Six-colour Hyb Probes - Jr, Tb, Cbc 2010 - Supp Info

7/28/2019 Six-colour Hyb Probes - Jr, Tb, Cbc 2010 - Supp Info

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Supporting Information

Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, 2010

Six-Colour HyBeacon Probes for Multiplex Genetic Analysis

James A. Richardson,[a] Marta Gerowska,[a] Montserrat Shelbourne,[a] David French,[b] and Tom Brown*[a]

cbic_201000623_sm_miscellaneous_information.pdf


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Experimental Section

Oligonucleotide synthesis

Standard DNA phosphoramidites, solid supports and additional reagents were purchased from Link Technologies Ltd unless

stated otherwise. Texas red, Alexa Fluor 350 and JOE NHS esters were purchased from Invitrogen Inc, Cy3, Cy3B and Cy5

NHS esters were purchased from GE Healthcare and DY 680 was purchased from Dyomics GmbH. 2-aminoethoxy dT was

synthesised using the procedure described below. All oligonucleotides were synthesised on an Applied Biosystems 394

automated DNA/RNA synthesizer using a standard 0.2 or 1.0 mole phosphoramidite cycles of acid-catalyzed detritylation,

coupling, capping and iodine oxidation. Stepwise coupling efficiencies and overall yields were determined by the automated

trityl cation conductivity monitoring facility and in all cases were >98.0%. All -cyanoethyl phosphoramidite monomers were

dissolved in anhydrous acetonitrile to a concentration of 0.1 M immediately prior to use. The coupling time for normal (A, G, C,

T) monomers was 25 s and the coupling time for the modified monomers was extended to 360 s. Cleavage of oligonucleotides

from the solid support and deprotection was achieved by exposure to concentrated aqueous ammonia for 60 min at room

temperature followed by heating in a sealed tube for 5 h at 55 C. Oligonucleotides were purified prior to labelling by ion-

exchange chromatography on a Gilson system using a Resource Q 6 mL column, after ion-exchange purification

oligonucleotides were desalted using NAP-25 Sephadex columns (GE Healthcare). The following protocol was used: run time

16 min, flow rate 5 mL per min, binary system, gradient: time in min (% buffer B);0 (0); 3 (0); 4 (40); 9.5 (80); 10 (100); 12 (100);

13 (0); 16 (0). Elution buffer A: 0.01 M sodium hydroxide, 0.05 M sodium chloride, pH 12.0, buffer B: 0.01 M sodium hydroxide,

1 M sodium chloride pH 12.0. Oligonucleotides were labelled post-synthetically: 50-150 nmol of the oligonucleotide in 70 L of

0.5 M Na2CO3/NaHCO3 buffer (pH 8.75) was incubated for 8 h at room temperature with 1 mg of the succinimidyl ester of the

dye in 30 L of DMSO. The crude oligonucleotides were purified by reversed-phase HPLC and desalted by NAP-10 gel-

filtration according to the manufacturers instructions (GE Healthcare). Reversed-phase HPLC purification was carried out on a

Gilson system using an ABI Aquapore column (C8), 8 mm x 250 mm, pore size 300 . The following protocols were used: run

time 30 min, flow rate 4 mL per min, binary system, gradient: time in min (% buffer B);0 (0); 3 (0); 5 (10); 21 (40); 21 (60)* 25

(100); 27 (0); 30 (0). Elution buffer A: 0.1 M ammonium acetate, pH 7.0, buffer B: 0.1 M ammonium acetate with 50%

acetonitrile pH 7.0. *The % buffer B at 21 min was 40% for all oligonucleotides except those containing the hydrophobic Texas

Red, DY 680, Cy3 or Cy5 dyes which required 70% buffer B. Elution of oligonucleotides was monitored by ultraviolet absorption

at 295 nm and in all cases the main peak corresponding to double labelled oligonucleotides was collected. After HPLC

purification oligonucleotides were desalted using NAP-10 Sephadex columns (GE Healthcare), aliquoted into eppendorf tubes

and stored at 20 C. All oligonucleotides were characterised by MALDI-TOF mass spectrometry and capillary electrophoresis

(Figure S5, table S3 and table S4).

