Supplemental Figures and Tables Supplemental Figures and Tables Figure S1. Phosphoamidate Analogues...

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Supplemental Figures and Tables

Figure S1. Phosphoamidate Analogues

Figure S2. Single Incorporation of modified nucleotides. (A) compound 11b, HIV-1 RT (0.025 U/μL), P1T2 (125 nM); (B) compound 11a,Therminator polymerase (0.05 U/μL), P1T2 (125 nM) ; (C) compound 11b, Therminator polymerase (0.05 U/μL), P1T2 (125 nM); (D) compound 11c, Therminator polymerase (0.05 U/μL), P1T1 (125 nM); (E) compound 18, Therminator polymerase (0.05 U/μL), P1T2 (125 nM)). dTTP (10 µM) and dATP (10 µM) were used as reference.

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Figure S3. Incorporation of dTTP into P1T2 (125 nM) by Therminator polymerase (0.00005 U µL-1).

Figure S4. Mass spectra of a single nucleotide incorporation experiment of compound 11a by Therminator

polymerase; (A) P1T2, compound 11a, Therminator polymerase. (B) P1T2, Therminator polymerase.

Five different oligonucleotides were detected in the reaction mixture: a primer elongated with two

10a (Mw 5860, peak 2); a primer elongated with one 10a and one dTMP (Mw 5840, peak 1); a

template degraded by two nucleotides (G and T) at the 3’ end and elongated with one 10a (Mw 6383,

peak 3); the degraded template elongated with two 10a (Mw 6707, peak 5); the degraded template

elongated with one 10a and one dTMP (Mw 6687, peak 4).

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Figure S5. Schemes for Polymerization and Pyrophosphorolysis by Therminator Polymerases using

Compound 11a and P1T2. (A) Chain Elongation of Primer; (B) Formation of dTTP and dGTP by

Template Degradation and Chain Elongation of Template. Symbol for Compound 10a (5-chloro-2’-

deoxyuridine 5’-monophosphonate) is X.

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Figure S6. Template Degradation by Pyrophosphorolysis. Compound 11b (1 mM), P1T2 (125 nM),

Therminator polymerase (0.05 U/μL), inorganic PPase (0.125 U/μL); Similar results are obtained

using 11c as substrate (data not shown)

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Figure S7. Elongation into P1T4 (125 nM, A) and P1T3 (125 nM, B) by Therminator polymerase (0.05 U/μL).

Time points: 15, 30, 60, 90 and 120 min. dTTP (50µM) and dATP (50µM) were used as reference.

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Figure S8. T-ROESY spectra of Compound 10b

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Figure S9. RMS deviation from normal B-DNA duplex for 20 ns simulations.

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Figure S10. Rdf, radial distribution function of solvent around O5’ and O4’’ in the dsDNA (A) and the

XNA:DNA duplex (B).

A)

B)

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Table S1. Elongation results of modified nucleotide analogues. Condition: P1T3 (125 nM) or P1T4

(125 nM), [Therminator polymerase] = 0.05 U/μL, at 500 μM substrate concentration in 120 min

incubation time; percentage of total radio-emitting oligonucleotides in the mixture.

11a 11b 11c 15a 15b 15c 16a 16b 16c 17a 17b 17c 18

P + 7 0 0 58 0 0 12.3 0 0 0 0 0 0 0 P + 6 0 21.4 27.8 0 0 37.6 5.3 0 0 0 0 0 0

P + 5 7.1 59.7 6.5 0 0 47.4 18.9 0 0 4.9 0 0 0

P + 4 84.3 18.9 0 0 0 2.7 20.8 0 0 9.8 0 0 0

P + 3 4.9 0 0 1.8 0 0 29.2 0 6.4 27.1 9 2.8 17.5

P + 2 3.7 0 0 21.7 0 0 20.6 17.7 48.4 35.1 33 28.2 72.6

P + 1 0 0 0 42 17.7 0 5.1 33.2 27.4 16 35.7 37.8 7.4

p 0 0 0 34.5 72.8 0 0 49 17.9 7.1 22.3 31.2 2.5

Primer P1 5'-CAGGAAACAGCTATGAC-3' Template T3 3'-GTCCTTTGTCGATACTGTTTTTTTGGAC-5' Template T4 3'-GTCCTTTGTCGATACTGAAAAACTGC-5'

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Table S2. Results from a Curves 5.3 calculation (Lavery and Skllenar, 1988) on 10 snapshot

structures from 15 to 20 ns. All values are averages over all nucleotide pairs in the helix, except for

the groove parameters that were taken from a central nucleotide pair in the helix (pair 6 A/T). * are

global parameters, relative to the helix axis, ** are local parameters calculated from one base pair to

the next one. Mgr is major groove, mgr = minor groove.

DNA:DNA

dx* -2.02 -2.63 -2.17 -0.88 -1.18 -1.94 -1.93 -2.14 -1.58 -1.77 dy* 0.25 0.10 0.23 -0.70 0.00 -0.36 0.61 -0.03 0.16 0.22

Inclin* 9.00 14.31 9.16 4.71 5.92 7.92 12.36 10.65 3.68 8.36

Tip* -4.23 -7.03 -3.34 2.89 -3.45 2.09 -8.42 -1.67 -2.76 0.60

Shift* 0.03 -0.05 0.13 -0.01 0.09 0.08 -0.03 0.04 0.17 0.14

Slide** -0.10 -0.16 -0.14 -0.13 -0.15 -0.16 -0.01 -0.12 -0.06 -0.13

Rise** 3.31 3.16 3.37 3.46 3.35 3.24 3.38 3.21 3.45 3.34

Tilt** -1.08 -1.43 0.24 1.01 -0.24 1.91 -2.49 0.07 -0.04 0.82

Roll** 0.87 1.58 2.88 0.51 2.59 1.27 -1.68 2.10 1.55 0.88

Twist** 32.88 32.48 31.75 33.09 32.35 33.90 35.13 32.48 32.95 33.40

Propeller -15.70 -14.69 -7.99 -11.75 -15.15 -16.82 -12.42 -17.80 -12.46 -15.49

mgr width 5 8 7 4 4 6 7 4 5 5

mgr depth 5 4 4 5 5 4 4 5 5 5

Mgr width 11 13 15 14 12 13 12 11 14 12

Mgr depth 6 8 9 1 6 7 7 4 5 6

helix diam 20 21 21 19 19 20 20 21 21 20

Sugar C2’endo + C1’exo

XNA:DNA

dx* -1.83 -2.07 -1.95 -2.92 -3.05 -2.25 -2.69 -2.75 -1.91 -1.34 dy* 0.63 0.37 0.07 0.05 0.02 0.93 0.63 0.84 -0.16 -0.03

Inclin* 13.19 17.66 18.85 15.58 7.08 7.41 10.28 10.78 8.91 10.93

Tip* -8.52 -2.0 1.99 -1.64 1.07 -6.69 -7.52 -4.51 -0.69 1.22

Shift* -0.03 0.12 0.19 0.10 0.16 0.12 0.08 0.16 0.14 0.16

Slide** 0.04 -0.16 -0.21 -0.15 -0.10 -0.92 -0.29 -0.11 -0.11 -0.28

Rise** 3.46 3.56 3.29 3.18 3.46 3.40 3.19 3.37 3.40 3.47

Tilt** -1.77 -1.16 1.47 -0.60 -1.47 0.89 -2.55 -4.12 -0.10 0.48

Roll** 2.58 0.01 -0.13 2.69 4.75 5.09 7.20 6.07 1.29 3.20

Twist** 27.07 31.38 31.82 29.66 25.51 27.66 27.15 25.35 31.38 29.99

Propeller -13.46 -11.48 -16.99 -6.62 1.09 -14.65 -12.04 -3.13 -12.76 -13.91

mgr width 4 7 7 9 8 5 7 7 7 9

mgr depth 5 4 4 2 3 6 4 3 4 3

Mgr width 17 17 15 14 17 15 10 12 14 13

Mgr depth 6 8 2 11 6 9 10 12 9 9

helix diam 20 21 21 21 22 21 22 21 20 20

Sugar See Table S3

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Table S3. Average ribose Sugar Phase angles (Altona and Sundaralingam, 1972) in the different

residues in the antiparallel XNA:DNA duplex during the last 5 ns of the trajectory. The circular

variance is close to 1 when the spread in the angles is high.

XNA DNA

Phase Circular Phase Circular

132.20 0.06 142.98 0.07 175.98 0.04 199.44 0.12

87.53 0.29 155.60 0.44

50.90 0.25 16.05 0.78

69.92 0.46 142.33 0.21

58.77 0.32 174.72 0.22

78.98 0.38 152.63 0.16

58.93 0.18 143.64 0.45

74.39 0.27 146.12 0.08

75.87 0.53 198.55 0.28

92.32 0.40 132.43 0.26

117.34 0.40 125.69 0.14

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Table S4. Atomic charges of XNA nucleotides used in the Amber MD simulations.

