Synthesis and biological evaluation of arabinose 5-phosphate mimics modified at position five

Article abstract of DOI:10.1016/j.carres.2014.01.004

A set of new metabolically stable arabinose 5-phosphate analogues possessing phosphate mimetic groups at position 5 was synthesised. Their ability to interact with arabinose 5-phosphate isomerase from Pseudomonas aeruginosa was evaluated by STD-NMR studie

Full text of DOI:10.1016/j.carres.2014.01.004

Accepted Manuscript
Synthesis and biological evaluation of arabinose 5-phosphate mimics modified
at position five
Laura Cipolla, Cristina Airoldi, Paola Sperandeo, Serena Gianera, Alessandra
Polissi, Francesco Nicotra, Luca Gabrielli
PII:
S0008-6215(14)00019-6
DOI:
http://dx.doi.org/10.1016/j.carres.2014.01.004
CAR 6644
Reference:
Carbohydrate Research
To appear in:
Received Date:
Revised Date:
Accepted Date:
13 September 2013
31 December 2013
8 January 2014
Please cite this article as: Cipolla, L., Airoldi, C., Sperandeo, P., Gianera, S., Polissi, A., Nicotra, F., Gabrielli, L.,
Synthesis and biological evaluation of arabinose 5-phosphate mimics modified at position five, Carbohydrate
Research (2014), doi: http://dx.doi.org/10.1016/j.carres.2014.01.004
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Title: Synthesis and biological evaluation of arabinose 5-phosphate mimics modified at
position five.
Laura Cipolla, Cristina Airoldi, Paola Sperandeo, Serena Gianera, Alessandra Polissi, Francesco
Nicotra and Luca Gabrielli*
Department of Biotechnology and Biosciences, Università degli Studi Milano-Bicocca, Piazza della
Scienza 2, Milan, Italy
Corresponding author: Dr. Luca Gabrielli, Department of Biotechnology and Biosciences,
Università degli Studi Milano-Bicocca, Piazza della Scienza 2, Milan, Italy. Tel: +39 0264483460;
fax: +39 02 64483565 E-mail l.gabrielli2@campus.unimib.it
Abstract. A set of new metabolically stable arabinose 5-phosphate analogues possessing phosphate
mimetic groups at position 5 was synthesised. Their ability to interact with arabinose 5-phosphate
isomerase from Pseudomonas aeruginosa was evaluated by STD-NMR studies. The synthesised
compounds were also characterised for their activity in vivo on P. aeruginosa and E. coli strains.
Unfortunately, none of the synthesised compounds was able neither to bind API nor to inhibit
bacterial growth.
Keywords. Arabinose 5-phosphate / sugar isomerase / NMR interaction studies / phosphate
analogues / Pseudomonas aeruginosa
Lipopolysaccharide (LPS) is a complex glycolipid essential for bacterial survival and responsible
of eliciting immune responses in the host. Among the LPS’s glycidic residues, 3-deoxy-D-manno-
octulosonic acid (Kdo) is an essential component of LPSs in all Gram-negative bacteria known to
date,1 therefore its biosynthesis is a significant target for new therapeutics design;2 it has been
shown that the breakdown of Kdo biosynthesis results in an accumulation of Lipid A precursors

inside the cell, affecting bacterial viability.3 Arabinose 5-phosphate isomerase (API) is the first
committed enzyme of the Kdo biosynthetic pathway that catalyses the reversible isomerization of
ribulose 5-phosphate (Ru5P) into arabinose 5-phosphate (A5P). Few API inhibitors have been
designed to date;4,5,6 among them the most effective compounds in vitro were described by
Woodard and co-workers in 2011.57 However, none of the reported inhibitors showed promising
,
activity in vivo, thus the search for potent inhibitors against arabinose 5-phosphate isomerase is still
open. We present herein the design, synthesis and biological evaluation of a set of new A5P
analogues, modified at position 5 by the introduction of sulphur- or carboxylate-based phosphate
mimetic groups (Fig. 1). Previous NMR studies revealed that an acidic group is fundamental for the
binding with the enzyme,8 due to its involvement in salt-bridging with basic residue in the catalytic
pocket.4 Since the phosphate group is known to have poor in vivo stability toward enzymatic
hydrolysis,9 the finding of a good and metabolically resistant phosphate mimetic is a key point for
the design of stable potential inhibitors of API.
Fig. 1. Set of A5P analogues with different acidic group at position 5.

