels. These results indicate that structural differences in

the alkoxy groups in these labels have little impact on the chemi-

luminescent reaction. For protein conjugates (Table 4), a similar

result was observed except for the conjugate of label 10b which

had slightly lower speciﬁc activity compared to 10a and 10b.

Total light output in the presence of the three surfactants was

enhanced for all three labels to a similar extent. Compound 10a

in particular, which has a more hydrophobic acridinium ring,

showed an enhancement in light output of 4–5-fold in the pres-

ence of all three surfactants. Enhancement was also higher in the

presence of micelles of CTPAC and DDAPS which are less

polar than CTAC micelles.^7dEnhancement in light output was

slightly attenuated (3–4-fold) for 10b and 10c with hydrophilic

acridinium rings both for free labels as well as the protein

conjugates.

3438 | Org. Biomol. Chem., 2012, 10, 3432–3447

This journal is © The Royal Society of Chemistry 2012

View Online

Table 5 Partition coefﬁcients of acridinium ester labels to CTAC

micelles (K_MW) and to the HPLC stationary phase (K_SW

)

Compound,

K_MW(pH 2.8)

Compound,

K_SW(pH 2.8)

Compound,

K_MW(pH 9)

Compound,

K_SW(pH 9)

10a, 578

10b, —

10c, 103

10d, 3486

10a, 30

10b, —

10c, 5

9a, (>10⁴)^a

9b, 3433

9a, —

9b, 141

9c, 58

9d, —

9c, 1895

10d, 256

9d, (>10⁴)^a

^aEstimated. K_MWof 10b was too weak to measure by HPLC.

disruption in the inﬂuence of the surfactant on light yield even

for acridinium esters with hydrophilic acridinium rings. Consist-

ent with this prediction, we observed similar enhancement in

light output for all three labels 10–10c and their protein conju-

gates in the presence of surfactants.

Fig. 10 Chemiluminescence stability of anti-TSH antibody conjugates

of acridinium ester labels in pH 8 buffer at 4 °C and 37 °C. Order of

decreasing stability at 37 °C are 10d > 10a ≥ 10c = 10b. Alkoxy-substi-

tuted acridinium ester conjugates were marginally less stable at 37 °C

but were equally stable at 4 °C after 4 weeks.

Micelle–water partition coefﬁcients

To provide support for our proposals that two different reaction

intermediates (ii and iii in Fig. 2) bind with differing strengths to

surfactant aggregates, we measured micelle–water partition

coefﬁcients (K_MW) of the alkoxy-substituted acridinium esters

(10a–10d and 9a–9d) to CTAC micelles. Peroxide adduct iii is

an unstable reaction intermediate and therefore we used the pseu-

dobase adduct i of the amine derivatives 9a–9d to simulate its

binding to CTAC micelles at pH = 9. At this pH, compounds

9a–9d exist solely as the pseudobase (Fig. 5 and Fig. S17–

S20†). Binding of the acridinium esters 10a–10d to CTAC

micelles was measured at pH 2.8. At this pH, the compounds

exist predominantly in the acridinium form as can be noted from

Fig. 5 and Fig. S17–S20.† The amine derivatives 9a–9d also do

not contain any charge in the phenolic ester leaving group at

pH = 9. Similarly, the NHS esters 10a–10d, have a charge-

neutral leaving group at pH = 2.8. Thus, any observed differ-

ences in binding for these compounds to CTAC micelles can be

attributed mainly to structural differences (hydrophobicity and

charge) in their acridine rings.

signiﬁcantly greater. In contrast to the weak binding observed at

low pH, the acridinium ester with a polar sulfobetaine zwitterion

in the acridinium ring (9b) showed strong partitioning into

CTAC micelles. A similar result was observed for compound 9c

whose K_MWat pH = 9 was approximately 20-fold higher com-

pared to pH 2.8. The two acridinium esters, 9a and 9d, with

hydrophobic acridinium rings showed very strong partitioning

into CTAC micelles at pH 9. Partition coefﬁcient plots (Fig. S28

and S31†) of these compounds actually had negative intercepts.

Partition coefﬁcients for these two compounds could not be

accurately estimated but appear to be >10⁴. The acridine ring of

peroxide intermediate iii (Fig. 2) contains two negative charges

and micelle binding is expected to be even stronger for this reac-

tive intermediate for all labels. In conclusion, our observations

on micellar partitioning of acridinium esters and their corre-

sponding pseudobases indicate that charge interactions can com-

pensate for weak hydrophobic binding. Thus, the micelle’s role

in enhancing light yield is only marginally affected for labels

with hydrophilic acridinium rings.

We used a chromatographic (HPLC) method pioneered by

Armstrong and Nome¹⁷for the measurement of partition coefﬁ-

cients of the acridinium esters to CTAC micelles as described in

the ESI.† Micelle–water partition coefﬁcients (K_MW) of acridi-

nium esters 10a–10d and the corresponding amine derivatives

9a–9d to CTAC micelles are listed in Table 5. For the acridinium

esters, at pH 2.8, K_MWdecreased in the order 10c < 10a < 10d.

As predicted, the acridinium esters with the more hydrophobic

rings (10a and 10d) partitioned more strongly than acridinium

esters with hydrophilic acridinium rings (10c). Compound 10b,

containing a very hydrophilic acridinium ring with a sulfobetaine

zwitterion, showed poor retention on the HPLC column which

also did not change as a function of surfactant concentration.

Micelle binding of this compound thus appears weak which is

consistent with chemiluminescence measurements that showed

only marginal acceleration in emission kinetics in the presence

of CTAC.

Chemiluminescence stability and non-speciﬁc binding

For practical applications, such as in clinical immunoassays,

acridinium ester labels must have low non-speciﬁc binding (a

requisite for assay sensitivity) and, must exhibit good chemilu-

minescence stability (necessary for long shelf life of reagents).

Chemiluminescence instability of acridinium esters is mostly

attributed to hydrolysis of the phenolic ester linkage,²but the

introduction of two, ﬂanking methyl groups dramatically

improves the stability of the phenolic ester.¹⁸The chemilumines-

cence stability of the anti-TSH antibody conjugates of the four

labels 10a–10d at 4 °C and at 37 °C in pH = 8 buffer is shown

in Fig. 10. As can be noted from the ﬁgure, the four labels

showed no loss of chemiluminescence even after 28 days at

4 °C. Increasing the temperature to 37 °C showed the loss of a

small amount (10–20%) of chemiluminescence for all labels in

the same timeframe. The alkoxy-substituted acridinium ester

labels 10a–10c were more or less equally stable at this

The pseudobases of the amine derivatives 9a–9d contain

negatively charged acridine rings and as a result, micelle–water

partition coefﬁcients for all compounds were observed to be

This journal is © The Royal Society of Chemistry 2012

Org. Biomol. Chem., 2012, 10, 3432–3447 | 3439

View Online

temperature. Because the C-2 alkoxy groups are remote from the

phenolic ester linkage, structural variations in these alkoxy

groups in 10a–10c are not expected to and did not affect chemi-

luminescence stability.

segregation of hydrophobic and hydrophilic structural features in

the acridinium ring and phenolic ester respectively, appears to be

an effective strategy in acridinium ester design for maximizing

the impact of the surfactant on chemiluminescence without com-

promising non-speciﬁc binding.

Acridinium dimethylphenyl ester labels are used in conjunc-

tion with magnetic microparticles in automated immunoassays in

Siemens Healthcare Diagnostics’ ADVIA Centaur® systems.

Assay sensitivity is a function of both light output of the label

and the background signal caused by non-speciﬁc binding of the

label. While a large number of studies¹⁹have outlined how to

devise protein-resistant surfaces either by PEG or zwitterion

modiﬁcation, similar studies on how to minimize non-speciﬁc

binding of labels such as chemiluminescent acridinium esters are

scant. In an earlier study,^1awe reported that PEG introduction in

the acridinium ring of alkoxy-substituted acridinium esters

reduces their non-speciﬁc binding to paramagnetic microparticles

(PMPs) and improves immunoassay performance. In the current

study, we observed that the alkoxy-substituted acridinium ester

10a with a more hydrophobic ring showed the maximum

enhancement of light output in the presence of surfactants. To

determine whether the polar sulfobetaine linker in the phenolic

ester leaving group of this compound could counterbalance the

hydrophobic acridinium ring, we measured the non-speciﬁc

binding of anti-TSH antibody conjugate of this label as well as

the conjugates of compounds 10c–10d to PMPs. The conjugates

were labeled to the same extent and contained 4–5 labels per

antibody molecule.