Ade Gua Thy Cyt

P 1.1694 P 1.1694 P 1.1694 P 1.1694 O1P -0.8028 O1P -0.8028 O1P -0.8028 O1P -0.8028

O2P -0.8028 O2P -0.8028 O2P -0.8028 O2P -0.8028

C5’ 0.2191 /-0.0775

C5’ 0.2191 /-0.0775

C5’ 0.2191 /-0.0775

C5’ 0.2191 /-0.0775

H5’1 -0.0746 /0.0879

H5’1 -0.0746 /0.0879

H5’1 -0.0746 /0.0879

H5’1 -0.0746 /0.0879

H5’2 -0.0746 /0.0879

H5’2 -0.0746 /0.0879

H5’2 -0.0746 /0.0879

H5’2 -0.0746 /0.0879

H5’3 /0.0879 H5T /0.0879 H5T /0.0879 H5T /0.0879

O5’ -0.3755 /-0.3008

O5’ -0.3755 /-0.3008

O5’ -0.3755 /-0.3008

O5’ -0.3755 /-0.3008

C4’ 0.0043 C4’ 0.0043 C4’ 0.0043 C4’ 0.0043

H4’ 0.1350 H4’ 0.1350 H4’ 0.1350 H4’ 0.1350

O4’ -0.2988 O4’ -0.2988 O4’ -0.2988 O4’ -0.2988

C1’ 0.0431 C1’ 0.0358 C1’ 0.0680 C1’ -0.0116

H1’ 0.1838 H1’ 0.1746 H1’ 0.1804 H1’ 0.1963

N9 -0.0268 N9 0.0577 N1 -0.0239 N1 -0.0339

C8 0.1607 C8 0.0736 C6 -0.2209 C6 -0.0183

H8 0.1877 H8 0.1997 H6 0.2607 H6 0.2293

N7 -0.6175 N7 -0.5725 C5 0.0025 C5 -0.5222

C5 0.0725 C5 0.1991 C7 -0.2269 H5 0.1863

C6 0.6897 C6 0.4918 H71 0.0770 C4 0.8439

N6 -0.9123 O6 -0.5699 H72 0.0770 N4 -0.9773

H61 0.4167 N1 -0.5053 H73 0.0770 H41 0.4314

H62 0.4167 H1 0.3520 C4 0.5194 H42 0.4314

N1 -0.7624 C2 0.7432 O4 -0.5563 N3 -0.7748

C2 0.5716 N2 -0.9230 N3 -0.4340 C2 0.7959

H2 0.0598 H21 0.4235 H3 0.3420 O2 -0.6548

N3 -0.7417 H22 0.4235 C2 0.5677

C4 0.3800 N3 -0.6636 O2 -0.5881

C4 0.1814

C3’ 0.3337 C3’ 0.3337 C3’ 0.3337 C3’ 0.3337

H3’ 0.0659 H3’ 0.0659 H3’ 0.0659 H3’ 0.0659

C2’ -0.0859 C2’ -0.0859 C2’ -0.0859 C2’ -0.0859

H2’1 0.0368 H2’1 0.0368 H2’1 0.0368 H2’1 0.0368

H2’2 0.0368 H2’2 0.0368 H2’2 0.0368 H2’2 0.0368

O3’ -0.6076 /0.6689

O3’ -0.6076 /0.6689

O3’ -0.6076 /0.6689

O3’ -0.6076 /0.6689

H3T /0.4340 H3T /0.4340 H3T /0.4340 H3T /0.4340

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Experimental Procedures

Procedures of chemical synthesis

General Experimental; For all reactions, analytical grade solvents were used. All moisture-sensitive

reactions were carried out in oven-dried glass-ware (135 °C). 1H and 13C NMR spectra were recorded

with a Bruker Advance 300 (1H NMR: 300 MHz, 13C NMR: 75 MHz) or 500 (1H NMR: 500 MHz, 13C

NMR: 125 MHz), using tetramethylsilane as internal standard for 1H NMR spectra and DMSO-d6 (39.5

ppm) or CDCl3 (77.2 ppm) for 13C NMR spectra. Abbreviations used are: s = singlet, d = doublet, t =

triplet, q = quartet, m = multiplet, br s = broad signal. Coupling constants are expressed in Hertz.

Mass spectra are obtained with a a quadrupole orthogonal acceleration time-of-flight mass

spectrometer (Synapt G2 HDMS, Waters, Milford, MA). Samples were infused at 3µL/min and spectra

were obtained in positive (or: negative) ionization mode with a resolution of 15000 (FWHM) using

leucine enkephalin as lock mass. Precoated aluminum sheets (Fluka Silica gel/TLC-cards, 254 nm)

were used for TLC. Column chromatography was performed on ICN silica gel 63-200, 60 Å.

Purification by HPLC was performed on a reverse phase C18 column by gradient elution (acetonitrile

and 100 mM triethylammonium bicarbonate (TEAB) buffer).

General method to synthesize compound 3

To a stirred solution of 2-deoxy-D-ribose (10.0 g, 74.6 mmol) in methanol (250 ml) was added HCl

(3.0 ml, 1.25 M in MeOH). The reaction mixture was stirred for 1 h at room temperature under an

Argon atmosphere then sodium bicarbonate (5.0 g) was added and the mixture was stirred for further

30 min. The solids were filtered and the solvent removed in vacuo to give as orange oil. The crude

residue 1 (11.0 g) was dissolved in pyridine (250 ml) and cooled to 0 °C and tert-butyldimethylsilyl

chloride (13.4 g, 89.1 mmol) was added. The mixture was stirred at room temperature until the

starting material 1 was completely consumed as monitored by TLC (~ 3 h). After conversion, benzoyl

chloride (9.48 ml, 81.7 mmol) and DMAP (2.27 g, 18.6 mmol) was added to the mixture and stirred for

2 h. The volatile was removed under reduced pressure and the crude residue was diluted with ethyl

acetate and washed with water, NaHCO3, brine, dried over Na2SO4 and concentrated under reduced

pressure. The crude residue 2 was dissolved in THF (250 ml) and cooled to 0 °C, acetic acid (1 ml)

and TBAF (96 ml, 1 M in THF, 96.5 mmol) were added. The mixture was stirred at room temperature

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for 2 h and quenched with MeOH (5 ml). The volatile was removed under reduced pressure, the crude

residue was diluted with ethyl acetate and washed with water, NaHCO3, brine, dried over Na2SO4 and

concentrated under reduced pressure. The crude residue was purified by column chromatography on

silica gel (EtOAc: Hexane = 1: 2) to afford compound 3 (13.0 g, 69.4 % for 4 steps) as a colorless

liquid.

(2R,3S,5R)-2-((Tert-butyldimethylsilyloxy)methyl)-5-methoxytetrahydrofuran-3-ol (1):

1H NMR (300 MHz, CDCl3, 25 °C): δ 5.04 (dd, J= 5.3 Hz, J= 1.9 Hz, 1H, H1), 4.40 (q, J= 4.4 Hz, 1H,

H3), 4.88-4.82 (m, 1H, H4), 3.76 (dd, J= 9.8 Hz, J= 5.2 Hz, 1H, H5), 3.53 (dd, J= 9.5 Hz, J= 8.1 Hz,

1H, H5), 3.30 (s, 3H, OCH3), 2.23-2.16 (m, 2H, H2, OH), 2.06 (dd, J= 12.0 Hz, J= 6.1 Hz, 1H, H2),

0.89 (s, 9H, C(CH3)3), 0.066 (s, 6H, SiCH3) ppm. 13 C NMR (75 MHz, CDCl3, 25 °C): δ 105.21, 85.89,

73.68, 65.13, 55.17, 41.11, 26.06, 18.46, -5.22, -5.27 ppm. HRMS: calcd for C12H26NaO4Si [M+Na]+

285.1493, found 285.1480.

(2R,3S,5S)-2-((Tert-butyldimethylsilyloxy)methyl)-5-methoxytetrahydrofuran-3-ol (1):

1H NMR (300 MHz, CDCl3, 25 °C): δ 5.09 (d, J= 4.5 Hz, 1H, H1), 4.18 (dd, J= 10.6 Hz, J= 5.6 Hz, 1H,

H3), 4.12 (t, J= 3.2 Hz, 1H, H4), 3.73 (dd, J= 10.9 Hz, J= 3.6 Hz, 1H, H5), 3.56 (dd, J= 10.9 Hz, J= 4.8

Hz, 1H, H5), 3.38 (s, 3H, OCH3), 2.81 (d, J= 10.6 Hz, 1H, OH), 2.17-2.09 (m, 1H, H2), 1.98 (d, J=

13.6 Hz, 1H, H2), 0.88 (s, 9H, C(CH3)3), 0.056 (s, 3H, SiCH3), 0.046 (s, 3H, SiCH3) ppm. 13 C NMR

(75 MHz, CDCl3, 25 °C): δ 105.83, 88.06, 63.95, 54.94, 41.25, 26.04, 18.46, -5.22, -5.32 ppm.

(2R,3S,5R)-2-((Tert-butyldimethylsilyloxy)methyl)-5-methoxytetrahydrofuran-3-yl benzoate (2)

1H NMR (300 MHz, CDCl3, 25 °C): δ 8.03 (d, J= 7.2 Hz, 2H, Ar-H), 7.59-7.54 (m, 1H, Ar-H), 7.43 (t, J=

7.7 Hz, 2H, Ar-H), 5.50 (quint, J= 3.9 Hz, 1H, H3), 5.20 (dd, J= 5.5 Hz, J= 3.2 Hz, 1H, H1), 4.28-4.23

(m, 1H, H4), 3.78 (dd, J= 10.4, Hz, J= 5.6 Hz, 1H, H5), 3.71 (dd, J= 10.5 Hz, J= 6.7 Hz, 1H, H5), 3.38

(s, 3H, OCH3), 2.45 (ddd, J= 14.2 Hz, J= 6.8 Hz, J= 3.1 Hz, 1H, H2), 2.25 (dt, J= 14.3 Hz, J= 5.3 Hz,

1H, H2), 0.88 (s, 9H, C(CH3)3), 0.079 (s, 6H, SiCH3) ppm. 13 C NMR (75 MHz, CDCl3, 25 °C): δ

166.12, 133.24, 130.16, 129.79, 128.51, 105.79, 84.67, 76.13, 64.56, 55.46, 39.42, 26.00, 18.43, -

5.22, -5.25 ppm. HRMS: calcd for C19H30NaO5Si [M+Na]+ 389.1760, found 389.1792.

(2R,3S,5S)-2-((Tert-butyldimethylsilyloxy)methyl)-5-methoxytetrahydrofuran-3-yl benzoate (2)


7.7 Hz, 2H, Ar-H), 5.38 (ddd, J= 7.6 Hz, J= 2.9 Hz, J= 1.7 Hz, 1H, H3), 5.16 (d, J= 5.2 Hz, 1H, H1),

4.28 (q, J= 3.1 Hz, 1H, H4), 3.91 (dd, J= 11.1, Hz, J= 3.3 Hz, 1H, H5), 3.80 (dd, J= 11.0 Hz, J= 3.2 Hz,

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1H, H5), 3.41 (s, 3H, OCH3), 2.44 (ddd, J= 14.4 Hz, J= 7.7 Hz, J= 5.4 Hz, 1H, H2), 2.13 (d, J= 14.4

Hz, 1H, H2), 0.89 (s, 9H, C(CH3)3), 0.089 (s, 3H, SiCH3), 0.074 (s, 3H, SiCH3) ppm. 13 C NMR (75

MHz, CDCl3, 25 °C): δ 166.70, 133.17, 130.34, 129.89, 128.49, 105.58, 84.52, 75.36, 63.73, 55.09,

39.61, 26.04, 18.46, -5.17, -5.29 ppm.

(2R,3S,5R)-2-(Hydroxymethyl)-5-methoxytetrahydrofuran-3-yl benzoate (3)


6.9 Hz, 2H, Ar-H), 5.58-5.55 (m, 1H, H3), 5.25 (dd, J= 5.7 Hz, J= 2.6 Hz, 1H, H1), 4.38 (q, J= 2.3 Hz,

1H, H4), 3.80-3.84 (m, 2H, H5), 3.45 (s, 3H, OCH3), 2.97 (t, J= 6.4 Hz, 1H, OH), 2.55-2.37 (m, 2H, H2)

ppm. 13 C NMR (75 MHz, CDCl3, 25 °C): δ 166.50, 133.42, 129.85, 129.79, 128.57, 105.87, 86.37,

76.18, 64.12, 55.72, 40.16 ppm. HRMS: calcd for C13H16NaO5 [M+Na]+ 275.0895, found 275.0891.