Fluorescence melting

All fluorescence melting of synthetic DNA target samples were prepared using a HyBeacon concentration of 0.15 M with a

target concentration of 0.5 M (with the exception of heterozygote samples, which contained 0.075 M of WT and 0.075 M MT

targets), containing Promega PCR buffer (Promega, UK) with a final salt concentration of 3 mM MgCl 2. Sample volumes for the

Rotor-Gene 3000/Q 6-plex were 20 L and sample volumes for analysis using the Perkin Elmer LS50-B spectrophotometer

were 200 L. Melting profiles for the Rotor-Gene 3000/Q 6-plex were as follows; 95 C (2 min), 35 C (13 min) and 35-90 C


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(0.3 C step size, 5 s step hold). Fluorescence melting experiments conducted using the spectrophotometer were performed

with the following melting profile; 30-80 C (1 C stepsize, 30 s step hold*), and were monitored using excitation and emission

wavelengths corresponding to the six channels of the Rotor-Gene Q 6-plex (*for heterozygote samples hold time was 120 s).

A

B

C

D


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E

FFig u re S1 . Melting curves (left) and melting peaks (right) of multiplex samples containing six HyBeacon probes (HyB-1, HyB-2,HyB-3, HyB-4, HyB-5 and HyB-6) and either WT or MT synthetic targets, using channel 1 ( A ex365/em460 nm), channel 2 (Bex470/em510 nm), channel 3 (C ex530/em557 nm), channel 4 (D ex585/em610 nm), channel 5 (E ex625/em660 nm) andchannel 6 (F ex680/em712 nm).

PCR analysisPCR amplification was performed in a Rotor-Gene 3000 using a mastermix purchased from 5 prime GmbH, forward primer

concentration of 0.5 M and a reverse primer concentration of 0.05 M. Each PCR reaction contained 2 ng human genomic

DNA and 0.15 M of each probe oligonucleotide. Amplification was performed using an initial activation step of 95 C for two

minutes, followed by 50 cycles of 95, 50 and 65C (25, 35, 35 seconds respectively), denaturation at 95

C for two minutes,

cooling to 35 C and then fluorescence melting analysis using the spectrophotometer and melting profile as above (samples

were combined to provide 200 L samples).


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Primer Sequence

Forward CCTGAGCGTGATTTGATAATGACCTA

Reverse GTGAAGGGTTCATATGCATAATCAAAAAGT

Table S1. Primer sequences for PCR amplification

Sequencing analysis

PCR amplified human genomic DNA was analyzed by 2 % agarose gel electrophoresis, amplicon bands were excised and the

amplicon DNA extracted using a QIAquick gel extraction kit (Qiagen). The extracted amplicon was sequenced using a CEQ

8000 genetic analysis system (Beckman Coulter).

TTTCCAGACTTCACTTCTAATGATGATTATGGGAGAACTGGAGCCTTCAGAGGGTAAAAT

TTTCCAGACTTCACTTCTAATGATGATTATGGGAGAACTGGAGCCTTCAGAGGGTAAAAT

************************************************************

TAAGCACAGTGGAAGAATTTCATTCTGTTCTCAGTTTTCCTGGATTATGCCTGGCACCAT

TAAGCACAGTGGAAGAATTTCATTCTGTTCTCAGTTTTCCTGGATTATGCCTGGCACCAT

************************************************************

TAAAGAAAATATCATCTTTGGTGTTTCCTATGATGAATATAGATACAGAAGCGTCATCAA

TAAAGAAAATATCATCTTTGGTGTTTCCTATGATGAATATAGATACAGAAGCGTCATCAA

************************************************************

AGCATGCCAACTAGAAGAGGTAAGAAACTATGTGAAAACTTTTTGATTATGCATATGAAC

AGCATGCCAACTAGAAGAGGTAAGAAACTATGTGAAAACTTTTTGATTATGCATATGAAC

************************************************************

CCTTCAC

CCTTCAC

*******

Fig u re S2 . Sequencing analysis of PCR amplified human genomic DNA, WT sequence (upper) and amplicon sequence (lower),* symbol denotes correct match.


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A

B

C


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Fig u re S4 . Example capillary electrophoresis and mass-spectra of a dual labelled HyBeacon (HyB-5m, Cy3b labelled).