The PMPs used for measurement of non-speciﬁc binding

were, 1–10 micron-sized iron(^III) oxide particles, coated with an

anti-TSH antibody on the amino-silanized particle surface using

commonly used glutaraldehyde coupling chemistry. The particles

were mixed with solutions of the acridinium ester-labeled conju-

gates and were then magnetically separated, washed twice with

water and then the chemiluminescence associated with the par-

ticles was measured. The ratio of this chemiluminescence value

in comparison to the total chemiluminescence input is referred to

asfractional non-speciﬁc binding (FNSB) and is a reﬂection of

the resistance of the conjugate towards non-speciﬁc adsorption

to the microparticles. The results of these measurements are

tabulated in Table 6 and details are described in the Experimental

section. The FNSB values in Table 6 indicate that the introduc-

tion of hydrophilic functional groups (sulfobetaine zwitterion or

PEG) in the acridinium ring does not confer any additional

advantage in the presence of a polar, zwitterionic linker in the

phenolic ester leaving group. Conjugates of all four labels had

similar values of non-speciﬁc binding with the conjugate of 10a

actually showing a two-fold decrease compared to 10d. Thus,

Conclusions

In the current study, we have investigated in detail for the ﬁrst

time how structural changes in the acridinium ring of chemilumi-

nescent acridinium esters affect light emission in the presence of

surfactants. Our results indicate that a relatively hydrophobic

acridinium ring is needed to maximize the impact of the surfac-

tant i.e. fast light emission and enhanced light output. The

hydrophobicity of the acridinium ring can be offset by introdu-

cing a polar sulfobetaine linker in the phenolic ester leaving

group so that non-speciﬁc binding of the label is not exacerbated.

Among the three C-2 alkoxy-substituted acridinium esters that

were synthesized in the current study, compound 10a with a C-2

isopropyloxy group in the acridinium ring and a sulfobetaine

linker in the phenolic ester, is likely to be a useful label for auto-

mated immunoassays. Not only does this compound exhibit fast

emission and high light output (double that of 10d with an

unsubstituted acridinium ring), the label also exhibits good che-

miluminescence stability and relatively low non-speciﬁc binding

to magnetic microparticles. An added bonus is that this com-

pound is the least bulky of the three alkoxy-substituted labels

with a relatively straightforward synthesis.

Experimental

General

Chemicals and surfactants were purchased from Sigma-Aldrich

(Milwaukee, Wisconsin, USA) unless indicated otherwise. Cetyl-

trimethylammonium chloride (CTAC) and hexa(ethylene)glycol

monomethyl ether were purchased from TCI America. Cetyltri-

propylammonium chloride (CTPAC) was synthesized using a lit-

erature procedure.²⁰

All ﬁnal acridinium esters and intermediates were analyzed

and/or puriﬁed by HPLC using a Beckman-Coulter HPLC

system. TLC analysis was performed using 250 μm analytical

silica plates from EM Separations Technologies. Flash chromato-

graphy was performed using glass columns or an ‘Autoﬂash’

system from ISCO. MALDI-TOF (matrix-assisted laser de-

sorption ionization-time of ﬂight) mass spectroscopy was per-

formed using a Voyager DE^TMBiospectrometry^TMWorkstation

from Perkin-Elmer. This is a benchtop instrument operating in

the linear mode with a 1.2 meter ion path length, ﬂight tube.

Spectra were acquired in positive ion mode. For small molecules,

α-cyano-4-hydroxycinnamic acid was used as the matrix and

spectra were acquired with an accelerating voltage of 20 000

volts and a delay time of 100 ns. For protein conjugates, sinapi-

nic acid was used as the matrix and spectra were acquired with

an accelerating voltage of 25 000 volts and a delay time of 85 ns.

For HRMS (high resolution mass spectra), samples were dis-

solved in HPLC-grade methanol and analyzed by direct-ﬂow

injection (injection volume = 5 μL) electrospray ionization (ESI)

on a Waters Qtof API US instrument in the positive ion mode.

Table 6 Non-speciﬁc binding of anti-TSH antibody conjugates of

acridinium esters to PMPs^a

Conjugate

FNSB^b

Relative FNSB^c

Anti-TSH-10a

Anti-TSH-10b

Anti-TSH-10c

Anti-TSH-10d

7.3 × 10⁻⁵

1.3 × 10⁻⁴

2.2 × 10⁻⁴

1.5 × 10⁻⁴

0.5

0.9

1.5

1.0^c

^aPMPs = paramagnetic particles. ^bFNSB = fractional non-speciﬁc

binding. ^cRelative FNSB of label 10d with an unsubstituted acridinium

ring was assigned a value of one.

3440 | Org. Biomol. Chem., 2012, 10, 3432–3447

This journal is © The Royal Society of Chemistry 2012

View Online

Optimized conditions were as follows: capillary = 3000 kV, cone

= 35, source temperature = 120 °C, desolvation temperature =

350 °C. NMR spectra were recorded on a Varian 500 MHz spec-

trometer. IR spectra of neat samples were recorded on a Bruker

TENSOR37 FT-IR spectrometer, ATR mode on ZnSe crystal.

UV-Visible spectra were recorded on a Beckman DU 7500 spec-

trophotometer. Chemiluminescence measurements were carried

out using a Berthold Technologies’ AutoLumat Plus LB 953

luminometer.

single spot of R_f= 0.4. HPLC analysis of the product was per-

formed using a Phenomenex, C₁₈, 10 micron, 3.9 × 300 mm

column and a 40 min gradient of 10 → 60% MeCN–water (each

with 0.05% TFA) at a ﬂow rate of 1.0 mL min⁻¹and UV detec-

tion at 260 nm. Product was observed eluting at 20 min. Yield =

1.98 g (91%). ν_max/cm⁻¹3347 (OH), 1686 (CO), 1599, 1435,

1320, 1277, 1183.

δ_H(500 MHz, CDCl₃) 2.27 (s, 6H), 3.87 (s, 3H), 7.70 (s, 2H);

MALDI-TOF MS m/z 180.9 (M + H)⁺; HRMS m/z 181.0873

(M + H)⁺(181.0865 calculated).

1. Synthesis of acridinium esters (Fig. 3 and Fig. S1–S16 ESI†)

Compound 4. Compound 3 (1.506 g, 8.36 mmoles) and p-

toluenesulfonyl chloride (2.390 g, 12.54 mmoles) were added to

a suspension of compound 2 (2.54 g, 10.03 mmoles) in anhy-

drous pyridine (80 mL). The reaction was stirred at room temp-

erature for 4 hours under a nitrogen atmosphere. The reaction

mixture was then concentrated by rotary evaporation. The

residue was extracted with dichloromethane (80 mL) and washed

with cold 1 N HCl (10 mL), brine (20 mL × 2), cold 1% NaOH

(20 mL) and brine (20 mL × 2) in that order. The organic layer

was dried over anhydrous magnesium sulfate, ﬁltered and con-

centrated by rotary evaporation. The product was puriﬁed by

ﬂash chromatography on silica using 20% ethyl acetate in

hexanes as eluent. Yield = 3.0 g (86%, yellow powder). TLC

analysis using 40% ethyl acetate in hexanes showed a single spot

of R_f= 0.63. HPLC analysis of the puriﬁed product was per-

formed using a Phenomenex, C₁₈, 10 micron, 3.9 × 300 mm

column and a 30 min gradient of 10 → 100% MeCN–water

(each with 0.05% TFA) at a ﬂow rate of 1.0 mL min⁻¹and UV

detection at 260 nm. Product was observed eluting at 23 min.

ν_max/cm⁻¹1739 and 1720 (CO), 1632, 1611, 1473, 1434, 1323,

1226, 1149. δ_H(500 MHz, CDCl₃) 2.49 (s, 6H), 3.95 (s, 3H),

3.97 (s, 3H), 7.57 (dd, 1H, J = 9.4, 2.7), 7.58 (d, J = 2.7), 7.67

(m, 1H), 7.79 (m, 1H), 7.92 (s, 2H), 8.21 (d, 1H, J = 9.4), 8.30

(d, 1H, J = 8.2), 8.42 (d, 1H, J = 8.5); MALDI-TOF MS m/z

416.6 (M + H)⁺; HRMS m/z 416.1497 (M + H)⁺(416.1498

calculated).

Compound 2. A solution of isatin (2.5 g, 0.017 mole) in

anhydrous DMF (50 mL) was cooled in an ice bath under a

nitrogen atmosphere and treated with sodium hydride (60% dis-

persion, 0.8 g, 1.5 equivalents). A purple solution was formed

which was stirred at 0 °C for 30 min and then warmed to room

temperature.

A

solution of 4-bromoanisole (2.13 mL,

0.017 mole) followed by copper(^I) iodide (6.46 g, 0.034 mole)

was added to this solution. The reaction was heated in an oil

bath at 145 °C for 6–7 hours. It was then cooled to room temp-

erature and diluted with an equal volume of ethyl acetate. The

resulting suspension was ﬁltered and the ﬁltrate was concentrated

under reduced pressure. TLC analysis using 25% ethyl acetate in

hexanes showed clean formation of the N-arylated isatin with

R_f= 0.4 (isatin R_f= 0.25).