(2R,3S,5S)-2-(Hydroxymethyl)-5-methoxytetrahydrofuran-3-yl benzoate (3)


7.8 Hz, 2H, Ar-H), 5.33 (ddd, J= 8.3 Hz, J= 3.9 Hz, J= 2.3 Hz, 1H, H3), 5.16 (d, J= 4.7 Hz, 1H, H1),

4.26 (q, J= 3.8 Hz, 1H, H4), 3.88-3.85 (m, 2H, H5), 3.41 (s, 3H, OCH3), 2.47 (ddd, J= 14.4 Hz, J= 8.3

Hz, J= 5.3 Hz, 1H, H2), 2.22 (br s, 1H, OH), 2.20 (d, J= 14.4 Hz, 1H, H2) ppm. 13 C NMR (75 MHz,

CDCl3, 25 °C): δ 166.88, 133.35, 129.96, 129.89, 128.52, 105.12, 83.74, 74.88, 62.81, 55.12, 39.46

ppm.

(2S,3S)-3-(Benzoyloxy)-5-methoxytetrahydrofuran-2-carboxylic acid (4)

The compound 3 (4.7 g, 18.6 mmol) was dissolved in acetonitrile (50 ml). PhI(OAc)2 (13.2 g, 41.0

mmol) was added to the solution followed by water (50 ml). The mixture was then treated with

TEMPO (0.58 g, 3.73 mmol) and stirred for 16 h. The mixture was then concentrated under reduced

pressure and the crude product was purified by column chromatography on silica gel (CH2Cl2: MeOH

= 50: 1) to afford the compound 4 (4.5 g, 90.7 %) as colorless liquid.

(2S,3S,5R)-3-(Benzoyloxy)-5-methoxytetrahydrofuran-2-carboxylic acid (4: C1-beta)

1H NMR (300 MHz, CDCl3, 25 °C): δ 11.47 (br s, 1H, CO2H), 8.04 (d, J= 7.1 Hz, 2H, Ar-H), 7.61-7.56

(m, 1H, Ar-H), 7.45 (t, J= 7.8 Hz, 2H, Ar-H), 5.92-5.89 (m, 1H, H3), 5.32 (dd, J= 5.2 Hz, J= 2.1 Hz,1H,

H1), 4.79 (d, J= 2.7 Hz, 1H, H4), 3.48 (s, 3H, OCH3), 2.57 (ddd, J= 14.2 Hz, J= 7.1 Hz, J= 2.1 Hz, 1H,

H2), 2.29 (dt, J= 14.1 Hz, J= 2.2 Hz, 1H, H2) ppm. 13 C NMR (75 MHz, CDCl3, 25 °C): δ 174.49,

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166.15, 133.57, 129.83, 129.35, 128.55, 106.69, 81.39, 55.89, 39.47 ppm. HRMS: calcd for C13H13O6

[M-H]- 265.0712, found 265. 0700.

(2S,3S,5S)-3-(Benzoyloxy)-5-methoxytetrahydrofuran-2-carboxylic acid (4: C1-alpha)


7.8 Hz, 2H, Ar-H), 5.55-5.51 (m, 1H, H3), 5.35 (d, J= 5.2 Hz, 1H, H1), 4.80 (d, J= 3.0 Hz, 1H, H4),

3.45 (s, 3H, OCH3), 2.48 (ddd, J= 14.5 Hz, J= 7.5 Hz, J= 5.3 Hz, 1H, H2), 2.28 (d, J= 14.5 Hz, 1H, H2)

ppm. 13 C NMR (75 MHz, CDCl3, 25 °C): δ 173.59, 166.74, 133.66, 130.06, 129.47, 128.61, 106.17,

81.38, 76.36, 55.70, 38.66 ppm.

(2R,3S)-2-Acetoxy-5-methoxytetrahydrofuran-3-yl benzoate (5)

The compound 4 (4.5 g, 16.9 mmol) was dissolved in dry THF (85 ml) and treated with pyridine (3.0

ml, 37.2 mmol). The reaction mixture was flushed with argon and then treated with lead tetraacetate

(8.25 g, 18.6 mmol). The reaction mixture was protected from light and stirred for 16 h. The solid was

filtered through celite and the filtrate was concentrated under reduced pressure. The residue was

diluted with ethyl acetate and washed with saturated NaHCO3, brine, dried over Na2SO4 and

concentrated under reduced pressure. The crude product was purified by column chromatography on

silica gel (EtOAc : Hexane = 1/ 6) to afford the compound 5 (3.4 g, 71 %) as colorless liquid.

(2R,3S,5R)-2-Acetoxy-5-methoxytetrahydrofuran-3-yl benzoate (5: C1 beta)

1H NMR (300 MHz, CDCl3, 25 °C): δ 8.01 (dd, J= 7.1 Hz, J= 1.4 Hz, 2H, Ar-H), 7.59-7.53 (m, 1H, Ar-

H), 7.43 (t, J= 7.3 Hz, 2H, Ar-H), 6.34 (s, 1H, H4), 5.53-5.50 (m, 1H, H1), 5.40-5.37 (m, 1H, H3), 3.42

(s, 3H, OCH3), 2.53-2.45 (m, 1H, H2), 2.39-2.32 (m, 1H, H2), 2.11 (s, 3H, COCH3) ppm. 13 C NMR (75

MHz, CDCl3, 25 °C): δ 169.63, 165.52, 133.44, 129.71, 129.32, 128.46, 107.44, 99.73, 77.77, 55.96,

37.49, 21.08 ppm. HRMS: calcd for C14H16NaO6 [M+Na]+ 303.0845, found 303.0844.

(2R,3S,5S)-2-Acetoxy-5-methoxytetrahydrofuran-3-yl benzoate (5: C1-alpha)

1H NMR (300 MHz, CDCl3, 25 °C): δ 8.05 (dd, J= 8.3 Hz, J= 1.2 Hz, 2H, Ar-H), 7.60-7.55 (m, 1H, Ar-

H), 7.45 (t, J= 7.8 Hz, 2H, Ar-H), 6.42 (s, 1H, H4), 5.36 (t, J= 1.4 Hz, 1H, H1), 5.34 (t, J= 1.3 Hz, 1H,

H3), 3.47 (s, 3H, OCH3), 2.59 (dt, J= 14.9 Hz, J= 6.1 Hz, 1H, H2), 2.17 (d, J= 14.7 Hz, 1H, H2), 2.11

(s, 3H, COCH3) ppm. 13C NMR (75 MHz, CDCl3, 25 °C): δ 169.48, 165.91, 133.48, 130.01, 129.68,

128.56, 107.08, 100.01, 76.36, 56.19, 36.37, 21.22 ppm.

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(2R,3S)-2-((Diisopropoxyphosphoryl)methoxy)-5-methoxytetrahydrofuran-3-yl benzoate (6)

The compound 5 (3.7 g, 13.2 mmol) was dissolved in dichloromethane (70 ml) and then cooled to -

20 °C. The cooled solution was then treated with phosphonate (3.89 g, 19.8 mmol) followed by

TMSOTf (4.78 ml, 26.4 mmol). The mixture was stirred for 16 h at -20 °C and then quenched with

triethylamine (4 ml). The mixture was washed with saturated NaHCO3, brine, dried over Na2SO4 and

concentrated under reduced pressure. The crude product was purified by column chromatography on

silica gel (EtOAc: Hexane = 1/ 1) to afford the compound 6 (4.0 g, 73 %) as colorless liquid.


7.5 Hz, 2H, Ar-H), 5.46 (dd, J= 5.7 Hz, J= 1.9 Hz, 1H, H1), 5.40-5.31 (m, 1H, H3), 5.10 (s, 1H, H4),

4.77 (septet, J= 4.7 Hz, 2H, CH), 3.99 (dd, J= 13.8 Hz, J= 8.5 Hz, 1H, PCH2), 3.78 (dd, J= 13.8 Hz, J=

8.8 Hz, 1H, PCH2), 3.47 (s, 3H, OCH3), 2.42-2.33 (m, 2H, H2), 1.34 (d, J= 4.7 Hz, 12H, CH3) ppm. 13

C NMR (75 MHz, CDCl3, 25 °C): δ 165.67, 133.47, 130.03, 129.83, 128.57, 107.35, 106.59 (t, J= 10.8

Hz), 78.28, 71.54 (d, J= 6.4 Hz), 71.35 (d, J= 6.7 Hz), 61.16 (d, J= 167.5 Hz), 56.40, 37.44, 24.24,

24.20, 24.12, 24.06 ppm. HRMS: calcd for C19H29NaO8P [M+Na]+ 439.1498, found 439.1494.

(2R,3S)-5-Acetoxy-2-((diisopropoxyphosphoryl)methoxy)tetrahydrofuran-3-yl benzoate (7)

A cold mixture of acetic anhydride, acetic acid, and conc. sulfuric acid was added to a solution of

compound 6 (4.0 g, 9.6 mmol) in dry CH2Cl2 at 0 °C and stirred for 15 min. The reaction mixture was

slowly poured into cold saturated NaHCO3 solution, and stirring was maintained for 3 h. The mixture

was extracted with CH2Cl2 from the water phase and dried over Na2SO4 and concentrated under

reduced pressure. The crude product was purified by column chromatography on silica gel (EtOAc:

Hexane = 1/ 1) to afford the compound 7 (4.0 g, 73 %) as colorless liquid.

1H NMR (300 MHz, CDCl3, 25 °C): δ 8.07-7.99 (m, 4H, Ar-H), 7.62-7.55 (m, 2H, Ar-H), 7.48-7.42 (m,

4H, Ar-H), 6.56 (dd, J= 6.0 Hz, J= 4.3 Hz, 1H, H1), 6.46 (d, J= 5.7 Hz, 1H, H1), 5.50 (dd, J= 5.8 Hz,

J= 1.8 Hz, 1H, H3), 5.44 (s, 1H, H4), 5.38 (d, J= 5.1 Hz, 1H, H3), 5.34 (s, 1H, H4), 4.77 (septet, J= 6.1

Hz, 4H, CH), 4.00-3.69 (m, 4H, PCH2), 2.71-2.43 (m, 3H, H2), 2.24 (d, J= 15.1 Hz, 1H, H2), 2.11 (s,

3H, OAc), 2.07 (s, 3H, OAc), 1.36-1.33 (m, 24H, CH3) ppm. 13 C NMR (75 MHz, CDCl3, 25 °C): δ

170.35, 169.94, 165.60, 133.62, 133.56, 130.00, 129.86, 129.38, 128.61, 128.58, 128.50, 107.93 (d,

J= 12.1 Hz), 107.49 (d, J= 12.3 Hz), 77.45, 75.70, 71.45 (d, J= 6.2 Hz), 71.36 (d, J= 6.3 Hz), 61.94 (d,

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J= 169.3 Hz), 61.62 (d, J= 169.4 Hz), 36.85, 35.87, 24.22, 24.16, 24.09, 21.23, 21.04 ppm. HRMS:

calcd for C20H29NaO9P [M+Na]+ 467.1447, found 467.1431.