H yB eacon Label E xpected Observed m ass

HyB-3m FAM 7598 7599

HyB-3m JOE 7853 7854

HyB-3m TxR 8285 8288

HyB-3m Cy3 7760 7764

HyB-3m Cy3b 7965 7969

HyB-3m Cy5 8157 8162

HyB-5m FAM 7598 7598

HyB-5m JOE 7853 7856

HyB-5m TxR 8285 8288

HyB-5m Cy3 7760 7762

HyB-5m Cy3b 7965 7969

HyB-5m Cy5 8157 8161

HyB-7m FAM 7598 7598

HyB-7m JOE 7853 7857

HyB-7m TxR 8285 8287

HyB-7m Cy3 7760 7762

HyB-7m Cy3b 7965 7970

HyB-7m Cy5 8157 8160

HyBeacon Label Expected Observed mass

HyB-3M FAM 7789 7792

HyB-3M JOE 8044 8049

HyB-3M TxR 8476 8471

HyB-3M Cy3 7951 7951

HyB-3M Cy3b 8156 8159

HyB-3M Cy5 8348 8351

HyB-5M FAM 7789 7791

HyB-5M JOE 8044 8054

HyB-5M TxR 8476 8473

HyB-5M Cy3 7951 7951

HyB-5M Cy3b 8156 8160

HyB-5M Cy5 8348 8350

HyB-7M FAM 7789 7791

HyB-7M JOE 8044 8051

HyB-7M TxR 8476 8477

HyB-7M Cy3 7951 7951

HyB-7M Cy3b 8156 8161

HyB-7M Cy5 8348 8352

Table S2. Mass spectrometry data from the HyBeacons used in the inital optimization experiments.

H yB eacon Label Expected Observed m ass

HyB-1 A350 7897 7897

HyB-2 FAM 7494 7496

HyB-3 JOE 8786 8788

HyB-4 TxR 8444 8446

HyB-5 Cy5 8157 8163

HyB-6 DY680 9761 9763

HyB-1b A350 7872 7872

HyB-5b Cy5 8142 8144

Table S3. Mass spectrometry data from the HyBeacons used in the multiplex experiments.


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Synthesis of 2-aminoethoxy T

Scheme 1. Synthesis of the 2-aminoethoxy T phosphoramidite monomer: (i) 5-methyluridine, diphenyl carbonate,

NaHCO3 cat., 100 C, DMF, 90%, (ii) DMTCl, pyridine, rt, 89%, (iii) ethylene glycol, Ti(OiPr)4, NaHCO3 cat., THF,

150 C, 79%, (iv) MsCl, Et3N, DCM, 57%, (v) NaN3, DMF, 18-crown-6, 89%, (vi) Ph3P, H2O, THF, 89%, (vii) Fmoc-

OSu, DCM, pyridine, rt, 82%, (viii) ( iPr)2NP(Cl)OCH2CH2CN, DIPEA, DCM, rt, 70%. DMT = 4,4-dimethoxytrityl, Fmoc

= 9-fluorenylmethyloxycarbonyl

General: All reagents used were purchased from Aldrich, Alfa Aesar or Fluka and used without purification with the

exception of the following solvents, which were purified by distillation: THF (over sodium wire), DCM, Et 3N, DIPEA and

pyridine (over calcium hydride). Most of the reactions were carried out under an argon atmosphere using oven-dried

glassware with purified and distilled solvents. Thin layer chromatography (TLC) was performed using Merck Kieselgel

60 F24 silica gel plates (0.22mm thickness, aluminium backed) and the compounds were visualised by irradiation at

254 nm or by staining with p-anisaldehyde solution. Column chromatography was carried out under air/argon

pressure using Fisher Scientific DAVISIL 60 (35-70 micron) silica.

1H NMR spectra were measured at 300 MHz on a Bruker AC300 spectrometer or at 400 MHz on a Bruker DPX400

spectrometer. 13C NMR spectra were measured at 75 MHz and 100 MHz on the same spectrometers respectively.

Chemical shifts are given in ppm relative to tetramethylsilane, and J values are quoted in Hz, correct to within 0.5 Hz.

All spectra were internally referenced to the appropriate residual undeuterated solvent signal.[1] Assignment was aided

by COSY (1H-1H) and HMQC/HMBC (1H-13C) experiments.