The crude N-aryl isatin derivative (11.7 g) was reﬂuxed in

10% KOH (150 mL) under a nitrogen atmosphere for 4 hours. It

was then ﬁltered while hot. The ﬁltrate was diluted with water

(150 mL) and ice. The resulting dark solution was then acidiﬁed

with concentrated HCl until a yellow precipitate of compound 2

separated out. The acridine carboxylic acid 2 was recovered by

ﬁltration and dried under vacuum over P₂O₅to give a yellowish-

brown powder. Yield = 1.84 g (43%). TLC analysis showed a

single spot of R_f= 0.15 using 25% methanol in chloroform as

eluent. HPLC analysis of the product was performed using a

Phenomenex, C₁₈, 10 micron, 3.9 × 300 mm column and a

40 min gradient of 10 → 40% MeCN–water (each with 0.05%

TFA, TFA = triﬂuoroacetic acid) at a ﬂow rate of 1.0 mL min⁻¹

and UV detection at 260 nm. Product was observed eluting at

6.2 min. ν_max/cm⁻¹3434 (OH), 1732 (CO), 1600, 1393, 1272,

1228. δ_H(500 MHz, CF₃COOD) 4.08 (s, 3H), 7.61 (br s, 1H),

7.99 (m, 1H), 8.01 (m, 1H), 8.23 (br t, 1H), 8.29 (br t, 2H), 8.41

(d, 1H, J = 8.8); MALDI-TOF MS m/z 253.9 (M + H)⁺; HRMS

m/z 254.0821 (M + H)⁺(254.0817 calculated).

Compound 5. A solution of boron tribromide in dichloro-

methane (1.0 M, 40 mL, 40.0 mmoles) was added to a solution

of compound 4 (2.84 g, 6.84 mmoles) in dichloromethane

(40 mL). The reaction was stirred at room temperature for

4 hours. The reaction mixture was cooled to 0 °C and MeOH

(200 mL) added slowly. The reaction mixture was allowed to

gradually warm to room temperature and was stirred for

16 hours. The reaction mixture was then cooled again to 0 °C

and solid sodium bicarbonate was added in portions until gas

evolution ceased. The reaction mixture was then concentrated by

rotary evaporation. The residue was partitioned between ethyl

acetate (150 mL) and de-ionized water (50 mL). The organic

layer was separated and washed with brine (50 mL). It was then

dried over anhydrous magnesium sulfate, ﬁltered and concen-

trated under reduced pressure. The product was puriﬁed by ﬂash

chromatography on silica (ISCO Autoﬂash system) using a

20 min gradient of 0 → 50% ethyl acetate in hexanes as eluent at

a ﬂow rate of 40 mL min⁻¹and UV detection at 260 nm. Con-

centration of the product fractions afforded an orange-yellow

solid. Yield = 2.46 g (90%). TLC analysis using 20% ethyl

acetate in hexanes showed a single spot of R_f= 0.14. HPLC

Compound 3. A solution of 3,5-dimethyl-4-hydroxybenzoic

acid (2 g, 0.012 mole) in methanol (50 mL) was cooled in an

ice-bath under a nitrogen atmosphere. Thionyl chloride (5 mL)

was added drop wise to the cold methanol solution. After com-

pletion of the addition, the reaction was warmed to room temp-

erature and stirred for 48 hours. The reaction was then

neutralized with the addition of solid sodium bicarbonate until

gas evolution ceased. The suspension was evaporated to dryness.

The residue was partitioned between ethyl acetate (100 mL) and

de-ionized water (50 mL). The ethyl acetate layer was washed

with brine, dried over anhydrous magnesium sulfate and concen-

trated under reduced pressure to give a tan powder. TLC analysis

of the product using 2% ethyl acetate in chloroform showed a

This journal is © The Royal Society of Chemistry 2012

Org. Biomol. Chem., 2012, 10, 3432–3447 | 3441

View Online

analysis of the puriﬁed product was performed using a Phenom-

enex, C₁₈, 10 micron, 3.9 × 300 mm column and a 30 min gradi-

ent of 10 → 70% MeCN–water (each with 0.05% TFA) at a ﬂow

rate of 1.0 mL min⁻¹and UV detection at 260 nm. Product was

observed eluting at 25.5 min. ν_max/cm⁻¹1746 and 1718 (CO),

1631, 1609, 1460, 1323, 1231, 1137. δ_H(500 MHz, CDCl₃)

2.30 (s, 6H), 3.91 (s, 3H), 7.54 (dd, 1H, J = 9.3, 2.6), 7.63 (m,

1H), 7.70 (d, 1H, J = 2.4), 7.76 (m, 1H), 7.81 (s, 2H), 8.27 (d,

1H, J = 9.4), 8.31 (d, 1H, J = 8.8), 8.38 (d, 1H, J = 8.8);

MALDI-TOF MS m/z 401.1 (M + H)⁺; HRMS m/z 402.1334

(M + H)⁺(402.1341 calculated).

2H), 2.47 (s, 12H), 2.74 (m, 2H), 3.94 (s, 3H), 4.18 (t, 2H, J =

6.1), 7.52 (dd, 1H, J = 9.3, 2.6), 7.56 (d, 1H, J = 2.5), 7.66 (m,

1H), 7.79 (m, 1H), 7.92 (s, 2H), 8.20 (d, 1H, J = 9.4), 8.29 (d,

1H, J = 8.2), 8.41 (d, 1H, J = 8.2); MALDI-TOF MS m/z 487.5

(M + H)⁺; HRMS m/z 487.2228 (M + H)⁺(487.2233

calculated).

Compound 6c. A solution of compound

5 (0.05 g,

0.125 mmole), triphenylphosphine (0.065 g, 0.25 mmole) and

hexa(ethylene)glycol monomethyl ether (0.055 g, 0.186 mmole)

in anhydrous tetrahydrofuran (5 mL) was treated with DIAD

(0.05 mL, 0.25 mmole) under a nitrogen atmosphere. The reac-

tion was stirred at room temperature for one hour by which time

TLC analysis using 5% methanol in ethyl acetate showed com-

plete conversion to a polar product of R_f= 0.5. No starting

material was present when the reaction was analyzed by TLC

using 1 : 1 ethyl acetate–hexanes. The product was puriﬁed by

ﬂash chromatography on silica using 2% methanol in ethyl

acetate as eluent. HPLC analysis of the puriﬁed product was per-

formed using a Phenomenex, C₁₈, 10 micron, 3.9 × 300 mm

column and a 30 min gradient of 10 → 70% MeCN–water (each

with 0.05% TFA) at a ﬂow rate of 1.0 mL min⁻¹and UV detec-

tion at 260 nm. Product was observed eluting at 29 min. Yield =

0.032 g (38%, yellow sticky solid). ν_max/cm⁻¹1747 and 1712

(CO), 1631, 1447, 1323, 1235, 1181, 1130, 1097. δ_H(500 MHz,

CDCl₃) 2.47 (s, 6H), 3.36 (s, 3H), 3.53 (m, 2H), 3.61–3.63 (m,

2H), 3.64 (br s, 12H), 3.69 (m, 2H), 3.75 (m, 2H), 3.95 (s, 3H),

3.96–3.98 (m, 2H), 4.30 (br t, 2H), 7.61 (d, 1H, J = 2.4),

7.86–7.91 (m, 2H), 7.93 (s, 2H), 8.09 (m, 1H), 8.49 (d, 1H, J =

8.9), 8.85 (m, 2H); MALDI-TOF MS m/z 679.7 (M + H)⁺;

HRMS m/z 680.3070 (M + H)⁺(680.3071 calculated).

Compound 6a. A solution of compound

5 (0.25 g,

0.62 mmole), triphenylphosphine (0.327 g, 1.25 mmoles) and 2-

propanol (0.096 mL, 1.13 mmoles) in anhydrous tetrahydrofuran

(10 mL) at 0 °C was treated with diisopropyl azodicarboxylate

(DIAD, 0.244 mL, 1.24 mmoles) under a nitrogen atmosphere.

The reaction was stirred at 0 °C for one hour by which time TLC

analysis using 20% ethyl acetate in hexanes showed complete

conversion to a less polar product of R_f= 0.4 (starting material

R_f= 0.14). The reaction was concentrated by rotary evaporation

to ∼5 mL and the product was puriﬁed by ﬂash chromatography

on silica using 10% ethyl acetate in hexanes as eluent. HPLC

analysis of the puriﬁed product was performed using a Phenom-

enex, C₁₈, 10 micron, 3.9 × 300 mm column and a 30 min gradi-

ent of 10 → 100% MeCN–water (each with 0.05% TFA) at a

ﬂow rate of 1.0 mL min⁻¹and UV detection at 260 nm. Product

was observed eluting at 30 min. Yield = 0.26 g (93%, yellow

sticky solid). ν_max/cm⁻¹1743 and 1716 (CO), 1633, 1612, 1466,

1323, 1228, 1210, 1132. δ_H(500 MHz, CDCl₃) 1.44 (d, 6H, J =

6.1), 2.47 (s, 6H), 3.95 (s, 3H), 4.74 (spt, 1H, J = 6.1), 7.52 (m,

1H), 7.58 (d, 1H, J = 2.8), 7.67 (m, 1H), 7.80 (m, 1H), 7.92 (s,

2H), 8.27 (br s, 1H), 8.34 (br s, 1H), 8.43 (d, 1H, J = 8.2);

MALDI-TOF MS m/z 445.8 (M + H)⁺; HRMS m/z 444.1805

(M + H)⁺(444.1811 calculated).