The sulphate derivative 1, the non-isosteric sulphonate 2, the non-isosteric carboxylate 3 and the
malonate analogue 4 (malonate group has been already used as a good phosphate mimetic)10 were
synthesised.
The key intermediate 8 was synthesised from D-arabinose (Scheme 1), by protection at the primary
hydroxyl group with t-butyldiphenylsilyl chloride (TBDPSCl) affording compound 5, and then with
acetone dimethyl acetal, in the presence of catalytic camphorsulphonic acid (CSA) (compound 6).
Benzylation of the hydroxyl group at position 3 (compound 7) and subsequent selective cleavage of
the silyl ether with TBAF, gave compound 8.

Scheme 1. Reagents and conditions. a) TBDPSCl, dry pyridine (4 °C, 75 %); b) DMP, CSA, dry
dichloromethane (4 °C, 76 %); c) BnBr, NaH, dry DMF (93 %); d) TBAF, dry THF (80 %); e)
.
SO3 Py, DMF (90 %); f) H2, Pd(OH)2, EtOH, (quant. yield); g) i: D2O (pH 4, 90 °C), ii: aq. 5 %
NaOH (quant. yield); h) I2, imidazole, PPh3, dry toluene (100 °C, 94 %); i) KSAc, DMF (98 %); j)
MeONa, dry MeOH (86 %); k) TEMPO, NaOCl, KBr, sat aq. NaHCO3 (85 %).
Reaction of intermediate 8 with SO3-Pyridine complex (Scheme 1) as sulphating agent11 in DMF,
followed by hydrogenolysis of the benzyl group to compound 10, and final acetal hydrolysis gave
compound 1. The final deprotection step was performed in a D2O solution in order to follow the
reaction by 1H-NMR.
Mitzunobu reaction of intermediate 8 with iodine, imidazole and triphenylphospine afforded iodide
11 (Scheme 1). Reaction of 11 with a DMF solution of potassium thioacetate to 12, followed by
Zemplèn methanolysis to thiol 13, and oxidation with TEMPO gave sulphonate 14. Final
deprotection steps in usual conditions afforded deprotected sulphonate 2.
On the other hand, direct oxidation of the intermediate 8 with TEMPO12 gave the corresponding
carboxylate derivative 16 (Scheme 2). Hydrogenolysis of the benzyl group to compound 17,
followed by deprotection of the dimethyl acetal as previously described afforded the non-isosteric
carboxylate analogue 3.

Scheme 2. Reagents and conditions. a) TEMPO, NaOCl, KBr, satd aq. NaHCO3 (95 %); b) H2,
Pd(OH)2, EtOH, (quant. yield); c) i: D2O (pH 2-4, rt-90 °C), ii: aq. 5 % NaOH (quant. yield); d)
Tf2O, DTBMP, dichloromethane, -78 °C; e) dibenzyl malonate, LiHMDS, dry THF (50 % over two
steps).
Finally, intermediate 8 was treated with triflic anhydride (Tf2O) and di-tert-butyl methyl pyridine
(DTBMP), obtaining the triflate 18 (Scheme 2).13 The triflate was immediately reacted with
dibenzylmalonate14 and LiHMDS as the base, in dry THF, giving malonate 19. Hydrogenolysis of
benzyl groups to compound 20, followed by acetal hydrolysis gave malonate 4.
Compounds 1-4 were evaluated for their ability to bind to API from Pseudomonas aeruginosa by
STD-NMR spectroscopy as previously described15,7 and to inhibit the growth of P. aeruginosa,
wild type strain E. coli (BW25113) and E. coli mutant strain (AS19).6,16 The absence of small
molecule-derived 1H NMR signals in STD spectra indicates the absence of enzyme–small molecule
associations. Unfortunately, none of the synthesised compounds was able to bind the target enzyme
nor to inhibit bacterial growth.
1. Experimental

