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Supplementary Information for:

Prebiotically plausible oligoribonucleotide ligation facilitated by chemoselective acetylation

Frank R. Bowler1, Christopher K. W. Chan1, Colm D. Duffy1, Béatrice Gerland2†,

Saidul Islam2§, Matthew W. Powner2‡, John D. Sutherland1,2★ & Jianfeng Xu1

1Medical Research Council Laboratory of Molecular Biology, Francis Crick

Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH, UK. 2School of

Chemistry, The University of Manchester, Oxford Road, Manchester M13 9PL,

UK. †Current address: Faculté de chimie,Université de Strasbourg,1 rue Blaise

Pascal,67008 Strasbourg Cedex, France. §Current address: School of Biological

and Chemical Sciences, Joseph Priestley Building, Queen Mary University

London, Mile End Road, London E1 4NS, UK. ‡Current address: Department of

Chemistry, University College London, Christopher Ingold Laboratories, 20

Gordon Street, London, WC1H 0AJ, UK. ★e-mail [email protected]

1

List of Schemes, Tables and Figures

Scheme S1 5

Scheme S2 9

Scheme S3 10

Figure S1 26

Table S1-S22 27-48

Figure S2-S11 49-58

Table S23-S27 61-63

Figure S12 64

Table S28-S29 64-65

Figure S13 66

Table S30 67

Figure S14 69

Table S31 69

Figure S15 72

Figure S16 73

Figure S17 73

Figure S18 74

Figure S19 76

Figure S20 77

Figure S21 77

Figure S22 79

Contents

General 3

Synthetic protocols 5

NMR investigations of chemoselective acetylation 24

Acetylation-ligation reactions and deacetylation 66

References 80

2

General

Reagents and solvents were obtained from Acros Organics, Alfa Aesar, Ambion,

BDH, Bio-Rad, ChemGenes, Fluka, Invitrogen, Lancaster, Link Technologies,

Molekula, New England Biolabs, Santa Cruz Biotechnology, Sigma-Aldrich, TCI

Europe, Toronto Research Chemicals, or VWR. DCl was prepared by the

addition of oxalyl chloride to D2O. NaOD was prepared by dissolving sodium

metal in D2O, or by dissolving NaOH in D2O and repeatedly evaporating the

solvent and redissolving in D2O (×3). Acetonitrile, pyridine and triethylamine

were distilled over calcium hydride before use. Saturated methanolic ammonia

was freshly prepared by bubbling ammonia gas (BOC) through ice-cooled

methanol. Ion-exchange resins purchased as the H+ form (Dowex® 50WX4-400

and 50WX2-400) were pre-washed with water until the filtrate was clear, then

converted to the Na+ form according to the following procedure: (i) stir in

1 M HCl aq. for 1 h then wash with H2O until pH neutral; (ii) stir in 1 M NaOH

aq. for 1 h then wash with H2O until pH neutral; (iii) stir in 1 M HCl aq. for 1 h

then wash with H2O until pH neutral; (iv) stir in 1 M NaOH aq. for 1 h then wash

with H2O until pH neutral. Ion-exchange resin purchased as the Na+ form (AG®

50W-X8) was pre-washed with 1 M NaOH aq. and then water until the filtrate

was pH neutral. All water was purified using a Millipore Milli-Q Plus 185

purification system.

TLC: Merck Kieselgel 60 F254; detection by UV, or by staining plates with

alkaline permanganate solution (KMnO4 (3 g), and K2CO3 (20 g) dissolved in

NaOH(aq.) (5% w/v, 5 mL) then made up to 300 mL with H2O), anisaldehyde

(EtOH/anisaldehyde/conc. H2SO4/AcOH 180:10:10:2 v/v), or ammonium

molybdate (5% w/v in H2SO4(aq.) (1.0 M)) followed by heating with a heat gun.

Flash chromatography: Sorbisil C60 silica gel. M.p.: Sanyo Gallenkamp;

uncorrected. FTIR: AT1 Mattson Genesis series spectrophotometer (KBr disc,

Nujol mull, film or soln.); alternatively, IR spectra of solids were recorded on a

Bruker Equinox 55/Bruker FRA 106/5 with a coherent 500 mW laser as

attenuated total reflectance (ATR) spectra using the ‘Golden Gate’ attachment

with a resolution of 2 cm-1; in cm-1. NMR: Bruker AVANCE 300, Bruker

AVANCE 400, Bruker AVANCE III 400, Bruker AVANCE II+ 500; in ppm, J

3

in Hz, assignments by COSY, HMBC, HMQC. Signal splittings are recorded as singlet (s), broad singlet (br. s), doublet (d), doublet of doublets (dd), double double doublet (ddd), triplet (t), doublet of triplets (dt), triplet of doublets (td), quartet (q), doublet of quartets (dq), triplet of quartets (tq), quintet (quin.), sextet (sex.), heptet (h), and multiplet (m). The notation (ABX) refers to a methylene spin system coupled to a unique adjacent proton. The notation (ABXP) refers to a methylene spin system coupled to a unique adjacent proton and phosphorus. The notation (AB) refers to an isolated methylene spin system. ESI-MS: Waters

Micromass Platform II Q-TOF. HRMS: ThermoFinnigan MAT95XP. MALDI-

TOF MS: Applied Biosystems Voyager-DE Pro, using a matrix of 3-

hydroxypicolinic acid and diammonium hydrogen citrate (25 and 35 mg/mL

respectively) in 30 % (v/v) acetonitrile in water; typically, 10 L of matrix

solution was mixed with 1-2 L of aqueous analyte solution, and 2 L of this

mixture spotted in duplicate; spectra were recorded in linear positive ionisation

mode, using a minimum of 200 shots/spectrum and calibrating to internal or

external synthetic RNA standards. RP-HPLC: Gilson semi-preparative system,

Rainin Dynamax-60A Si 83-141-C, C18 Microsorb (250 4.6 mm) and Atlantis

Prep OBD T3 (250 x 10 mm) 10 µm columns, Gilson 115 UV detector, 255 nm,

samples were filtered through 0.22 m syringe tip filter units. SAX-HPLC:

Varian 940-LC analytical to preparative HPLC with 445-LC scale-up module,

440-LC fraction collector and ProStar column valve module, DNAPac PA-200

(250 4 mm, with a 50 4 mm guard column) analytical and DNAPac PA-100

(250 22 mm) preparative columns. Gel electrophoresis: Life Technologies

XCell SureLock® Mini-Cell system, denaturing (8 M urea) 20 % polyacrylamide

(8 cm long) gels prepared using 1.0 mm Novex® cassettes, typically run at 120-

200 V in TBE buffer; fluorescence imaging performed using a GE Typhoon Trio

Variable Mode Imager, and quantified using ImageQuant TL software; gel

staining with SYBR® Gold was performed for 15-45 min; UV imaging using a

Syngene transilluminator and GeneSnap software.

4

Synthetic protocols

Cyanogen 6

Cyanogen 6 was prepared by thermal elimination of acetic acid from

diacetylglyoxime according to a literature procedure1, and was dissolved in D2O

to make up a 1.0 M solution, which was used immediately (the volatility of

cyanogen means that its concentration in this solution decreases rapidly when the

solution is exposed to the atmosphere).

Nucleoside 3/2-monophosphates, N3P’s and N2P’s

N3P’s and N2P’s were commercially available, or readily prepared from

commercially available protected phosphoramidites, as illustrated below

(Scheme S1) for I3P (14).

Scheme S1: Synthesis of inosine 3-monophosphate (14) from fully protected

phosphoramidite. i. HO(CH2)2CN, tetrazole, MeCN, 3 Å molecular sieves;

ii. tBuOOH; iii. DCA, CH2Cl2 (82 % over 3 steps); iv. TMG, TMSCl, MeCN;

v. NH3/MeOH; vi. CsF, MeOH; vii. Na+ Dowex® (95 % over 4 steps).

5

2-tert-Butyl-dimethylsilyl--D-inosine-3-(bis)cyanoethyl phosphate 13

Cyanoethanol (77 L, 1.13 mmol), 3 Å molecular sieves (250 mg, dried in vacuo

at 300°C for 24 h), anhydrous MeCN (4 mL) and 5-O-(4,4-dimethoxytrityl)-

2-O-tert-butyl-dimethylsilyl--D-inosine-3-O-(N,N-di-iso-propyl-cyanoethyl

phosphoramidite) (200 mg, 0.23 mmol) were stirred under an atmosphere of

nitrogen at r.t. for 30 min. 1H-Tetrazole (0.45M in MeCN, 3.15 mL, 1.13 mmol)

was then added and the reaction mixture stirred for 8 h. tert-Butyl hydroperoxide

(6.0M in decane, 225 L, 1.13 mmol) was added and the mixture stirred for 1 h.

The reaction mixture was then filtered through a pad of Celite® and concentrated

in vacuo. The residue was dissolved in saturated NaHCO3 solution (3 mL) and

the resultant solution extracted with EtOAc (3 x 5 mL). The combined organic

layers were washed with brine (5 mL), dried over MgSO4 and then concentrated

in vacuo. The residue was dissolved in CH2Cl2 (5 mL) followed by addition of

dichloroacetic acid (298 L, 3.62 mmol), and the reaction mixture was stirred for

15 min. After quenching the reaction with saturated NaHCO3 solution (4 mL),

the crude product was extracted into CH2Cl2 (3 5 mL). The combined organic

layers were dried over MgSO4 and concentrated in vacuo. The crude yellow

product was purified by silica gel flash column chromatography eluting with a

gradient of CH2Cl2:MeOH (100:0 to 90:10) to yield 2-tert-butyl-dimethylsilyl--

D-inosine-3-(bis)cyanoethyl phosphate 13 (105 mg, 82%) as a white foam. Rf

0.3 (CH2Cl2:MeOH 9:1). M.p. 160-170°C (decomp.). IR (solid, cm-1): 3735

(weak, N-H), 3405 (broad, O-H), 2931 (weak, C-H), 2360 (sharp, medium,

CN), 2341 (sharp, medium, CN), 1699 (strong, C=O), 1255 (medium, P=O),

1034 (shoulder, strong, P=O), 1005 (shoulder, strong, P=O). 1H-NMR (400 MHz,

MeOD) 8.38 (s, 1H, H-(C8)); 8.12 (s, 1H, H-(C2)); 6.07 (d, J = 6.1 Hz, 1H, H-

(C1)); 4.98-5.05 (m, 2H, H-(C2), H-(C3)); 4.48 (app. d, J = 1.0 Hz, 1H, H-

6

(C4)); 4.34-4.43 (m, 4H, -OCH2CH2CN); 3.91 (dd, J = 12.6, 3.0 Hz, 1H, H-

(C5)); 3.86 (dd, J = 12.6, 2.5 Hz, 1H, H-(C5)); 2.96 (td, J = 5.8, 0.8 Hz, 4H, -

OCH2CH2CN); 0.77 (s, 9H, -SiC(CH3)3); 0.00 (s, 3H, -Si(CH3)2); –0.18 (s,

3H, -Si(CH3)2). 13C-NMR (100 MHz, MeOD) 158.8 (C4); 149.8 (C6); 147.4

(C2); 141.2 (C8); 126.4 (C5); 118.7 (2C, CN); 89.8 (C1); 86.5 (C4); 80.2 (C3);

76.5 (C2); 64.9 (2C, -OCH2CH2CN); 62.8 (C5); 26.2 (3C, -SiC(CH3)3); 20.3

(2C, -OCH2CH2CN); 19.0 (-SiC(CH3)3); –4.7 (-Si(CH3)2); –5.3 (-Si(CH3)2). 31P-NMR (162 MHz, MeOD, 1H-decoupled) –3.11 (br. s.). ESI-MS (neg. m/z):

567 (100%, [M–H+]–). ESI-MS (pos. m/z): 591 (100%, [M+Na+]+). ESI-HRMS

(pos.): [M+Na+]+ calculated for C22H33N6O8SiPNa, 591.1759; found, 591.1770.

-D-Inosine-3-monophosphate di-sodium salt 14

2-tert-Butyl-dimethylsilyl--D-inosine-3-biscyanoethyl phosphate 13 (105 mg,

0.19 mmol) was dissolved in anhydrous MeCN (6 mL). Trimethylsilyl chloride

(93 μL, 0.74 mmol) and N,N,N,N-tetramethylguanidine (116 μL, 0.92 mmol)

were added, and the solution was stirred at r.t. under a nitrogen atmosphere for

16 h. The solution was concentrated in vacuo and the residue dissolved in

anhydrous MeOH (0.6 mL) to which was added a saturated solution of

methanolic ammonia (0.5 mL). After 5 min, CsF (70 mg, 0.46 mmol) was added

and the suspension was refluxed for 24 h. The reaction mixture was concentrated

in vacuo and the residue redissolved in H2O (2 mL) to which Ba(OAc)2 (87 mg,

0.34 mmol) was added. Agitation caused the precipitation of BaF2, which was

removed by centrifugation. EtOH (1 mL) was added to the supernatant to

precipitate the product as a mono-barium salt in three batches which were

isolated by centrifugation. The solid was washed with EtOH:H2O (8:1) to

give-D-inosine-3'-phosphate mono-barium salt (70 mg, 90%), which was

dissolved in H2O (1 mL) and Dowex® 50WX4-400 resin (20 mg, Na+-form, pre-

washed) was added. The suspension was stirred for 3 h, filtered and the filtrate

lyophilised to give -D-inosine-3-phosphate di-sodium salt 14 (67 mg, 95% over

7

4 steps) as a white solid. IR (solid, cm-1): 3311 (weak, O-H), 1686 (strong, C=O),

1588 (strong), 1418, 1218 (weak), 1090 (strong-broad, P=O). 1H-NMR (400

MHz, D2O) 8.28 (s, 1H, H-(C8)); 8.13 (s, 1H, H-(C2)); 6.03 (d, J = 6.1 Hz, 1H,

H-(C1)); 4.70 (t, J = 5.5 Hz, 1H, H-(C2)); 4.59-4.66 (m, 1H, H-(C3)); 4.32 (q,

J = 3.3 Hz, 1H, H-(C4)); 3.81 (app. d, J = 3.0 Hz, 2H, H-(C5), H-(C5)). 13C-

NMR (100 MHz, D2O) 158.8 (C4); 148.5 (C6); 146.2 (C2); 140.2 (C8); 124.1

(C5); 88.3 (C1); 85.2 (C4); 73.9 (C2); 72.8 (C3); 61.2 (C5). ESI-MS (neg.

m/z): 347 (100%, [M–2Na++H+]–). TOF-ESI-HRMS (neg.): [M–2Na++H+]–

calculated for C10H12N4O8P, 347.0398; found, 347.0399.

Oligonucleotides (AGA2P/3P trimer and CC2P/3P dimer) for NMR studies

Trimeric AGA3P (18, Scheme S2) and dimeric CC3P (22, Scheme S3)

oligonucleotides were derived from fully protected phosphoramidite starting

materials. Conversion to mixed 2/3P’s was achieved by hydrolysis of the

analogous cyclic phosphate in aqueous solution at pH 4.0; cyclic phosphates

AGA>P (19, Scheme S2) and CC>P (23, Scheme S3) were obtained as described

below.

8

Scheme S2: Synthesis of AGA3′P (18) and AGA>P (19) from fully protected

phosphoramidites. i. HO(CH2)2CN, tetrazole, MeCN, 3 Å molecular sieves;

ii. tBuOOH; iii. DCA, CH2Cl2 (94 % over 3 steps); iv. Ac-G-CE

phosphoramidite, tetrazole, MeCN, 3 Å molecular sieves; v. tBuOOH; vi. DCA,

CH2Cl2 (74 % over 3 steps); vii. Ac-A-CE phosphoramidite, tetrazole, MeCN, 3 Å

molecular sieves; viii. tBuOOH; ix. DCA, CH2Cl2 (87 % over 3 steps); x.

NH3/MeOH; xi. CsF, MeOH; xii. Na+ Dowex® (50 % over 3 steps); xiii. MeNC,

pH 6.0 (quant.).

