Simultaneous Quantification of Multiple Nucleic Acid ...

Simultaneous Quantification of Multiple Nucleic Acid Targets Using Chemiluminescent Probes Kenneth A. Browne,1,* Dimitri D. Deheyn,2 Gamal A. El-Hiti,3 Keith Smith3 and Ian Weeks4,† 1

Gen-Probe Incorporated, 10210 Genetic Center Drive, San Diego, CA 92121 *e-mail: [email protected]

2

Marine Biology Research Division, Scripps Institution of Oceanography, University of California at San Diego, La Jolla, CA 92093

3

School of Chemistry, Cardiff University, Main Building, Park Place, Cardiff, Wales, UK CF10 3AT 4

Molecular Light Technology Research Limited (now Gen-Probe Cardiff Ltd.), 5 Chiltern Close, Cardiff Industrial Park, Cardiff, Wales, UK CF14 5DL

†Present address: School of Medicine, Cardiff University, Tenovus Building, Heath Park, Cardiff, Wales, UK CF14 4XN

Supporting Information

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Scheme S1. Reactions between AE (I) and hydroxide or hydroxide/peroxide. HO OH -

O

N

-

+

OH

N

N

N

N

+ HO OO

-

O

O

AE (I) -

O O OH

OH

O

-

O O O-

(III)

9-hydroperoxy adduct (II)

O O

O

O

O

O

O

1,2-dioxetane (IV)

1,2-dioxetan3-one (V)

phenolate

-

OH

OH -

O

N

N

+

N+

N+

N

O

OH O

pseudobase or carbinol (VII)

-

O

OH O

(VIII)

*

+ CO2

+ -

O

OH

9-carboxylic acid (IX)

OH O

O-

O

excited acridone (VI*)

N

+ h O

acridone (VI)

AE pathways: Using an unsubstituted, N-methyl AE (I) as a minimally complex model compound to describe AE pathways, with the understanding that the AEs in the current work share these pathways, Scheme S1 outlines many of the transformations and equilibria anticipated en route towards and away from the light emitting species. Elevated pH from addition of detect 2 reagent (see Experimental Section) shifts the equilibrium of H2O2 from detect 1 reagent towards its anion form (ionic strengthcorrected pKa1 = 11.9).1 This facilitates formation of a 9-hydroperoxy adduct (II) and, in turn, a 9hydroperoxy adduct anion (III) and a 1,2-dioxetane (IV), thereby comprising the observed rate limiting reactions. Loss of a phenolate results in a high-energy 1,2-dioxetan-3-one (V) that rapidly eliminates CO2 to form an excited N-methylacridone (VI*). When VI* relaxes to VI, it emits light (or heat, though not measured in the current studies). The intramolecular transformations from IV to VI are anticipated 2

to be rapid. The increased pH afforded upon addition of detect 2 solution also drives competing processes that are not detected by light emission (dark reactions). Like the hydroperoxy anion, hydroxide can react at the acridinium 9 position, in this case forming a pseudobase (VII). Product analysis of I by Kaltenbach and Arnold indicated that after 60 min at pH 9 in the absence of H2O2, ca. 90% of the reaction was composed of VII.2 Thus, as other reactions deplete I, a substantial reserve of VII can equilibrate back to I. The alkaline solution additionally supports formation of a hydrated ester intermediate (VIII) and its essentially irreversible hydrolysis to a 9-carboxylic acid (IX) and a phenolate. King et al. calculated the pseudo-first-order base hydrolysis rate constant (kHO-[HO-]) for a model AE in which kHO- was 220 M-1 s-1.3 Using this constant as a reasonable approximation in our work at pH 9 ([HO-] = 1e-5 M), kHO-[HO-] corresponds to 0.0022 s-1 (½ = 315 s) for I  VII  IX and, therefore, ester hydrolysis is not expected to contribute substantially to the dark processes on the timescales of the light-emitting reactions for most of the AE probes currently used.

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Table S1. Constants for target amounts (x) versus emission responses (y) by

EcoB1932-1947(-)HICS18 and CalA1185-1206(-)HICS87.

(a) PMT 1 (≤450 nm; 0-12 s), 200 amol – 200 fmol EcoB1921– 1958(+)

200 amol – 200 fmol EcoB1921– 1958(+)

0 CalA1174–1217(+)

50 fmol CalA1174–1217(+)

a

y = 0.944x + 4.72 (0.997)

y = 0.928x + 4.74 (0.999)

b

y = 0.914x + 4.96 (0.997)

y = 0.936x + 4.92 (0.997)

b

y = 0.959x + 4.88 (0.999)

y = 0.948x + 4.90 (0.999)

200 amol – 200 fmol CalA1174– 1217(+)

200 amol – 200 fmol CalA1174– 1217(+)

0 EcoB1921–1958(+)

50 fmol EcoB1921–1958(+)

a

y = 0.749x + 4.03 (0.987)

y = 0.750x + 4.04 (0.992)

b

y = 0.898x + 3.84 (0.994)

y = 0.818x + 3.98 (0.998)

b

y = 0.741x + 3.95 (0.991)

y = 0.690x + 4.04 (0.990)

EcoB1932-1947(-)HICS18 calibration curves

(b) PMT 2 (≥550 nm; 12-184 s), CalA1185-1206(-)HICS87 calibration curves

a

Linear equations containing constants to best fits of target amounts versus cps data in Figure 3; values

in parentheses are coefficients of determination (R2). bLinear equations for repeat experiments.

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Additional experimental details.