Compound 7a. A mixture of compound 6a (0.213 g,

0.48 mmole), 1,3-propane sultone (0.88 g, 7.2 mmoles, 15

equivalents)

and

2,6-di-tert-butylpyridine

(0.79

mL,

3.5 mmoles) in 1-butyl-3-methylimidazolium hexaﬂuorophos-

phate [BMIM][PF₆] (3 mL) was heated at 155 °C in a round

bottom ﬂask under argon with vigorous stirring. After 16 hours,

the reaction was cooled to room temperature. A small portion of

the reaction (0.002 mL) was withdrawn, diluted with MeCN

(0.1 mL) and analyzed by HPLC using a Phenomenex, C₁₈,

10 micron, 3.9 × 300 mm column and a 30 min gradient of 10

→ 100% MeCN–water (each with 0.05% TFA) at a ﬂow rate of

1.0 mL min⁻¹and UV detection at 260 nm. The N-sulfopropyl

acridinium ester (methyl ester of compound 7a) was observed

eluting at 19.5 min (90% conversion). The reaction was diluted

with ethyl acetate (5 mL) and loaded onto a silica column equili-

brated with ethyl acetate. The column was eluted with ethyl

acetate (500 mL) to elute unreacted starting material and base

followed by 40% methanol in ethyl acetate to elute product. The

fractions containing the acridinium ester were concentrated

under reduced pressure. The N-sulfopropyl acridinium ester

(0.25 g) was reﬂuxed in 20 mL of 2 N HCl for 3 hours. It was

then cooled to room temperature. HPLC analysis as described

above showed complete conversion to compound 7a eluting at

15.5 min. The reaction mixture was concentrated by rotary evap-

oration to ∼10 mL and diluted with MeCN (10 mL). The

product 7a was puriﬁed by preparative HPLC using a YMC,

C₁₈, 10 micron, 30 × 250 mm column and 30 min gradient of 10

Compound 6b. A solution of compound

5 (0.025 g,

0.062 mmole), triphenylphosphine (0.033 g, 0.13 mmole) and 3-

dimethylamino-1-propanol (0.015 mL, 0.13 mmole) in anhy-

drous tetrahydrofuran (3 mL) at room temperature was treated

with DIAD (0.025 mL, 0.124 mmole) under a nitrogen atmos-

phere. The reaction was stirred for one hour by which time TLC

analysis using 1 : 1, methanol–ethyl acetate showed complete

conversion to a polar product of R_f= 0.2. Using ethyl acetate as

eluent, starting material was completely consumed. The reaction

was diluted with ethyl acetate (25 mL) and 1 N HCl (25 mL).

The HCl layer was separated and washed with ethyl acetate (2 ×

25 mL). It was then cooled in ice and treated with 5% KOH until

the product precipitated out. The resulting suspension was

extracted twice with ethyl acetate (2 × 25 mL). The combined

ethyl acetate extracts were dried over anhydrous magnesium

sulfate, ﬁltered and concentrated under reduced pressure to

afford a yellow solid. HPLC analysis of the product was per-

formed using a Phenomenex, C₁₈, 10 micron, 3.9 × 300 mm

column and a 30 min gradient of 10 → 70% MeCN–water (each

with 0.05% TFA) at a ﬂow rate of 1.0 mL min⁻¹and UV detec-

tion at 260 nm. Product was observed eluting at 25 min. Yield =

24.5 mg (80%). ν_max/cm⁻¹1741 and 1715 (CO), 1631, 1611,

1442, 1321, 1225, 1146, 1128. δ_H(500 MHz, CDCl₃) 2.20 (m,

3442 | Org. Biomol. Chem., 2012, 10, 3432–3447

This journal is © The Royal Society of Chemistry 2012

View Online

→ 100% MeCN–water (each with 0.05% TFA) at a solvent ﬂow

rate of 20 mL min⁻¹and UV detection at 260 nm. The HPLC

fractions containing product 7a were combined and concentrated

under reduced pressure to give a yellow, sticky solid. Yield =

0.12 g (45%). TLC analysis (1 : 4, MeOH–ethyl acetate with a

drop of acetic acid) showed product with R_f= 0.4. ν_max/cm⁻¹

3477 and 3415 (OH), 1743 and 1705 (CO), 1633, 1556, 1465,

1385, 1307, 1148 1097. δ_H(500 MHz, CF₃COOD) 1.48 (d, 6H,

J = 6.1), 2.53 (s, 6H), 2.89 (m, 2H), 3.78 (t, 2H, J = 6.0), 4.88

(spt, 1H, J = 6.0), 5.79 (m, 2H), 7.83 (d, 1H, J = 2.4), 8.04 (m,

3H), 8.16 (m, 1H), 8.38 (m, 1H), 8.67 (d, 1H, J = 8.6), 8.80 (m,

2H); MALDI-TOF MS m/z 553.2 (M + H)⁺; HRMS m/z

552.1688 (M + H)⁺(552.1692 calculated).

21.5 min. The reaction was diluted with ethyl acetate and

puriﬁed by ﬂash chromatography as described previously for

compound 7a. Yield = 0.187 g (54%). The acridinium methyl

ester (120 mg) was reﬂuxed in 1 N HCl (5 mL) for 1.5 hours. It

was then cooled to room temperature and analyzed by HPLC

which showed complete conversion to compound 7c eluting at

18.5 min. The product was puriﬁed by preparative HPLC using a

YMC, C₁₈, 10 micron, 30 × 250 mm column and 30 min gradi-

ent of 10 → 70% MeCN–water (each with 0.05% TFA) at a

solvent ﬂow rate of 20 mL min⁻¹and UV detection at 260 nm.

The HPLC fractions containing product 7c were combined and

concentrated under reduced pressure to give a yellow, sticky

solid. Yield = 78 mg (66% from acridinium methyl ester). TLC

analysis (2 : 1, MeOH–ethyl acetate with a drop of acetic acid)

showed product with R_f= 0.40. ν_max/cm⁻¹3420 (OH), 1750 and

1709 (CO), 1628, 1554, 1473, 1448, 1379, 1305, 1143, 1102,

1034. δ_H(500 MHz, CF₃COOD) 2.52 (s, 6H), 2.88 (m, 2H),

3.53 (s, 3H), 3.78 (t, 2H, J = 6.0), 3.87–3.89 (br s, 14H),

3.91–3.94 (m, 2H), 4.01–4.07 (m, 2H), 4.20 (br s, 2H), 4.47 (br

s, 2H), 5.81 (br s, 2H), 7.82 (br s, 1H), 8.03 (s, 2H), 8.06 (m,

1H), 8.17 (m, 1H), 8.41 (m, 1H), 8.71 (d, 1H, J = 8.7), 8.84 (m,

2H); MALDI-TOF MS m/z 787.9 (M + H)⁺; HRMS m/z

788.2950 (M + H)⁺(788.2952 calculated).

Compound 7b. A mixture of compound 6b (0.025 g,

0.050 mmole), 1,3-propane sultone (0.125 g, 1.02 mmoles, 20

equivalents) and 2,6-di-tert-butylpyridine (0.079 mL,

0.35 mmole) in [BMIM]PF₆] (0.5 mL) was heated at 150 °C

under argon for 24 hours. It was then cooled to room temperature

and analyzed by HPLC using a Phenomenex, C₁₈, 10 micron,

3.9 × 300 mm column and a 30 min gradient of 10 → 70%

MeCN–water (each with 0.05% TFA) at a ﬂow rate of 1.0 mL

min⁻¹and UV detection at 260 nm. The di-alkylated product

(methyl ester of compound 7b) was observed eluting at 18 min

(>80% conversion). The crude reaction mixture was partitioned

between water and ethyl acetate (25 mL each). The aqueous

layer containing product was separated and washed once with

ethyl acetate (50 mL). It was then concentrated under reduced

pressure. The residue was reﬂuxed in 1 N HCl (10 mL) for

2 hours under a nitrogen atmosphere. The reaction was cooled to

room temperature and analyzed by HPLC as described above

which showed complete conversion to compound 7b eluting at

14.3 min. The product 7b was puriﬁed by preparative HPLC

using a YMC, C₁₈, 10 micron, 30 × 250 mm column and 30 min

gradient of 10 → 70% MeCN–water (each with 0.05% TFA) at a

solvent ﬂow rate of 20 mL min⁻¹and UV detection at 260 nm.

The HPLC fractions containing product 7b were combined and

concentrated under reduced pressure to give a yellow, sticky

solid. Yield = 0.032 g (88%). TLC analysis (2 : 1, MeOH–ethyl

acetate with a drop of acetic acid) showed product with R_f=

0.17. ν_max/cm⁻¹3421 (OH), 1750 and 1702 (CO), 1630, 1555,

1468, 1378, 1307, 1186, 1147, 1035. δ_H(500 MHz, CF₃COOD)

2.51 (s, 6H), 2.55 (m, 4H), 2.88 (m, 2H), 3.24 (s, 6H), 3.42 (t,

2H, J = 5.7), 3.70–3.80 (m, 6H), 4.38 (br s, 2H), 5.82 (m, 2H),

7.82 (br s, 1H), 8.04 (s, 2H), 8.08 (m, 1H), 8.17 (br d, 1H), 8.43

(m, 1H), 8.75 (br d, 1H), 8.83 (d, 1H, J = 9.2), 8.89 (d, 1H, J =

9.5); MALDI-TOF MS m/z 717.1 (M + H)⁺; HRMS m/z

717.2161 (M + H)⁺(717.2152 calculated).