9

Scheme S3: Synthesis of CC3′P (22) and CC>P (23) from fully protected

phosphoramidites. i. HO(CH2)2CN, tetrazole, MeCN, 3 Å molecular sieves;

ii. tBuOOH; iii. DCA, CH2Cl2 (85 % over 3 steps); iv. Ac-C-CE

phosphoramidite, tetrazole, MeCN, 3 Å molecular sieves; v. tBuOOH; vi. DCA,

CH2Cl2 (87 % over 3 steps); vii. TMG, TMSCl, MeCN; viii. NH3/MeOH; ix. CsF,

MeOH; x. Na+ Dowex® (62 % over 4 steps); xi. MeNC, pH 5.0 (quant.).

N6-Acetyl-2-tert-butyl-dimethylsilyl--D-adenosine-3-(bis)cyanoethyl phosphate

15

Cyanoethanol (341 L, 5.0 mmol), 3 Å molecular sieves (1 g, dried in vacuo at

300°C for 17 h) and anhydrous MeCN (16 mL) were stirred under an atmosphere

of nitrogen at r.t. for 30 min. N6-Acetyl-5-O-(4,4-dimethoxytrityl)-2-O-tert-

10

butyl-dimethylsilyl--D-adenosine-3-O-(N,N-di-iso-propyl-cyanoethyl

phosphoramidite) (926 mg, 1.0 mmol) was added and the reaction was stirred for

30 min. 1H-Tetrazole (0.45M in MeCN, 11.1 mL, 5.0 mmol) was then added and

the reaction mixture stirred for 18 h. tert-Butyl hydroperoxide (6.0M in decane,

1 mL, 5.0 mmol) was added and the mixture stirred for 1.5 h. The reaction was

then filtered through a pad of Celite® and concentrated in vacuo. The residue was

dissolved in EtOAc (20 mL), and the resultant solution washed with saturated

NaHCO3 (20 mL), brine (20 mL), dried (MgSO4) and then concentrated in vacuo.

The residue was dissolved in CH2Cl2 (10 mL) followed by addition of

dichloroacetic acid (1.32 mL, 16.0 mmol), and the reaction mixture was stirred

for 15 min. After quenching the reaction with saturated NaHCO3 solution, the

crude product was extracted into CH2Cl2 (3 20 mL), and the combined organic

layers dried over MgSO4, and concentrated in vacuo. The crude yellow product

was purified by silica gel column chromatography eluting with

cyclohexane:EtOAc:MeOH (10:10:1 to 10:10:3) to yield N6-acetyl-2-tert-butyl-

dimethylsilyl--D-adenosine-3-(bis)cyanoethyl phosphate 15 (570 mg, 94%) as

a white foam. Rf 0.14 (EtOAc:CH2Cl2:MeOH 10:10:1). IR (solid, cm-1): 3339

(broad, O-H), 2933 (weak, C-H), 2360 (weak, CN), 1613 (strong, C=O, 1587,

1460 (medium), 1253 (strong, P=O), 1031 (strong, P=O). 1H-NMR (400 MHz,

CDCl3) 9.34 (s, 1H, -NH(CO)CH3); 8.67 (s, 1H, H-(C8)); 8.19 (s, 1H, H-(C2));

6.07 (d, J = 10.6 Hz, 1H, HO-(C5)); 5.92 (d, J = 7.8 Hz, 1H, H-(C1)); 5.22

(ddd, J = 7.6, 4.7, 2.8 Hz, 1H, H-(C2)); 5.05 (dd, J = 7.3, 4.8 Hz, 1H, H-(C3));

4.54 (app. s, 1H, H-(C4)); 4.30-4.44 (m, 4H, -OCH2CH2CN); 3.97 (app. d, J =

13.1 Hz, 1H, H-(C5)); 3.78-3.89 (m, 1H, H-(C5)); 2.83 (t, J = 5.9 Hz, 4H, -

OCH2CH2CN); 2.64 (s, 3H, -NH(CO)CH3); 0.71 (s, 9H, -SiC(CH3)3); –0.16 (s,

3H, -Si(CH3)2); –0.38 (s, 3H, -Si(CH3)2). 13C-NMR (100 MHz, CDCl3) 170.7 (-

NH(CO)CH3); 151.9 (C8); 150.1 (C4); 149.9 (C6); 143.3 (C2); 123.0 (C5); 116.3

(2C, CN); 90.2 (C1); 86.2 (C4); 79.4 (C3); 73.1 (C2); 62.6 (2C, -

OCH2CH2CN); 62.5 (C5); 25.9 (-NH(CO)CH3); 25.3 (3C, -SiC(CH3)3); 19.8

(2C, -OCH2CH2CN); 17.8 (-SiC(CH3)3); –5.3 (-Si(CH3)2); –5.8 (-Si(CH3)2). 31P-

NMR (162 MHz, CDCl3, 1H-decoupled) –2.55 (s). 31P-NMR (162 MHz, CDCl3)

–2.55 (m). ESI-MS (pos. m/z): 610 (50%, [M+H+]+), 632 (100%, [M+Na+]+).

ESI-HRMS (m/z): [M+Na+]+ calculated for C24H36O8N7NaPSi, 632.2024; found

632.2025.

11

N2-Acetyl-2-tert-butyl-dimethylsilyl--D-guanosine-3-cyanoethylphosphoryl-N6-

acetyl-2-tert-butyl-dimethylsilyl--D-adenosine-3-(bis)cyanoethyl phosphate 16

N4-Acetyl-2-tert-butyl-dimethylsilyl--D-adenosine-3-

(bis)cyanoethylphosphate 15 (550 mg, 0.9 mmol), 3 Å molecular sieves (1 g,

dried in vacuo at 300°C for 17 h), N2-Acetyl-5-O-(4,4-dimethoxytrityl)-2-O-

tert-butyl-dimethylsilyl--D-guanosine-3-O-(N,N-di-iso-propyl-cyanoethyl

phosphoramidite) (1 g, 1.08 mmol) and anhydrous MeCN (15 mL) were stirred

under an atmosphere of nitrogen at r.t. for 30 min. 1H-tetrazole (0.45M in MeCN,

10 mL, 4.5 mmol) was then added and the reaction mixture stirred for 18 h. tert-

Butyl hydroperoxide (6.0M in decane, 1 mL, 6.0 mmol) was added and the

mixture stirred for 1.5 h. The reaction mixture was then filtered through a pad of

Celite® and the filtrate concentrated in vacuo. The residue was dissolved in

EtOAc (20 mL), and the resultant solution washed with saturated NaHCO3

solution (20 mL), brine (20 mL), dried over MgSO4 and then concentrated in

vacuo. The residue was dissolved in CH2Cl2 (20 mL) followed by addition of

dichloroacetic acid (371 L, 4.5 mmol), and the reaction mixture was stirred for

15 min. After quenching the reaction with saturated NaHCO3 solution, the crude

product was extracted into CH2Cl2 (3 20 mL), and the combined organic layers

dried over MgSO4 and concentrated in vacuo. The crude yellow product was

purified by silica gel flash column chromatography eluting with

cyclohexane:EtOAc:MeOH (10:10:4) to yield 16 (776 mg, 74%) as a white

foam, and as a 1:1.8 mixture of diastereoisomers. Rf 0.1 (CH2Cl2:MeOH 95:5).

M.p. 162°C (decomp.). 1H-NMR (400 MHz, CDCl3) 12.12-12.37 (2 x br. s.,

1H, NH); 10.23-10.59 (2 x br. s., 1H, NH); 9.62-9.65 (2 x br. s, 1H, NH); 8.68-

8.75 (2 x s, 1H, H-(C8A)); 8.29-8.38 (2 x s, 1H, H-(C2A)); 7.98-8.12 (2 x s, 1H,

12

H-(C8G)); 6.09 (d, J = 4.3 Hz, 1H, H-(C1A)); 5.78 (d, J = 6.8 Hz, 1H, H-

(C1Gminor)); 5.74 (d, J = 6.6 Hz, 1H, H-(C1Gmajor)); 5.08-5.29 (m, 3H, H-(C3G),

H-(C2A), H-(C3A)); 4.91-5.06 (m, 1H, H-(C2G)); 4.50-4.67 (m, 3H, H-(C4A),

H-(C5A), H-C(5A)); 4.25-4.48 (m, 7H, -OCH2CH2CN, H-(C4G)); 3.91-4.03

(m, 1H, H-(C5G)); 3.74-3.89 (m, 1H, H-(C5G)); 2.76-2.93 (m, 6H, -

OCH2CH2CN)); 2.25-2.63 (3 x s, 6H, -NH(CO)CH3); 0.65-0.84 (m, 18H, -

SiC(CH3)3); –0.32-0.10 (m, 12H, -Si(CH3)2). 13C-NMR (100 MHz, CDCl3)

173.1 (-NH(CO)CH3); 171.6 ((-NH(CO)CH3)); 155.5 (C4G); 152.5 (C8A); 151.1;

149.5 (C6A); 148.1 (C6G); 147.9; 142.2; 142.1 (C2A); 139.2 (C8G); 122.3 (C5A);

121.6 (C5G); 116.9 (2C, CN); 116.7 (CN); 89.0 (C1A); 88.4 (C1G); 84.1 (C4G);

80.7 (C4A); 78.3 (C3G); 75.4 (C2A); 73.9 (C2G); 73.7 (C3A); 66.3 (C5A); 63.1

(-OCH2CH2CN); 62.8 (2C, -OCH2CH2CN); 61.1 (C5G); 25.5 (3C, -SiC(CH3)3);

25.4 (3C, -SiC(CH3)3); 24.4 (-NH(CO)CH3); 24.3 (-NH(CO)CH3); 19.8 (-

OCH2CH2CN); 19.8 (-OCH2CH2CN); 18.0 (-SiC(CH3)3); 17.9 (-SiC(CH3)3); –5.0

(-Si(CH3)3); –5.1 (-Si(CH3)3); –5.4 (-Si(CH3)3); –5.5 (-Si(CH3)3). 31P-NMR (162

MHz, CDCl3, 1H decoupled) –2.45 (s), –2.55 (s), –2.64 (s), –2.99 (s). ESI-MS

(pos. m/z): 1164 (100 %, [M+H+]+), 1186 (100 %, [M+Na+]+).

Fully protected AGA trimer 17

13

N2-Acetyl-2-tert-butyl-dimethylsilyl--D-guanosine-3-cyanoethylphosphoryl-

N6-acetyl-2-tert-butyl-dimethylsilyl--D-adenosine-3-(bis)cyanoethyl

phosphate 16 (776 mg, 0.66 mmol), 3 Å molecular sieves (1 g, dried in vacuo at

300°C for 17 h), N6-Acetyl-5-O-(4,4-dimethoxytrityl)-2-O-tert-

butyldimethylsilyl--D-adenosine--3-O-(N,N-di-iso-propyl-cyanoethyl

phosphoramidite) (728 mg, 0.79 mmol) and anhydrous MeCN (10 mL) were

stirred under an atmosphere of nitrogen at r.t. for 30 min. 1H-Tetrazole (0.45M in

MeCN, 13 mL, 5.85 mmol) was then added and the reaction mixture stirred for

18 h. tert-Butyl hydroperoxide (6.0M in decane, 0.66 mL, 3.96 mmol) was added

and the mixture stirred for 1.5 h. The reaction mixture was then filtered through a

pad of Celite® and the filtrate concentrated in vacuo. The residue was dissolved

in EtOAc (20 mL), and the resultant solution washed with saturated NaHCO3 (20

mL) and brine (20 mL). The combined organic layers were dried over MgSO4

and then concentrated in vacuo. The residue was dissolved in CH2Cl2 (20 mL)

followed by addition of dichloroacetic acid (865 L, 10.5 mmol), and the

reaction mixture was stirred for 15 min. After quenching the reaction with

saturated NaHCO3 solution, the crude product was extracted into CH2Cl2 (3 20

mL), and the combined organic layers were dried over MgSO4 and concentrated

in vacuo. The crude yellow product was purified by silica gel flash column

chromatography eluting with CH2Cl2:MeOH (100:0 to 95:5) to yield the fully

protected trimer 17 (967 mg, 87%) as a white foam, and as a complex mixture of

diastereoisomers and possibly rotamers. Rf 0.5 (CH2Cl2:MeOH 95:5). NMR

14

assignments are given where possible. 1H-NMR (400 MHz, CDCl3) 8.27-9.07

(m, 4H, H-(C2A1), H-(C2A3), H-(C8A1), H-(C8A3)); 7.75 (m, 1H, H-(C8G)); 5.84-

6.53 (m, 3H, H-(C1A1), H-(C1A3), H-(C1G)); 3.71-4.04 (m, 2H, H-(C5A3), H-

(C5A3)); 2.75-2.90 (m, 8H, -OCH2CH2CN); 2.20-2.68 (m, 9H, CH3); 0.58-0.91

(m, 27H, SiC(CH3)3); –0.44-0.15 (m, 18H, Si(CH3)2). 13C-NMR (400 MHz,

CDCl3) 173.0; 155.6; 152.5; 151.9; 151.2; 151.1; 150.4; 150.2; 150.0; 149.6;

148.2; 148.1; 147.6; 140.3; 123.3; 122.8; 122.6; 122.3; 116.9; 116.8; 116.7;

116.7; 116.5; 90.1; 89.9; 89.0; 88.8; 86.4; 74.7; 73.4; 63.0; 62.8; 62.6; 26.1; 25.8;

25.7; 25.5; 25.5; 25.4; 25.4; 24.1; 24.0; 19.8; 18.0; 18.0; 17.9; 17.9; 17.9; 17.8; –

4.88; –4.94; –5.07; –5.14; –5.2; –5.4; –5.5; –5.6; –5.6. 31P-NMR (162 MHz,

CDCl3, 1H-decoupled) –1.97 (s); –2.12 (s); –2.35 (s); –2.46 (s); –2.55 (s); –2.66

(s); –3.23 (s); –3.31 (s); –3.41 (s); –3.56 (s); –3.65 (s); –3.91 (s). ESI-MS (pos. m/z): 1725 (90%, [M+Na+]+.

-D-Adenyl-(53)--D-guanyl-(53)--D-adenosine-3-monophosphate

tetra-sodium salt 18

Fully protected AGA trimer 17 (546 mg, 0.32 mmol) was dissolved in saturated

methanolic ammonia (10 mL) and heated in a closed vessel at 60°C for 16 h. The

solution was then concentrated in vacuo and the residue dissolved in MeOH (5

mL). CsF (365 mg, 2.4 mmol) was added and the solution was refluxed for 24 h.

The solution was concentrated in vacuo and the residue purified by RP-HPLC (eluting with H2O:MeOH (85:15), Tretention= 2.0 min). The aqueous fraction with Tretention= 2.0 min was concentrated in vacuo to give a white residue, which was dissolved in H2O (3 mL) and stirred

15

with Dowex® 50WX2-400 (3 g, Na+ form, pre-washed) for 24 h. The suspension was filtered, and the filtrate lyophilised to give -D-adenyl-(53)--D-guanyl-(53)--D-adenosine-3-monosphosphate

tetra-sodium salt 18 (170 mg, 50%, over 4 steps) as a white solid. IR (solid, cm-1): 3181 (broad, O-H), 1635 (broad, C=O), 1233 (strong, P=O),

1069 (strong, P=O). 1H-NMR (400 MHz, D2O) 8.16 (s, 1H, H-(C8A3)); 7.97 (s,

1H, H-(C8A1)); 7.82 (s, 1H, H-(C2A3)); 7.72 (br. s., 2H, H-(C2A1), H-(C8G)); 5.89

(d, J = 5.0 Hz, 1H, H-(C1A3)); 5.73 (d, J = 4.8 Hz, 1H, H-(C1A1)); 5.50 (d, J =

4.5 Hz, 1H, H-(C1G)); 4.66-4.73 (obs. HOD, 1H, H-(C3G)); 4.56-4.64 (m, 2H,

H-(C2A3), H-(C3A3)); 4.54-4.63 (m, 2H, H-(C3A1), H-(C2A1)); 4.47-4.52 (m,

1H, H-(C2G)); 4.42 (br. s, 1H, H-(C4A3)); 4.32 (br. s, 1H, H-(C4G)); 3.99-4.26

(m, 5H, H-(C4A1), H-(C5A3), H-(C5A3), H-(C5G), H-(C5G); 3.61-3.75 (m,

2H, H-(C5A1), H-(C5A1). 13C-NMR (100 MHz, D2O) 157.9 (C4G); 154.7

(C4A1); 154.6 (C4A3); 153.2 (C2G); 152.3 (C2A1); 151.8 (C2A3); 150.6 (C6G); 148.3

(C6A3); 147.3 (C6A1); 140.0 (C8A1); 139.1 (C8A3); 136.5 (C8G); 118.4 (C5A1);

117.8 (C5A3); 115.8 (C5G); 88.8 (C1A1); 87.4 (C1G); 86.9 (C1A3); 84.1 (C4A1);

83.0 (C4A3); 82.0 (C4G); 73.9 (C2A3); 72.6 (C2A1); 72.6 (C2G); 65.2 (C5G);

64.6 (C5A3); 60.9 (C5A1). 31P-NMR (162 MHz, D2O, 1H-decoupled) 3.59 (s), –

0.62 (s), –0.81 (s). ESI-MS (neg. m/z): 509 ([M–4Na++2H+]2–, 100%). TOF-ESI-

HRMS (neg. m/z): [M–4Na++2H+]2– calculated for C30H36O20N15P32– 509.5742,

found 509.5746.