Low-resolution mass spectra were recorded using electrospray ionisation on a Fisons VG platform instrument or a

Waters ZMD quadrupole mass spectrometer in acetonitrile or methanol (HPLC grade). High-resolution mass spectra

were recorded in acetonitrile or methanol (HPLC grade) using electrospray ionisation on a Bruker APEX III FT-ICR

mass spectrometer.

In all cases, the best yields have been stated.


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2,2-Anhydro-5-methyluridine (2)[2-4]

O

OH

HO NO

N

O

1'2'3'

4'

5' 6

1

2

34

57

5-Methyluridine (1) (42.0 g, 162.6 mmol), diphenyl carbonate (41.8 g, 195.2 mmol) and sodium hydrogen carbonate

(1.37 g, 16.3 mmol) were dissolved in anhydrous DMF (46 mL). The reaction mixture was stirred at 100 C for 4 hrs,

then left to cool to room temperature and the solvent was concentrated in vacuo. Diethyl ether (700 mL) was added,

and the reaction stirred for 30 mins forming a thick gum. The ether was decanted, and the gum was recrystallised from

EtOH three times to give compound 2 as a white powder (36.5 g, 152.0 mmol, 90%). M w = 240.07, C10H12N2O5, Rf

(20% MeOH/DCM): 0.24

1H NMR (400 MHz, DMSO-d6) 7.74 (d, J= 1.28 Hz, 1H, H

6), 6.29 (d, J = 5.7 Hz, 1H, H1), 5.86 (d, J= 4.3 Hz, 1H, 3-

OH), 5.17 (d, J= 5.7 Hz, 1H, H2), 4.95 (t, J= 5.3 Hz, 1H, 5-OH), 4.40 - 4.34 (m, 1H, H3), 4.10 - 4.0 (m, 1H, H4), 3.30 -

3.11 (m, 2H, H5), 1.79 (d, J= 1.27 Hz, 3H, CH3).

13C NMR (100 MHz, DMSO-d6): 171.4 (C

4), 159.2 (C2), 132.0 (C6), 116.4 (C5), 90.0 (C1), 88.9 (C4), 88.3 (C2), 74.5

(C3), 60.6 (C5), 13.3 (CH3).

LRMS: [ES+, MeOH] m/z(%): 263.1 ([M+Na]+, 100%), 503.2 ([2M+Na]+, 50%).

HRMS: calc. for C10H12N2O5 (M) 240.0746, [M+Na]+ = 263.0644, found 263.0638, [2M+Na]+ = 503.1390, found

503.1363.


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5-O-(4,4-Dimethoxytrityl)-2,2-anhydro-5-methyluridine (3)[4]

To a stirred solution of 2 (36.3 g, 151.2 mmol) in distilled pyridine (504 mL), 4,4-dimethoxytrityl chloride (71.7 g, 211.6

mmol) was added portion-wise over a period of 1 hr. The reaction mixture was stirred at rt for 5 hrs. Methanol (160

mL) and Et3N (10 mL) were added and the reaction was stirred for 10 min, before solvent removal in vacuo. The

crude mixture was dissolved in DCM (500 mL) and extracted with saturated aq NaHCO 3. The organic layers were

combined, dried over anhydrous Na2SO4 and the solvent was removed in vacuo. The crude product was purified by

column chromatography (0-15% MeOH/DCM, 0.5% pyridine) to give compound 3 as a white solid (73.5 g, 135.6 mmol,

89%). Mw = 542.21, C31H30N2O7, R f (10% MeOH/DCM) = 0.29

1H NMR (400 MHz, DMSO-d6) 7.84 (s, 1H, H

6), 7.30 - 7.22 (m, 4H, ArH), 7.17 - 7.21 (m, 1H, ArH), 7.13 (dd, J = 9.0,

3.0 Hz, 4H, H10), 6.82 (2xd, J = 8.2 Hz, 4H, H11), 6.32 (d, J = 5.6 Hz, 1H, H1), 5.93 (d, J = 4.4 Hz, 1H, 3-OH), 5.18 (d,

J = 5.8 Hz, 1H, H2), 4.31 - 4.27 (m, 1H, H3), 4.26 - 4.20 (m, 1H, H4), 3.73 (d, J = 1.6 Hz, 6H, 2OCH3), 2.92 (dd, J =