Compound 9a. A solution of compound 7a (0.120 g,

0.217 mmole) in DMF (4 mL) was treated with diisopropylethyl-

amine (0.076 mL, 0.44 mmole) and N,N,N′,N′-tetramethyl-O-(N-

succinimidyl)uronium tetraﬂuoroborate (TSTU) (0.079 g,

0.26 mmole). The reaction was stirred at room temperature. After

15 min the reaction was analyzed by HPLC using a Phenom-

enex, C₁₈, 10 micron, 3.9 × 300 mm column and a 30 min gradi-

ent of 10 → 100% MeCN–water (each with 0.05% TFA) at a

ﬂow rate of 1.0 mL min⁻¹and UV detection at 260 nm. The

NHS ester of compound 9a was observed eluting at 18.5 min.

This solution was added drop wise to a chilled solution of com-

pound 8 (0.4 g, 0.89 mmole, HBr salt) dissolved in a mixture of

0.25 M sodium bicarbonate (8 mL) and DMF (2 mL). The

resulting reaction was stirred at room temperature. After 30 min,

HPLC analysis indicated complete conversion to compound 9a

eluting at 12 min. Using a gradient of 10 → 70% MeCN–water

(each with 0.05% TFA), product eluted at 14 min. The product

was puriﬁed by preparative HPLC using

a YMC, C₁₈,

10 micron, 30 × 250 mm column and 30 min gradient of 10 →

100% MeCN–water (each with 0.05% TFA) at a solvent ﬂow

rate of 20 mL min⁻¹and UV detection at 260 nm. The HPLC

fractions containing product 9a were combined and concentrated

under reduced pressure to give a yellow, sticky solid. Yield =

0.13 g (65%, TFA salt). ν_max/cm⁻¹3416 (NH), 1750 and 1706

(CO), 1630, 1554, and 1468, 1378, 1307, 1186, 1147, 1035. δ_H

(500 MHz, CF₃COOD) 1.49 (d, 6H, J = 6.0), 2.36 (m, 2H), 2.52

(s, 10H), 2.89 (m, 2H), 3.20 (s, 3H), 3.42 (m, 4H), 3.61 (m, 4H),

3.77 (br m, 6H), 4.87 (m, 1H), 5.79 (br t, 2H), 7.67 (s, 2H), 7.79

(s, 1H), 8.04 (br t, 1H), 8.17 (br d, 1H), 8.39 (br t, 1H), 8.64 (d,

1H, J = 8.7), 8.80 (br d, 2H); MALDI-TOF MS m/z 802.7 (M +

H)⁺; HRMS m/z 823.3028 (M + Na)⁺(823.3023 calculated).

Compound 7c. A mixture of compound 6c (0.295 g,

0.434 mmole), 1,3-propane sultone (0.795 g, 6.51 mmoles) and

2,6-di-tert-butylpyridine (0.72 mL, 3.25 mmoles) in [BMIM]

[PF₆] (2.6 mL) was heated at 150 °C under argon. After

24 hours, the reaction was cooled to room temperature and ana-

lyzed by HPLC using a Phenomenex, C₁₈, 10 micron, 3.9 ×

300 mm column and a 30 mine gradient of 10 → 70% MeCN–

water (each with 0.05% TFA) at a ﬂow rate of 1.0 mL min⁻¹and

UV detection at 260 nm. The N-sulfopropyl acridinium ester

(methyl ester of compound 7c) was observed eluting at

Compound 9b. A solution of compound 7b (0.032 g,

0.045 mmole) in DMF (3.4 mL) and de-ionized water (0.6 mL)

was treated with diisopropylethylamine (0.039 mL, 0.22 mmole)

Org. Biomol. Chem., 2012, 10, 3432–3447 | 3443

View Online

and TSTU (0.067 g, 0.22 mmole). The reaction was stirred at

room temperature. After 15 min, the reaction was analyzed by

HPLC using a Phenomenex, C₁₈, 10 micron, 3.9 × 300 mm

column and a 30 min gradient of 10 → 70% MeCN–water (each

with 0.05% TFA) at a ﬂow rate of 1.0 mL min⁻¹and UV detec-

tion at 260 nm. The NHS ester of compound 9b was observed

eluting at 17 min. The NHS ester was puriﬁed by preparative

HPLC using a YMC, C₁₈, 10 micron, 30 × 250 mm column and

30 min gradient of 10 → 70% MeCN–water (each with 0.05%

TFA) at a solvent ﬂow rate of 20 mL min⁻¹and UV detection at

260 nm. The HPLC fractions containing the NHS ester were

frozen at −80 °C and lyophilized to afford a yellow powder.

Yield = 25 mg (69%). A solution of this NHS ester (0.01 g,

0.012 mmole) in DMSO (1 mL) was added drop wise to a

chilled solution of compound 8 (0.027 g, 0.06 mmole, HBr salt)

dissolved in 0.25 M sodium bicarbonate (0.6 mL). The resulting

solution was stirred at room temperature. After 30 minutes the

reaction was analyzed by HPLC using a Phenomenex, C₁₈,

10 micron, 3.9 × 300 mm column and a 30 min gradient of 10

→ 70% MeCN–water (each with 0.05% TFA) at a ﬂow rate of

1.0 mL min⁻¹and UV detection at 260 nm. Product 9b was

observed eluting at 13.5 min. The product was puriﬁed by pre-

parative HPLC using a YMC, C₁₈, 10 micron, 30 × 250 mm

column and 30 min gradient of 10 → 70% MeCN–water (each

with 0.05% TFA) at a solvent ﬂow rate of 20 mL min⁻¹and UV

detection at 260 nm. The HPLC fractions containing 9b were

concentrated under reduced pressure. Yield = 8.5 mg (71%).

ν_max/cm⁻¹3408 (NH), 1745 and 1670 (CO), 1629, 1554, 1467,

1377, 1159, 1036. δ_H(500 MHz, CF₃COOD) 2.38 (br s, 2H),

2.54 (s, 6H), 2.57 (m, 4H), 2.92 (m, 2H), 3.24 (s, 3H), 3.29 (s,

6H), 3.41 (br s, 6H), 3.47 (br s, 4H), 3.65 (br s, 4H), 3.70–3.90

(br m, 10H), 4.42 (br s, 2H), 5.82 (m, 2H), 7.17 (br s, 1H), 7.72

(br s, 2H), 7.83 (br s, 1H), 8.13 (m, 1H), 8.22 (d, 1H, J = 9.8),

8.48 (m, 1H), 8.78 (d, 1H, J = 8.6), 8.85 (d, 1H, J = 9.8), 8.92

(d, 1H, J = 10.0); MALDI-TOF MS m/z 965.5 (M + H)⁺;

HRMS m/z 966.3660 (M + H)⁺(966.3663 calculated).

CF₃COOD) 2.38 (m, 2H), 2.54 (s, 6H), 2.91 (m, 2H), 3.22 (m,

3H), 3.32–3.50 (m, 4H), 3.56 (s, 3H), 3.63 (m, 4H), 3.69–3.83

(m, 6H), 3.85 (m, 2H), 3.88–3.96 (br s, 14H), 3.97 (m, 2H),

4.08 (m, 2H), 4.23 (br s, 2H), 4.49 (br s, 2H), 5.83 (m, 2H),

7.69 (s, 2H), 7.83 (d, 1H, J = 2.1), 8.03 (s, 2H), 8.10 (m, 1H),

8.21 (dd, 1H, J = 9.9, 2.0), 8.45 (m, 1H), 8.72 (d, 1H, J = 8.9),

8.86 (m, 2H); MALDI-TOF MS m/z 1036.4 (M + H)⁺; HRMS

m/z 1037.4460 (M + H)⁺(1037.4463 calculated).

Compound 10a. A solution of compound 9a (0.05 g,

0.053 mmole, TFA salt) in 1 : 1 DMF–methanol (5 mL) was

treated with diisopropylethylamine (0.054 mL, 0.31 mmole) and

glutaric anhydride (0.036 g, 0.32 mmole). The reaction was

stirred at room temperature. After one hour the reaction was ana-

lyzed by HPLC using a Phenomenex, C₁₈, 10 micron, 3.9 ×

300 mm column and a 30 min gradient of 10 → 70% MeCN–

water (each with 0.05% TFA) at a ﬂow rate of 1.0 mL min⁻¹and

UV detection at 260 nm. The glutarate derivative of compound

9a was observed eluting at 16 min (complete conversion). The

reaction was diluted with toluene (5 mL) and concentrated under

reduced pressure. The crude glutarate derivative was dissolved in

DMF (6 mL) and treated with diisopropylethylamine (0.109 mL,

0.63 mmole) and TSTU (0.187 g, 0.62 mmole). The reaction

was stirred at room temperature. After 30 min the reaction was

analyzed by HPLC which indicated >80% conversion to 10a

eluting at 17 min. The NHS ester was puriﬁed by preparative

HPLC using a YMC, C₁₈, 10 micron, 30 × 250 mm column and

30 min gradient of 10 → 70% MeCN–water (each with 0.05%

TFA) at a solvent ﬂow rate of 20 mL min⁻¹and UV detection at

260 nm. The HPLC fractions containing the NHS ester were

frozen at −80 °C and lyophilized to afford a yellow powder.