-D-Adenyl-(53)--D-guanyl-(53)--D-adenosine-2,3-cyclic phosphate

tri-sodium salt 19

16

The cyclization of -D-Adenyl-(53)--D-guanyl-(53)--D-adenosine-3-

monosphosphate tetra-sodium salt 18 was carried out according to a literature

procedure2. The trimer 18 (51 mg, 0.046 mmol) was dissolved in H2O (0.5 mL),

and the pH was adjusted to pH 6 with DCl (1.0M). Methyl isocyanide 7 (10 l,

0.2 mmol) was added and the mixture was vigorously stirred at 40ºC for 16 h.

The solution was concentrated in vacuo, the residue dissolved in H2O (1 mL) and

the resultant solution extracted with EtOAc (3 x 5 mL). The combined organic

layers were washed with H2O (3 x 1 mL). The combined aqueous layers were

lyophilised to give -D-adenyl-(53)--D-guanyl-(53)--D-adenosine-

2,3-cyclic phosphate tri-sodium salt 19 (55 mg) as a yellow powder which was

not further purified. 1H-NMR (400 MHz, D2O) 8.13 (s, 1H, H-(C8A)); 8.08 (s,

1H, H-(C8A)); 7.95 (s, 1H, H-(C2A)); 7.91 (s, 1H, H-(C2A)); 7.86 (s, 1H, H-

(C8G)); 6.10 (d, J = 4.3 Hz, 1H, H-(C1A3)); 5.87 (d, J = 5.8 Hz, 1H, H-(C1A1));

5.66 (d, J = 5.8 Hz, 1H, H-(C1G)); 5.26 (ddd, J = 9.7, 6.9, 4.3 Hz, 1H, H-

(C2A3)); 5.08 (ddd, J = 8.4, 7.1, 4.7 Hz, 1H, H-(C3A3)); 4.64-4.72 (m, 3H, H-

(C3G), H-(C2A1), H-(C3A1)); 4.61 (t, J = 5.4 Hz, 1H, H-(C2G)); 4.50-4.56 (m,

1H, H-(C4A3)); 4.36-4.41 (m, 1H, H-(C4G)); 4.29-4.33 (m, 1H, H-(C4A1)); 4.21

(dt, J = 11.3, 5.3 Hz, 2H, H-(C5A3), H-(C5A3)); 4.10-4.16 (m, 1H, H-(C5G), H-

(C5G)); 3.75 (m, 2H, H-(C5A1), H-(C5A1)). 31P-NMR (162 MHz, D2O, 1H-

decoupled) 20.15 (s), –0.62 (s), –0.67 (s). ESI-MS (neg. m/z): 500 ([M–3Na+

+H+]2–, 40%), 511 ([M–2Na+]2–, 100%).

N4-Acetyl-2-tert-butyl-dimethylsilyl--D-cytidine-3-(bis)cyanoethyl phosphate 20

17

Cyanoethanol (341 L, 5.0 mmol), 3 Å molecular sieves (600 mg, dried in vacuo

at 300°C for 24 h), anhydrous MeCN (16 mL) and N4-acetyl-5-O-(4,4-

dimethoxytrityl)-2-O-tert-butyl-dimethylsilyl--D-cytidine-3-O-(N,N-di-iso-

propyl-cyanoethyl phosphoramidite) (900 mg, 1.0 mmol) were stirred under an

atmosphere of nitrogen at r.t. for 30 min. 1H-Tetrazole (0.45M in MeCN, 11.1

mL, 5.0 mmol) was then added and the reaction mixture stirred for 4.5 h. tert-

Butyl hydroperoxide (5.0M in H2O, 1 mL) was added and the mixture stirred for

1 h. The reaction mixture was filtered through Celite®, the filtrate concentrated in

vacuo, and the residue dissolved in EtOAc (20 mL). The organic phase was

washed with saturated NaHCO3 solution (10 mL), brine (10 mL), and then

concentrated in vacuo. The resultant oil was dissolved in CH2Cl2 (40 mL) and the

resultant solution was stirred with dichloroacetic acid (1.4 mL, 17.0 mmol) for

15 min before the addition of saturated NaHCO3 solution until no further

effervescence was observed. The mixture was extracted into CH2Cl2 (20 mL), and

the aqueous layer was washed with CH2Cl2 (2 x 20 mL). The organic layers were

combined, dried over MgSO4 and concentrated in vacuo to give a crude pale

yellow oil (1.04 g) which was purified by silica gel column chromatography

eluting with EtOAc:cyclohexane:MeOH (10:10:0 to 10:10:1) to yield N4-acetyl-

2-tert-butyl-dimethylsilyl--D-cytidine-3-(bis)cyanoethyl phosphate 20 (500

mg, 85%) as a white foam. Rf 0.18 (EtOAc:cyclohexane:MeOH 10:10:1). M.p.

74-78°C. IR (solid, cm-1): 2360 (strong, C≡N), 1653 (broad, medium, C=O),

1495 (medium), 1253 (strong, P=O), 1041 (strong, P=O). 1H-NMR (400 MHz,

CDCl3) 9.90 (br. s, 1H, NH); 8.38 (d, J = 7.1 Hz, 1H, H-(C6)); 7.32 (d, J = 7.6

Hz, 1H, H-(C5)); 5.61 (app. s, 1H, H-(C1)); 5.04 (m, 1H, H-(C3)); 4.73 (br. s,

1H, H-(C2)); 4.30-4.47 (m, 5H, H-(C4), -OCH2CH2CN); 4.18 (br. s., 1H, H-

O(C5)); 4.11 (app. d, J = 12.6 Hz, 1H, H-(C5)); 3.92 (dd, J = 12.5, 2.9 Hz, 1H,

H-(C5)); 2.73-2.88 (m, 4H, -OCH2CH2CN); 2.26 (s, 3H, CH3); 0.92 (s, 9H, -

SiC(CH3)3); 0.20 (s, 3H, -Si(CH3)2); 0.18 (s, 3H, -Si(CH3)2). 13C-NMR (100

18

MHz, CDCl3) 171.3 (HN(CO)CH3); 162.9 (C2); 155.3 (C4); 145.3 (C6); 116.6

(2C, CN); 96.5 (C5); 92.0 (C1); 81.9 (C4); 74.5 (C3); 74.0 (C2); 62.8 (2C, -

OCH2CH2CN); 59.5 (C5); 25.6 (3C, -SiC(CH3)3); 24.9 (HN(CO)CH3); 19.6 (2C,

-OCH2CH2CN); 18.0 (-SiC(CH3)3); –4.6 (-Si(CH3)2); –5.2 (-Si(CH3)2). 31P-NMR

(162 MHz, CDCl3, 1H-decoupled) –1.84 (s). 31P-NMR (162 MHz, CDCl3) –

1.85 (d, J = 7.8 Hz). ESI-MS (neg. m/z): 620 (100%, [M+Cl–]–). ESI-MS (pos.

m/z): 586 (100%, [M+H+]+). ESI-HRMS (m/z): [M+H+]+ calculated for

C23H37O9N5PSi, 586.2093; found 586.2087.

N4-Acetyl-2-tert-butyl-dimethylsilyl--D-cytidine-3-cyanoethylphosphoryl-N4-

acetyl-2-tert-butyl-dimethylsilyl--D-cytidine-3-(bis)cyanoethyl phosphate 21

N4-Acetyl-2-tert-butyl-dimethylsilyl--D-cytidine-3-(bis)cyanoethyl phosphate

20 (500 mg, 0.86 mmol), 3 Å molecular sieves (600 mg, dried in vacuo at 300°C

for 24 h), anhydrous MeCN (16 mL) and N4-acetyl-5-O-(4,4-

dimethoxytrityl)-2-O-tert-butyl-dimethylsilyl--D-cytidine-3-O-(N,N-di-iso-

propyl-cyanoethyl phosphoramidite) (1.15 g, 1.28 mmol) were stirred under an

atmosphere of nitrogen at r.t. for 30 min. 1H-Tetrazole (0.45M in MeCN, 15.2

mL, 6.84 mmol) was then added and the reaction mixture stirred for 6 h. tert-

Butyl hydroperoxide (5.0M in H2O, 0.56 mL) was added and the mixture stirred

for 1 h. The reaction mixture was filtered through Celite®, the filtrate

concentrated in vacuo, and the residue was dissolved in EtOAc (20 mL). The

organic phase was washed with saturated NaHCO3 solution (10 mL), brine (10

mL), and then concentrated in vacuo. The resultant oil was dissolved in CH2Cl2

(40 mL) and stirred with dichloroacetic acid (1.13 mL, 13.7 mmol) for 15 min

before the addition of saturated NaHCO3 solution until no further effervescence

19

was observed. The mixture was extracted into CH2Cl2 (20 mL), and the aqueous

layer was washed with CH2Cl2 (2 x 20 mL). The organic layers were combined,

dried over MgSO4 and concentrated in vacuo to yield a crude pale yellow foam

which was purified by silica gel column chromatography eluting with

EtOAc:cyclohexane:MeOH (10:10:0 to 10:10:1) to yield the fully protected

dimer 21 (822 mg, 87%) as a white foam and as a 1:1 mixture of 2

diastereoisomers. Rf 0.14 (EtOAc:cyclohexane:MeOH 10:10:1). 1H-NMR (400

MHz, CDCl3, integrations for two diastereoisomers) 10.04-10.39 (m, 4H, H-

(C6), NH); 8.47 (br. s, 2H, H-(C6), NH); 7.79-8.01 (m, 2H, H-(C6), NH); 7.27-

7.40 (m, 4H, H-(C5)); 5.68 (br. s, 4H, H-(C1)); 4.16-5.16 (m, 32H, H-(C2), H-

(C3), H-(C4); H-(C5); H-(C5), -OCH2CH2CN); 2.63-2.90 (m, 12H, -

OCH2CH2CN); 2.08-2.38 (m, 12H, CH3CO); 0.90 (br. s, 9H, -SiC(CH3)3); 0.89

(br. s, 9H, -SiC(CH3)3); 0.88 (br. s, 9H, -SiC(CH3)3); 0.88 (br. s, 9H, -

SiC(CH3)3); 0.01-0.21 (m, 24H, -Si(CH3)2. 13C-NMR (100 MHz, CDCl3) 171.4;

171.2; 163.3; 163.0; 162.9; 155.5; 155.3; 155.1; 155.0; 145.9; 144.4; 117.1;

116.9; 116.8; 116.8; 96.9; 93.3; 92.5; 91.6; 82.4; 79.3; 76.8; 74.5; 73.4; 65.8;

63.2; 63.1; 62.9; 60.4; 59.6; 26.9; 26.0; 25.7; 25.5; 24.9; 21.1; 19.7; 19.7; 18.4;

18.1; 17.7; 14.2; –4.6; –4.7; –4.9; –5.0; –5.1; –5.1. 31P-NMR (162 MHz, CDCl3, 1H-decoupled) –1.47 (br. s), –1.56 (br. s), –2.31 (br. s), –2.37 (br. s). ESI-MS

(neg. m/z): 1099 (100%, [M–H+]–), 1135 (100%, [M+Cl–]–). ESI-MS (pos. m/z):

1122 (100%, [M+Na+]+).

-D-Cytidinyl-(53)--D-cytidine-3-monophosphate tri-sodium salt 22

20

Fully protected dimer 21 (420 mg, 0.38 mmol) was dissolved in anhydrous

MeCN (8 mL). Trimethylsilyl chloride (290 μL, 2.29 mmol) and N,N,N,N-

tetramethylguanidine (360 μL, 2.87 mmol) were added, and the solution was then

stirred at r.t. under a nitrogen atmosphere for 16 h. The solution was

concentrated in vacuo and the residue dissolved in anhydrous MeOH (3 mL) to

which was added a saturated solution of methanolic ammonia (3 mL). After 5

min, CsF (290 mg, 1.90 mmol) was added and then the supension was refluxed

for 24 h. The reaction mixture was concentrated and the residue redissolved in

H2O (5 mL). Dowex® 50WX4-400 resin (1 g, Na+-form, pre-washed) was added

and the suspension was stirred for 3 h, filtered, concentrated in vacuo, and this

procedure repeated until the 1H-NMR signal corresponding to N,N,N,N-

tetramethylguanidine (H= 2.88 ppm) had disappeared,to give -D-cytidinyl-

(53)--D-cytidine-3-monophosphate 22 (165 mg, 62%) as a white solid.). 1H-NMR (400 MHz, D2O) 7.85 (m, 2H, H-(C6C1), H-(C6C2)); 5.91-5.98 (m,

3H, H-(C5C1), H-(C5C2), H-(C1C2)); 5.78 (d, J = 2.8 Hz, 1H, H-(C1C1)); 4.52

(dt, 1H, H-(C3C2)); 4.38-4.47 (m, 2H, H-(C3C1), H-(C2C1)); 4.32-4.37 (m, 1H,

H-(C4C2)); 4.24-4.31 (m, 3H, H-(C4C1), H-(C2C2), H-(C5C2)); 4.13 (dt, J =

11.5, 3.1 Hz, 1H, H-(C5C2)); 3.93-3.99 (dd, J = 13.1, 2.5 Hz, 1H, H-(C5C1));

3.82 (dd, J = 13.1, 4.0 Hz, 1H, H-(C5)). 13C-NMR (100 MHz, D2O) 165.9

(C4); 165.8 (C4); 157.5 (C2); 157.3 (C2); 141.0 (C6); 140.7 (C6); 96.4 (C5);

96.0 (C5); 90.4 (C1C1); 89.4 (C1C2); 82.5 (C4C1); 81.9 (C4C2); 74.0 (C3C2);

73.1 (C2C1); 72.2 (C3C1); 71.7 (C2C2); 64.4 (C5C2); 59.9 (C5C1). 31P-NMR (162

MHz, D2O, 1H-decoupled) 3.74 (br. s.), –0.67 (s). ESI-MS (neg. m/z): 627

(95%, [M–3Na++2H+]–), 313 (100%, [M–3Na++H+]2–).

-D-Cytidinyl-(53)--D-cytidine-2,3-cyclic phosphate di-sodium salt 23

21

-D-Cytidinyl-(53)--D-cytidine-3-phosphate 22 (100 mg, 0.15 mmol) was

dissolved in H2O (1.5 mL), and the pH was adjusted to pH 5.0 with HCl (1.0M).