10.1, 4.3 Hz, 1H, H5), 2.78 (dd, J= 10.1, 7.5 Hz, 1H, H5), 1.79 (s, 3 H, CH3)

13C NMR (100MHz, DMSO-d6): 171.4 (C

4), 158.9 (C2), 158.1 (C12), 144.6 (ArC), 135.2 (C9), 135.1 (C9), 132.1 (C6),

129.4 (ArC), 127.8 (ArC), 127.4 (ArC), 126.6 (ArC), 116.9 (C 5),113.2 (C11), 90.0 (C1), 88.1 (C2), 86.9 (C3), 85.3 (C8),

74.8 (C4), 63.0 (C5), 55.0 (2OCH3), 13.5 (CH3).

LRMS: [ES+, MeOH] m/z(%): 565.2 ([M+Na]+, 100%), 1108.1 ([2M+Na]+, 50%).

HRMS: calc. for C31H30N2O7 (M) 542.2053, [M+H]+ = 543.2126, found 543.2129.


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5-O-(4,4-Dimethoxytrityl)- 2-O-(2-hydroxyethyl)-5-methyluridine (4)[5]

This compound has been synthesised before but using a different method see Reference. [5]

Sodium hydrogen carbonate (0.39 g, 4.62 mmol) and anhydrous ethylene glycol (2.60 mL, 46.2 mmol) were dissolved

in anhydrous DMF (24 mL), to which titanium (IV) isopropoxide (5.50 mL, 18.5 mmol) was added and the reaction

mixture was stirred at 160 C for 3 hrs. Compound 3 (5.01 g, 9.24 mmol) was added and the reaction was refluxed at150 C for 24 hrs. The reaction was left to cool to room temperature, then it was centrifuged to remove the solid

titanium complex, decanted and the solvent was removed in vacuo. The crude oil was purified by column

chromatography (1-5% MeOH/DCM, 1% pyridine) to give compound 4 as a white solid (4.40 g, 7.29 mmol, 79%). Mw =

604.24, C33H36N2O9, Rf (8% MeOH/DCM) = 0.32

1H NMR (400 MHz, DMSO-d6) 11.37 (s, 1H, NH), 7.49 (s, 1H, H

6), 7.36 - 7.43 (m, 2H, ArH), 7.32 (t, 2H, J = 7.6 Hz,

ArH), 7.29 - 7.22 (m, 5H, ArH), 6.90 (d, 4H, J = 8.9 Hz, H11), 5.86 (d, 1H, J = 4.8 Hz, H1'), 5.10 (d, 1H, J = 6.1 Hz, 3-

OH), 4.74 (s, 1H, OH20), 4.25 (q, 1H, J = 5.5 Hz, H3'), 4.10 (t, 1H, J = 4.9 Hz, H2'), 4.01 - 3.95 (m, 1H, H4'), 3.74 (s, 6H,

2OCH3), 3.70 - 3.57 (m, 2H, H19or H18), 3.57 - 3.51 (m, 2H, H19or H18), 3.26 (dd, 1H, J = 10.8, 4.3 Hz, H5), 3.20 (dd,

1H, J= 10.6, 2.1 Hz, H5), 1.41 (s, 3H, CH3).

13C NMR (100 MHz, DMSO-d6) 163.7 (C

4), 158.2 (C12), 150.4 (C2), 144.6 (ArC), 135.6 (ArC), 135.3 (ArC), 135.1 (C6),

129.7 (ArC), 127.9 (ArC), 127.7 (ArC), 126.8 (ArC), 113.3 (C 11), 109.5 (C5), 86.6 (C1'), 85.9 (C8), 82.9 (C4'), 81.0(C2),

71.7 (C18), 69.0 (C3'), 63.2 (C5'), 60.3 (C19), 55.0 (2OCH3), 11.7 (CH3).

HRMS: calc. for C33H36N2O9 (M) 604.2421, [M+Na]+ = 627.2319, found 627.2321.


