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

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 ionexchange 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 gelfiltration 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).

D
S3

F
Figure 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 (B ex470/em510 nm), channel 3 (C ex530/em557 nm), channel 4 (D ex585/em610 nm), channel 5 (E ex625/em660 nm) and channel 6 (F ex680/em712 nm).

PCR analysis PCR 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 65 C (25, 35, 35 seconds respectively), denaturation at 95 C for two minutes, were combined to provide 200 L samples).

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

S4

Primer Forward Reverse

Sequence CCTGAGCGTGATTTGATAATGACCTA 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 *******

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

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S6

F
Figure S3. Melting curves (left) and melting peaks (right) of multiplex PCR samples containing six HyBeacon probes (HyB-1, HyB-2, HyB-3, HyB-4, HyB-5 and HyB-6) amplifying human genomic DNA or a water negative control, using channel (A ex365/em460 nm), channel 2 (B ex470/em510 nm), channel 3 (C ex530/em557 nm), channel 4 (D ex585/em610 nm), channel 5 (E ex625/em660 nm) and channel 6 (F ex680/em712 nm) performed using a spectrofluorimeter.

S7

Figure S4. Example capillary electrophoresis and mass-spectra of a dual labelled HyBeacon (HyB-5m, Cy3b labelled).
HyBeacon HyB-3m HyB-3m HyB-3m HyB-3m HyB-3m HyB-3m HyB-5m HyB-5m HyB-5m HyB-5m HyB-5m HyB-5m HyB-7m HyB-7m HyB-7m HyB-7m HyB-7m HyB-7m Label FAM JOE TxR Cy3 Cy3b Cy5 FAM JOE TxR Cy3 Cy3b Cy5 FAM JOE TxR Cy3 Cy3b Cy5 Expected 7598 7853 8285 7760 7965 8157 7598 7853 8285 7760 7965 8157 7598 7853 8285 7760 7965 8157 Observed mass 7599 7854 8288 7764 7969 8162 7598 7856 8288 7762 7969 8161 7598 7857 8287 7762 7970 8160 HyBeacon HyB-3M HyB-3M HyB-3M HyB-3M HyB-3M HyB-3M HyB-5M HyB-5M HyB-5M HyB-5M HyB-5M HyB-5M HyB-7M HyB-7M HyB-7M HyB-7M HyB-7M HyB-7M Label FAM JOE TxR Cy3 Cy3b Cy5 FAM JOE TxR Cy3 Cy3b Cy5 FAM JOE TxR Cy3 Cy3b Cy5 Expected 7789 8044 8476 7951 8156 8348 7789 8044 8476 7951 8156 8348 7789 8044 8476 7951 8156 8348 Observed mass 7792 8049 8471 7951 8159 8351 7791 8054 8473 7951 8160 8350 7791 8051 8477 7951 8161 8352

Table S2. Mass spectrometry data from the HyBeacons used in the inital optimization experiments.
HyBeacon HyB-1 HyB-2 HyB-3 HyB-4 HyB-5 HyB-6 HyB-1b HyB-5b Label A350 FAM JOE TxR Cy5 DY680 A350 Cy5 Expected 7897 7494 8786 8444 8157 9761 7872 8142 Observed mass 7897 7496 8788 8446 8163 9763 7872 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) FmocOSu, 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, Et3N, 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.
1

Column chromatography was carried out under air/argon

pressure using Fisher Scientific DAVISIL 60 (35-70 micron) silica. H NMR spectra were measured at 300 MHz on a Bruker AC300 spectrometer or at 400 MHz on a Bruker DPX400
13

spectrometer.

C 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.

S9

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

O 3 HO 5' O 4' 3' OH 2' 4 N N 1' 1 5 6 7

2 O

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%). Mw = 240.07, C10H12N2O5, Rf (20% MeOH/DCM): 0.24

H NMR (400 MHz, DMSO-d6) 7.74 (d, J = 1.28 Hz, 1H, H6), 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).
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C NMR (100 MHz, DMSO-d6): 171.4 (C4), 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, Rf (10% MeOH/DCM) = 0.29
1

H NMR (400 MHz, DMSO-d6) 7.84 (s, 1H, H6), 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)
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C NMR (100MHz, DMSO-d6): 171.4 (C4), 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 (C5),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 at 150 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
1

H NMR (400 MHz, DMSO-d6) 11.37 (s, 1H, NH), 7.49 (s, 1H, H6), 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, 3OH), 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).
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C NMR (100 MHz, DMSO-d6) 163.7 (C4), 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 (C11), 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-methanesulfonylethyl)-5-methyluridine (5)

Compound 4 (41.0 g, 67.9 mmol) was dissolved in distilled DCM (700 mL). The solution was cooled to 70 C before Et3N (47.0 mL, 34.3 mmol) was added followed by methanesulfonyl chloride (6.30 mL, 81.4 mmol in 60 mL DCM) slowly over a period of 10 mins. The reaction mixture was stirred at 70 C for 3 hrs, then quenched with MeOH (200 mL) and was concentrated in vacuo. The crude mixture was dissolved in DCM (500 mL) and washed with saturated KCl (2 450 mL). The organic layer was dried over anhydrous Na2SO4 and the solvent was removed in vacuo. The crude was twice-purified by column chromatography (1-10% iPrOH/DCM, 0.5% pyridine) to give compound 5 as a white solid (26.8 g, 39.2 mmol, 57%). Mw = 682.22, C34H38N2O11S, Rf (9% MeOH/DCM) = 0.48
1