Methyl isocyanide 7 (168 l, 1.49 mmol) was added and the suspension was

vigorously stirred at 40°C for 16 h. The solution was concentrated in vacuo, the

residue redissolved in H2O (1 mL) and the resultant solution washed with EtOAc

(3 x 3 mL). The combined organic layers were extracted with H2O (3 x 2 mL)

and the combined aqueous layers were concentrated in vacuo to give -D-

cytidinyl-(53)--D-cytidine-2,3-cyclic phosphate 23 (quant.) as a yellow

powder. 1H-NMR (400 MHz, D2O) 7.75 (d, J = 7.6 Hz, 1H, H-(C6C1)); 7.63 (d,

J = 7.6 Hz, 1H, H-(C6C2)); 5.96 (d, J = 7.6 Hz, 1H, H-(C5C2)); 5.87-5.92 (m, 2H,

H-(C5C1), H-(C1C2)); 5.80 (d, J = 4.5 Hz, 1H, H-(C1C1)); 4.93-5.04 (m, 2H, H-

(C2C2), H-(C3C2)); 4.35-4.46 (m, 2H, H-(C3C1), H-(C4C2)); 4.29 (t, J = 4.7 Hz,

1H, H-(C2C1)); 4.19-4.26 (m, 2H, H-(C4C1), H-(C5C2)); 4.05-4.13 (m, 1H, H-

(C5C2)); 3.81 (dd, J = 13.1, 2.8 Hz, 1H, H-(C5C1)); 3.72 (dd, J = 12.9, 3.8 Hz,

1H, H-(C5C1). 13C-NMR (100 MHz, D2O) 166.2 (C4C1); 165.9 (C4C2); 157.4

(C2C1); 156.9 (C2C2); 142.8 (C6C2); 141.3 (C6C1); 96.2 (C5C1); 96.2 (C5C2); 93.0

(C1C2); 89.8 (C1C1); 83.9 (C4C2); 82.9 (C4C1); 81.5 (C2C2); 77.1 (C3C2); 73.1

(C2C1); 73.0 (C3C1); 64.8 (C5C1); 60.2 (C5C2). 31P-NMR (162 MHz, D2O,

decoupled) 19.80 (s), –0.85 (s). 31P-NMR (162 MHz, D2O) 19.80 (dd, J =

11.7, 5.9 Hz), –0.75 (m). TOF-ESI-MS (neg. m/z): 609 (30%, [M–2Na++H+]–).

631 (20%,[M–Na+]–). TOF-HRESI-MS (neg.): [M–2Na++H+]– calculated. for

C18H23N6O14P2, 609.0748; found, 609.0759.

Methyl phosphate (bis)cyclohexylammonium salt 243

22

Methyl phosphate (bis)cyclohexylammonium salt 24 was prepared according to a

previously reported procedure3. To a stirring mixture of crystalline phosphoric

acid (1.0 g, 10.2 mmol) in dry pyridine (4.15 mL, 51 mmol) was added dry

MeOH (102 mmol) followed by dry triethylamine (2.8 mL, 20.4 mmol) via a

dropping funnel. After complete dissolution, acetic anhydride (1.93 mL, 20.4

mmol) was added dropwise. The reaction mixture was stirred for 3 h at 90°C

under reflux, and then cooled to r.t. After addition of H2O (5 mL), the reaction

mixture was stirred at 90°C for a further 1 h and cooled to r.t. The solution was

diluted with H2O (12 mL) and the aqueous phase extracted with diethyl ether (3 x

25 mL). The combined organic layers were concentrated in vacuo, and the

resultant oily liquid was dissolved in acetone/H2O (9:1) to which

cyclohexylamine (2.1 mL, 30.6 mmol) was added. The mixture was cooled to

4°C for 12 h, and the white solid formed was collected by filtration and dried

under vacuum. The solid was heated in EtOH to dissolve the most part, an

insoluble residue was filtered off, and the filtrate was cooled for 12 h at 4°C to

allow crystallization. The crystalline solid was filtered, washed with EtOH, and

dried in vacuo to give methyl phosphate (bis)cyclohexylammonium salt 24 (1.58

g, 51%) as colourless needles. M.p 198°C (Lit.4 195-198°C). IR (solid, cm-1):

3345 (weak, N-H), 2929 (weak, C-H), 2854 (weak, C-H), 2826 (weak, C-H),

1055 (strong, P=O). 1H-NMR (400 MHz, D2O) 3.32 (d, 3H, J = 10.1 Hz, P-

OCH3); 2.86-3.15 (m, 2H, H-(C1)); 1.79-1.93 (m, 4H, H-(C2)); 1.61-1.73 (m,

4H, H-(C3)); 1.48-1.56 (m, 2H, H-(C4)); 1.12-1.29 (m, 8H, H-(C2΄), H-(C3΄));

0.97-1.11 (m, 2H, H-(C4΄)). 13C-NMR (100 MHz, D2O) 51.2 (d, J = 4.6 Hz,

POCH3); 50.2 (C1); 30.3 (C2); 24.2 (C4); 23.7 (C3). 31P-NMR (162 MHz, D2O, 1H-decoupled) 4.67 (s). 31P-NMR (162 MHz, D2O) 4.67 (q, J = 9.8 Hz). TOF-

ESI-HRMS (pos. m/z): 311 (100%, [M+H+]+).

Methyl phosphate di-sodium salt 25

23

To methyl phosphate (bis)cyclohexylammonium salt 24 (500 mg, 5.1 mmol) in

H2O (5 mL) was added freshly prepared Dowex® 50WX4-400 resin (5 g, Na+-

form, pre-washed) and the resultant suspension stirred for 24 h at r.t. The

suspension was filtered, and the filtrate was lyophilised. The process was

repeated until 1H-NMR signals corresponding to cyclohexylamine had

disappeared, to give methyl phosphate di-sodium salt 25 (700 mg, 88%) as a

white powder. 1H-NMR (400 MHz, D2O) 3.45 (d, 3H, J = 10.6 Hz, POCH3). 31P-NMR (162 MHz, D2O, 1H-decoupled) 2.72 (br. s). 31P-NMR (162 MHz,

D2O) q, J = 9.8 Hz). ESI-MS (neg. m/z): 111 (50%, [M–2Na++H+]–).

TOF-ESI-HRMS (neg. m/z): [M–2Na++H+]–) calculated for CH4O4P 110.9852;

found 110.9850.

NMR investigations of chemoselective acetylation

Characterisation of products of acetylation reactions

The formation (and quantification) of acetyl-nucleoside phosphate(s) was

observed by NMR spectroscopic analysis. Characteristic downfield shifted

H-(C2), H-(C3) or H-(C5) were detected by 1H-NMR and 1H-1H COSY

analysis, in tandem with 31P-NMR spectroscopy. In some cases, ESI and ESI-

HRMS were used for further evidence of the presence of various species in

mixtures. Reaction mixtures were additionally spiked with authentic samples to

confirm the presence of: sodium acetate (1.79 ppm (s, 3H, Me)), thioacetic acid

(2.42 ppm (s, 3H, Me)), and nucleoside-2',3'-cyclic phosphate to give

corresponding multiple signal enhancements. Deacetylation was performed by

treatment of the reaction sample with conc. NH3 solution (2 drops), and upon

incubation, signals assigned to acetylated nucleoside phosphates became

replaced by those of nucleoside phosphates.

General procedure for the reaction of nucleoside phosphates with

cyanoacetylene 4 and sodium thioacetate 3

24

Nucleoside phosphates and thioacetic acid were dissolved in D2O at r.t., and the

pD was adjusted to the desired value with NaOD (1.0 M). Cyanoacetylene 4

(0.98 M) was added which resulted in the formation of a white precipitate of

tetradeuterio-β,β-dicyanovinyl-thioether D4-5, and a rapid increase in pD (to 10-

10.5). The pD was readjusted to 6.5 by the careful addition of DCl (1.0 M), and

the reaction mixture was monitored at r.t. by 1H- and 31P-NMR spectroscopy over

24 h. The results of these reactions are tabulated in Table S1 and the data used to

characterise the given products is given in Tables S2-S22.

Table legends

n.d., not detectable. tr., trace – specifically, < 1 % yield. n.a.., not assignable due

to overlapping of signals/not applicable. dec., decoupled. -, not obtained. obs.,

obscured. part. obs., partially obscured. calcd., calculated. n.r., not run. obs.

HOD, signal obscured by HOD peak.

25

Figure S1: Nomenclature for species from the acetylation reactions of

monomeric nucleotides. Shown are the -ribonucleoside derivatives that

constituted the majority of nucleotides in this study, a -descriptor is omitted for

brevity.

26

Table S1: Reactions of nucleoside phosphates with sodium thioacetate 3 and cyanoacetylene 4.

Entry Nucleotide (100 mM)

AcSNa/mM

DCCCN/mM pD

Products and residual starting material(s)/%

N3'P N2'P N2'P,3'OAc

N3'P,2'OAc N>p

N3'P,2'OAc,5'OAc

N2'P,3'OAc,5'OAc

N3'P, 5'OAc

N3'P(OAc), 2'OAc

N2'P(OAc),3'OAc

1 C3'P 100 200 6.5 70 - - 20 8 - - - 2 -2 C3'P 500 500 6.5 55 - - 26 - 11 - 8 - -3 C2'P 100 200 6.5 - 79 8 - 4 - - - - tr.4 C5'P 100 200 6.5 C5'P, 70; C5'P,3'OAc, 8; C5'P,2'OAc, 7; C5'P,2'OAc,3'OAc, 75 C3'(2')P (63:37) 100 200 6.5 38 34 4 17 3 - - - - -6 A3'P 100 200 6.5 47 - - 52 - - - - - -7 A2'P 100 200 6.5 - 80 10 - 8 - - - - -8 A3'(2')P (50:50) 100 200 6.5 20 42 4 28 2 2 - - - -9 A3'(2')P (80:20) 100 200 6.5 43 20 - 35 - - - - - -10 A3'(2')P (80:20) 100 200 7.5 53 20 - 23 - - - - - -11 A3'(2')P (80:20) 250 500 6.5 37 19 - 38 tr. 5 - - - -12 A3'(2')P (80:20) 500 500 6.5 8 19 tr. 63 tr. 7 - - - -13 U2'P 100 200 6.5 - 86 9 - 5 - - - - -14 U3'P 100 200 6.5 73 - - 19 4 - - - - -15 U3'(2')P (66:34) 100 200 6.5 34 25 6 24 10 - - - tr. tr.16 G2'P 100 200 6.5 - 70 13 - 10 - 3 - - 317 G3'P 100 200 6.5 65 - - 35 - - - - - -18 G3'(2')P (63:37) 100 200 6.5 30 31 8 22 6 - - - 2 -19 I3'P 100 200 6.5 45 - - 54 - - - - - -20 A3'P+C3'P (47:53) 100 200 6.5 20 : 44 - - 27 : 5 - - - - - -21 A3'P+U3'P (50:50) 100 200 6.5 24 : 40 - - 24 : 8 - - - - - -

22 A3'P+I3'P (64:36) 100 200 6.5 29 : 23 - - 35 : 13 - - - - - -

23 A3'P+G3'P (72:28) 100 200 6.5 49 : 21 - - 19 : 6 4 : 1 - - - - -24 CC3'P 100 200 6.5 51 - - 42 - 5 - - - -25 CC3'(2')P (62:38) 100 200 6.5 n.a. n.a. n.d. 20 4 - - - - -

N.B – Ratios in brackets for entries 5, 8-12, 15, 18, 20-23, and 25 are the starting ratios of nucleotides.

27

Table S2: Characterisation of products from the reaction described in Table S1, entries 1 and 2.

C3P C3P,2OAc C3P(OAc),2'OAc C> P

/ppm; multiplicity,

J/Hz

H-(C6) 7.78(d, J = 7.6)

7.72(d, J = 7.6) n.a. 7.58

(d, J = 7.6)

H-(C5) 5.97(d, J = 7.6)

5.97(d, J = 7.6) n.a. 5.94

(d, J = 7.6)

H-(C1) 5.86(d, J = 4.1)

5.92(d, J = 4.4) n.a. 5.75

(d, J = 2.5)

H-(C2) 4.30(t, J = 4.7)

5.31(dd, J = 5.5, 4.3) n.a. 5.07

(td, J = 6.5, 2.6)

H-(C3) 4.41(ddd, J = 7.8, 5.6)

4.63(ddd, J = 9.1, 6.0,

5.5)n.a. 4.86

(dt, J = 12.4, 5.8)

H-(C4) 4.15(dt, J = 6.0, 3.3) n.a. n.a. n.a.

H-(C5) 3.84(ABX, J = 12.9, 2.8) n.a. n.a. n.a.

H-(C5)

3.78(ABX, J = 12.9, 4.1) n.a. n.a. n.a.

P 2.75(d, J = 7.8) n.a. n.a. 20.13

(dd, J = 12.7, 6.5)P (dec.) 2.75 (s) 1.52 (s) -8.91 (s) 20.13 (s)

ESI-MS(neg. m/z)

322(95%, [M–Na+]–)

364(45%, [M–Na+]–)

406 (5%, [M–Na+]–)

304(2%, [M–Na+]–)

HRMS(neg. m/z)

Calcd. 322.0446 364.0551 406.0657 304.0340Found 322.0464 364.0546 406.0651 304.0323

28

Table S3: Characterisation of products from the reaction described in Table S1, entry 2.

C3P C3P,2OAc C3P,2'OAc,5'-OAc C3'P,5'OAc

/ppm; multiplicity,

J/Hz

H-(C6) 7.77(d, J = 7.6)

7.72(d, J = 7.6)

7.64(d, J = 7.6)

7.61(d, J = 7.6)

H-(C5) 5.97(d, J = 7.6)

5.97-6.01 (obs.) 5.97-6.00 (obs.) 5.76-6.09

(obs.)

H-(C1) 5.85(d, J = 4.0)

5.92(d, J = 4.0)

5.89(d, J = 4.5) n.a.

H-(C2) 4.30(t, J = 4.7)

5.31(dd, J = 5.7, 4.2) 5.30-5.34 (obs.) n.a.

H-(C3) 4.40(dt, J = 7.8, 5.7)

4.62(dt, J = 9.1, 6.1)

4.66-4.72 (obs. HOD) n.a.

H-(C4) 4.09-4.20 (m)

4.14-4.20(part. obs.) 4.38-4.45 (obs.) n.a.

H-(C5) 3.81-3.87(dd, J = 12.9, 3.0)

3.71-3.89 (obs.) 4.25-4.33 (obs.) n.a.

H-(C5)

3.74-3.80(dd, J = 12.9, 4.0)

3.71-3.89 (obs.) 4.25-4.33 (obs.) n.a.

29


C2P C2P,3OAc C2P(OAc),3OAc C >P

/ppm; multiplicity,

J/Hz

H-(C6) 7.71(d, J = 7.6)

7.58(d, J = 7.6) n.a. 7.60

(d, J = 7.6)

H-(C5) 5.96(d, J = 7.6)

5.97(d, J = 7.6) n.a. 5.98 - 6.03

(obs.)

H-(C1) 5.91(d, J = 5.3)

5.87(d, J = 4.3) n.a. 5.76

(d, J = 2.5)

H-(C2) 4.58(dt, J = 7.1, 5.3)

4.74(obs. HOD) n.a. 5.07

(td, J = 6.5, 2.6)

H-(C3) 4.26(t, J = 5.2)

5.23(dd, J = 5.7, 3.9)

5.27(dd, J = 5.8, 4.0)

4.85(obs. HOD)

H-(C4) 4.04(td, J = 4.7, 3.2)

4.17(q, J = 3.6) n.a. n.a.

H-(C5) 3.78(ABX, J = 12.9, 3.3) n.a. n.a. n.a.

H-(C5) 3.71(ABX, J = 12.6, 4.5) n.a. n.a. n.a.

P 3.05(d, J = 5.9)

1.76(d, J = 9.8) n.a. 20.13

(dd, J = 11.7, 5.9)P (dec.) 3.05 (s) 1.76 (s) -8.65 (s) 20.13 (s)

ESI-MS(neg. m/z)

322 (100%, [M–Na+]–), 344 (100%, [M–H+]–) 364 (45%, [M–Na+]–) 406 (5%, [M–Na+]–) 305 (4%, [M–Na+]–)

HRMS(neg. m/z)

Calcd. 322.0446 364.0551 406.0657 -Found 322.0438 364.0564 406.0641 -

30


C5P C5P,2OAc C5P,3OAc C5P,2OAc,3OAc

/ppm; multiplicity,

J/Hz

H-(C6) 7.95(d, J = 7.6) n.a. n.a. n.a.

H-(C5) 6.02(d, J = 7.6) n.a. n.a. n.a.

H-(C1) 5.90(d, J = 3.3) n.a. 5.99

(d, J = 3.8)6.14

(d, J = 5.3)

H-(C2) 4.20 - 4.30(m)

5.24(dd, J = 5.5, 4.4)

4.49(dd, J = 6.4, 5.4

5.33-5.43(m)

H-(C3) 4.20 - 4.30(m)

4.45(t, J = 5.5)

5.21(dd, J = 5.3, 3.0)

5.33-5.43(m)

H-(C4) 4.13 - 4.19(m) n.a. 4.42

(dt, J = 5.5, 2.7)4.34

(dt, J = 5.1, 2.4)

H-(C5) 4.03(ABXP, J = 11.6, 3.8, 2.5) n.a. n.a. n.a.