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5-O-(4,4-Dimethoxytrityl)- 2-O-(2-azidoethyl)-5-methyluridine (6)

Sodium azide (12.8 g, 196.1 mmol) and 18-crown-6 (0.10 g, 0.39 mmol) were added to a solution of 5 (26.8 g, 39.2

mmol) in anhydrous DMF (290 mL). The solution was stirred at 60 C for 14 hrs. The solvent was removed under

vacuum, and the residual colourless oil was partitioned between DCM (450 mL) and water (450 mL). The organic layer

was separated and the aqueous layer was extracted with DCM (2 350 mL). The combined organic extracts were

washed with brine (450 mL) and dried over anhydrous Na 2SO4. Following concentration in vacuo, the pale yellow foam

was quickly purified by column chromatography (1-15%

i

PrOH/DCM, 0.5% pyridine) to give compound 6 as a whitefoam (22.21 g, 35.3 mmol, 89%). Mw = 629.25, C33H35N5O8, Rf (70% EtOAc/toluene) = 0.34

1H NMR (400 MHz, DMSO-d6): 11.37 (s, 1H, NH), 7.51 (d, 1H, J= 1.0 Hz, H

6), 7.43 - 7.38 (m, 2H, ArH), 7.35 - 7.21

(m, 7H, ArH), 6.90 (d, 4H, J= 8.0 Hz, H11), 5.89 (d, 1H, J = 5.0 Hz, H1'), 5.25 (d, 1H, J = 6.5 Hz, 3-OH), 4.26 (q, 1H, J

= 5.5 Hz, H3'), 4.12 (t, 1H, J = 4.8 Hz, H2'), 4.04 - 3.98 (m, 1H, H4'), 3.90 - 3.84 (m, 1H, H18 or 19), 3.79 - 3.71 (m, 7H,

2OCH3 and H18 or 19), 3.52 - 3.36 (m, 2H, H18 or 19), 3.28 (dd, 1H, J = 10.5, 4.5 Hz, H5'), 3.23 (dd, 1H, J = 10.5, 2.5 Hz,

H5'), 1.42 (s, 3H, CH3).

13C NMR (100 MHz, DMSO-d6): 163.5 (C

4) 158.0 (C12), 150.2 (C2), 144.4 (ArC), 135.2 (C6), 135.2 (C9), 134.9 (C9),

129.5 (ArC), 127.7 (ArC), 127.5 (ArC), 126.6 (ArC), 113.0 (C 11), 109.4 (C5), 86.4 (C1'), 85.7 (C8), 82.7 (C4'), 80.7 (C2'),

68.7 (C18), 68.5 (C3'), 62.9 (C5'), 54.8 (2OCH3), 49.8 (C19), 11.5 (CH3).

LRMS: [ES+, MeCN]; m/z(%) 652.4 ([M+Na]+, 100%).



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5-O-(4,4-Dimethoxytrityl)- 2-O-(2-aminoethyl)-5-methyluridine (7)[6]

This compound has been synthesised before but using a different method see Reference. [6]

Compound 6 (4.09 g, 6.50 mmol) was dissolved in THF (65 mL) before triphenyl phosphine (3.41 g, 13.0 mmol) was

added followed by distilled water (0.6 mL, 32.5 mmol). The resulting mixture was stirred at 45 C for 14 hrs. The

solvent was removed in vacuo, and the residual colourless oil was purified by column chromatography (30-80%iPrOH/DCM, 0.2% Pyridine) to give compound 7 as a white foam (3.49 g, 5.79 mmol, 89%) Mw = 603.26, C33H37N3O8,

Rf (20% MeOH: DCM) = 0.05

1H NMR (400 MHz, CDCl3): 7.59 (s, 1H, H

6), 7.41 - 7.33 (m, 2H, ArH), 7.30 - 7.15 (m, 7H, ArH), 6.79 (d, 4H, J = 9.0

Hz, H11), 5.99 (d, 1H, J= 4.0 Hz, H1'), 4.38 (t, 1H, J = 5.0 Hz, H3'), 4.14 - 4.08 (m, 1H, H4'), 4.03 (t, 1H, J= 4.5 Hz, H2'),

3.95 - 3.87 (m, 1H, H18), 3.74 (s, 6H, 2OCH3), 3.63 - 3.55 (m, 2H, 3-OH, H18), 3.48 (dd, 1H, J = 11.0, 3.0 Hz, H5'),

3.37 (dd, 1H, J= 11.0, 3.0 Hz, H5'), 2.95 - 2.84 (m, 2H, H19), 1.34 (s, 3H, CH3).