1.1. General methods
Solvents were dried over molecular sieves, for at least 24 h prior to use, when required. When dry
conditions were required, the reaction was performed under Ar or N2 atmosphere. Thin-layer
chromatography (TLC) was performed on silica gel 60F254 coated glass plates (Merck) with UV
detection when possible, charring with a conc. H2SO4/EtOH/H2O solution (10:45:45 v/v/v), or with
a solution of (NH4)6Mo7O24 (21 g), Ce(SO4)2 (1 g), conc. H2SO4 (31 mL) in water (500 mL) and
then heating to 110°C for 5 min. Flash column chromatography was performed on silica gel 230-
400 mesh (Merck). Platinum oxide was filtered by Acrodis® Premium 25 mm Syringe Filter, with
0.45 um Nylon membrane. Routine 1H and 13C NMR spectra were recorded at 400 MHz (1H) and at
100.57 MHz (13C) or on a Varian Mercury instrument. Chemical shifts are reported in parts per
million downfield from TMS as an internal standard; J values are given in Hz. Mass spectra were
recorded on a System Applied Biosystems MDS SCIEX instrument (Q TRAP, LC/MS/MS, turbon
ion spray) or on a System Applied Biosystem MDS SCIEX instrument (Q STAR elite nanospray).
ESI full MS were recorded on a Thermo LTQ instrument by direct inlet; relative percentages are
shown in brackets. Elementar analysis (C, H, N) were performed with Perkin-Elmer series II 2400
analyse.
1.2. (3S,4S,5R)-5-(((tert-butyldiphenylsilyl)oxy)methyl)tetrahydrofuran-2,3,4-triol (5). To a
stirred suspension of D-arabinose (1.83 g, 12.24 mmol, 1 equiv) in dry Py (30 mL), TBDPSCl (3.83
mL, 14.9 mmol, 1.2 equiv) was added dropwise at 0 °C under argon atmosphere. Afer 10 min the
reaction was stirred at 4 °C overnight. EtOH was added to quench the reaction and the mixture was
stirred for 15 min. The mixture was concentrated under reduced pressure. The product was purified
by flash column chromatography (5:5, PE:EtOAc) affording 5 (3.547 g, 75 % yield) as a yellow oil
(mixture of α- and β- anomers). NMR and mass data are in agreement with those reported.17
1.3. (3aS,5R,6R,6aS)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-2,2-dimethyltetrahydrofuro[2,3-
d][1,3]dioxol-6-ol (6). To a stirred solution of 5 (3.216 g, 8.278 mmol, 1 equiv) in dry CH2Cl2 (190
mL), 2,2- dimethoxypropane (2.18 mL, 17.79 mmol, 2 equiv) and camphorsulphonic acid (0.3 g,

0.828 mmol, 0.1 equiv) were added at 4 °C under argon atmosphere. The reaction mixture was
stirred overnight at 4 °C. Then Et3N was added to neutralize the reaction and the mixture was
stirred for 15 min. The mixture was concentrated under reduced pressure. The product was purified
by flash column chromatography (8:2, PE:EtOAc) giving 6 (2.69 g, 76 % yield) as a yellow oil.
NMR and mass data are in agreement with those reported.17
1.4. (((3aS,5R,6R,6aS)-6-(benzyloxy)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-
yl)methoxy)(tert-butyl)diphenylsilane (7). To a stirred solution of 6 (1.250 g, 2.919 mmol, 1
equiv) in dry THF (20 mL), benzyl bromide (1.84 mL, 11.676 mmol, 4 equiv) and sodium hydride
60 % (584 mg, 14.595 mmol, 5 equiv) were slowly added portion-wise. The reaction was stirred for
three hours at room temperature and then the mixture was concentrated under reduced pressure. The
product was purified by flash column chromatography (100 % PE →9:1, PE:EtOAc) giving 7 (1.40
g, 93 % yield) as a yellowish oil. 1H NMR (400 MHz, CDCl3) δ 7.62 – 7.54 (m, 5H, H Ph), 7.38 –
7.17 (m, 10H, H Ph), 5.82 (d, J1,2 = 4.1 Hz, 1H, H1), 4.59 (d, J2,1 = 4.1 Hz, 1H, H2), 4.54 (d, J = 1.5
Hz, 2H, CH2 Bn), 4.21 – 4.11 (m, 2H, H3, H4), 3.80 – 3.67 (m, 2H, H5), 1.27 (s, 3H, Me), 1.22 (s,
3H, Me), 0.97 (s, 9H, tBu). 13C NMR (101 MHz, CDCl3) δ 137.77 (1C, Cquat C Ph), 135.93 (1C, C
Ph), 133.50 (1C, Cquat C Ph), 133.38 (1C, Cquat C Ph), 130.09 (1C, C Ph), 130.03 (1C, C Ph), 128.83
(1C, C Ph), 128.18 (1C, C Ph), 128.07 (1C, C Ph), 128.04 (1C, C Ph), 128.02 (1C, C Ph), 112.77
(1C, Cquat), 106.07 (1C, C1), 85.52 (1C, C2), 85,43(1C, C4), 189 83.15 (1C, C3), 71.99 (1C, CH2
Bn), 63.69 (1C, C5), 27.28 (1C, Me), 27.14 (3C, Me tBu), 26.45 (1C, Me), 19.52 (1C, Cquat tBu).
MS: m/z = 541.4 [M+Na]+, 557.4 [M+K]+. C31H38O5Si (518,72): calcd C, 71.78; H, 7.38, found C,
71.69; H, 7.39.
1.5. ((3aS,5R,6R,6aS)-6-(benzyloxy)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-
yl)methanol (8). To a stirred solution of 7 (1.29 g, 2.49 mmol, 1 equiv) in dry THF (20 mL),
tetrabutyl ammonium fluoride (1 M THF solution, 7.47 ml, 7.47 mmol, 3 equiv) was added. The
reaction mixture was stirred for 1 hour and then concentrated under reduced pressure. The product
was purified by flash column chromatography (PE:EtOAc 7:3→6:4) giving 8 (560 mg, 80 % yield).