H-(C5)

3.93(ABXP, J = 11.9, 5.0, 3.0) n.a. n.a. n.a.

P (dec.) 2.09 (s) 2.09 (s) 2.09 (s) 2.09 (s)ESI-MS

(neg. m/z) 322 (100%, [M–Na+]–) 364 (20%, [M–Na+]–) 364 (20%, [M–Na+]–) 406 (5%, [M–Na+]–)

HRMS(neg. m/z)

Calcd. 322.0446 364.0551 364.0551 406.0657Found 322.0439 364.0564 364.0564 406.0663

31


C2P C3P C2P,3OAc C3P,2OAc C2P,5OAc C>P

/ppm; multiplicity,

J/Hz

H-(C6) n.a. n.a. n.a. n.a. n.a. n.a.H-(C5) n.a. n.a. n.a. n.a. n.a. n.a.

H-(C1) 5.90-5.92 (obs.)

5.86 (d, J = 4.0)

5.90-6.13 (obs.)

5.90-5.92 (obs.)

5.89 (d, J = 4.3)

5.77 (d, J = 2.5)

H-(C2)4.57

(dt, J = 6.8, 5.3)

4.30 (dd, J = 5.0,

4.3)4.78-4.88

(obs. HOD)5.32

(dd, J = 5.7, 3.9)

4.57-4.59 (obs.)

5.08 (td, J = 6.6,

2.8)

H-(C3) 4.26 (t, J = 5.2)

4.40 (dt, J = 7.8,

5.7)

5.24 (dd, J = 5.5,

3.5)4.54-4.67

(obs.) n.a. 4.86-4.94 (obs. HOD)

H-(C4)4.05

(td, J = 4.7, 3.2)

4.10 - 4.19 (m) n.a. 4.12-4.25

(obs.) n.a. n.a.

H-(C5) 3.59-3.92 (obs.)

3.59-3.92 (obs.) n.a. 3.59-3.92

(obs.) n.a. 3.59-3.92 (obs.)

H-(C5) 3.59-3.92 (obs.)

3.59-3.92 (obs.) n.a. 3.59-3.92

(obs.) n.a. 3.59-3.92 (obs.)

P 3.49(d, J = 5.9)

3.70 (d, J = 5.9) n.a. 3.20

(d, J = 9.8) n.a. n.a.P (dec.) 3.49 (s) 3.70 (s) 2.75 (s) 3.20 (s) n.a. 20.14 (s)

ESI-MS(neg. m/z)

322 (100%, [M–

Na+]–)

322(100%, [M–

Na+]–)

364 (50%, [M–

Na+]–)

364 (50%, [M–Na+]–)

406 (10%, [M–Na+]–)

304 (5%, [M–Na+]–)

HRMS(neg. m/z)

Calcd. 322.0445 322.0445 364.0551 364.0551 - -Found 322.0428 322.0428 364.0540 364.0540 - -

32


A3P A3P,2OAc

/ppm; multiplicity,

J/Hz

H-(C8) 8.20 (s) 8.20 (s)H-(C2) 8.00 (s) 8.01 (s)

H-(C1) 5.96 (d, J = 6.3)

6.15 (d, J = 5.5)

H-(C2) 4.71 (t, J = 5.8)

5.57 (t, J = 5.4)

H-(C3) 4.63 (ddd, J = 7.9, 5.2, 3.0)

4.83 (ddd, J = 9.1, 5.5, 4.3)

H-(C4) 4.35 (q, J = 3.0)

4.40 (q, J = 2.8)

H-(C5) 3.83 (ABX, J = 12.4, 3.5)

3.89 (ABX, J = 13.1, 2.8)

H-(C5)

3.80 (ABX, J = 12.9, 3.3)

3.82 (ABX, J = 13.1, 3.2)

P 3.62(d, J = 7.8)

2.57(d, J = 9.8)

P (dec.) 3.62 (s) 2.57 (s)ESI-MS

(neg. m/z)346

(100%, [M–Na+]–)388

(45%, [M–Na+]–)HRMS

(neg. m/z)Calcd. - 388.0664Found - 388.0651

N.B. A diacetylated species was additionally detected by ESI-MS (neg. m/z): 430 (2%, [M–Na+]–), but could not be detected by NMR spectroscopy or further characterised.

34


A2P A2P,3OAc A>p

/ppm; multiplicity,

J/Hz

H-(C8) 8.16 (s) n.a. n.a.H-(C2) 7.91 (s) n.a. n.a.

H-(C1) 5.98(d, J = 6.1)

6.03(d, J = 6.8)

6.06(d, J = 4.5)

H-(C2) 4.91(q, J = 5.5)

5.13(ddd, J = 9.3, 6.8, 6.1)

5.24(ddd, J = 10.6, 6.7, 4.4)

H-(C3) 4.42(dd, J = 4.9, 3.4)

5.36(dd, J = 5.5, 2.5)

4.98(td, J = 7.3, 2.3)

H-(C4) 4.16(q, J = 3.3)

4.27 (m)

4.32 (q, J = 3.6)

H-(C5) 3.77(ABX, J = 12.9, 2.8) n.a. n.a.

H-(C5)

3.68(ABX, J = 12.9, 3.5) n.a. n.a.

P

(d, J = 7.8)0.88

(d, J = 9.8) -

P (dec.) 2.27 (s) 0.88 (s) 19.94 (s)ESI-MS

(neg. m/z)346

(100%, [M–Na+]–)388

(10%, [M–Na+]–)328

(5%, [M–Na+]–)HRMS

(neg. m/z)Calcd. 346.0558 388.0664 328.0452Found 346.0554 388.0669 328.0444

N.B. A diacetylated species was additionally detected by ESI-MS (neg. m/z): 430 (2%, [M–Na+]–), but could not be detected by NMR spectroscopy or further characterised.

35

Table S9: Characterisation of products from the reactions described in Table S1, entries 8-12.

A2P A3P A2P,3OAc A3P,2OAc A3P,2OAc,5OAc A>P

/ppm; multiplicity,

J/Hz


H-(C1) 6.05(d, J = 6.1)

5.98(d, J = 6.3)

6.10(d, J = 6.8)

6.18(d, J = 5.5)

6.09 - 6.37(obs.)

6.14(d, J = 4.8)

H-(C2)5.00

(dt, J = 7.5, 5.7)

4.69 - 4.75(obs. HOD)

5.21(ddd, J = 9.3, 6.7,

5.7)5.58

(t, J = 5.4)5.65

(dd, J = 9.6)5.32

(ddd, J = 10.6, 6.7, 4.4)

H-(C3)4.49

(dd, J = 5.0, 3.3)

4.65(ddd, J = 7.9, 5.2,

3.0)5.44

(dd, J = 5.5, 2.5)4.86

(ddd, J = 9.0, 5.4, 3.8)

4.97-5.12(obs.)

5.02 - 5.10(obs.)

H-(C4) 4.23(q, J = 3.2)

4.37(q, J = 3.1)

4.31-4.38(obs.)

4.42(q, J = 2.9)

4.48-4.61(part. obs.) n.a.

H-(C5) 3.71-3.97(m)

3.72-3.94(m)

3.63-3.94(m)

3.72-3.99(m)

4.23-4.41(obs.) n.a.

H-(C5)

3.71-3.97(m)

3.72-3.94(m)

3.63-3.94(m)

3.72-3.99(m)

4.23-4.41(obs.) n.a.

P 1.94(d, J = 7.8)

2.80(d, J = 7.8)

0.53(d, J = 7.8)

1.29(d, J = 7.8)

2.42(d, J = 7.8)

19.95(dd, J = 11.7, 7.8)

P (dec.) 1.94 (s) 2.80 (s) 0.53 (s) 1.29 (s) 2.42 (s) 19.93 (s)ESI-MS

(neg. m/z)346

(90%, [M-Na+]–)346

(90%, [M-Na+]–)388

(100%, [M-Na+]–)388

(100%, [M-Na+]–)430

(5%, [M-Na+]–)328

(10%, [M-Na+]–)HRMS

(neg. m/z)Calcd. 346.0558 346.0558 388.0663 388.0663 430.0769 328.0452Found 346.0555 346.0555 388.0669 388.0669 430.0768 328.0443

36

Table S10: Characterisation of products from the reaction described in Table 1, entry 13.

U2P U2P,3OAc U>P

/ppm; multiplicity,

J/Hz

H-(C6) 7.75(d, J = 8.1)

7.77(d, J = 8.1)

7.64(d, J = 8.1)

H-(C5) 5.81(d, J = 8.1) n.a. 5.78

(d, J = 8.1)

H-(C1) 5.90(d, J = 5.3)

5.97(d, J = 6.3) n.a.

H-(C2) 4.61(dt, J = 7.6, 5.5)

4.79 (obs. HOD)

5.09(td, J = 6.8, 2.8)

H-(C3) 4.26(t, J = 5.0)

5.22(dd, J = 5.8, 3.8)

4.97(obs. HOD)

H-(C4) 4.04(dd, J = 7.8, 4.3)

4.19(q, J = 3.5)

4.19-4.23 (obs)

H-(C5) 3.78 (ABX, J = 12.6, 3.0)

3.7-3.78 (obs.)

3.71-3.78 (obs.)

H-(C5)

3.70 (ABX, J = 12.6, 4.5)

3.71-3.78 (obs.)

3.71-3.78 (obs.)

P 2.24(d, J = 5.9)

1.12(d, J = 7.8) n.a.

P (dec.) 2.24 (s) 1.12 (s) 20.03 (s)ESI-MS

(neg. m/z)323

(100%, [M–Na+]–) n.a. 305 (5%, [M–Na+]–)

HRMS(neg. m/z)

Calcd. 323.0286 n.a. 305.0180Found 323.0282 n.a. 305.0206

37


U3P U3P,2OAc U>p

/ppm; multiplicity,

J/Hz

H-(C6) 7.82(d, J = 8.1)

7.77(d, J = 8.1)

7.65(d, J = 8.1)

H-(C5) 5.82(d, J = 8.1)

5.82(obs.)

5.79(d, J = 8.1)

H-(C1) 5.86(d, J = 4.5)

5.94(d, J = 4.3)

5.75-5.91(obs.)

H-(C2) 4.28 - 4.37(t, J = 4.8)

5.33(dd, J = 5.9, 4.3)

5.10(td, J = 6.9, 2.8)

H-(C3) 4.44(dt, J = 7.9, 5.4)

4.66(dt, J = 9.1, 5.9)

4.87(ddd, J = 12.1, 7.1, 5.5)

H-(C4) 4.17(td, J = 4.5, 3.5)

4.16-4.23 (obs.)

4.21-4.26 (m)

H-(C5) 3.83(ABX, J = 12.9, 3.0)

3.86(ABX, J = 12.4, 2.8) n.a.

H-(C5)

3.72(ABX, J = 13.1, 4.0)

3.76-3.82 (m) n.a.

P 2.95(d, J = 5.9)

1.73(d. J = 7.8) n.a.

P (dec.) 2.95 (s) 1.73 (s) 20.04ESI-MS

(neg. m/z)323

(100%, [M–Na+]–)365

(10%, [M–Na+]–)305

(5%, [M–Na+]–)HRMS

(neg. m/z)Calcd. 323.0286 365.0392 305.0180Found 323.0288 365.0381 305.0208

N.B. A diacetylated species was detected by ESI and ESI-HRMS (ESI-MS (neg. m/z): 430 (2%, [M-Na+]–); ESI-HRMS (neg. m/z): [M-Na+]– calculated for C13H16N2O11P1,

407.0497, found 407.0486), but could not be detected by NMR spectroscopy or further characterised.

38


U2P U3P U2P,3OAc U3P,2OAc U2P(OAc),3OAc

U3P(OAc),2OAc U>p

/ppm; multiplicity,

J/Hz

H-(C6)7.78

(d, J = 8.1)

7.83(d, J = 8.1)

7.74-7.80(obs.)

7.77(d, J = 8.1) n.a. n.a. 7.66

(d, J = 8.1)

H-(C5)5.83

(d, J = 8.1)

5.83(d, J = 8.1)

5.81-5.83(obs.)

5.83(d, J = 8.1) n.a. n.a. 5.80

(d, J = 8.1)

H-(C1)5.91-5.97(obs.)

5.88(d, J = 4.5)

5.88(d, J = 4.5)

5.90-6.02(obs.) n.a. 6.02

(d, J = 5.5)5.82-5.87

(obs.)

H-(C2)4.57-4.69(m)

4.35(t, J = 4.9)

4.78-4.82(obs. HOD)

5.35(t, J = 5.0) n.a. 5.35-5.42

(obs)5.11

(td, J = 6.9, 2.9)

H-(C3)4.29(t, J = 4.9)

4.41-4.50 (obs.)

5.25(dd, J = 5.7, 3.9)

4.54-4.75(obs.) n.a. n.a. 4.77-4.82

(obs. HOD)

H-(C4)4.07

(q, J = 3.8)

4.15-4.26 (obs.)

4.11-4.13(obs.)

4.14-4.36(obs.) n.a. n.a. 4.13-4.16

(obs.)

H-(C5)3.64-3.91(obs.)

3.64-3.91 (obs.)

3.64-3.91(obs.)

3.64-3.91(obs.) n.a. n.a. 3.64-3.91

(obs.)

H-(C5)

3.64-3.91(obs.)

3.64-3.91 (obs.)

3.64-3.91(obs.)

3.64-3.91(obs.) n.a. n.a. 3.64-3.91

(obs.)

P0.42

(d, J = 7.8)

1.77(d, J = 8)

0.06(d, J = 9.8)

1.40(d, J = 5.9) n.a. -8.92

(d, J = 9.8)20.04

(dd, J = 11.7, 5.9)

P (dec.) 0.42 (s) 1.77 (s) 0.06 (s) 1.40 (s) –9.12 (s) –8.92 (s) 20.04 (s)

39


G2P G2P,3OAc G2P,3OAc,5-OAc

G2P(OAc),3OAc G>p

/ppm; multiplicity,

J/Hz

H-(C8) n.a. n.a. n.a. n.a. n.a.

H-(C1) 5.94(d, J = 5.3)

6.00(d, J = 5.8)

6.00 (obs.) n.a. 6.06

(d, J = 3.3)

H-(C2) 4.94-5.15(app. q, J = 5.5)

5.25(dd, J = 13.4,

6.3)5.25

(obs.) n.a.5.37

(ddd, J = 8.7, 6.7, 3.8)

H-(C3) 4.50(app. t, J = 4.0)

5.43(m)

5.46-5.49 (part. obs.) n.a. 5.06-5.15

(m)H-(C4) 4.18

(app. d, J = 3.3)4.25-4.34

(obs.) n.a. n.a. 4.34-4.42(obs.)

H-(C5) 3.63-3.97(obs.)

3.63-3.97(obs.) n.a. n.a. 3.63-3.97

(obs.)H-(C5) 3.63-3.97

(obs.)3.63-3.97

(obs.) n.a. n.a. 3.63-3.97(obs.)

P 2.89(br. s)

1.78 (br. s) n.a. –9.34

(d, J = 7.8 Hz)19.98

(t, J = 7.8)P (dec.) 2.89

(br. s)1.78

(br. s) n.a. –9.34 (s) 19.98(br. s)

41


G3P G3P,2OAc

/ppm; multiplicity,

J/Hz

H-(C8) 7.96 (s) 7.93 (s)

H-(C1) 5.89 (d, J = 6.1)

6.08 (d, J = 5.6)

H-(C2) 4.67-4.70 (obs. HOD)

5.65 (t, J = 5.7)

H-(C3) 4.56-4.61 (obs HOD)

4.94-5.01 (obs.)

H-(C4) 4.32-4.39 (m)

4.29-4.34 (obs.)

H-(C5) 3.76-3.94 (obs.)

3.76-3.93 (obs.)

H-(C5)

3.76-3.94 (obs.)

3.76-3.93 (obs.)