(2R,3S,5R)-5-(5-Chloro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-

((diisopropoxyphosphoryl)methoxy)tetrahydrofuran-3-yl benzoate (8a)

To a solution of 5-chlorouracil (0.33 g, 2.25 mmol) in CH3CN (5 ml), N,O-bis(trimethylsilyl)acetamide

(1.65 ml, 6.75 mmol) was added and the mixture stirred for 20 min at room temperature. To the

mixture a solution of the compound 7 (0.50 g, 1.13 mmol) in CH3CN (2 ml) was added. After cooling at

0 °C TMSOTf (0.41 ml, 2.25 mmol) was added dropwise and the reaction mixture was stirred

overnight at 4 °C. The mixture was poured into a mixture of CH2Cl2 and saturated NaHCO3 solution

(5/1). The CH2Cl2 layer was removed and the water layer was extracted with CH2Cl2. The combined

CH2Cl2 extracts were washed with brine and dried over Na2SO4. After the solvent was removed the

residue was purified by column chromatography (CH2Cl2/MeOH = 50:1) on silica gel to give the

compound 8a (beta isomer 0.15 g, 25 %, alpha isomer 0.25 g, 42 %) as a white solid.

1H NMR (300 MHz, CDCl3, 25 °C): δ 9.24 (br s, 1H, NH), 8.04 (d, J = 7.2 Hz, 2H, Ar-H), 7.81 (s, 1H,

H6), 7.64-7.59 (m, 1H, Ar-H), 7.47 (t, J = 7.5 Hz, 2H, Ar-H), 6.71 (t, J = 7.8 Hz, 1H, H1’), 5.53 (t, J =

4.6 Hz, 1H, H3’), 5.37 (s, 1H, H4’), 4.82 (septet, J = 6.2 Hz, 2H, OCH(CH3)2), 4.03-3.85 (m, 2H, PCH2),

2.70 (dd, J1= 14.8 Hz, J2= 6.4 Hz, 1H, H2’), 2.40-2.37 (m, 1H, H2’), 1.37 (d, J = 6.2 Hz, 12H,

OCH(CH3)2) ppm. 31P NMR (121.5 MHz, CDCl3, 25 °C): 17.43 ppm. 13 C NMR (75 MHz, CDCl3, 25

°C): δ 165.53, 158.93, 149.97, 136.46, 133.85, 129.95, 128.95, 128.69, 110.68, 107.30 (d, Jc-p= 11.0

Hz), 86.64, 76.99, 71.89 (d, Jc-p= 6.4 Hz), 71.81 (d, Jc-p= 6.4 Hz), 62.64 (d, Jc-p= 169.5 Hz), 35.26,

24.17, 24.12 ppm. HRMS: calcd for C22H27ClN2O9P [M-H]- 529.1143, found 529.1134.

Diisopropyl ((2R,3S,5R)-5-(5-chloro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-

hydroxytetrahydrofuran-2-yloxy)methylphosphonate (9a)

The compound 8a (0.25 g, 0.47 mmol) was dissolved in 7 N ammonia in methanol (5 ml) and the

reaction mixture was stirred overnight at room temperature. The reaction mixture was concentrated

under reduced pressure and the crude residue was purified by column chromatography

(CH2Cl2/MeOH = 20:1) on silica gel to give the compound 9a (0.19 g, 94 %) as a white solid.

1H NMR (300 MHz, CDCl3, 25 °C): δ 9.93 (br s, 1H, NH), 7.71 (s, 1H, H6), 6.67 (t, J = 7.4 Hz, 1H, H1’),

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5.23 (s, 1H, H4’), 4.78 (septet, J = 6.5 Hz, 2H, OCH(CH3)2), 4.43 (br s, 1H, H3’), 4.29 (br s, 1H, OH),

3.94 (dd, J1= 14.1 Hz, J2= 9.1 Hz, 1H, PCH2), 3.83 (dd, J1= 14.1 Hz, J2= 7.6 Hz, 1H, PCH2), 2.51 (dd,

J1= 13.9 Hz, J2= 6.2 Hz, 1H, H2’), 2.13-2.04 (m, 1H, H2’), 1.35 (br s, 12H, OCH(CH3)2) ppm. 31P NMR

(121.5 MHz, CDCl3, 25 °C): 18.09 ppm. 13 C NMR (75 MHz, CDCl3, 25 °C): δ 159.25, 150.10, 137.05,

110.36, 110.31 (d, Jc-p= 7.8 Hz), 87.17, 74.97, 72.19 (d Jc-p= 6.7 Hz), 72.04 (d Jc-p= 6.7 Hz), 62.57

(d, Jc-p= 170.3 Hz), 37.73, 24.19 ppm. HRMS: calcd for C15H23ClN2O8P [M-H]- 425.0881, found

425.0875.

((2R,3S,5R)-5-(5-Chloro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-

yloxy)methylphosphonic acid (10a)

The compound 9a (0.26 g, 0.61 mmol) in dry DCM (5 ml) were treated with HMDS (1.9 ml, 9.14 mmol),

and then TMSI (0.52 ml, 3.66 mmol) was added dropwise under stirring at 0 °C. The mixture was

stirred for 1h at 0 °C. The reaction was quenched with 1.0 M TEAB solution (2 ml). The mixture was

concentrated, and the residue was purified by RP-HPLC running a gradient of CH3CN in 0.1 M TEAB

buffer solution to afford the compound 10a (0.11 g, 53 %) as a white solid.

1H NMR (300 MHz, D2O, 25 °C): δ 7.94 (s, 1H, H6), 6.51 (t, J = 7.7 Hz, 1H, H1’), 5.21 (s, 1H, H4’),

4.47(d, J = 3.5 Hz, 1H, H3’), 3.70 (dd, J1= 13.0 Hz, J2= 8.0 Hz, 1H, PCH2), 3.49 (dd, J1= 13.0 Hz, J2=

8.5 Hz, 1H, PCH2), 2.41-2.26 (m, 2H, H2’) ppm. 31P NMR (121.5 MHz, D2O, 25 °C): 12.65 ppm. 13 C

NMR (75 MHz, D2O, 25 °C): δ 161.79, 151.05, 138.32, 109.67 (d, Jc-p= 10.5 Hz), 109.32, 86.19,

73.87, 64.42 (d, Jc-p= 154.2 Hz), 36.02 ppm. HRMS: calcd for C9H11ClN2O8P [M-H]- 340.9942, found

340.9944.

((2R,3S,5R)-5-(5-Chloro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-

yloxy)methylphosphonic diphosphoric anhydride (11a)

The compound 10a (40 mg, 0.07 mmol) was dissolved in anhydrous DMF (1 ml) and Bu3N (53 µl,

0.22 mmol) was added. The mixture was stirred at room temperature for 30 min. and concentrated in

vacuo and then redissolved in anhydrous DMF (1 ml) and treated with N,N-carbonyldiimidazole (0.12

g, 0.73 mmol). The mixture was stirred for 30 min and tris(tetrabutylammonium) hydrogen

pyrophosphate (0.53 g, 0.59 mmol) in DMF (2 ml) was added. The mixture was stirred overnight.

Excess aqueous ammonia (1 ml) was added and the mixture concentrated in vacuo. The residue was

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purified by chromatography on DEAE-cellulose (gradient: water/ 1M TEAB (v/v); 1/0, 1/0.2, 1/0.5, 1/1).

Further purification was performed by RP-HPLC by gradient elution to afford the compound 11a (12

mg, ~23 %) as a white solid.


4.56 (d, J = 4.0 Hz, 1H, H3’), 3.96 (d, J= 8.8 Hz, 2H, PCH2), 2.43-2.38 (m, 2H, H2’) ppm. 31P NMR

(121.5 MHz, D2O, 25 °C): δ 7.53 (d, J= 26.2 Hz, Pα), -10.67 (d, J= 20.1 Hz, Pγ), -23.39 (dd, J= 26.2 Hz,

J= 20.1 Hz, Pβ) ppm. 13 C NMR (75 MHz, D2O, 25 °C): δ 161.48, 150.79, 138.43, 109.67 (d, Jc-p=

10.2 Hz), 109.36, 86.32, 73.82, 63.49 (d, Jc-p= 163.1 Hz), 35.77 ppm. HRMS: calcd for

C9H13ClN2O14P3 [M-H]- 500.9268, found 500.9284.

P-(((2R,3S,5R)-5-(5-Chloro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-

yloxy)methyl)-N,N-bis(3-methoxy-3-oxopropyl)phosphonamidic acid (12a)

In a two-neck flask, the compound 10a (40 mg, 0.07 mmol) and dimethyl iminodipropionate

hydrochloride (66 mg, 0.29 mmol) were suspended in a mixture of water (0.5 ml) and t-BuOH (1.5 ml).

One drop of Et3N was added to the solution and then the mixture was heated at 80 °C. A solution of

DCC (0.106 g, 0.51 mmol) in t-BuOH (1 ml) was added and the reaction mixture was stirred for 1h at

80 °C. After cooling, the solvent was removed under reduced pressure. The resulting crude residue

was purified by chromatography on silica gel (gradient : CHCl3/ MeOH/ H2O (v/v/v) 5:1:0, 5:2:0.25,

5:3: 0.25) to yield the compound 12a (16 mg, 42 %) as a white solid.

1H NMR (300 MHz, MeOD, 25 °C): δ 7.93 (s, 1H, H6), 6.50 (t, J = 6.5 Hz, 1H, H1’), 5.07 (s, 1H, H4’),

4.35 (d, J = 3.9 Hz, 1H, H3’), 3.77 (dd, J = 13.0 Hz, J = 6.9 Hz, 1H, PCH2), 3.64 (s, 6H, OCH3), 3.54

(dd, J = 13.0 Hz, J = 9.1 Hz, 1H, PCH2), 3.32-3.30 (m, 4H, NCH2), 2.60 (t, J= 7.5 Hz, 4H, CH2CO),

2.31-2.24 (m, 2H, H2’) ppm. 31P NMR (121.5 MHz, MeOD, 25 °C): δ 16.11 ppm. 13 C NMR (125 MHz,

MeOD, 25 °C): δ 174.63, 161.82, 151.85, 139.08, 112.16 (d, Jc-p= 9.7 Hz), 110.19, 88.15, 75.62,

66.56 (d, Jc-p= 145.4 Hz), 51.94, 43.50 (d, Jc-p= 4.7 Hz), 38.52, 36.03 ppm. HRMS: calcd for

C17H24ClN3O11P [M-H]- 512.0837, found 512.0848.