1H NMR (400 MHz, CDCl3) δ 7.42 – 7.03 (m, 5H, H Ph), 5.83 (d, J1,2 = 3.9 Hz, 1H, H1), 4.60 (d,
J2,1 = 3.9 Hz, 1H, H2), 4.58 – 4.45 (m, 2H, CH2 Bn), 4.15 – 4.08 (m, 1H, H4), 3.89 (d, J = 2.5 Hz,
1H, H3), 3.72 – 3.59 (m, 2H, H5), 2.71 (bs, 1H, OH), 1.44 (s, 3H, Me), 1.25 (s, 3H, Me). 13C NMR
(101 MHz, CDCl3) δ 137.39 (1C, Cquat C Ph), 128.73 (1C, C Ph), 128.20 (1C, C Ph), 127.98 (1C, C
Ph), 113.06 (1C, Cquat), 105.78 (1C, C1), 85.79 (1C, C4), 85.38 (1C, C2), 82.92 (1C, C3), 72.01 (1C,
CH2 Bn), 62.79 (1C, C5), 27.29 (1C, Me), 26.52 (1C, Me). MS: m/z = 303.4 [M+Na]+, 319.3
[M+K]+, 583.3 [2M+Na]+. C15H20O5 (280,32): calcd C, 64.27; H, 7.19; found C, 64.19; H, 7.18.
1.6. Sodium ((3aS,5R,6R,6aS)-6-(benzyloxy)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-
yl)methyl sulfate (9). To a stirred solution of 8 (80 mg, 0.285 mmol, 1 equiv) in dry DMF (3 mL),
SO3-Py (454 mg, 2.854 mmol, 10 equiv) was added. The reaction was stirred for 1 hour and then it
was neutralized adding NaOH 5 % solution. The mixture was extracted 6 times with brine and
EtOAc; the organic phase was dried with Na2SO4, filtered and concentrated under reduced pressure.
The product was purified by flash column chromatography (95:0.5, EtOAc:MeOH) giving 9 (85
mg, 90 % yield). 1H NMR (400 MHz, CD3OD) δ 7.46 – 7.17 (m, 5H, H Ph), 5.89 (d, J1,2 = 3.9 Hz,
1H, H1), 4.69 (d, J2,1 = 3.9 Hz, 1H, H2), 4.60 (s, 2H, CH2 Bn), 4.35 (td, J4,5 = 6.7 Hz, J4,3 = 1.8 Hz,
1H, H4), 4.15 (d, J5,4 = 6.7 Hz, 2H, H5), 4.09 (d, J3,4 = 1.8 Hz, 1H, H3), 1.51 (s, 1H, Me), 1.30 (s,
1H, Me). 13C NMR (101 MHz, CD3OD) δ 138.87 (1C, Cquat Ph), 129.37 (1C, C Ph), 129.00 (1C, C
Ph), 128.79 (1C, C Ph), 113.70 (1C, Cquat), 107.32 (1C, C1), 85.77 (1C, C2), 84.67 (1C, C4), 84.13
(1C, C3), 72.58 (1C, CH2 Bn), 68.46 (1C, C5), 27.18 (1C, Me), 26.23 (1C, Me). MS: m/z = 359.2
[M-H]-. C15H19NaO8S (382,36): calcd C, 47.12; H, 5.01, found C, 47.16; H, 5.02.
1.7. Sodium ((3aS,5R,6R,6aS)-6-(benzyloxy)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-
yl)methyl sulfate (10). Compound 9 (70 mg, 0.212 mmol) was dissolved in MeOH (4 mL).
Palladium hydroxide (70 mg) was added, the mixture was stirred under vacuum in order to remove
the residual gas, and then H2 was added. The reaction was stirred at room temperature for 24 hours
and then the catalyst was filtered on a syringe filter and washed with MeOH. The solvent was
evaporated giving 10 (53 mg, 98 % yield). 1H NMR (400 MHz, CD3OD) δ 5.90 (d, J1,2 = 3.8 Hz,