P 0.88 (d, J = 7.8)

–0.19(d, J = 9.8)

P (dec.) 0.88 (s) –0.19 (s)

42


G2P G3P G2P,3OAc G3P,2OAc G>P

/ppm; multiplicity,

J/Hz

H-(C8) 7.89 (s) 7.91 (s) n.a. 7.88 (s) 7.84 (s)

H-(C1) 5.90(d, J = 5.3)

5.82 (d, J = 5.4)

5.97 (d, J = 6.6)

5.99 (d, J = 4.5)

6.02 (d, J = 3.5)

H-(C2) 5.00(dt, J = 7.4, 5.4)

4.68 (t, J = 5.4)

5.40 (dd, J = 6.5, 3.0)

5.58 (dd, J = 5.5, 4.5)

5.33 (ddd, J = 8.6, 6.8, 3.8)

H-(C3) 4.47(dd, J = 5.0, 4.3)

5.00 (dt, J = 7.4, 5.4)

5.22 (dt, J = 8.8, 6.6)

4.91 (dt, J = 8.8, 5.5)

5.06 (ddd, J = 9.5, 6.9, 4.5)

H-(C4) 4.09-4.18 (m)

4.21-4.34 (m) n.a. 4.15

(dd, J = 7.1, 4.0)4.30-4.36

(m)

H-(C5) 3.75-3.92 (m)

3.75-3.92 (m)

3.75-3.92 (m)

3.75-3.92 (m)

3.75-3.92 (m)

H-(C5)

3.70(ABX, J = 12.8, 4.0)

3.75-3.92 (m)

3.75-3.92 (m)

3.75-3.92 (m)

3.75-3.92 (m)

P 1.59(d, J = 7.8)

1.96 (d, J = 7.8)

0.19(d, J = 9.8)

0.58(d, J = 7.8)

19.97(t, J = 9.8)

P (dec.) 1.59 (s) 1.96 (s) 0.19 (s) 0.58 (s) 19.97 (s)ESI-MS

(neg. m/z)384

(20%, [M–Na+]–)384

(20%, [M–Na+]–)404

(25%, [M–Na+]–)404

(25%, [M–Na+]–)344

(5%, [M–Na+]–)HRMS

(neg. m/z)Calcd. 384.0327 384.0327 404.0613 404.0613 -Found 384.0315 384.0315 404.0611 404.0611 -

N.B. A diacetylated species was detected by ESI and ESI-HRMS (ESI-MS (neg. m/z): 446 (2%, [M-Na+]–); ESI-HRMS (neg. m/z): [M-Na+]– calculated for C14H17N5O10P1,

446.0719, found 446.0740), but could not be detected by NMR spectroscopy or further characterised.

43


I3P I3P,2OAc

/ppm; multiplicity,

J/Hz

H-(C8) 8.30 (s) 8.29 (s)H-(C2) 8.16 (s) 8.16 (s)H-(C1) 6.06

(d, J = 6.1)6.25

(d, J = 5.3)H-(C2) 4.70

(obs. HOD)5.64

(t, J = 5.4)H-(C3) 4.63-4.69

(obs. HOD)4.89-4.98

(m)H-(C4) 4.40

(q, J = 3.8)4.36

(q, J = 3.4)H-(C5) 3.78-3.85

(m)3.85-3.92

(m)H-

(C5)3.78-3.85

(m)3.85-3.92

(m)P 2.12

(d, J = 7.8)0.60

(d, J = 9.8)P (dec.) 2.12 (s) 0.60 (s)

ESI-MS(neg. m/z)

347 (25%, [M–Na+]–)

389 (100%, [M–Na+]–)

HRMS(neg. m/z)

Calcd. 347.0398 389.0503Found 347.0396 389.0491

N.B. A diacetylated species was detected by ESI MS (neg. m/z): 431 (10%, [M-Na+]–), but could not be detected by NMR spectroscopy or further characterised.

44


C3P C3P,2OAc A3P A3P,2OAc

/ppm; multiplicity,

J/Hz

H-(C1) 5.85(d, J = 4.3)

5.90(d, J = 3.8)

6.00(d, J = 6.3)

6.18 (d, J = 5.5)

H-(C2) 4.26-4.32 (t, J = 4.7)

5.31 (dd, J = 5.7, 3.9)

4.74-4.76(obs. HOD)

5.59 (t, J = 5.3)

H-(C3) 4.39-4.45 (obs.)

4.58-4.62 (obs.)

4.64(ddd, J = 7.8, 5.0,

2.9)

4.79-4.88 (obs. HOD)

H-(C4) 4.12-4.17 (obs.)

4.17-4.21 (obs.)

4.33-4.39 (obs.)

4.39-4.44 (obs.)

H-(C5) 3.73-3.98 (obs.)

3.73-3.98 (obs.)

3.73-3.98 (obs.)

3.73-3.98 (obs.)

H-(C5) 3.73-3.98 (obs.)

3.73-3.98 (obs.)

3.73-3.98 (obs.)

3.73-3.98 (obs.)

P 3.42 (d, J = 7.8)

2.61(d, J = 9.8)

3.59(d, J = 7.8)

2.48 (d, J = 7.8)

P (dec.) 3.42 (s) 2.61 (s) 3.59 (s) 2.48 (s)

45


U3P U3P,2OAc A3p A3P,2OAc

/ppm; multiplicity,

J/Hz

H-(C1) 5.84(d, J = 4.5)

5.92(d, J = 4.3)

5.99(d, J = 6.3)

6.19(d, J = 5.8)

H-(C2) 4.33(t, J = 4.9)

5.32(t, J = 4.9)

4.69-4.73(obs. HOD)

5.60(t, J = 5.5)

H-(C3) 4.18-4.41(obs.)

4.58-4.62(obs. HOD)

4.62-4.65(obs. HOD)

4.80-4.95 (obs. HOD)

H-(C4) 4.11-4.15(obs.)

4.14-4.19(obs.)

4.32-4.35(obs.)

4.35-4.54 (obs.)

H-(C5) 3.48-4.01(obs.)

3.48-4.01(obs.)

3.48-4.01(obs.)

3.48-4.01 (obs.)

H-(C5) 3.48-4.01(obs.)

3.48-4.01(obs.)

3.48-4.01(obs.)

3.48-4.01 (obs.)

P 2.49(d, J = 7.8)

1.12(d, J = 9.8)

2.49(d, J = 7.8)

0.94(d, J = 9.8)

P (dec.) 2.49 (s) 1.13 (s) 2.49 (s) 0.95 (s)ESI-MS

(neg. m/z)323

(70%, [M–Na+]–)365

(45%, [M–Na+]–)346

(40%, [M–Na+]–)388

(85%, [M–Na+]–

HRMS(neg. m/z)

Calcd. 323.0285 365.0391 346.0558 388.0663Found 323.0276 365.0401 346.0541 388.0635

N.B. Trace signals observed:

A>P, U>P 31P (dec): 20.04, 19.93 ppm (A>P, U>P).

A3P(OAc),2OAc, U3P(OAc),2OAc31P (dec): –8.82, –8.93 ppm. ESI-MS (neg. m/z): 407 (20%, [U3P(OAc),2OAc–Na+]–). ESI-HRMS (neg. m/z): [M–Na+]–

calculated for C13H16N2O11P1, 407.0497, found 407.0529.ESI-MS (neg. m/z): 430 (40%, [A3P(OAc),2OAc–Na+]–). ESI-HRMS (neg. m/z): [M–Na+]–

calculated for C14H17N5O9P1, 430.0769, found 430.0768.

46


I3P I3P,2OAc A3P A3P,2OAc

/ppm; multiplicity,

J/Hz

H-(C1) 6.01(d, J = 5.8 )

6.19(d, J = 5.5)

5.97(d, J = 6.3)

6.17(d, J = 6.1)

H-(C2) 4.69-4.73(obs. HOD)

5.58(t, J = 5.5 Hz)

4.69-4.73(obs. HOD)

5.58(t, J = 5.5)

H-(C3) 4.57-4.69(obs.)

4.83-4.94(obs.)

4.57-4.69(obs.)

4.82-4.93(obs.)

H-(C4) 4.20-4.49(obs.)

4.30-4.45(obs.)

4.20-4.49(obs.)

4.26-4.46 (obs.)

H-(C5) 3.72-3.93(obs.)

3.73-3.95 (obs.)

3.72-3.93(obs.)

3.73-3.95 (obs.)

H-(C5) 3.72-3.93(obs.)

3.73-3.95 (obs.)

3.72-3.93(obs.)

3.73-3.95 (obs.)

P - - - -

P (dec.) 2.37 (s) 0.88 (s) 2.31 (s) 0.78 (s)ESI-MS

(neg. m/z)347

(60%, [M–Na+]–)389

(100%, [M–Na+]–)346

(100%, [M–Na+]–)388

(95%, [M–Na+]–)HRMS

(neg. m/z)Calcd. 347.0398 389.0503 346.0558 388.0663Found 347.0428 389.0530 346.0540 388.0638

N.B. Trace signals observed:

A>P, I>P 31P (dec): 19.96, 19.93 ppm.

A3P(OAc),2OAc, I3P(OAc),2OAc31P (dec): –8.82, –8.86 ppm. ESI-MS (neg. m/z): 431 (2%, [I3P(OAc),2OAc–Na+]–). ESI-HRMS (neg. m/z): [M-Na+]–

calculated for C14H16N4O10P1, 431.0609, found 431.0603.ESI-MS (neg. m/z): 430 (2%, [A3P(OAc),2OAc–Na+]–). ESI-HRMS (neg. m/z): [M-Na+]–

calculated for C14H17N5O9P1, 430.0769, found 430.0767.

47


A3P G3P A3P,2OAc G3P,2OAc A>p G>P

/ppm; multiplicity,

J/Hz


H-(C1) 6.00(d, J = 6.6)

5.83(d, J = 6.1)

6.20(d, J = 6.1)

6.02(d, J = 5.3)

6.16(d, J = 4.3) n.a.

H-(C2) 4.72-4.74(obs. HOD)

4.64-4.71(m)

5.61(t, J = 5.8)

5.57-5.61(obs.)

5.34(ddd, J = 10.5, 6.7, 4.4) n.a.

H-(C3) 4.65-4.71(m)

4.64-4.71(m)

4.90(ddd, J = 8.9, 5.4, 3.7) n.a. 5.07

(dt, J = 10.8, 4.0) n.a.

H-(C4) 4.39(q, J = 2.9)

4.31(q, J = 3.3)

4.43(q, J = 3.0) n.a. 4.39

(obs.) n.a.

H-(C5) 3.74-3.94(obs.)

3.74-3.94(obs.)

3.74-3.94(obs.) n.a. 3.73-3.94

(obs.) n.a.

H-(C5) 3.74-3.94(obs.)

3.74-3.94(obs.)

3.74-3.94(obs.) n.a. 3.73-3.94

(obs.) n.a.

P - - - - - -P (dec.) 1.78 (br. s) 1.78 (br. s) 0.36 (s) 0.39 (br. s) 19.93 (s) 19.99 (s)

ESI-MS(neg. m/z)

346(100%, [M–Na+]–)

362(35%, [M–Na+]–)

388(30%, [M–Na+]–)

404(5%, [M–Na+]–)

328(5%, [M–Na+]–)

344(2%, [M–Na+]–)

HRMS(neg. m/z)

Calcd. 346.0558 362.0507 388.0663 404.0612 - -Found 346.0573 362.0507 388.0638 404.0585 - -

48


CC3P CC3P,2OAc CC3P2OAc,5-OAc

/ppm; multiplicity,

J/Hz

H-(C6) 7.85(d, J = 7.6)

7.76 (d, J = 7.8)

7.71 (d, J = 7.8)

H-(C6) 7.82 (d, J = 7.6)

7.63 (d, J = 7.6)

7.50 (d, J = 7.6)

H-(C5) 5.88-5.90 (obs.)

5.96 (d, J = 7.6)

5.94-5.98 (obs.)

H-(C5) 5.88-5.90 (obs.)

5.91 (d, J = 7.8)

5.91-5.94 (obs.)

H-(C1C1)5.92

(d, J = 2.3)5.88 - 5.90

(obs.)5.88-5.90

(obs.)

H-(C1 C2)5.73

(d, J = 2.3)5.78

(d, J = 4.0)5.78

(d, J = 4.0)H-(C2 C1) n.a. n.a. n.a.

H-(C2 C2) n.a. 5.23 (dd, J = 5.8, 4.0)

5.23 (dd, J = 5.8, 4.0)

H-(C3 C1) n.a. n.a. n.a.

H-(C3 C2) n.a. 4.69-4.71 (obs. HOD)

4.69-4.71 (obs. HOD)

P n.a. n.a. n.a.

P (dec.) 2.10 (obs. s), –0.80 (s)

2.10 (obs. s), –0.96 (s)

2.10 (obs. s), –1.08 (obs. s)

ESI-MS(neg. m/z)

313 (100%, [M–2Na+]2–)

334 (75%, [M–2Na+]2–)

355 (10%, [M–2Na+]2–)

49


CC2P CC3P CC3P,2OAc CC>P

/ppm; multiplicity,

J/Hz

H-(C6) n.a. n.a. n.a. n.a.H-(C6) n.a. n.a. n.a. n.a.H-(C5) n.a. n.a. n.a. n.a.H-(C5) n.a. n.a. n.a. n.a.

H-(C1 C1) n.a. n.a. n.a. n.a.

H-(C1 C2)5.77

(d, J = 3.3)5.75

(d, J = 2.5)6.01 - 6.03

(obs.) n.a.

H-(C2 C1) n.a. n.a. n.a. n.a.

H-(C2 C2) n.a. n.a. 5.27(m) n.a.

H-(C3 C1) n.a. n.a. n.a. n.a.H-(C3 C2) n.a. n.a. n.a. n.a.

P n.a. n.a. n.a. n.a.

P (dec.) 2.32-3.13 (obs.), –0.75 (obs.)

2.32-3.13 (obs.), –0.75 (obs.)

2.32-3.13 (obs.),

–0.75 (obs.)

19.80 (s), 1.34 (br. s), 0.92 (br.

s)ESI-MS

(neg. m/z)

313(100%, [M–

2Na+]2–)

313(100%, [M–2Na+]2–)

334(60%, [M–2Na+]2–)

631(25%, [M–Na+]–)

HRMS(neg. m/z)

Calcd. - - 691.0784 -Found - - 691.0788 -

50

Figure S2: 1H-NMR spectrum of the products of the reaction described in Table

S1, entry 1.

51


S1, entry 3.

52


S1, entry 4.

53


S1, entry 6.

54


S1, entry 7.

55


S1, entry 10.

56


S1, entry 14.

57


S1, entry 18.

58


S1, entry 24.

59

Figure S11: 1H-NMR spectrum of CC3'P 22, the starting material of the reaction

described in Table S1, entry 24.

General procedure for the reaction of nucleoside phosphates with methyl

isocyanide 7 and sodium thioacetate 3

60


pD was adjusted to the value stated (Table S23) with NaOD (1.0 M). Methyl

isocyanide 7 was added and the pD was readjusted with DCl/NaOD (1.0 M)

whilst stirring vigorously. The reactions were monitored by NMR spectroscopy

at r.t. The characterisation of acetylation products was by 1H, 1H-1H COSY and 31P-NMR spectroscopy, and yields are given in Table S23. Chemical shifts

corresponded to those found for species characterised from the reaction of

nucleoside phosphates with cyanoacetylene 4 and sodium thioacetate 3 (Table

S1) and are omitted to avoid repetition.

General procedure for the reaction of nucleoside phosphates with N-

cyanoimidazole (NCI) 8 and sodium thioacetate 3


pD was adjusted to 6 with NaOD (1.0 M). N-Cyanoimidazole 8 was added and

the pD was readjusted to 6 with DCl/NaOD (1.0 M) whilst stirring vigorously.

The reactions were monitored by NMR spectroscopy at r.t. for 24 h, yields are

given in Table S24 with characterisation as above and additionally in Table S25.

General procedure for the reaction of nucleoside phosphates with sodium

thioacetate 3 and potassium ferricyanide 10

Nucleoside phosphates (100 mM final concentration) were dissolved in D2O at

r.t. and the pD was adjusted to 6.5 with NaOD (1.0 M). The solution was

sparged with Ar (g) for 15 min, then potassium ferricyanide 10 (200 mM) was

added to give an orange solution, followed by potassium thioacetate (250 mM).

The reaction mixture was stirred vigorously at r.t. in the dark for 1 h, after which

time it had turned colourless and an off-white precipitate had formed. A pD of

5.4 was recorded, then readjusted to 6.5 with NaOD (1.0 M). The reactions were

monitored by NMR spectroscopy at r.t. after 5 h and 24 h, and yields are given

in Table S26 with characterisation as above.