(b) Bis(4-methoxyphenyl)amine (5). Compound 5 was prepared according to a standard literature procedure.4 A mixture of 4-anisidine (2.46 g, 20 mmol), 4-iodoanisole (6.12 g, 30 mmol), K2CO3 (5.53 g, 40 mmol), CuI (0.38 g, 2 mmol), and L-proline (0.46 g, 4 mmol) in DMSO (30 mL) was heated at 90 °C for 48 h under dry conditions. The cooled mixture was partitioned between water and ethyl acetate. The organic layer was separated, and the aqueous layer was extracted with ethyl acetate. The combined organic layers were washed with brine, dried (MgSO4), and concentrated under reduced pressure. The residue obtained was purified by column chromatography (silica gel; Et2O–hexane, 1:4) to give 5 (3.43 g, 15.0 mmol; 75% yield) as a crystalline material: mp 102-103 C (lit. 102-103 C;5; 99.5-101.5 C6); 1

H NMR (CDCl3)  6.98 (d, J = 8.8 Hz, 4H), 6.86 (d, J = 8.8 Hz, 4H), 5.33 (s, exch, 1H), 3.81 (s, 6H);

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C NMR (CDCl3)  154.3, 138.0, 119.6, 114.8, 55.7; HRMS (EI) calcd for C14H15NO2 (M+) 229.1103,

found 229.1106. (f) Succinimidyl 4-iodobutanoate. 4-Iodobutanoic acid (0.48 g, 2.24 mmol) in dry THF (10 mL) was cooled to 0 C, and N-hydroxysuccinimide (0.25 g, 2.17 mmol) in dry THF (2 mL) and dicyclohexylcarbodiimide (DCC; 0.52 g, 2.50 mmol) in THF (4 mL) were added, successively. The mixture was stirred at 0 C for 3 h, and then at room temperature overnight. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue obtained was purified by column chromatography (silica gel; Et2O–hexane, 1:2) to give succinimidyl 4-iodobutanoate (0.59 g, 1.89 mmol; 84% yield) as a colorless solid: mp 86-87 C (lit. 86-87C7); 1H NMR (CDCl3)  3.03 (t, J = 7.0 Hz, 2H), 2.62 (s, 4H), 2.53 (t, J = 7.0 Hz, 2H), 2.05 (apparent quintet, J = 7.0 Hz, 2H); HRMS (APCI) calcd for C8H11NO4I (MH+) 311.9733, found 311.9736.

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Table S2. MALDI-TOF MS of oligonucleotides. Name

Nucleic

acid calcd

obsda

backbone EcoB1932-1947(-)HICS16

DNA/OMe

11,484.54

n/ab

EcoB1932-1947(-)HICS17

DNA/OMe

11,535.48

11,537.30

EcoB1932-1947(-)HICS18

DNA/OMe

11,535.48

11,534.30

EcoB1921–1958(+)

RNA

12,163.23

12,162.44

CalA1185-1206(-)HICS86

DNA

12,889.34

12,889.04

CalA1185-1206(-)HICS87

DNA

13,000.33

12,998.05

CalA1174–1217(+)

RNA

13,529.24

13,526.49

CtrB1452-1465(+)HICS51

OMe

9,391.20

9,392.20

CtrB1447-1470(-)

OMe

8,176.33

8,173.24

NgoA133-145(+)HICS62

OMe

9,076.96

9,077.42

NgoA128-150(-)

OMe

7,783.08

7,783.34

a

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS was performed on a

Waters Micromass MALDI micro MX spectrometer (Milford, MA; positive ion detection mode; pulse voltage = 950 – 1,300 V; nitrogen laser (337 nm) intensity = 500). One µL of 1 – 16 pmol of the oligonucleotides was incubated with ca. 50 beads of Dowex® 50WX8 H+ form, 100-200 mesh cationexchange resin in a solution containing 1 µL of 3 mM ammonium citrate (pH 9.4) and 5 µL of a mixture of 431 mM 3-hydroxypicolinic acid and 122 mM 2-picolinic acid in 1:1 ACN:H2O; the desalted oligonucleotides (1.5 µL) were air-dried on a gold-plated sample plate for analysis. Masses were calibrated with a DNA oligonucleotide as an internal standard. bInsufficient quantities of this oligonucleotide were available for MALDI-TOF MS analysis.

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References

(1) Evans, M. G.; Uri, N. Trans. Faraday Soc. 1949, 45, 224-230. (2) Kaltenbach, M. S.; Arnold, M. A. Mikrochim. Acta 1992, 108, 205-219. (3) King, D. W.; Cooper, W. J.; Rusak, S. A.; Peake, B. M.; Kiddle, J. J.; O'Sullivan, D. W.; Melamed, M. L.; Morgan, C. R.; Theberge, S. M. Anal. Chem. 2007, 79, 4169-4176. (4) Zhang, H.; Cai, Q.; Ma, D. J. Org. Chem. 2005, 70, 5164-5173. (5) McNulty, J.; Cheekoori, S.; Bender, T. P.; Coggan, J. A. Eur. J. Org. Chem. 2007, 1423-1428. (6) Wolfe, J. P.; Timori, J.; Sadighi, J. P.; Yin, J.; Buchwald, S. L. J. Org. Chem. 2000, 65, 11581174. (7) Brown, R. C.; Li, Z.; Rutter, A. J.; Mu, X.; Weeks, O. H.; Smith, K.; Weeks, I. Org. Biomol. Chem. 2009, 7, 386-394.

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