Yield = 61 mg (quantitative). ν_max/cm⁻¹3290 (NH), 1815, 1782

and 1733 and 1653 (CO), 1553, 1449, 1375, 1199, 1157, 1069,

1037. δ_H(500 MHz, CF₃COOD) 1.49 (d, 6H, J = 6.0), 2.36 (m,

2H), 2.52 (s, 10H), 2.89 (m, 2H), 3.20 (s, 3H), 3.42 (m, 4H),

3.61 (m, 4H), 3.77 (br m, 6H), 4.87 (m, 1H), 5.79 (br t, 2H),

7.67 (s, 2H), 7.79 (s, 1H), 8.04 (br t, 1H), 8.17 (br d, 1H), 8.39

(br t, 1H), 8.64 (d, 1H, J = 8.7), 8.80 (br d, 2H); MALDI-TOF

MS m/z 1014.2 (M + H)⁺; HRMS m/z 1034.3501 (M + Na)⁺

(1034.3503 calculated).

Compound 9c. A solution of compound 7c (0.0082 g,

0.01 mmole) in DMF (1.5 mL) was treated with

diisopropylethylamine (0.0036 mL, 0.02 mmole) and TSTU

(0.0047 g, 0.016 mmole). The reaction was stirred at room temp-

erature. After 15 min, the reaction was analyzed by HPLC using

a Phenomenex, C₁₈, 10 micron, 3.9 × 300 mm column and a

30 min gradient of 10 → 70% MeCN–water (each with 0.05%

TFA) at a ﬂow rate of 1.0 mL min⁻¹and UV detection at

260 nm. The NHS ester of compound 9c was observed eluting at

20.5 min. This solution was added drop wise to a chilled sol-

ution of compound 8 (0.035 g, 0.078 mmole, HBr salt) dissolved

in DMSO (0.28 mL) and 0.25 M sodium bicarbonate (0.6 mL).

The resulting solution was stirred at room temperature. After

30 min, HPLC analysis indicated complete conversion to com-

pound 9c eluting at 13.5 min. The product was puriﬁed by pre-

parative HPLC using a YMC, C₁₈, 10 micron, 30 × 250 mm

column and 30 min gradient of 10 → 70% MeCN–water (each

with 0.05% TFA) at a solvent ﬂow rate of 20 mL min⁻¹and UV

detection at 260 nm. The HPLC fractions containing 9c were

concentrated under reduced pressure. Yield = 11.2 mg (85%,

TFA salt). ν_max/cm⁻¹3396 (NH), 1747 and 1677 (CO), 1630,

1553 and 1474, 1195, 1161, 1125, 1038. δ_H(500 MHz,

Compound 10b. A solution of compound 9b (8.5 mg,

0.008 mmole) in 10% aqueous methanol (3.5 mL) was treated

with diisopropylethylamine (0.0077 mL, 0.044 mmole) and glu-

taric anhydride (4.5 mg, 0.04 mmole). The reaction was stirred

at room temperature. After 15 min the reaction was analyzed by

HPLC using a Phenomenex, C₁₈, 10 micron, 3.9 × 300 mm

column and a 30 min gradient of 10 → 100% MeCN–water

(each with 0.05% TFA) at a ﬂow rate of 1.0 mL min⁻¹and UV

detection at 260 nm. The glutarate derivative of compound 9b

was observed eluting at 12 min (∼90% conversion). The reaction

was diluted with toluene (2 mL) and concentrated under reduced

pressure. The glutarate derivative was dissolved in 10% aqueous

DMF (3 mL) and treated with diisopropylethylamine

(0.0154 mL, 0.088 mmole) and TSTU (26.4 mg, 0.088 mmole).

The reaction was stirred at room temperature. After 10 min,

HPLC analysis indicated complete conversion to 10b eluting at

13 min. The NHS ester was puriﬁed by preparative HPLC using

a YMC, C₁₈, 10 micron, 30 × 250 mm column and 30 min

3444 | Org. Biomol. Chem., 2012, 10, 3432–3447

View Online

gradient of 10 → 70% MeCN–water (each with 0.05% TFA) at a

solvent ﬂow rate of 20 mL min⁻¹and UV detection at 260 nm.

The HPLC fractions containing the NHS ester were frozen at

−80 °C and lyophilized to afford a yellow powder. Yield =

7.5 mg (73%). ν_max/cm⁻¹3290 (NH), 1809, 1782, 1734 and

1674 (CO), 1557, 1467, 1442, 1379, 1196, 1160, 1131, 1036. δ_H

(500 MHz, CF₃COOD) 2.10–2.39 (m, 6H), 2.39–2.71 (m, 10H),

2.78 (br s, 4H), 2.90 (m, 4H), 3.03 (s, 3H), 3.09–3.35 (m, 12H),

3.40 (m, 4H), 3.56 (m, 4H), 3.65–3.99 (m, 12H), 4.41 (br s,

2H), 5.84 (br s, 2H), 7.72 (br s, 2H), 7.81 (br s, 1H), 8.09 (br t,

1H), 8.18 (d, 1 H, J = 9.2), 8.44 (br t, 1H), 8.74 (d, 1H, J = 8.5),

8.84 (d, 1H, J = 10), 8.91 (d, 1H, J = 8.5); MALDI-TOF MS

m/z 1179.1 (M + H)⁺; HRMS m/z 1177.4152 (M + H)⁺

(1177.4144 calculated).

0.2 mL of 0.1 M sodium carbonate, pH 9. The protein solution

was treated with DMSO solutions of acridinium esters 10a–10c

as follows: for labeling with 7.5 equivalents of 10a, 0.0051 mL

of a 10 mg mL⁻¹DMSO was added; for labeling with 10

equivalents of 10b, 0.0157 mL of a 5 mg mL⁻¹DMSO solution

was added; and, for labeling with 10 equivalents of 10c,

0.0166 mL of a 5 mg mL⁻¹DMSO solution was added. The

labeling reactions were stirred at 4 °C for 16 hours and were then

transferred to 4 mL Amicon^TMﬁlters (MW 30 000 cutoff) and

diluted with 3.5 mL de-ionized water. The volume was reduced

to ∼0.1 mL by centrifuging at 4000 G for 10 min. The concen-

trated conjugate solutions were diluted with 4 mL de-ionized

water and centrifuged again to reduce the volume. This process

was repeated a total of four times. Finally, the concentrated con-

jugates were diluted with 0.1 mL de-ionized water. The conju-

gates were analyzed by MALDI-TOF mass spectrometry, using

the Voyager-DE instrument from Perkin-Elmer, to measure acri-

dinium ester incorporation. This entailed measuring the molecu-

lar weight of the unlabeled protein and the labeled protein. The

acridinium ester label contributed to the observed difference in

mass between these two measurements. By knowing the molecu-

lar weight of the speciﬁc acridinium ester label, the extent of

label incorporation for that speciﬁc acridinium ester could thus

be calculated. Label incorporation in each protein is given in

Table 7.

Compound 10c. A solution of compound 9c (11.2 mg,

0.01 mmole) in methanol (1 mL) was treated with diisopropyl-

ethylamine (0.0086 mL, 0.049 mmole) and glutaric anhydride

(5.6 mg, 0.049 mmole). The reaction was stirred at room temp-

erature. After 15 min the reaction was analyzed by HPLC using

a Phenomenex, C₁₈, 10 micron, 3.9 × 300 mm column and a

30 min gradient of 10 → 70% MeCN–water (each with 0.05%

TFA) at a ﬂow rate of 1.0 mL min⁻¹and UV detection at

260 nm. The glutarate derivative of compound 9c was observed

eluting at 15.4 min. The glutarate derivative was puriﬁed by pre-

parative HPLC using a YMC, C₁₈, 10 micron, 30 × 250 mm

column and 30 min gradient of 10 → 70% MeCN–water (each

with 0.05% TFA) at a solvent ﬂow rate of 20 mL min⁻¹and UV

detection at 260 nm. The product fraction was concentrated

under reduced pressure. The glutarate (9 mg, 0.0072 mmole)

was dissolved in DMF (1 mL) and treated with diisopropylethyl-

amine (0.0063 mL, 0.036 mmole) and TSTU (10.8 mg,

0.036 mmole). The reaction was stirred at room temperature.