General procedure for the reaction of nucleoside phosphates with N-

acetylimidazole (NAI) 9

Nucleoside phosphates (100 mM final concentration) were dissolved in D2O at

r.t. and the pD was adjusted to 6.5 with NaOD (1.0 M). N-acetylaimidazole 9

61

was added (200 mM) and reaction continued for 1 h. The pD was then readjusted

from ~ 7 to 6.5 with DCl (1.0 M). The reactions were monitored by NMR

spectroscopy at r.t. after 5 h and 24 h, and yields are given in Table S27 with

characterisation as above.

62

Table S23: Reactions of nucleotides with sodium thioacetate 3 and methyl isocyanide 7.

Entry Nucleotide(100 mM)

AcSNa/mM

CH3NC/mM

Time/h pD


N3P N2P N2P,3'OAc N3P,2'OAc N>PN3P,2'OAc,5'OAc

N3'P(OAc),2'OAc

N2'P(OAc),3'OAc

1 A3P 100 400 24 5 37 - - 18 42 - 2 -2 A3P 100 400 24 6 52 - - 40 6 - 3 -3 A3P 100 400 24 7 81 - - 18 2 - - -4 A3P 200 400 48 6 22 - - 47 15 3 13 -5 A2P 200 400 48 6 - 24 37 - 24 - - 166 C3P 200 400 48 6 27 - - 45 18 - 9 -7 A3(2)P (80:20) 200 400 48 6 9 6 8 43 14 - 16 48 G3(2)P (63:37) 200 400 48 6 13 18 20 20 21 - 4 4

Table S24: Reactions of nucleotides with sodium thioacetate 3 and N-cyanoimidazole 8.

Entry Nucleotide(100 mM)

AcSNa/mM

N-cyanoimidazole/

mMpD


N3P N2P N

2P,3'OAcN

3P,2'OAc N>PN3P,2'OAc,5'OAc

N2'P,3',OAc,5'OAc

N3'P(OAc),2'OAc

N2'P(OAc),3'OAc

N>P,5'OAc

1 A3'P 200 200 6 14 - - 53 14 8 - 8 - 32 A2'P 200 200 6 - 47 28 - 20 - 5 - - -3 A3'(2')P (80:20) 100 100 6 20 16 3 47 14 - - - - -4 G3'(2')P (63:37) 100 100 6 11 25 7 37 15 - - - - 25 U2'P 200 200 6 - 27 23 - 43 - - - 5 26 C3'(2')P (56:44) 200 200 6 8 17 12 29 23 6 - 2 - 37 AGA3'P 200 200 6 6 - - 64 25 5 - - - -8 AGA(3')2'P (66:34) 100 100 6 n.a. n.a. n.d. 17 27 - - - - -

Table S25: Characterisation of products from the reaction described in Table S24, entries 7 & 8.

63

AGA3P AGA3P,2OAc AGA3P,2OAc,5OAc AGA>P

/ppm; multiplicity,

J/Hz

H-(C1A1)5.72

(d, J = 5.0)5.79

(d, J = 5.3) n.a. 5.79-5.81(part. obs.)

H-(C1G) n.a. 5.57(d, J = 5.0) n.a. 5.59

(d, J = 5.6)

H-(C1A3)5.91

(d, J = 5.5)6.07

(d, J = 4.5)6.07-6.09

(part. obs.)6.04

(d, J = 4.0)H-(C2A1) n.a. n.a. n.a. n.a.H-(C2G) n.a. n.a. n.a. n.a.

H-(C2A3) n.a. 5.44(t, J = 5.2)

5.50(t, J = 5.3)

5.21(ddd, J = 9.7, 6.9, 4.3)

H-(C3A1) n.a. n.a. n.a. n.a.H-(C3G) n.a. n.a. n.a. n.a.

H-(C3A3) n.a. 4.89(dt, J = 8.8, 5.3)

4.93-4.98(m)

5.03(ddd, J = 8.6, 7.1, 4.8)

H-(C4A1) n.a. n.a. n.a. n.a.H-(C4G) n.a. n.a. n.a. n.a.

H-(C4A3) n.a. 4.48(br. s.) n.a. 4.48 (br. s.)

ESI-MS(neg. m/z) - 530

(50%, [M–2Na+]2–)563

(40%, [M–2Na+]2–)501

(100%, [M–2Na+]2–)HRMS

(neg. m/z)Calcd. - 530.5795 562.5785 500.5690Found - 530.5787 562.5751 500.5685

64

Table S26: Reactions of nucleotides with sodium thioacetate 3 and potassium ferricyanide 10.

Entry Nucleotide (100 mM)

Reactiontime (h)



N3'P,2'OAc N>p

N3'P,2'OAc,5'OAc

N2'P,3'OAc,5'OAc

N3'P, 5'OAc

N2'P, 5'OAc

N3'P(OAc), 2'OAc

N2'P(OAc),3'OAc

N3'P(OAc), 2'OH

N2'P(OAc), 3'OH

1 A3'P 5 41 - - 42 10 2 - tr. - 4 - 1 -2 A2'P 5 - 62 11 - 7 - - - 1 - 2 - 16

3 A3'(2’)P (67:33) 5 25

(37)22

(68)4

(11)29

(43) 9 1(2) - tr. tr. 3

(4) tr. 1(1)

4(14)

4 A3'P 24 42 - - 42 10 1 - 1 - 3 - - -5 A2'P 24 - 62 16 - 14 - 1 - 2 - 1 - 2

6 A3'(2’)P (67:33) 24 25

(37)23

(71)5

(15)29

(43) 12 1(2) - tr. tr. 2

(3) tr. - tr.

Table S27: Reactions of nucleotides with N-acetylimidazole (NAI) 9.

Entry Nucleotide (100 mM) Reactiontime (h)



N3'P,2'OAc N>p N>p,

5’OAc

N3'P,2'OAc,5'OAc

N2'P,3'OAc,5'OAc

N3'P, 5'OAc

N2'P, 5'OAc N3'P(OAc), 2'OAc N2'P(OAc),

3'OAc

1 A3'P 5 4 - - 83 4 tr. 6 - - - tr. -2 A2'P 5 - 60 23 - 12 1 - 2 - 2 - -

3 A3'(2’)P (67:33) 5 2(3)

23(70)

6(18)

57 (85) 6 1 5

(7) - - tr. - -

4 A3'P 24 14 - - 73 4 tr. 6 - tr. - - -5 A2'P 24 - 61 23 - 12 1 - 1 - 2 - -6 A3'(2’)P (67:33) 24 9 22 6 48 6 tr. 5 - tr. tr. - -

N.B. Representative 31P chemical shifts (ppm) assigned to low level products are as follows: A>P,5OAc, 19.46; A2′P,3′OAc,5′OAc, -0.40; A3′P,5′OAc, 1.15; A2′P,5′OAc, 0.41;

A3′P(OAc),2′OAc, -8.73; A2′P(OAc),3′OAc, -9.29; A3′P(OAc),2′OH, -8.19; A2′P(OAc),3′OH, -8.81.

65

Figure S12: Potential mechanism for the acetylation of A3’P by thioacetate 3

and cyanoacetylene 4 in D2O.

Reactions of methyl phosphate di-sodium salt 25 with cyanoacetylene 4 and

sodium thioacetate 3

The reaction of methyl phosphate 25 with cyanoacetylene 4 in the presence and

absence of thioacetate 3 was undertaken to aid with the identification and

quantification of the change in chemical shifts that may arise from the

cyanovinylation, or acetylation of a phosphate group.

Methyl phosphate di-sodium salt 25 (16 mg, 0.1 mmol) and thioacetic acid (7.5

mg, 0.1 mmol) were dissolved in D2O (0.8 mL) and the pD was adjusted to 6.5

using DCl/NaOD (1.0 M). Cyanoacetylene 4 (1.0 M, 0.2 mL) was added and the

formation of a white precipitate was observed. The pD was readjusted from 8 to

6.5 using DCl (1.0 M) and the reaction was monitored by 1H- and 31P-NMR

spectroscopy at r.t. The formation of acetyl methyl phosphate 28 was observed

and monitored over 8 d. The ratio of methyl phosphate 25 to acetyl methyl

phosphate 28 remained constant from day 2 to 8 at r.t. (Table S28). No

cyanovinylated mono-methyl phosphate was observed.

Time/dMethyl

phosphate 25 (%)

Acetyl methyl phosphate 28

(%)0 95 51 85 152 80 208 80 20

Table S28. Reaction of methyl phosphate 25 with thioacetate 3 and

cyanoacetylene 4.

66

A control reaction of methyl phosphate 25with cyanoacetylene 4, in the absence

of thioacetate 3, was carried out and monitored for 52 d at r.t. Cyanovinylation

to give up to 72 % of dideutero-cyanovinyl methyl phosphate 29 was observed

(Table S29).

Time (d) Methyl phosphate 25 (%)Dideutero-cyanovinyl

methyl phosphate D2-29 (%)

1 96 42 91 83 87 136 80 2052 28 72

Table S29. Control reaction of methyl phosphate 25 with cyanoacetylene 4.

Data for acetyl methyl phosphate sodium salt 28

1H-NMR (400 MHz, D2O) 3.58 (d, J = 11.3 Hz, 3H, POCH3), 2.10

(d, J = 1.3 Hz, 3H, CH3CO). 31P-NMR (162 MHz, D2O, 1H-decoupled) –6.08

(s). 31P-NMR (162 MHz, D2O) –6.08 (app. d, J = 11.7 Hz). ESI-MS (neg. m/z):

153 (10%, [M–Na+]–). TOF-ESI-HRMS (neg. m/z): [M–Na+]– calculated for

C3H6O5P 152.9958; found 152.9947.

Data for dideutero-cyanovinyl methyl phosphate sodium salt D2-29

1H-NMR (400 MHz, D2O) 3.59 (d, J = 11.1 Hz, 3H, POCH3). 31P-NMR (162

MHz, D2O, 1H-decoupled) –2.65 (s). 31P-NMR (162 MHz, D2O) –2.65 (q, J =

10.8 Hz). ESI-MS (neg. m/z): 164 (20%, [M–Na+]–). TOF-ESI-HRMS (neg.

m/z): [M–Na+]–) calculated for C4H3D2NO4P1 164.0118; found 164.0121.

67

Figure S13: Highly chemoselective acetylation of the 2'-terminal hydroxyl group

of a trinucleotide in aqueous medium, using thioacetate 3 and N-cyanoimidazole

8. Ade, N9-linked adenine; Gua, N9-linked guanine.

Acetylation-ligation reactions and deacetylation

RNA oligomers

Unless otherwise stated, oligomers were purchased in HPLC-purified Na+ form

from Integrated DNA Technologies.

Additional non-acetylated oligomers (Table S30, entry 1-5) were synthesised by

solid-phase synthesis using a BioAutomation MerMade4 or MerMade6

automated synthesiser and standard RNA phosphoramidites and reagents from

ChemGenes or Link Technologies. 2'-5'-linkages and terminal 2'-phosphates

were introduced using 3'-TBDMS phosphoramidites from ChemGenes.

Oligomers bearing terminal (2'- or 3'-) phosphates were synthesised on 3'-

phosphate SynBase CPG from Link Technologies. Oligomers were purified by

SAX-HPLC (flow rate 15 mLmin-1; detection 260 nm; gradient 0 to 1 M NaCl

in 10 mM pH 11.5 sodium phosphate buffer over 30 min, then isocratic at 1 M

NaCl for 5 min; target fractions collected directly into an equal volume of 1 M

TEAA pH 7 buffer), then desalted (Waters Sep-Pak® C18 12 cc Vac Cartridge;

cartridge pre-washed with water (10 mL) then MeOH (10 mL) before sample

68

loading, then washed with 50 mM TEAA pH 7 aqueous buffer (12 mL) before

sample elution with 30 % (v/v) MeOH in 50 mM TEAA pH 7 aqueous buffer (5

× 2 mL fractions)), lyophilised, redissolved in water and re-lyophilised (to

remove traces of TEAA). Oligomers employed in ligation reactions were

additionally converted to the Na+ form using pre-washed AG® 50W-X8 resin; an

excess (100 mg) of resin was added to an aqueous solution (1 mL) of the RNA

oligomer and the mixture agitated for a minimum of 4 h, before the resin was

removed by filtration (washing with 2 × 250 L water) and the treatment

repeated with fresh resin. Oligomer quantification was by UV absorbance at

260 nm, and characterisation by MALDI-TOF MS.

Acetylated RNA standards (Table S30, entry 6-8) were synthesised by solid-

phase synthesis on a BioAutomation MerMade4 using a protocol and set of

phosphoramidites newly developed in our laboratory (CKWC, CDC, JX and

JDS, manuscript in preparation). 17 nt standards (Table S30, entry 6-7) were

additionally purified by SAX-HPLC (flow rate 15 mLmin-1, detection 280 nm,

gradient 0 to 0.4 M NaCl in 10 mM pH 8.0 Tris aqueous buffer with 25 % (v/v)

formamide over 40 min, then isocratic at 0.4 M NaCl 5 min), and desalted by

dialysis (Thermo Scientific Slide-A-Lyzer Dialysis Cassettes, 2K MWCO)

against 10 mM TEAA buffer pH 7 at 4 °C (Thermo Scientific Slide-A-Lyzer

Dialysis Cassettes, 2K MWCO).

Entry RNA Sequence (5'-3')Average Mass for

[M+H] + (Da)Observed Calculated

1 UGUGCCAGUA-2'p 3241.85 3241.942 GCCAGUA-2'p 2282.97 2284.403 GCCAGUA-3'p 2283.53 2284.404 UGUGCCAGUA-2',5'-GGUUCUC 5378.58 5381.265 UGUGCCAGUA-3',5'-GGUUCUC 5378.89 5381.266 UGUGCCAGUA-2',5'(3'OAc)-GGUUCUC 5424.00 5423.247 UGUGCCAGUA-3',5'(2'OAc)-GGUUCUC 5422.51 5423.248 GCAGUA-3',5'(2'OAc)-GGUUCUC 4160.56 4160.52

Table S30: Synthesised RNA oligomers with MALDI-TOF MS characterisation

data.

69

Procedure for acetylation-ligation (Fig. 3b)

A mixture (L) of 80 M primer and 50 mM N-acetylimidazole 9 was

incubated at 21 °C for 5 h. A templated ligation reaction (10 L) was then

conducted using 4 M of acetylated primer from this mixture, 25 M template,

30 M ligator, 200 mM imidazole nitrate buffer (pH 6.2), 10 mM MnCl2, and

100 mM N-cyanoimidazole 8 (added last as a freshly prepared 1 M aqueous

solution). Unused acetylation mixture was analysed by MALDI-TOF MS (Fig.

S14). The ligation reaction mixture was incubated at 21 °C for 19 h, then diluted

with an equal volume of Ambion Gel Loading Buffer II (95 % formamide, 18

mM EDTA, 0.025 % SDS, with xylene cyanol and bromophenol blue tracking

dyes) and heated at 95 °C for 4 min before cooling briefly on ice and loading

onto an 8 M urea denaturing PAGE gel. Control reactions were performed as

above but without the indicated reaction component, keeping all other

concentrations constant, with the exception of the ‘no N-acetylimidazole 9’

control; this was conducted using 4 M of non-acetylated primer, and with 2.5

mM of hydrolyzed NAI 9 (from an aqueous solution (L) of 50 mM NAI 9 that

had been left at 21 °C for 5 h). The ladder was a miRNA marker from New

England BioLabs, and the gel was stained using SYBR® Gold and imaged by

UV transillumination.

Ligation product was extracted from an excised gel band as follows: the excised

band was frozen at -82 °C, crushed, diluted with 50 mM TEAA pH 7 buffer

(800 L), re-frozen at -82 °C, then defrosted and heated at 90 °C for 5 min. The

resulting mixture was agitated for 3 h (on a Stuart SB3 rotator) before the gel

was removed by filtration. The filtrate was desalted using a Waters Sep-Pak®

C18 Plus Short Cartridge (cartridge pre-washed with water (10 mL) then MeOH

(10 mL) before sample loading, then washed with 50 mM TEAA pH 7 aqueous

buffer (3 mL) before sample elution with 30 % (v/v) MeOH in 50 mM TEAA

pH 7 aqueous buffer (5 mL)). The eluent was then lyophilised and redissolved in

water (10 L) and analysed by MALDI-TOF MS.