3,3'-((((2R,3S,5R)-5-(5-Chloro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-

yloxy)methyl)(hydroxy)phosphorylazanediyl)dipropanoic acid (16a)

The compound 10a (16 mg, 0.03 mmol) was dissolved in 0.4 M NaOH/ MeOH (1/1, 1 ml) and stirred

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at room temperature for 3 h. The reaction solution was cooled at 0 °C and 1 N HCl (0.2 ml) was added.

The mixture was purified by RP-HPLC by gradient elution to the compound 16a (12 mg, ~56 %) as a

white solid.

1H NMR (500 MHz, DMSO-d6, 25 °C): δ 7.77 (s, 1H, H6), 6.36 (t, J = 6.6 Hz, 1H, H1’), 5.07 (s, 1H,

H4’), 4.14 (d, J = 4.4 Hz, 1H, H3’), 3.51 (dd, J = 13.0 Hz, J = 7.7 Hz, 1H, PCH2), 3.41 (dd, J = 13.0 Hz,

J = 7.9 Hz, 1H, PCH2), 3.17-3.13 (m, 4H, NCH2), 2.39 (t, J = 6.8 Hz, 4H, CH2CO), 2.17 (dd, J = 13.9

Hz, J = 6.5 Hz, 1H, H2’), 2.08-2.04 (m, 1H, H2’) ppm. 31P NMR (202.5 MHz, DMSO-d6, 25 °C): δ

14.64 ppm. 13 C NMR (125 MHz, DMSO-d6, 25 °C): δ 173.76, 158.99, 149.78, 137.31, 109.56 (d, Jc-

p= 7.5 Hz), 108.16, 86.00, 73.61, 64.56 (d, Jc-p= 142.5 Hz), 42.63, 37.56, 36.23 ppm. HRMS: calcd

for C15H20ClN3O11P [M-H]- 484.0524, found 484.0537.

P-(((2R,3S,5R)-5-(5-Chloro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-

yloxy)methyl)-N-((S)-1,4-dimethoxy-1,4-dioxobutan-2-yl)phosphonamidic acid (13a)

This compound was synthesized according to the procedure for the synthesis of compound 12a,

using L-aspartic acid hydrochloride in a yield of 67 % (24 mg).


4.30 (d, J = 4.2 Hz, 1H, H3’), 4.29-4.22 (m, 1H, CH), 3.79 (dd, J = 12.7 Hz, J = 7.8 Hz, 1H, PCH2),

3.72 (s, 3H, OCH3), 3.64 (s, 3H, OCH3), 3.58 (dd, J = 12.8 Hz, J = 9.1 Hz, 1H, PCH2), 2.83 (t, J= 5.4

Hz, 2H, CH2CO), 2.35-2.20 (m, 2H, H2’) ppm. 31P NMR (121.5 MHz, MeOD, 25 °C): δ 14.10 ppm. 13 C

NMR (125 MHz, MeOD, 25 °C): δ 175.46 (d, Jc-p= 4.3 Hz), 173.16, 164.93, 154.40, 138.78, 111.68 (d,

Jc-p= 9.9 Hz), 110.58, 88.19, 75.79, 67.45 (d, Jc-p= 147.9 Hz), 52.79, 52.19, 40.82 (d, Jc-p= 3.4 Hz),

38.67 ppm. HRMS: calcd for C15H26ClN3O11P [M-H]- 484.0524, found 484.0517.

(2S)-2-((((2R,3S,5R)-5-(5-Chloro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-

2-yloxy)methyl)(hydroxy)phosphorylamino)succinic acid (17a)

This compound was synthesized according to the procedure for the synthesis of compound 16a, from

the compound 13a in a yield of 49 % (16 mg).

1H NMR (500 MHz, DMSO-d6, 25 °C): δ 7.76 (s, 1H, H6), 6.37 (t, J = 6.7 Hz, 1H, H1’), 5.08 (s, 1H,

H4’), 4.15 (d, J = 4.5 Hz, 1H, H3’), 3.79-3.76 (m, 1H, CH), 3.53 (dd, J = 12.9 Hz, J = 7.9 Hz, 1H,

PCH2), 3.41 (dd, J = 12.9 Hz, J = 8.0 Hz, 1H, PCH2), 2.49-2.47 (m, 2H, CH2CO), 2.18 (dd, J = 14.0 Hz,

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J = 6.6 Hz, 1H, H2’), 2.08-2.04 (m, 1H, H2’) ppm. 31P NMR (202.5 MHz, DMSO-d6, 25 °C): δ 13.57

ppm. 13 C NMR (125 MHz, DMSO-d6, 25 °C): δ 175.54, 173.50, 159.01, 149.82, 137.43, 109.50 (d,

Jc-p= 8.1 Hz), 108.22, 86.00, 73.73, 65.46 (d, Jc-p= 146.0 Hz), 51.43, 37.60 ppm. HRMS: calcd for

C13H16ClN3O11P [M-H]- 456.0211, found 456.0215.

Bis(((2R,3S,5R)-5-(5-chloro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-

yloxy)methyl)diphosphonic acid (15a)


4.49 (d, J = 4.0 Hz, 1H, H3’), 4.03-3.96 (m, 1H, PCH2), 3.86-3.79 (m, 1H, PCH2), 2.45-2.32 (m, 2H,

H2’) ppm. 31P NMR (121.5 MHz, D2O, 25 °C): δ 6.86 ppm. 13 C NMR (75 MHz, D2O, 25 °C): δ 162.03,

151.14, 138.26, 109.59, 109.54 (d, Jc-p= 7.7 Hz), 109.36, 86.32, 73.82, 63.4986.20, 73.83, 63.78 (dd,

Jc-p= 168.4, Jc-p= 4.8 Hz), 35.86 ppm. HRMS: calcd for C18H21Cl2N4O15P2 [M-H]- 664.9856, found

664.9850.

((2R,3S,5R)-5-(6-Amino-9H-purin-9-yl)-3-hydroxytetrahydrofuran-2-yloxy)methylphosphonic acid (10c)

To a solution of 6-chloropurine (0.27 g, 1.76 mmol) in CH3CN (5 ml), N,O-bis(trimethylsilyl)acetamide

(0.66 ml, 2.70 mmol) was added and the mixture stirred for 20 min at room temperature. To the

mixture a solution of the compound 7 (0.60 g, 1.35 mmol) in CH3CN (2 ml) was added. After cooling at

0 °C TMSOTf (0.27 ml, 1.49 mmol) was added dropwise and the reaction mixture was stirred for 3 h

at room temperature. The mixture was poured into a mixture of CH2Cl2 and saturated NaHCO3

solution (5/1). The CH2Cl2 layer was removed and the water layer was extracted with CH2Cl2. The

combined CH2Cl2 extracts were washed with brine and dried over Na2SO4. After the solvent was

removed the residue was dissolved in 7 N ammonia in MeOH (10 ml) and stirred at room temperature

for overnight. After removing the solvent under reduced pressure, the crude residue was dissolved in

dry DCM (5 ml). HMDS (1.9 ml, 9.14 mmol), and then TMSI (0.52 ml, 3.66 mmol) were added

dropwise under stirring at 0 °C. The mixture was stirred for 1h at 0 °C. The reaction was quenched

with 1.0 M TEAB solution (2 ml). The mixture was concentrated, and the residue was purified by RP-

HPLC running a gradient of CH3CN in 0.1 M TEAB buffer solution to afford the compound 10c (0.08 g,

10 %) as a white solid.

S24

1H NMR (500 MHz, D2O, 25 °C): δ 8.28 (s, 1H, H2), 8.10 (s, 1H, H8), 6.55 (t, J = 7.1 Hz, 1H, H1’),

4.59 (d, J= 1H, H4’), 4.47(d, J = 4.7 Hz, 1H, H3’), 3.54 (dd, J1= 13.0 Hz, J2= 8.8 Hz, 1H, PCH2), 3.47

(dd, J1= 13.0 Hz, J2= 9.8 Hz, 1H, PCH2), 2.80 (ddd, J= 14.9 Hz, J= 7.3 Hz, J= 5.2 Hz, 1H, H2’), 2.61

(dd, J= 14.8 Hz, J= 7.1 Hz, 1H, H2’) ppm. 31P NMR (121.5 MHz, D2O, 25 °C): 14.57 ppm. 13 C NMR

(125 MHz, D2O, 25 °C): δ 155.27, 152.53, 148.67, 140.12, 118.15, 109.77 (d, Jc-p= 10.0 Hz), 83.54,

74.39, 63.75 (d, Jc-p= 130.0 Hz), 37.21 ppm. HRMS: calcd for C10H13N5O6P [M-H]- 330.0603, found

330.0603.

((2R,3S,5R)-5-(6-Amino-9H-purin-9-yl)-3-hydroxytetrahydrofuran-2-yloxy)methylphosphonic

diphosphoric anhydride (11c)


the compound 10c in a yield of 22 % (10 mg).


5.40 (s, 1H, H4’), 4.74 (d, J = 5.1 Hz, 1H, H3’), 3.90-3.74 (m, 2H, PCH2), 2.99-2.87 (m, 1H, H2’), 2.66

(dd, J1= 15.0 Hz, J2= 7.1 Hz, 1H, H2’) ppm. 31P NMR (121.5 MHz, D2O, 25 °C): δ 7.72 (d, J= 26.2 Hz,

Pα), -10.06 (d, J= 20.2 Hz, Pγ), -23.23 (dd, J= 25.7 Hz, J= 20.3 Hz, Pβ) ppm. 13 C NMR (125 MHz,

D2O, 25 °C): δ 155.33, 152.46, 148.78, 140.14, 118.11, 109.62 (d, Jc-p= 11.3 Hz), 83.37, 74.11,

62.89 (d, Jc-p= 163.7 Hz), 36.68 ppm. HRMS: calcd for C10H15N5O12P3 [M-H]- 489.9930, found

489.9943.

P-(((2R,3S,5R)-5-(6-amino-9H-purin-9-yl)-3-hydroxytetrahydrofuran-2-yloxy)methyl)-N,N-bis(3-

methoxy-3-oxopropyl)phosphonamidic acid (12c)



1H NMR (500 MHz, MeOD, 25 °C): δ 8.42 (s, 1H, H2), 8.20 (s, 1H, H8), 6.66 (t, J = 7.1 Hz, 1H, H1’),

5.09 (s, 1H, H4’), 4.48 (d, J = 4.2 Hz, 1H, H3’), 3.64 (dd, J1= 12.9 Hz, J2= 7.4 Hz, 1H, PCH2), 3.63 (s,

6H, OCH3), 3.44 (dd, J1= 12.8 Hz, J2= 9.3 Hz, 1H, PCH2), 3.31-3.29 (m, 4H, NCH2), 2.78-2.73 (m, 1H,

H2’), 2.57 (t, J= 7.6 Hz, 4H, CH2CO), 2.51 (ddd, J1= 14.1 Hz, J2= 6.8 Hz, J3= 0.9 Hz) ppm. 31P NMR

(121.5 MHz, MeOD, 25 °C): δ 16.40 ppm. 13 C NMR (75 MHz, MeOD, 25 °C): δ 174.64, 157.34,

153.91, 150.71, 141.04, 120.03, 112.03 (d, Jc-p= 11.3 Hz), 85.31, 76.00, 66.14 (d, Jc-p= 146.3 Hz),

S25

51.94, 43.52 (d, Jc-p= 4.8 Hz), 39.70, 36.00 ppm. HRMS: calcd for C18H26N6O9P [M-H]- 501.1499,

found 501.1502.