13C NMR (101 MHz, CDCl3): 163.9 (C

4), 158.7 (ArC), 150.5 (C2), 144.4 (ArC), 135.5 (ArC), 135.3 (C6), 130.1 (ArC),

128.2 (ArC), 128.0 (ArC), 127.1 (ArC), 113.3 (C 11), 111.1 (C5), 87.4 (C1), 86.9 (C8), 83.9 (C4), 82.9 (C2), 72.0 (C18),

69.5 (C3), 62.7 (C5), 55.2 (2OCH3), 41.2 (C19), 11.8 (CH3).

LRMS: [ES+, MeOH] m/z(%) 626.2 ([M+Na]+, 30%).



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5-O-(4,4-Dimethoxytrityl)-2-O-(2-(9- luorenylmethoxyamido)ethyl)-5-methyluridine (8)

Compound 7 (1.63 g, 2.71 mmol) was dissolved in distilled DCM (25 mL) before anhydrous pyridine (0.55 mL, 6.76

mmol) was added followed by 9-Fluorenylmethyl N-succinimidyl carbonate (3.19 g, 9.47 mmol). The resulting mixture

was stirred at rt for 12 hrs, then diluted with DCM (150 mL) and partitioned with saturated ammonium chloride (150

mL). The organic layer was washed with saturated NaHCO3 (2 150 mL), then dried over anhydrous Na2SO4 and

concentrated in vacuo to give a pale yellow foam. Purification by column chromatography (1-10% EtOH/DCM, 0.5%

pyridine) gave compound 8 as a white foam (1.84 g, 2.23 mmol, 82%). Mw = 825.33, C48H47N3O10, Rf (90%

EtOAc/Hexane) = 0.78

1H NMR (400 MHz, CDCl3): 7.72 (d, 2H, J = 7.5 Hz, ArH), 7.69 - 7.63 (m, 1H, ArH), 7.56 (d, 2H, J = 7.5 Hz, ArH),

7.42 - 7.32 (m, 4H, ArH), 7.31 - 7.14 (m, 9H, ArH), 6.82 (d, 4H, J = 8.5 Hz, H11), 5.92 (d, 1H, J = 2.0 Hz, H1'), 4.45 -

4.39 (m, 1H, H3'), 4.39 - 4.29 (m, 2H, J= 6.5 Hz, H21), 4.18 (t, 1H, J= 6.8 Hz, H22), 4.09 - 4.02 (m, 2H, H4'), 3.97 (br. s,

1H, H2'), 3.94 - 3.88 (m, 1H, H18 or 19), 3.86 - 3.72 (m, 7H, 2OCH3 and H18 or 19), 3.47 - 3.49 (m, 1H, H5'), 3.48 - 3.31 (m,

3H, H5' and H18 or 19), 1.37 (s, 3H, CH3).

13C NMR (100 MHz, CDCl3): 163.6 (C

4), 158.4 (ArC), 156.6 (C20), 149.3 (ArC), 144.0 (ArC), 143.6 (ArC), 140.9 (ArC),

135.1 (C6), 134.9 (ArC), 134.7 (ArC), 129.8 (ArC), 128.8 (ArC), 127.8 (ArC), 127.6 (ArC), 127.4 (ArC), 127.3 (ArC),

126.8 (ArC), 126.7 (ArC), 124.7 (ArC), 119.6 (ArC), 112.9 (C11), 112.8 (ArC), 110.8 (C5), 87.4 (C1'), 86.5 (C8), 83.1 (C4'),

82.6 (C2'), 70.2 (C18), 68.5 (C3'), 66.5 (C22), 61.5 (C5'), 54.9 (2OCH3), 46.8 (C22), 40.5 (C19), 11.5 (CH3).

LRMS: [ES+, MeCN] m/z(%) 848.6 ([M+Na]+, 100%).