H NMR (400 MHz, CDCl3) 7.67 (s, 1H, H6), 7.37 (d, 2H, J = 7.3 Hz, ArH), 7.31 - 7.14 (m, 7H, ArH), 6.79 (d, 4H, J =

8.3 Hz, H11), 5.87 (d, 1H, J = 1.5 Hz, H1'), 4.53 - 4.42 (m, 2H, H3 & 18 or 19), 4.37 (ddd, 1H, J = 11.8, 5.1, 2.2 Hz, H18 or 19), 4.20 (ddd, 1H, J = 11.8, 5.1, 2.2 Hz, H18 or 19), 4.05 (m, 1H, H4), 4.00 (dd, 1H, J = 5.1, 1.5 Hz, H2), 3.94 (ddd, 1H, J = 11.8, 7.1, 2.2 Hz, H18 or 19), 3.74 (2 s, 6H, 2OCH3), 3.49 - 3.55 (m, 1H, H5'), 3.42 (dd, 1H, J = 10.9, 2.6 Hz, H5'), 2.98 - 3.05 (s, 3H, H20), 1.32 (s, 3H, CH3).
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C NMR (100 MHz, CDCl3): 163.9 (C4), 158.7 (C12), 150.5 (C2), 144.3 (ArC), 135.4 (C9), 135.3 (C9), 135.0 (C6), ), 68.6 (C19 or 18, C3), 67.1 (C19 or 18), 61.4 (C5'), 55.2 (2OCH3), 37.9 (C20), 11.8 (CH3).

130.1 (ArC), 128.1 (ArC), 128.0 (ArC), 127.1 (ArC), 113.3 (C11), 111.1 (C5), 87.7 (C1'), 86.8 (C8), 83.2 (C4' or 2), 83.0 (C4'
or 2

LRMS: [ES+, MeOH] m/z (%): 705.3 ([M+Na]+, 100%). HRMS: calc. for C34H38N2O11S (M) 682.2196, [M+Na]+ = 705.2094, found 705.2096.

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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 Na2SO4. Following concentration in vacuo, the pale yellow foam was quickly purified by column chromatography (1-15% iPrOH/DCM, 0.5% pyridine) to give compound 6 as a white foam (22.21 g, 35.3 mmol, 89%). Mw = 629.25, C33H35N5O8, Rf (70% EtOAc/toluene) = 0.34
1

H NMR (400 MHz, DMSO-d6): 11.37 (s, 1H, NH), 7.51 (d, 1H, J = 1.0 Hz, H6), 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).
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C NMR (100 MHz, DMSO-d6): 163.5 (C4) 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 (C11), 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%). HRMS: calc. for C33H35N5O8 (M) 629.2486, [M+Na]+ = 652.2383, found 652.2368.

S14

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%
i

PrOH/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

1

H NMR (400 MHz, CDCl3): 7.59 (s, 1H, H6), 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).
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C NMR (101 MHz, CDCl3): 163.9 (C4), 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 (C11), 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 (C 19), 11.8 (CH3). LRMS: [ES+, MeOH] m/z (%) 626.2 ([M+Na]+, 30%). HRMS: calc. for C33H37N3O8 (M) 603.2581, [M+Na]+ = 626.2478, found 626.2456.

S15

5-O-(4,4-Dimethoxytrityl)-2-O-(2-(9-fluorenylmethoxyamido)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
1

H 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).
13

C NMR (100 MHz, CDCl3): 163.6 (C4), 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%). HRMS: calc. for C48H47N3O10 (M) 825.3261, [M+Na]+ = 848.3159, found 848.3164.

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5-O-(4,4-Dimethoxytrityl)-2-O-(2-(9-fluorenylmethoxyamido)ethyl) -5-methyluridine- 3-O-(2-cyanoethyl-N,Ndiisopropyl)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 the combined 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
1

H 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).
31

P 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] [2] [3] [4] [5] [6] H. E. Gottlieb, V. Kotlyar, A. Nudelman, J. Org. Chem. 1997, 62, 7512. C. B. S. Reese, Q., WO 00/56747 2000. U. Legorburu, C. B. Reese, Q. L. Song, Tetrahedron 1999, 55, 5635. J. Sheng, J. S. Jiang, J. Salon, Z. Huang, Org. Lett. 2007, 9, 749. M. Manoharan, P. D. Cook, T. P. Prakash, A. M. Kawasaki US2003/008807941, 8th May, 2003. 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.

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NMR Spectra

O N HO O O OH 2 N

O N HO O O OH 2 N

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O N DMTO O O OH 3 N

O N DMTO O O OH 3 N

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O NH DMTO O OH O 4 OH N O

O NH DMTO O OH O 4 OH N O

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O NH DMTO O OH O 5 O O S O N O

O NH DMTO O OH O 5 O O S O N O

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O NH DMTO O OH O 6 N3 N O

O NH DMTO O OH O 6 N3 N O

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O NH DMTO O OH O 7 NH 2 N O

O NH DMTO O OH O 7 NH 2 N O

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O NH DMTO O OH O 8 N H Fmoc N O

O NH DMTO O OH O 8 N H Fmoc N O

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O NH DMTO O NC O P N O O 9 N H Fmoc N O

O NH DMTO O NC O P N O O 9 N H Fmoc N O

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