70

Figure S14: MALDI-TOF mass spectrum of the 10 nt 3'P primer following

acetylation. Peaks attributed to >P (numerical mass in black), 3'-P,2'-OH

starting material (blue), mono- (green) and bisacetylated (red) primer and their

adducts are labelled. See Table S31 for calculated masses.

RNA Species Calculated Average Mass (Da)[M+H]+ [M+NH4]+ [M+Na]+

10 nt 2',3'>P 3223.93 3240.96 3245.9110 nt 3'P/2'P, 2'OH/3'OH 3241.94 3258.97 3263.92

Monoacetylated 10 nt 3'P/2'P, 2'OH/3'OH

3283.98 3301.01 3305.96

Bisacetylated 10 nt 3'P/2'P, 2'OH/3'OH

3326.02 3343.05 3348.00

7 nt 2',3'>P 2266.38 2283.41 2288.367 nt 3'P/2'P, 2'OH/3'OH 2284.40 2301.43 2306.38

Monoacetylated 7 nt 3'P/2'P, 2'OH/3'OH

2326.44 2343.47 2348.42

Bisacetylated 7 nt 3'P/2'P, 2'OH/3'OH

2368.48 2385.51 2390.46

Table S31: Calculated masses for 7 nt and 10 nt primers and their derivatives.

71

Procedure for acetylation-ligation using dye-labelled primer (Fig. 4a)

Templated ligations and gel analysis were performed as described above (for

Fig. 3b), but using 5'-(6FAM)-labelled primer and without gel (SYBR® Gold)

staining. Control reactions were performed without the indicated reaction

component, keeping all other concentrations constant. The gel was imaged by

fluorescence scanning. For accurate quantification, the reaction was repeated

× 6, and 5% of the quenched reaction mixture (containing 2 pmoles of FAM-

labelled RNA) was loaded for gel analysis as above. For quantification, tracker-

dye-free gel loading buffer (95 % formamide, 18 mM EDTA, 0.025 % SDS) was

used for reaction quenching and gel analysis. Control reactions were performed

in triplicate and quantified in the same way.

Procedure for time-course analysis of acetylation-ligation using dye-labelled

primer and acetylation of total RNA (Fig. 4c and d)

A mixture ( 2 L) of 6.2 M dye-labelled primer, 38.5 M template, 46.2 M

ligator and 50 mM N-acetylimidazole 9 was incubated at 21 °C for 5 h. This

acetylation mixture was then brought to a volume of 72L with additional

ligation reaction components, namely imidazole nitrate buffer (pH 6.2; 16 L of

a 1 M solution) and MnCl2 (4 L of a 200 mM solution). To obtain a ‘0 h’ time

point, a 7.2L aliquot was removed, diluted with water (0.8L) and gel

loading buffer (24L; 95 % formamide, 18 mM EDTA, 0.025 % SDS), and

immediately frozen in N2 (l) and stored at -82 °C. To the remaining reaction

mixture (64.8L) was added 100 mM N-cyanoimidazole 8 (as a freshly

prepared 1 M aqueous solution) to give a final reaction volume of 72 L

(primer, template and ligator concentrations of 4, 25 and 30 M respectively;

final imidazole nitrate and MnCl2 concentrations 200 mM and 10 mM

respectively). The ligation reaction was incubated at 21 °C. At the time points

indicated (Fig. 4c), 8 L aliquots were removed and quenched by addition of

gel loading buffer (24 L) followed by immediate freezing in N2 (l) and

storage at -82 °C. Frozen samples were defrosted, then 2 L of each (containing

2 pmoles of FAM-labelled RNA) were diluted to 20 L with additional gel

loading buffer and heated at 95 °C for 4 min before gel loading and

72

electrophoresis as described above. The gel was imaged and quantified by

fluorescence scanning. The reaction was performed in triplicate.

Procedure for acetylation-ligation reaction to assess selectivity for 3'P vs 2'P

ligation (Fig 5a-c)

A mixture (L) containing 40 M primer (or 40 M each of 2'P and 3'P primer

for the competition experiments) and 50 mM N-acetylimidazole was incubated at

21 °C for 5 h. A templated ligation reaction (10 L) was then conducted using

4 M of acetylated primer (or 4 M each of acetylated 2'P and 3'P primer for the

competition experiments) from this mixture, 4 M template, 4 M 3'-(6FAM)

ligator, 200 mM imidazole nitrate buffer (pH 6.2), 10 mM MnCl2, and 100 mM

N-cyanoimidazole (added last as a freshly prepared 1 M aqueous solution). The

ligation reaction mixture was incubated at 21 °C for 18 h, then diluted with an

equal volume of Ambion Gel Loading Buffer II. Gel loading and electrophoresis

was performed as described above. The gel was imaged by fluorescence

scanning, before staining with SYBR® Gold and re-scanning.

Remainder acetylation reaction mixtures were analysed by MALDI-TOF MS

(Fig. S15 and S16). A total of 3 spectra were accumulated for each sample.

Accumulated spectra were baseline corrected and peaks integrated using ABI

Data Explorer software, to estimate relative yields of the acetylation reaction

(Fig. S16).

For accurate quantification, the reaction was repeated × 3, and 5% of the

quenched reaction mixture (containing 2 pmoles of FAM-labelled RNA) was

loaded for gel analysis as above. For quantification, tracker-dye-free gel loading

buffer (95 % formamide, 18 mM EDTA, 0.025 % SDS) was used for reaction

quenching and gel analysis.

73

Figure S15: a-f, MALDI-TOF mass spectra of acetylated primers reveals

selective acetylation of oligonucleotides terminating with 3'-P over those with 2'-

P. The primer(s) present are as labelled in each spectrum. The acetylation

mixtures contained 40 M of (each of) the indicated primer(s) and 50 mM NAI

9. Calculated masses for primers and their derivatives are presented in Table

S31; estimated yields are given in Figure S16.

74

Figure S16: Relative percentages of oligonucleotide primers and derivatives

thereof, as estimated by integration of the peak areas of the MALDI-TOF mass

spectra presented in Fig. S15.

Figure S17: Lane a, FAM-labelled 10 nt 3'P primer was acetylated and ligated

using the standard conditions (for Fig. 3b) and stoichiometric amounts of

primer, ligator and template (4 M in each). Lane b, as per lane a, but a mixture

of FAM-labelled 10 nt 3'P and 2'P primers was used (2 M in each). Lane c, as

per lane b, but without ligator. Lane d, as per lane b, but without template. Lane

e, as per lane b, but without NCI. Lane f, as per lane b, but without acetylation

(5 mM hydrolysed NAI was used in the ligation mixture). Left-hand lane,

unmodified FAM-labelled 10 nt 3'P primer. Gel loading and analysis as

described above (for Fig. 3b). The presence of a second major product band that

only appears when primer, NAI and NCI are all present, and that still appears

when ligator and template are absent, is consistent with primer dimerisation.

This dimerisation is essentially absent when the concentrations of ligator and

template strands exceed the primer concentration (compare Fig. 4a). NAI,

N-acetylimidazole. NCI, N-cyanoimidazole; P 3'/2'p, 10 nt 3'/2'-phosphate

primer; T, 13 nt template; L, 7 nt ligator.

75

Figure S18: MALDI-TOF mass spectra showing loss of acetate from an

acetylated 13 nt RNA strand, 5’-GCAGUA(2'OAc)GGUUCUC-3', by

ammonolysis. a and b, crude 13 nt oligomer. c and d, 13 nt oligomer after

treatment with ~ 5 M aqueous ammonia at 40 °C for 1 h, revealing a loss of

42 Da as consistent with deacetylation. e and f, 13 nt oligomer after

ammonolysis under milder conditions, namely ~ 1 M aqueous ammonia at 21 °C

for 48 h, again indicating deacetylated 13 nt RNA as the major product. (m/z) 13

nt RNA+OAc [M+H]+, calcd average mass for C125H156O92N47P12 requires

4160.52; 13 nt RNA [M+H]+, calcd average mass for C123H154O91N47P12 requires

4118.48.

76

Procedure for deacetylation and HPLC analysis to establish linkage isomers

(Fig. 6a and b)

A mixture (50 L) of 80 M primer (10 nt 3'P or 2'P) and 50 mM N-

acetylimidazole was incubated at 21 °C for 5 h. A templated ligation reaction

(200 L) was then conducted using 20 M of acetylated primer from this

mixture, 20 M template, 20 M ligator, 200 mM imidazole nitrate buffer (pH

6.2), 10 mM MnCl2, and 100 mM N-cyanoimidazole 8 (added last as a freshly

prepared 1 M aqueous solution). The ligation reaction mixture was incubated at

21 °C for 18 h, then diluted with water (300 L) and 1 M TEAA buffer (pH 7,

0.5 mL) and desalted using a Waters Sep-Pak® C18 Plus Short Cartridge

(cartridge pre-washed with water (10 mL) then MeOH (10 mL) before sample

loading, then washed with 50 mM TEAA pH 7 aqueous buffer (3 mL) before

sample elution with 30 % (v/v) MeOH in 50 mM TEAA pH 7 aqueous buffer (5

mL)). The eluent was lyophilised, then redissolved in water (200 L) and

divided into aliquots (5 × 40 L). Three aliquots were treated separately with an

equal volume of ~ 5 M aqueous ammonia at pH 9.2 and heated at 40 °C for 1 h,

before diluting to 100 L with water and analysing immediately by SAX-HPLC

(flow rate 1 mLmin-1; detection 260 nm; gradient isocratic 0 M NaCl in 10 mM

pH 11.5 sodium phosphate buffer for 1 min, then 0 to 1 M NaCl over 29 min,

then isocratic at 1 M NaCl for 5 min). The first aliquot was analysed directly,

whilst the other aliquots were analysed together with a co-injection of 17 nt

standard (200 pmoles for the reaction with 10 nt 3'P primer, and 40 pmoles for

the reaction with 10 nt 2'P primer) bearing either a 2',5'- or 3',5'-linkage at the

ligation junction (see Table S30, entry 4 and 5 respectively). Reaction yields

were estimated by peak integration using Varian Galaxie software, comparing

product and ligator peak integrals and correcting for calculated extinction

coefficients (at 260 nm). In the case of the 10 nt 2'P ligation, additional

correction was made for the hydrolysis of 17 nt 2',5'-linked acetyl RNA product

(see Fig. S21e and f). MALDI-TOF MS of a desalted (10 nt 3'P primer) reaction

aliquot pre- and post-ammonolysis (Fig. S19) was also performed.

77

Figure S19: MALDI-TOF MS spectra of a desalted acetylation-ligation reaction

of a 10 nt 3'P primer (RNA sequences as per Fig. 3a), pre- (a, b) and post- (c, d)

ammonolysis. a, spectrum showing 7 nt ligator (m/z calcd 2158.34), 10 nt primer

>P (m/z calcd 3223.93), 13 nt template (m/z calcd 4164.60) and 17 nt ligation

product (monoacetylated, m/z calcd 5423.24; bisacetylated, m/z calcd 5465.28).

b, spectrum (a) magnified to better show the ligation product(s). c, spectrum

post ammonolysis, showing an increased signal for 10 nt 2'/3'P (m/z calcd

3241.94) and deacetylated 17 nt ligation product (m/z calcd 5381.26). d,

spectrum (c) magnified to better show deacetylated ligation product.

78

Figure S20: SAX-HPLC traces of RNA standards (conditions as described above

for Fig. 6); see Fig. 3a for strand sequences. a, 0.8 nmoles of 7 nt ligator. b, 0.8

nmoles of each of 7 nt ligator and 10 nt 3'P primer, and 1.6 nmoles of 10 nt 2'P

primer. c, 0.8 nmoles of each of 7 nt ligator and 10 nt 3'P primer. d, 0.8 nmoles

of each of 7 nt ligator, 10 nt 3'P primer, 10 nt 2'P primer and 13 nt template.

Figure S21: a-d, SAX-HPLC traces (conditions as described above for Fig. 6) of

17 nt RNA standards with the following sequences (all sequences are reported

5'-3'):

a UGUGCCAGUA-2',5'(3'OAc)-GGUUCUC (0.4 nmoles);

79

b UGUGCCAGUA-2',5'(3'OAc)-GGUUCUC (0.4 nmoles) +

UGUGCCAGUA-2',5'-GGUUCUC (0.8 nmoles);

c UGUGCCAGUA-3',5'(2'OAc)-GGUUCUC (0.4 nmoles);

d UGUGCCAGUA-3',5'(2'OAc)-GGUUCUC (0.4 nmoles) +

UGUGCCAGUA-3',5'-GGUUCUC (0.8 nmoles).

The basic (pH 11.5) elution solvent causes some deacetylation of the acetylated

standards, which can be seen most clearly from the split peak of trace a. e-f,

SAX-HPLC traces (conditions as described above for Fig. 6) of 0.8 nmoles of

each of:

e UGUGCCAGUA-2',5'(3'OAc)-GGUUCUC + GGUCAUCCAAGAG

pre-ammonolysis;

f UGUGCCAGUA-2',5'(3'OAc)-GGUUCUC + GGUCAUCCAAGAG

post-ammonolysis;

g UGUGCCAGUA-3',5'(2'OAc)-GGUUCUC + GGUCAUCCAAGAG

pre-ammonolysis;

h UGUGCCAGUA-3',5'(2'OAc)-GGUUCUC + GGUCAUCCAAGAG

post-ammonolysis.

Ammonlysis was with ~ 5 M ammonia at pH 9.2 and 40 °C for 1 h. For the 17 nt

standard containing a single 2',5'-linkage (e and f), new peaks appear at lower

retention times, consistent with partial (29 % estimated from integration of

ligator and 17 nt standard peaks, correcting for extinction coefficients at 260

nm) hydrolysis of the deacetylated 2',5'-linkage to afford 7 nt ligator (‘L’) and

10 nt primer >P (‘>P’), the latter of which is hydrolysed further to afford 10 nt

primer (‘P’) as a mixture of the 2'P and 3'P. No backbone hydrolysis is

detectable for the 17 nt standard containing only 3',5'-linkages (g and h). The

presence of a template strand in each case promotes 2',5'- over 3',5'-

phosphodiester bond hydrolysis. To control for any deacetylation and

subsequent hydrolysis of 2',5'-linkages that may have biased the observed

ligation regioselectivity, acetylated 17 nt standards were submitted to the

ligation reaction conditions and analysed by gel electrophoresis (Supplementary

Fig. S22). No RNA hydrolysis was observed, thus providing further evidence that

the observed selectivity resulted from chemoselective acetylation of

phosphorylated primers.

80

Figure S22: Synthetic monoacetylated 17 nt standards of the ligation products

with 2',5'- (a and c) or 3',5'- (b and d) ligation junctions were mixed with 13 nt

template (40 pmoles of each) and either analysed directly by gel electrophoresis

(a and b), or subjected to the standard ligation reaction conditions (5 mM

hydrolysed NAI 9, 200 mM imidazole nitrate buffer (pH 6.2), 10 mM MnCl2, and

100 mM NCI 8, at 21 °C for 18 h; c and d). No hydrolysis to the 10 nt primer

was detected (40 pmoles of primer and template were electrophoresed for

comparison). Gel loading and analysis was identical to that used for ligation

reactions (described above), and imaging was by fluorescence scanning after

treatment with SYBR® Gold nucleic acid gel stain.

81

References

1. Park, D. J., Stern, A. G. & Wilier, R. L. A convenient laboratory Preparation

of cyanogen. Synth. Commun. 20, 2901–2906 (1990).

2. Mullen, L. B. & Sutherland, J. D. Simultaneous nucleotide activation and

synthesis of amino acid amides by a potentially prebiotic multi-component

reaction. Angew. Chem. Int. Ed. 46, 8063–8066 (2007).

3. Dueymes, C., Pirat, C. & Pascal, R. Facile synthesis of simple mono-alkyl

phosphates from phosphoric acid and alcohols. Tet. Lett. 49, 5300–5301 (2008).

4. Moffatt, J. G. & Khorana, H. G. Carbodiimides VII. Tetra-p-nitrophenyl

pyrophosphate, a new phosphorylating agent. J. Am. Chem. Soc. 79, 3741–3746

(1957).

82

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