After 15 min, HPLC analysis indicated 80% conversion to com-

pound 10c eluting at 16.5 min. The NHS ester was puriﬁed by

preparative HPLC using a YMC, C₁₈, 10 micron, 30 × 250 mm

column and 30 min gradient of 10 → 70% MeCN–water (each

with 0.05% TFA) at a solvent ﬂow rate of 20 mL min⁻¹and UV

detection at 260 nm. The HPLC fractions containing the NHS

ester were frozen at −80 °C and lyophilized to afford a yellow,

sticky solid. Yield = 5 mg (57%). ν_max/cm⁻¹3216 (NH), 1815,

1782 and 1725 (CO), 1430, 1362, 1200, 1070, 1049. δ_H

(500 MHz, CF₃COOD) 2.06–2.40 (m, 6H), 2.54 (br s, 6H), 2.62

(br s, 2H), 2.78 (br s, 2H), 2.85–2.97 (m, 4H), 3.04 (br s, 4H),

3.17 (br s, 4H), 3.31 (m, 6H), 3.41 (br s, 4H), 3.55 (s, 6H), 3.79

(m, 6H), 3.85 (br s, 2H), 3.89 (br s, 10H), 3.96 (br s, 2H), 4.07

(br s, 2H), 4.22 (br s, 2H), 4.48 (br s, 2H), 5.82 (br s, 2H), 7.71

(br s, 2H), 7.82 (br s, 2H), 8.08 (br t, 1H), 8.19 (d, 1H, J = 9.6),

8.44 (br t, 1H), 8.70 (br d, 1H), 8.85 (br t, 2H); MALDI-TOF

MS m/z 1247.5 (M + H)⁺; HRMS m/z 1270.4802 (M + Na)⁺

(1270.4773 calculated).

3. Emission wavelength measurements (Fig. 4)

Visible wavelength emission spectra of the acridinium esters

10a–10d were measured using a FSSS (Fast Spectral Scanning

System) camera (Spectra Scan Model 704) from Photo Research

Inc. In a typical measurement, 50–100 μL of a 1 mg mL⁻¹sol-

ution of the acridinium ester in a 2 : 1 mixture of water–MeCN

(with 0.05% TFA) was diluted with 0.3 mL of reagent 1 com-

prising 0.5% hydrogen peroxide in 0.1 M nitric acid. Just prior

to the addition of reagent 2 comprising 7 mM CTAC in 0.25 M

sodium hydroxide, the shutter of the camera was opened and

light was collected for 5 seconds. The output of the instrument is

a graph of light intensity versus wavelength.

4. UV-Visible spectrophotometric measurements

(Fig. 5 and Fig. S17–S20 ESI†)

UV-Visible spectrophotometry of acridinium esters 10a–10d for

determination of the pK_aof acridinium to pseudobase transition

was carried out by dissolving the HPLC-puriﬁed acridinium

esters in DMSO (∼1 mg mL⁻¹). These solutions were further

diluted 20-fold into 0.2 M phosphate buffer at pH 2, 2.6, 3.0,

Table 7 Acridinium ester label incorporation in protein conjugates

Observed

mass

Observed increase

in mass

# Of

labels

2. Synthesis of anti-TSH antibody conjugates

Protein/conjugate

A murine anti-TSH monoclonal antibody with an acidic pI = 5.6

(TSH = thyroid stimulating hormone) was used for labeling with

the acridinium ester labels 10a–10c.

Unlabeled anti-TSH

Mab

Anti-TSH Mab-10a

Anti-TSH Mab-10b

Anti-TSH Mab-10c

150 486

—

154 279

155 419

155 376

3793

4933

4890

4.2

4.6

4.3

The anti-TSH murine monoclonal antibody (1 mg, 6.67 nano-

moles, stock solution 5 mg mL⁻¹, 0.2 mL) was diluted with

Org. Biomol. Chem., 2012, 10, 3432–3447 | 3445

View Online

3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5 and 7.0. A solution of 0.1 M HCl

was used for pH 1. The diluted acridinium ester solutions were

allowed to stand for one hour at room temperature and then

UV-Visible spectra were recorded from 220–500 nm. The

absorption intensity of the acridinium band at 371 nm for com-

pound 10d and 382–384 nm for compounds 10a–10c was

measured as a function of pH. A plot of this data is shown in

Fig. 5. UV-Visible spectra of the acridinium esters 10a–10d and

their amine precursors 9a–9d were also recorded in 0.1 M phos-

phate pH 2.8 and 0.1 M phosphate pH 9 respectively and are

illustrated in Fig. S17–S20 (ESI†).

in the ESI.† Partition coefﬁcient plots of the acridinium esters

are illustrated in Fig. S25–S31.

7. Measurement of chemiluminescence stability (Fig. 10)

Acridinium ester anti-TSH conjugates were diluted to a concen-

tration of 0.2 nM in a buffer of 10 mM sodium phosphate, 8 mM

sodium azide and 15 μM BSA at pH 8.0. The diluted conjugates

were kept at 4 and 37 °C for four weeks. The residual chemi-

luminescence of each diluted conjugate was assessed over the

course of the four weeks by periodically averaging the chemi-

luminescence measurements of ﬁve 10 μL samples on the Auto-

lumat LB953 Plus luminometer with the change in

5. Chemiluminescence measurements (Tables 1–4, Fig. 6–9

and Fig. S21–S24 ESI†)

chemiluminescence gauged against initial values as

percentage.

a

Chemiluminescence of acridinium esters 10a–10c and their anti-

TSH antibody conjugates was measured on an Autolumat

LB953 Plus luminometer from Berthold Technologies. HPLC-

puriﬁed acridinium esters 10a–10c were initially dissolved in a

mixture of methanol and 10 mM phosphate, pH 8 (1–2 mg

mL⁻¹) and were further diluted for chemiluminescence measure-

ments in an aqueous buffer, pH = 8, of 10 mM disodium hydro-

gen phosphate, 0.15 M NaCl, 8 mM sodium azide and

0.015 mM bovine serum albumin (BSA). Protein conjugates,

13–28 μM, based on protein concentration measured using the

BCA protein assay from Pierce, were serially diluted 10⁵-fold for

chemiluminescence measurements. Similarly, solutions of acridi-

nium ester labels (∼0.5 mM) were serially diluted 10⁶-fold for

chemiluminescence measurements. A 0.010 mL volume of each

diluted acridinium ester sample was dispensed into the bottom of

a cuvette. Cuvettes were placed into the primed LB953 and the

chemiluminescence reaction was initiated with the sequential

addition of 0.3 mL of Reagent 1, a solution of 0.5% hydrogen

peroxide in 0.1 M nitric acid followed by the addition of 0.3 mL

of Reagent 2, a solution of 0.25 M sodium hydroxide with or

without surfactant. Each chemiluminescence ﬂash curve was

measured in 240 intervals of 0.5 seconds (2 minutes total time)

from the point of chemiluminescence initiation with the addition

of 0.25 M NaOH. Each chemiluminescence reaction was carried

out a minimum of three times, averaged and converted to a per-

centage of the chemiluminescence accumulated up to each time

interval. Chemiluminescence values for 2 min collection times

were also normalized for comparison to reactions without surfac-

tant with reactions with the various surfactants. The output from

the luminometer instrument was expressed as RLUs (relative

light units). For evaluation of surfactants, the surfactant was

either omitted or included in Reagent 2 with one of the following

surfactants at ﬁve times its reported CMC in water: (a) cetyltri-

methylammonium chloride (CTAC), CMC = 1.4 mM,²⁰(b)

cetyltripropylammonium chloride (CTPAC), CMC = 0.65 mM,²⁰

and (c) N,N-dimethyldodecylammonio-1,3-propane sulfonate

(DDAPS), CMC = 3.17 mM.²¹

8. Measurement of non-speciﬁc binding (Table 6)

Acridinium ester anti-TSH conjugates were diluted to a concen-

tration of 2 nM in a buffer of 0.1 M HEPES (4-(2-hydro-

xyethyl)-1-piperazineethanesulfonic acid), 0.15

M sodium

chloride, 10 mM Triton-X100 and 0.1 mM BSA at pH 7.7. To

100 μL of each diluted conjugate was added and mixed, 200 μL

of TSH-free serum and 0.25 mL of ADVIA:Centaur® (Siemens

Healthcare Diagnostics) TSH3 assay solid phase containing

magnetically separable, paramagnetic microparticles (PMPs)

covalently derivatized with sheep anti-TSH polyclonal antibody.

The mixed reagents were kept at room temperature for 15 min.

The solid phase was magnetically collected and washed twice

with 1 mL of water to remove unbound acridinium ester conju-

gate. Each test was carried out in sets of ﬁve replicates. The che-

miluminescence of the washed magnetic particles was measured

on the Autolumat LB953 Plus luminometer averaging the ﬁve

replicate values. FNSB (fractional non-speciﬁc binding) was

measured as the ratio of the chemiluminescence associated with

the washed magnetic particles to the total chemiluminescence

from 100 μL of each diluted conjugate mixed into the solid

phase.

Acknowledgements

We thank Ms Yuanfang Deng for providing us with IR spectra of

the synthetic compounds described in the manuscript.