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S17

5-O-(4,4-Dimethoxytrityl)-2- O-(2-(9- luorenylmethoxyamido)ethyl) -5-methyluridine- 3-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (9)

To a solution of nucleoside 8 (1.75 g, 2.12 mmol) in distilled DCM (10 mL) and distilled DIPEA (0.85 mL, 4.89 mmol),

strictly under an argon atmosphere and excluding moisture, was added chloro-phosphitylating reagent (0.56 mL, 2.55

mmol) dropwise, and the reaction was stirred at rt for 4 hours. The reaction mixture was transferred to a separating

funnel containing distilled DCM (25 mL), and washed with saturated aq KCl (25 mL). The organic layers were

combined, and dried over anhydrous Na2SO4. The inorganics were washed with degassed DCM (2

10 mL) and thecombined fractions were transferred under argon and concentrated in vacuo. Purification by column chromatography

(65-70% EtOAc/hexane, 0.5% pyridine) under argon pressure, gave the desired product 9 as a diastereomeric mixture

(ca. 2:3), as an air-sensitive, powdery white solid (1.52 g, 1.48 mmol, 70%). Mw = 1025.43, C57H64N5O11P, Rf (75%

EtOAc/Hexane) = 0.6

1H NMR (400 MHz, CD3CN) : 9.12 (br. s, 1H, NH), 8.6 - 8.53 (m, 1H, NH), 7.82 (d, 2H, J= 7.5 Hz, ArH), 7.64 (d, 2H,

J = 7.0 Hz, ArH), 7.53 (s, 1H, H6), 7.50 - 7.21 (m, 13H, ArH), 6.87 (dd, 4H, J = 7.8, 6.8 Hz, H11), 5.90 (dd, 1H, J = 9.3,

3.3 Hz, H1'), 4.58 - 4.41 (m, 1H, H3'), 4.34 (dd, 2H, J = 13.1, 6.5 Hz, H21), 4.25 - 4.12 (m, 3H, H4', H2' and H22), 3.64 -

3.90 (m, 9H, 2OCH3, H23 and H18 or 19), 3.63 - 3.50 (m, 3H, H25 and H18 or 19), 3.49 - 3.39 (m, 1H, H18 or 19), 3.38 - 3.22

(m, 3H, H5 and H18 or 19), 2.61 (br. s, 1H, H24), 2.53 - 2.41 (m, 1H, H24), 1.39 (d, 3H, J= 8.0 Hz, H7), 1.24 - 0.91 (m, 12H,

H26).

31P NMR (300 MHz, CD3CN) 150.96, 150.06 (isomers).

LRMS: [ES+, MeCN] m/z(%) 1048.6([M+Na]+, 100%) and 1026.6 ([M+H]+, 10%).

References

[1] H. E. Gottlieb, V. Kotlyar, A. Nudelman, J. Org. Chem. 1997, 62, 7512.[2] C. B. S. Reese, Q., WO 00/567472000.[3] U. Legorburu, C. B. Reese, Q. L. Song, Tetrahedron 1999, 55, 5635.[4] J. Sheng, J. S. Jiang, J. Salon, Z. Huang, Org. Lett. 2007, 9, 749.[5] M. Manoharan, P. D. Cook, T. P. Prakash, A. M. Kawasaki US2003/008807941, 8th May, 2003.[6] M. Manoharan, T. P. Prakash, I. Barber-Peoc'h, B. Bhat, G. Vasquez, B. S. Ross, P. D. Cook, J. Org. Chem.

1999, 64, 6468.


18/25

S18

NMR Spectra

O

OH

HO NO

2

N

O

O

OH

HO NO

2

N

O


19/25

S19

O

OH

DMTO NO

3

N

O

O

OH

DMTO NO

3

N

O


20/25

S20

O

OOH

DMTO N

NH

O

O

4OH

O

OOH

DMTO N

NH

O

O

4

OH


21/25

S21

5

O

OOH

DMTO N

NH

O

O

O S

O

O

5

O

OOH

DMTO N

NH

O

O

O S

O

O


22/25

S22

O

OOH

DMTO N

NH

O

O

6

N3

O

OOH

DMTO N

NH

O

O

6

N3


23/25

S23

O

OOH

DMTO N

NH

O

7NH2

O

O

OOH

DMTO N

NH

O

7

NH2

O


24/25

S24

O

OOH

DMTO N

NH

O

O

NH

Fmoc

8

O

OOH

DMTO N

NH

O

O

NH

Fmoc

8


25/25

S25

O

OO

DMTO N

NH

O

NHPN

O

NC

O

Fmoc

9

O

OO

DMTO N

NH

O

NH

P

N

ONC

O

Fmoc

9

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Six-colour Hyb Probes - Jr, Tb, Cbc 2010 - Supp Info