3,3'-((((2R,3S,5R)-5-(6-Amino-9H-purin-9-yl)-3-hydroxytetrahydrofuran-2-

yloxy)methyl)(hydroxy)phosphorylazanediyl)dipropanoic acid (16c)



1H NMR (300 MHz, DMSO-d6, 25 °C): δ 8.34 (s, 1H, H2), 8.15 (s, 1H, H8), 7.27 (br s, 2H, CO2H), 6.54

(t, J = 6.9 Hz, 1H, H1’), 5.02 (s, 1H, H4’), 4.26 (d, J = 4.1 Hz, 1H, H3’), 3.45 (dd, J1= 12.5 Hz, J2= 8.8

Hz, 1H, PCH2), 3.32 (dd, J1= 12.5 Hz, J2= 8.2 Hz, 1H, PCH2), 3.18-3.11 (m, 4H, NCH2), 2.41 (t, J= 6.6

Hz, 4H, CH2CO), 2.50-2.39 (m, 2H, H2’) ppm. 31P NMR (121.5 MHz, DMSO-d6, 25 °C): δ 14.98 ppm.

13 C NMR (75 MHz, DMSO-d6, 25 °C): δ 173.68, 155.91, 152.64, 149.31, 138.91, 118.35, 109.22 (d,

Jc-p= 10.8 Hz), 82.78, 73.99, 63.95 (d, Jc-p= 143.5 Hz), 42.56, 36.12 ppm. HRMS: calcd for

C16H22N6O9P [M-H]- 473.1186, found 473.1191.

P-(((2R,3S,5R)-5-(6-amino-9H-purin-9-yl)-3-hydroxytetrahydrofuran-2-yloxy)methyl)-N-((S)-1,4-

dimethoxy-1,4-dioxobutan-2-yl)phosphonamidic acid (13c)


the compound 10c, using L-aspartic acid hydrochloride in a yield of 83 % (20 mg).


5.20 (s, 1H, H4’), 4.61 (d, J = 4.8 Hz, 1H, H3’), 4.19-4.13 (m, 1H, CH), 3.69 (s, 3H, OCH3), 3.66 (s, 3H,

OCH3), 3.62-3.51 (m, 2H, PCH2), 2.93-2.86 (m, 1H, H2’), 2.83-2.80 (m, 2H, CH2CO), 2.70 (dd, J1=

14.7 Hz, J2= 7.1 Hz, 1H, H2’) ppm. 31P NMR (202.5 MHz, D2O, 25 °C): δ 15.70 ppm. 13 C NMR (125

MHz, D2O, 25 °C): δ 175.09 (d, Jc-p= 3.9 Hz), 173.15, 155.30, 152.52, 148.68, 139.86, 118.22,

109.78 (d, Jc-p= 12.2 Hz), 83.55, 74.08, 65.15 (d, Jc-p= 146.7 Hz), 52.52, 52.02, 50.95, 38.98 (d, Jc-

p= 4.1 Hz), 36.89 ppm. HRMS: calcd for C16H22N6O9P [M-H]- 473.1186, found 473.1202.

(2S)-2-((((2R,3S,5R)-5-(6-Amino-9H-purin-9-yl)-3-hydroxytetrahydrofuran-2-

yloxy)methyl)(hydroxy)phosphorylamino)succinic acid (17c)


S26



5.19 (s, 1H, H4’), 4.65 (d, J = 4.7 Hz, 1H, H3’), 3.92-3.85 (m, 1H, CH), 3.61-3.46 (m, 2H, PCH2), 2.95-

2.85 (m, 1H, H2’), 2.69-2.57 (m, 2H, CH2CO), 2.46 (dd, J1= 14.5 Hz, J2= 8.0 Hz, 1H, H2’) ppm. 31P

NMR (202.5 MHz, DMSO, 25 °C): δ 16.29 ppm. 13 C NMR (125 MHz, DMSO, 25 °C): δ 175.25,

173.11, 155.92, 152.66, 149.31, 118.32, 109.25 (d, Jc-p= 9.9 Hz), 82.74, 73.99, 64.79 (d, Jc-p= 144.3

Hz), 51.38, two peaks are hidden under solvent signals. ppm. HRMS: calcd for C14H18N6O9P [M-H]-

445.0873, found 445.0882.

((2R,3S,5R)-5-(6-Amino-9H-purin-9-yl)-3-hydroxytetrahydrofuran-2-yloxy)methyl((5-(6-amino-9H-

purin-9-yl)-3-hydroxytetrahydrofuran-2-yloxy)methyl)diphosphonic acid (15c)


5.27 (s, 1H, H4’), 4.63 (d, J = 4.4 Hz, 1H, H3’), 4.20-4.13 (m, 1H, PCH2), 3.73-3.66 (m, 1H, PCH2),

2.77-2.68 (m, 1H, H2’), 2.58 (dd, J1= 14.6 Hz, J2= 7.1 Hz, 1H, H2’) ppm. 31P NMR (121.5 MHz, D2O,

25 °C): δ 8.06 ppm. 13 C NMR (125 MHz, D2O, 25 °C): δ 154.79, 152.32, 148.28, 139.85, 117.66,

109.74 (d, Jc-p= 13.7 Hz), 109.739, 83.41, 74.37, 63.70 (d, Jc-p= 171.3 Hz), 37.68 ppm. HRMS:

calcd for C20H25N10O11P2 [M-H]- 643.1179, found 643.1160.

(2R,3S,5R)-2-((Diisopropoxyphosphoryl)methoxy)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-

yl)tetrahydrofuran-3-yl benzoate (8b)

This compound was synthesized according to the procedure for the synthesis of compound 8a, using

thymine in a yield of 87 % (α/β = 1:1).

1H NMR (300 MHz, CDCl3, 25 °C): δ 8.98 (br s, 1H, NH), 8.045 (d, J = 7.1 Hz, 2H, Ar-H), 7.65-7.59 (m,

1H, Ar-H), 7.51-7.46 (m, 3H, Ar-H, H6), 6.83 (dd, J = 8.4 Hz, J = 6.4 Hz, 1H, H1’), 5.53 (d, J = 4.6 Hz,

1H, H3’), 5.26 (s, 1H, H4’), 4.81 (septet, J = 6.2 Hz, 2H, OCH(CH3)2), 3.98 (dd, J = 13.2. Hz, J = 9.5

Hz, 1H, PCH2), 3.79 (dd, J = 13.2. Hz, J = 10.4 Hz, 1H, PCH2), 2.63 (dd, J1= 14.7 Hz, J2= 6.4 Hz, 1H,

H2’), 2.46-2.41 (m, 1H, H2’), 2.05 (d, J = 0.8 Hz, 3H, CH3), 1.40-1.34 (m, 12H, OCH(CH3)2) ppm. 31P

NMR (121.5 MHz, CDCl3, 25 °C): 17.67 ppm. 13 C NMR (75 MHz, CDCl3, 25 °C): δ 165.66, 163.64,

150.95, 135.24, 133.91, 130.01, 129.07, 128.75, 113.22, 106.93 (d, Jc-p= 14.3 Hz), 85.94, 71.81 (d,

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Jc-p= 7.2 Hz), 71.68 (d, Jc-p= 6.4 Hz), 62.49 (d, Jc-p= 171.7 Hz), 34.73, 24.23, 12.55 ppm. HRMS:

calcd for C23H32N2O9P [M+H]+ 511.1845, found 511.1854.

Diisopropyl ((2R,3S,5R)-3-hydroxy-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-

yl)tetrahydrofuran-2-yloxy)methylphosphonate (9b)


the compound 8b in a yield of 90 % .

1H NMR (300 MHz, CDCl3, 25 °C): δ 10.05 (br s, 1H, NH), 7.36 (s, 1H, H6), 6.79 (t, J = 6.7 Hz, 1H,

H1’), 5.11 (s, 1H, H4’), 4.80-4.74 (m, 2H, OCH(CH3)2), 4.69 (br s, 1H, H3’), 4.43 (br s, 1H, OH), 3.92

(dd, J = 13.6. Hz, J = 9.3 Hz, 1H, PCH2), 3.75 (dd, J = 13.5. Hz, J = 9 Hz, 1H, PCH2), 2.44 (dd, J1=

13.9 Hz, J2= 6.3 Hz, 1H, H2’), 2.17-2.08 (m, 1H, H2’), 1.98 (s, 3H, CH3), 1.36-1.32 (m, 12H,

OCH(CH3)2) ppm. 31P NMR (121.5 MHz, CDCl3, 25 °C): 18.61 ppm. 13 C NMR (75 MHz, CDCl3, 25

°C): δ 164.21, 151.31, 135.70, 112.54, 109.85 (d, Jc-p= 11.6 Hz), 86.30, 75.03, 71.84 (d, Jc-p= 6.4

Hz), 71.78 (d, Jc-p= 6.5 Hz), 62.16 (d, Jc-p= 171.2 Hz), 37.19, 24.14, 12.56 ppm. HRMS: calcd for

C16H28N2O8P [M+H]+ 407.1583, found 407.1566.

((2R,3S,5R)-3-Hydroxy-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-2-

yloxy)methylphosphonic acid (10b)


the compound 9b in a yield of 42 % (0.15 g).

1H NMR (500 MHz, D2O, 25 °C): δ 7.58 (d, J = 1 Hz, 1H, H6), 6.57 (t, J = 7.5 Hz, 1H, H1’), 5.51 (s, 1H,

H4’), 4.48 (t, J = 3.0 Hz, 1H, H3’), 3.73 (dd, J1 = 12.5 Hz, J2 = 8.5 Hz, 1H, PCH2), 3.52 (dd, J1= 12.5

Hz, J2= 8.5 Hz, 1H, PCH2), 2.36-2.34 (m, 2H, H2’), 1.90 (d, J = 1 Hz, 3H, CH3) ppm. 31P NMR (121.5

MHz, D2O, 25 °C): 13.68 ppm. 13 C NMR (75 MHz, D2O, 25 °C): δ 167.10, 152.71, 138.13, 113.07,

110.39 (d, Jc-p= 10.6 Hz), 86.35, 74.96, 65.56 (d, Jc-p= 153.2 Hz), 36.59, 12.27 ppm. HRMS: calcd

for C10H14N2O8P [M-H]- 321.0488, found 321.0486.