References

1 (a) A. Natrajan, D. Sharpe, J. Costello and Q. Jiang, Anal. Biochem.,

2010, 406, 204–213; (b) S.-J. Law, C. S. Leventis, A. Natrajan, Q. Jiang,

P. B. Connolly, J. P. Kilroy, C. R. McCudden and S. M. Tirrell, US Pat

5 656 426, 1997; (c) A. Natrajan, D. Sharpe and Q. Jiang, US Pat

6 664 043 B2, 2003; (d) A. Natrajan, D. Sharpe and D. Wen, Org.

Biomol. Chem., 2011, 9, 5092–5103.

2 (a) I. Weeks, I. Behesti, F. McCapra, A. K. Campbell and J.

S. Woodhead, Clin. Chem., 1983, 29 (8), 1474–1479; (b) P.

W. Hammond, W. A. Wiese, A. A. Waldrop III, N. C. Nelson and L.

J. Arnold Jr, J. Biolumin. Chemilumin., 1991, 6, 35–43.

3 (a) F. McCapra, Acc. Chem. Res., 1976, 9 (6), 201–208; (b) F. McCapra,

D. Watmore, F. Sumun, A. Patel, I. Beheshti, K. Ramakrishnan and

J. Branson, J. Biolumin. Chemilumin., 1989, 4, 51–58.

4 (a) J. Rak, P. Skurski and J. Błażejowski, J. Org. Chem., 1999, 64, 3002–

3008; (b) K. Krzymiński, A. Ożóg, P. Malecha, A. D. Roshal,

6. Micelle–water partition coefﬁcients (Table 5 and

Fig. S25–S31 ESI†)

Theory and experimental protocol for measurement of partition

coefﬁcients of acridinium esters to CTAC micelles is described

3446 | Org. Biomol. Chem., 2012, 10, 3432–3447

View Online

A. Wróblewska, B. Zadykowicz and J. Błażejowski, J. Org. Chem., 2011,

76, 1072–1085.

13 J. W. Bunting, V. S. F. Chew, S. B. Abhyankar and Y. Goda,

Can. J. Chem., 1984, 62, 351–354.

5 (a) J. Toullec and M. Moukawim, Chem. Commun., 1996, 221–222;

(b) V. K. Balakrishnan, E. Buncel and G. W. Vanloon, Environ. Sci.

Technol., 2005, 39, 5824–5830; (c) C. A. Bunton and H. J. Foroudian,

Langmuir, 1993, 9, 2832–2835; (d) C. A. Bunton, M. M. Mhala, J.

R. Moffat, D. Monarres and G. Savelli, J. Org. Chem., 1984, 49, 426–

430.

6 (a) E. D. Hughes and C. K. Ingold, J. Chem. Soc., 1935, 244–255;

(b) E. D. Hughes, Trans. Faraday Soc., 1941, 37, 603–631; (c) E.

D. Hughes and C. K. Ingold, Trans. Faraday Soc., 1941, 37, 657–685;

(d) K. A. Cooper, M. L. Dhar, E. D. Hughes, C. K. Ingold, B.

J. MacNulty and L. I. Woolf, J. Chem. Soc., 1948, 2043–2049.

7 (a) P. D. Proﬁo, R. Germani, G. Savelli, G. Cerichelli, N. Spreti and C.

A. Bunton, J. Chem. Soc., Perkin Trans. 2, 1996, 1505–1509;

(b) G. Cerichelli, L. Luchetti, G. Mancini, M. N. Muzzioli, R. Germani,

P. P. Ponti, N. Spreti, G. Savelli and C. A. Bunton, J. Chem. Soc., Perkin

Trans. 2, 1989, 1081–1085; (c) G. Cerichelli, G. Mancini, L. Luchetti,

G. Savelli and C. A. Bunton, J. Phys. Org. Chem., 1991, 4, 71–76;

(d) G. Cerichelli, L. Luchetti, G. Mancini, G. Savelli and C. A. Bunton,

Langmuir, 1996, 12, 2348–2352.

8 (a) L. Brinchi, P. D. Proﬁo, R. Germani, G. Savelli and C. A. Bunton,

Langmuir, 1997, 13, 4583–4587; (b) L. Brinchi, R. Germani, G. Savelli,

N. Spreti, R. Ruzziconi and C. A. Bunton, Langmuir, 1998, 14, 2656–

2662; (c) L. Brinchi, R. Germani, G. Savelli and C. A. Bunton, J. Phys.

Org. Chem., 1999, 12, 890–894.

9 (a) C. A. Bunton and L. Sepulveda, J. Phys. Chem., 1979, 83, 680–683;

(b) O. Duman, S. Tunç and B. Kanci, Fluid Phase Equilib., 2011, 301,

56–61; (c) M. F. Nazar, S. S. Shah and M. A. Khosa, J. Surfactants

Deterg., 2010, 13, 529–537; (d) R. Hosseinzadeh, R. Maleki, A.

A. Matin and Y. Nikkhahi, Spectrochim. Acta, Part A, 2008, 69, 1183–

1187.

14 (a) C. A. Bunton, F. Nome, F. H. Quina and L. S. Romsted, Acc. Chem.

Res., 1991, 24, 357–364; (b) P. Scrimin, P. Tecilla, U. Tonellato and C.

A. Bunton, Colloids Surf., A, 1998, 144, 71–79; (c) L. S. Romsted, C.

A. Bunton and J. Yao, Curr. Opin. Colloid Interface Sci., 1997, 2, 622–628.

15 (a) C. A. Bunton, M. M. Mhala and J. R. Moffat, J. Org. Chem., 1987,

52, 3832–3835; (b) A. Natrajan and C. N. Sukenik, J. Org. Chem., 1988,

53, 3559–3563.

16 H. Gharibi, B. M. Razavizadeh and M. Hashemianzaheh, Colloids Surf.,

A, 2000, 174, 375–386.

17 D. W. Armstrong and F. Nome, Anal. Chem., 1981, 53, 1662–1666.

18 S.-J. Law, T. Miller, U. Piran, C. Klukas, S. Chang and J. Unger, J. Bio-

lumin. Chemilumin., 1989, 4, 88–98.

19 (a) R. C. Chapman, E. Ostuni, L. Yan and G. M. Whitesides, Langmuir,

2000, 16, 6927–6936; (b) R. C. Chapman, E. Ostuni, M. N. Liang,

G. Meluleni, E. Kim, L. Yan, G. Pier, H. S. Warren and G.

M. Whitesides, Langmuir, 2001, 17, 1225–1233; (c) S. J. Soﬁa,

V. Premnath and E. W. Merrill, Macromolecules, 1998, 31, 5059–5070;

(d) Y.-H. Zhao, B.-K. Zhu, L. Kong and Y.-Y. Xu, Langmuir, 2007, 23,

5779–5786; (e) A. R. Denes, E. B. Somers, A. C. L. Wong and F. Denes,

J. Appl. Polym. Sci., 2001, 81, 3425–3438; (f) J. Ladd, Z. Zhang,

S. Chen, J. C. Hower and S. Jiang, Biomacromolecules, 2008, 9, 1357–

1361; (g) Y. Chang, S. Chen, Z. Zhang and S. Jiang, Langmuir, 2006, 22,

2222–2226; (h) Z. Zhang, T. Chao, S. Chen and S. Jiang, Langmuir,

2006, 22, 10072–10077; (i) W. K. Cho, B. Kong and I. S. Choi, Lang-

muir, 2007, 23, 5678–5682; ( j) G. Cheng, Z. Zhang, S. Chen, J.

D. Bryers and S. Jiang, Biomaterials, 2007, 28, 4192–4199;

(k) H. Kitano, A. Kawasaki, H. Kawasaki and S. Morokoshi, J. Colloid

Interface Sci., 2005, 282, 340–348.

20 R. Bacaloglu, C. A. Bunton and F. Ortega, J. Phys. Chem., 1989, 93,

1497–1502.

10 (a) J. F. M. daSilva, S. J. Garden and A. C. Pinto, J. Braz. Chem. Soc.,

2001, 12, 273–324; (b) A. Natrajan, Q. Jiang, D. Sharpe and J. Costello,

US Pat 7 309 615 B2, 2007.

11 (a) O. Mitsunobu, Synthesis, 1981, 1–28; (b) D. L. Hughes, Organic

Reactions, Wiley, New York, 1992, vol. 42, pp. 335–656.

21 Y. Wang, X. Huang, Y. Li, J. Wang and Y. Wang, Colloids Surf., A, 2009,

333, 108–114.

22 D. W. Armstrong and G. Y. Stine, J. Am. Chem. Soc., 1983, 105, 2962–

2964.

23 A. Berthod, J. Chromatogr., A, 1997, 780, 191–206.

12 A. Natrajan and D. Wen, Green Chem., 2011, 13, 913–921.

24 M. Arunyanart and L. J. Cline Love, Anal. Chem., 1984, 56, 1557–1561.

Org. Biomol. Chem., 2012, 10, 3432–3447 | 3447

Article Doi

Chemiluminescence from alkoxy-substituted acridinium dimethylphenyl ester labels

DOI: 10.1039/c2ob00022a

Source and publish data:

Authors:

Article abstract of DOI:10.1039/c2ob00022a

Full text of DOI:10.1039/c2ob00022a

Products guided by the article

R&D Labs maybe for 1373514-02-3

Relevant to this article

Hot Product