Diphosphoric ((2R,3S,5R)-3-hydroxy-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-

yl)tetrahydrofuran-2-yloxy)methylphosphonic anhydride (11b)


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the compound 10b in a yield of 15 % (10 mg).


4.55 (br s, 1H, H3’), 3.94 (dd, J1 = 13.5 Hz, J2 = 8.5 Hz, 1H, PCH2), 3.88 (dd, J1= 13.5 Hz, J2= 8.5 Hz,

1H, PCH2), 2.39-2.37 (m, 2H, H2’), 1.94 (s, 3H, CH3) ppm. 31P NMR (121.5 MHz, D2O, 25 °C): δ 7.72

(d, J= 26.4 Hz, Pα), -11.03 (d, J= 19.9 Hz, Pγ), -23.23 (t, J= 33.7 Hz, Pβ) ppm. 13 C NMR (75 MHz,

D2O, 25 °C): δ 166.14, 151.78, 137.10, 112.20, 109.32 (d, Jc-p= 11.1 Hz), 85.56, 73.95, 63.22 (d, Jc-

p= 162.9 Hz), 35.36, 11.35 ppm. HRMS: calcd for C10H16N2O14P3 [M-H]- 480.9814, found 480.9839,

calcd for C10H15N2O11P2 [M-PO3H]- 401.0151, found 401.0165,.

P-(((2R,3S,5R)-3-hydroxy-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-2-

yloxy)methyl)-N,N-bis(3-methoxy-3-oxopropyl)phosphonamidic acid (12b)




4.34 (br s, 1H, H3’), 3.74 (dd, J= 12.1 Hz, J= 8.3 Hz, 1H, PCH2), 3.64 (s, 6H, OCH3), 3.48 (dd, J= 11.2

Hz, J= 9.1 Hz, 1H, PCH2), 3.34 (t, J = 7.4 Hz, 4H, NCH2), 2.59 (t, J = 7.4 Hz, 4H, C(O)CH2), 2.26-2.24

(m, 2H, H2’), 1.96 (s, 3H, CH3) ppm. 31P NMR (121.5 MHz, D2O, 25 °C): δ 16.36 ppm. 13 C NMR (75

MHz, D2O, 25 °C): δ 174.69, 166.37, 152.71, 138.11, 112.78, 111.60 (d, Jc-p= 11.5 Hz), 87.21, 75.87,

66.2 (d, Jc-p= 146.6 Hz), 51.95, 43.42 (d, Jc-p= 4.6 Hz), 38.00, 35.93, 12.64 ppm. HRMS: calcd for

C18H27N3O11P [M-H]- 492.1383, found 492.1381.

3,3'-(Hydroxy(((2R,3S,5R)-3-hydroxy-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-

yl)tetrahydrofuran-2-yloxy)methyl)phosphorylazanediyl)dipropanoic acid (16b)




4.49 (br s, 1H, H3’), 3.76 dd, J= 13.0 Hz, J= 7.6 Hz, 1H, PCH2), 3.58 (dd, J= 12.8 Hz, J= (.1 Hz, 1H,

PCH2), 3.26 (t, J = 8.1 Hz, 4H, NCH2), 2.50 (t, J = 8.1 Hz, 4H, C(O)CH2), 2.40-2.38 (m, 2H, H2’), 1.95

(s, 3H, CH3) ppm. 31P NMR (202.5 MHz, DMSO, 25 °C): δ 14.78 ppm. 13 C NMR (125 MHz, DMSO,

25 °C): δ 173.82, 163.81, 150.76, 135.91, 111.00, 109.04 (d, Jc-p= 11.3 Hz), 84.91, 73.93, 64.28 (d,

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Jc-p= 143.8 Hz), 42.57, 37.04, 36.20, 11.94 ppm. HRMS: calcd for C16H23N3O11P [M-H]- 464.1070,

found 464.1076.

(2S)-2-(Hydroxy(((2R,3S,5R)-3-hydroxy-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-

yl)tetrahydrofuran-2-yloxy)methyl)phosphorylamino)succinic acid (17b)




4.49 (br s, 1H, H3’), 3.89-3.82 (m, 1H, CH), 3.76 (dd, J1= 12.5 Hz, J2= 8.3 Hz, 1H, PCH2O), 3.55 (dd,

J1 = 12.5 Hz, J2 = 9.6 Hz, 1H, PCH2O), 2.57 (dd, J= 14.2 Hz, J= 4.6 Hz, 1H, PCH2), 2.44- 2.36 (m, 3H,

H2’, PCH2), 1.96 (s, 3H, CH3) ppm. 31P NMR (202.5 MHz, DMSO, 25 °C): δ 14.16 ppm. 13 C NMR

(125 MHz, DMSO, 25 °C): δ 175.32, 173.21, 163.79, 150.75, 135.90, 111.01, 109.21 (d, Jc-p= 11.3

Hz), 84.96, 73.93, 65.09 (d, Jc-p= 144.6 Hz), 51.52, one peak is hidden under solvent signals, 37.13,

12.03 ppm. HRMS: calcd for C14H19N3O11P [M-H]- 436.0763, found 436.0765.

Bis(((2R,3S,5R)-3-hydroxy-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-2-

yloxy)methyl)diphosphonic acid (15b)


4.48 (d, J = 4.5 Hz, 1H, H3’), 3.94 (dt, J1 = 13.0 Hz, J2 = 4.0 Hz, 1H, PCH2), 3.77 (dt, J1= 13.0 Hz, J2=

4.0 Hz, 1H, PCH2), 2.35 (dd, J1 = 14.5 Hz, J2 = 6.5 Hz, 1H, H2’), 2.31-2.25 (m, 1H, H2’), 1.92 (s, 3H,

CH3) ppm. 31P NMR (121.5 MHz, D2O, 25 °C): δ 6.78 ppm. 13 C NMR (75 MHz, D2O, 25 °C): δ 166.29,

151.78, 136.92, 112.19, 109.26 (t, Jc-p= 5.7 Hz), 85.55, 63.76 (dd, Jc-p= 168.3 Hz, Jc-p= 4.6 Hz),

35.65, 11.39 ppm. HRMS: calcd for C20H27N4O15P2 [M-H]- 625.0948, found 625.0938.

N-(3-(Dimethoxyphosphoryl)-1-methoxy-1-oxopropan-2-yl)-P-(((2R,3S,5R)-3-hydroxy-5-(5-methyl-2,4-

dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-2-yloxy)methyl)phosphonamidic acid (14)


4.24 (br s, 1H, H3’), 4.31-4.21 (m, 1H, CH), 3.78 (dd, J1 = 12.5 Hz, J2 = 8.3 Hz, 1H, PCH2O), 3.75 (d,

J= 0.9 Hz, 3H, OCH3), 3.74 (s, 3H, OCH3), 3.71 (d, J= 0.9 Hz, OCH3), 3.51 (dd, , J1 = 12.5 Hz, J2 =

9.3 Hz, 1H, PCH2O), 2.38 (dd, J= 17.6 Hz, J= 6.1 Hz, 2H, PCH2), 2.25-2.22 (m, 2H, H2’), 1.97 (s, 3H,

S30

CH3) ppm. 31P NMR (121.5 MHz, D2O, 25 °C): δ 30.91, 13.47 ppm. 13 C NMR (75 MHz, MeOD, 25

°C): δ 174.88, 166.35, 152.71, 138.16, 112.83, 111.38 (d, Jc-p= 11.3 Hz), 87.15, 75.90, 67.41 (d, Jc-

p= 146.8 Hz), 53.34 (d, J= 4.5 Hz), 53.26 (d, J= 4.7 Hz), 52.81, 51.66 (d, J= 3.2 Hz), 38.07, 31.48 (d,

Jc-p= 134.3 Hz), 12.62 ppm. HRMS: calcd for C16H27N3NaO12P2 [M+Na]+ 538.0968, found 538.0875.

P-(((2R,3S,5R)-3-hydroxy-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-2-

yloxy)methyl)-N-(1-methoxy-1-oxo-3-phosphonopropan-2-yl)phosphonamidic acid (14-a)


4.48 (br s, 1H, H3’), 4.49-3.99 (m, 1H, CH), 3.78 (dd, , J1 = 13.1 Hz, J2 = 8.0 Hz, 1H, PCH2O), 3.73 (s,

3H, OCH3), 3.62 (dd, , J1 = 12.8 Hz, J2 = 8.7 Hz, 1H, PCH2O), 2.39-2.37 (m, 2H, H2’), 1.96 (s, 3H,

CH3), 1.96-1.83 (m, 2H, PCH2) ppm. 31P NMR (121.5 MHz, D2O, 25 °C): δ 16.37, 16.23 ppm. 13 C

NMR (75 MHz, D2O, 25 °C): δ 177.10, 166.51, 152.00, 137.14, 112.09, 109.48 (d, Jc-p= 10.7 Hz),

85.56, 73.94, 64.88 (d, Jc-p= 145.2 Hz), 52.19, 51.52, 35.66, 33.73 (d, Jc-p= 132.1 Hz), 11.44 ppm.

HRMS: calcd for C14H22N3O12P2 [M-H]- 486.0679, found 486.0673.

2-(Hydroxy(((2R,3S,5R)-3-hydroxy-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-

yl)tetrahydrofuran-2-yloxy)methyl)phosphorylamino)-3-phosphonopropanoic acid (18)

1H NMR (600 MHz, DMSO, 25 °C): δ 7.47 (s, 1H, H6), 6.45 (t, J = 7.2 Hz, 1H, H1’), 4.97 (s, 1H, H4’),

4.15 (d, J = 4.8 Hz, 1H, H3’), two peaks are hidden under water signal (CHCOOH, PCH2O), 2.12-1.98

(m, 4H, PCH2O, PCH2CH), 1.83 (s, 3H, CH3) ppm. 31P NMR (121.5 MHz, D2O, 25 °C): δ 20.56, 16.69

ppm. 13 C NMR (150 MHz, DMSO, 25 °C): δ 171.24, 163.85, 150.81, 136.00, 110.99, 109.12, 84.97,

73.94, 64.56 (d, Jc-p= 151.5 Hz), 50.24, 39.19, 30.09 (d, Jc-p= 123 Hz), 12.10 ppm. HRMS: calcd for

C13H20N3O12P2 [M-H]- 472.0522, found 472.0525.

DNA polymerase reactions

Oligonucleotides P1, T1-T4 were purchased from Sigma Genosys. The concentrations were deter

mined with a Varian Cary-300-Bio UV Spectrophotometer. The primer was 5’-33P-labeled with 5’-

[γ33P]-ATP (Perkin Elmer) using T4 polynucleotide kinase (New England Biolabs) according to the

procedure provided by the supplier. The labeled oligonucleotide was further purified using Illustra

TM Microspin TM G-25 columns (GE Healthcare).

S31

End-labelled primer was annealed to its template by combining primer and template in a molar ratio of

1:2 and heating the mixture to 75 °C for 10 min followed by slow cooling to room temperature over a

period of 1.5 h. For the incorporation of compound 11a-c, 15a-c, 16a-c, 17a-c and 18, a series of 20

µL-batch reactions were performed with the enzyme (HIV-1 RT, pol III, Taq, Vent (exo-), Therminator

DNA polymerase). The final mixture contained 125 nM primer template complex, reaction buffer (50

mM Tris.HCl, 50 mM KCl, 10 mM MgCl2, 0.5 mM spermidine, 10 mM dithiothreitol (DTT); pH 8.3 for

HIV-1 RT; 20 mM Tris.HCl, 10 mM KCl, 2 mM MgSO4, 0.1 % Triton X-100, pH 8.8 for thermostable

polymerase; 20 mM Tris.HCl, 10 mM MgCl2, 10 mM MDTT, 20 μg/mL BSA, 4% glycerol, pH 7.5 for

polymerase III), appropriate concentration of enzyme, and different concentrations of modified

nucleotide building blocks (1 mM, 500 µM, 200 µM and 100 µM). In the control reaction a 10 µM

dATP or dTTP were used as reference. The mixture was incubated at 37 °C or 75 °C respectively,

aliquots were quenched after 10, 20, 30, 60 and 120 min. For elongation experiments, the same

mixture with appropriate primer:template hybrid was incubated at 37 °C or 75 °C and aliquots were

quenched after 15, 30, 60, 90, 120 min. In the reaction with the natural nucleotides, dATP (50 µM) and

dTTP (50 µM) were used as positive control.

For mass analysis, unlabelled primer P1 is annealed to template T2 by adding 10.2 μL of a 90 μM

primer solution to 10 μL of a 110 μM template solution, in a final volume of 20.2 μL. The mixture is

heated at 75 °C for 10 min and cooled down slowly to room temperature during 2 hours. The final

concentration of P1T2 hybrid is 45.4 μM. Compound 11a (2 mM solution) is added to an equal volume

of mixture containing H2O, Thermopol buffer, 4 units of Therminator DNA polymerase and 13.6 μM of

P1T2, in a final volume of 20 μL, the reaction is performed at 75 °C and is quenched after 1.5 hours

by putting the mixture at 95 °C for 5 min. Furthermore, the sample desalted using IllustraTM

MicrospinTM G-25 columns.

For template degradation reaction, template was 5’-33P-labeled with 5’-[γ33P]-ATP using the same

procedure as labeling primer. End labeled template annealed to the corresponding primer by

combining primer and template in a molar radio of 1:2, and heated the mixture at 75 °C for 5 min

followed by slowly cooling to room template over a period of 1.5 h.

Steady-state kinetics of single nucleotide incorporation

The steady-state kinetics of single nucleotide incorporation of compound 16a and the natural

nucleoside triphosphate (dTTP) were determined by a gel-based polymerase assay. In all the

S32

experiments, the template T2 and the primer P1 were used. The primer and template in a 1:2 molar

ration were hybridized in a buffer containing 20 mM Tris.HCl, 10 mM KCl, 2 mM MgSO4, 0.1 % Triton

X-100, pH 8.8 and used in an amount to provide 125 nM concentration of the primer in each 20 µL

reaction. A range of building block concentrations between 10 µM to 1 mM of compound 16a and

between 0.1 µM to 10 µM for the natural building block were used. The final concentration of

Therminator DNA polymerase for the single incorporation of compound 16a is 0.0005 U/µL and for

dTTP is 0.00005 U/µL. Reaction mixtures were incubated at 75 °C and aliquots were drawn at 6

different time intervals. Reactions were quenched by addition of a buffer containing 80 % formamide,

2 mM EDTA and 1X TBE buffer, and putting in 95 °C for 5 min. Analysis of polymerase reaction was

performed by polyacrylamide gel electrophoresis. The incorporation rates (V) were calculated based

on the percentage of the extended oligonucleotide in the mixture (P+1 band). The kinetic parameters

(Vmax and KM) were determined by plotting V (nM/min) versus substrate concentration (µM) and fitting

the data to a non-linear Michaelis-Menten regression using GraphPad Prism Software 5.0.

NMR conformation determination

A Bruker Avance II 600 spectrometer equipped with 5 mm TCI HCN Z gradient cryoprobe was used to

measure 3JHH coupling constants and NOE interactions used to determine a predominant

conformation of the five membered ring. Spectra were processed using Topspin (version 2.1, Bruker

Biospin). The 3JHH coupling constants were measured from 1D proton spectra of a spinning sample at

5, 10, 15 and 20 °C, with 64K points over a spectral width of 12000 Hz. Coupling constant data were

used to determine the ring conformation using the MATLAB pseudorotation GUI (Hendrickx and

Martins, 2008). Two hundred milliseconds of T-ROESY (Hwang and Shaka,1992) spectrum with

200ms mixing time was collected with a spectral width of 5400 Hz in F1 and F2 with 2048 complex

points in F2 and 512 increments in F1. Shifted sinebell function was used in both dimensions. Zero

filling was applied to end up with 2048 · 1024 points.

Molecular modelling of a DNA pyrophosphate-phosphonate nucleotide analogues hybrid

duplex

Amber 11 was used in all simulations (Case et al, 2005).

Electrostatic charges. Atomic electrostatic charges of the p5 molecules, to be used in the amber

software package (Case et al, 2005) were calculated from the electrostatic potential at the 6-31G*

level using the package Gamess (Schmidt et al, 1993) and a RESP fitting procedure (Bayly et al,

S33

1993). charges on the bases were restrained to keep the same atomic charges for the nucleosides as

in the amber 94 library (Table S4).

Amber parameters. The force field parameters for amino acids and nucleic acids used in the amber

simulations are those from the ff99bsc0 dataset (Pérez et al, 2007). The phosphonate angle and

bond parameters were taken from Baaden 2001 (Baaden et al, 2001) (see http://ambermd.org/,

contributed parameters).

Model building. Pdb structure 1BNA, a DNA 12-mer duplex with sequence

CGCGAATTCGCG:CGCGAATTCGCG (Drew et al, 1981) was used as reference structure

(DNA:DNA) as well as a template to construct the antiparallel DNA pyrophosphate-phosphonate

nucleotide analogues (pN) duplex pN:DNA, with sequence

pCpGpCpGpApApTpTpCpGpCpG:CGCGAATTCGCG. In the latter, the C5’ and O5’ were switched

In one chain to get a phosphonate:dna duplex.

Molecular dynamics simulations. Solvated molecular dynamics was used to verify the stability of the

hybrid p5:dna duplex as well as a normal DNA:DNA 12-mer. The duplexes pN:DNA and DNA:DNA

were solvated in a truncated octahedron TIP3P water box (Jorgensen et al, 1983). 22 Na+ counter-

ions were added to get an electrostatic neutral system. The water molecules and counter-ions were

then allowed to relax their positions while keeping the solute fixed. Initially, for 20 ps, the systems

were heated up to 300K with constant-T, constant-V conditions while constraining the position of the

solute and using a Langevin temperature equilibration scheme. The molecular dynamics was then

continued for 200 ps at constant T and constant pressure to equilibrate the system further. The

simulation temperature was 300 K. Molecular dynamics simulations were initiated with no restraints,

with periodic boundary conditions, using a cut-off distance of 10 Å for the non-bonded interactions and

the particle-mesh-Ewald method for the summation of the coulombic interactions(Cheatham et al,

1995), MD time step = 0.002 ps. Total simulation times were 20 ns.

Supplemental References

Altona, C. and Sundaralingam, M. (1972) Conformational analysis of the sugar ring in nucleosides

and nucleotides. New description using the concept of pseudorotation. J. Am. Chem. Soc. 94, 8205-

8212.

S34

Baaden, M.; Burgard, M.; Boehme, C. and Wipff, G. (2001) Lanthanide cation binding to a phosphoryl-

calix[4]arene: the importance of solvent and counterions investigated by molecular dynamics and

quantum mechanical simulations. Phys. Chem. Chem. Phys. 3, 1317-1325.

Bayly, C.I.;Cieplak, P.; Cornell, W. and Kollman, P.A. (1993) A Well-Behaved Electrostatic Potential

Based Method Using Charge Restraints For Determining Atom-Centered Charges: The RESP Model.

J. Phys. Chem. 97, 10269-10280.

Case, D.A.; Cheatham T.E. 3rd; Darden T.; Gohlke H.; Luo R.; Merz K.M. Jr.; Onufriev A.; Simmerling

C.; Wang B.and Woods R.J. (2005) The AMBER biomolecular simulation programs. J. Comput. Chem.

26, 1668-1688.

Cheatham, T.E. III; Miller, J.L.; Fox, T.; Darden, T.A.; Kollman, P.A. (1995) Molecular dynamics

simulations on solvated biomolecular systems–The particle mesh Ewald method leads to stable

trajectories of DNA, RNA and proteins. J. Am. Chem. Soc. 117, 4193-4194.

Drew, H.R.; Wing R.M.; Takano T.; Broka C.; Tanaka S.; Itakura K. and Dickerson R.E. (1981)

Structure of a B-DNA dodecamer: conformation and dynamics. Pro. Natl. Acad. Sci. U S A. 78, 2179-

2183.

Hendrickx, P.M. and Martins, J.C. (2008) A user-friendly Matlab program and GUI for the

pseudorotation analysis of saturated five-membered ring systems based on scalar coupling constants.

Chem. Cent. J. Oct 24, 2:20.

Hwang, T.-L. and Shaka, A.J. (1992) Cross relaxation without TOCSY: transverse rotating-frame

Overhauser effect spectroscopy. J. Am. Chem. Soc. 114, 3157-3159.

Jorgensen, W.L.; Chandrasekhar, J.; Madura, J.; Impey, R.W.; Klein, M.L. (1983) Comparison of

simple potential functions for simulating liquid water. J. Chem. Phys. 79, 926-935.

Lavery, R. and Sklenar, H. (1988). The definition of generalized helicoidal parameters and of axis

curvature for irregular nucleic acids. J. Biomol. Struct. Dyn. 6, 63-91.

Pérez, A.; Marchán I.; Svozil D.; Sponer J.; Cheatham T.E. 3rd; Laughton C.A. and Orozco M. (2007)

Refinement of the AMBER force field for nucleic acids. Improving the description of alpha/gamma

conformers. Biophys. J. 92, 3817-3829.

Schmidt, M.W.; Baldridge, K.K.; Boatz, J.A.; Elbert, S.T.; Gordon, M.S.; Jensen, J.H.; Koseki, S.;

Matsunaga, N.; Nguyen, K.A.; Su, S.; Windus, T.L.; Dupuis, M. and Montgomery, J.A. (1993) General

Atomic and Molecular Electronic Structure System. J. Comput. Chem. 14, 1347-1363.

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