1H, H1), 4.53 (d, J2,1 = 3.8 Hz, 1H, H2), 4.22 (s, 1H, H3), 4.20 – 4.09 (m, 3H, H4, H5), 1.50 (s, 3H,
Me), 1.29 (s, 3H, Me). 13C NMR (101 MHz, CD3OD) δ 113.51 (1C, Cquat), 107.34 (1C, C1), 87.86
(1C, C2), 87.30 (1C, C4), 76.52 (1C, C3), 68.60 (1C, C5), 27.03 (1C, Me), 26.10 (1C, Me). MS:
m/z = 269.1 [M-H]-. C8H13NaO8S (292,24): calcd. C, 32.88; H, 4.48, found C, 31.91; H, 4.49.
1.8. Sodium ((2R,3S,4S)-3,4,5-trihydroxytetrahydrofuran-2-yl)methyl sulfate (1). Compound
10 (40 mg, 0.137 mmol) was dissolved in D2O (0.7 mL) and the solution at pH 4 was transferred
into an NMR tube. The reaction was followed by 1H-NMR until completion. Then the pH was
neutralized adding NaOH 5 % and the solvent was lyophilised to give 1 (34 mg, quantitative yield).
1H NMR (400 MHz, D2O) δ 5.13 (bs, 1H, H1β), 5.08 (d, J = 2.4 Hz, 1H, H1α), 4.12 – 4.02 (m, 3H,
H5'α, H5'β, H3a), 3.99 – 3.91 (m, 3H, H5''α, H5''β, H2β), 3.90 – 3.81 (m, 3H, H2α, H3β, H4α). 13C
NMR (101 MHz, D2O) δ 100.93 (1C, C1α), 95.19 (1C, C1β), 80.97 (1C, C2α), 80.44 (1C, 3α),
78.62 (1C, C3β), 75.76 (1C, C2β), 75.27 (1C, C4α), 73.92 (1C, C4β), 68.77 (1C, C5β), 67.38 (1C,
C5α). MS: m/z = 229.1 [M-H]-. C5H9NaO8S (252,17): calcd. C, 23.81; H, 3.60, found: C, 23.88; H,
3.59.
1.9. (3aS,5S,6S,6aS)-6-(benzyloxy)-5-(iodomethyl)-2,2-dimethyltetrahydrofuro[2,3-
d][1,3]dioxole (11). To a stirred solution of 8 (600 mg, 2.140 mmol, 1 equiv) in dry toluen (20 mL),
tryphenylphosphine (2.95 g, 10.7 mmol, 5 equiv), imidazole (0.459 mg, 6.42 mmol, 3 equiv) and
then iodine (2.28 g, 8.56 mmol, 4 equiv) were added. The reaction mixture was stirred for 1 hour at
100 °C, then 20 mL of NaHCO3 saturated solution were added. Under vigorous stirring, iodine was
added until the mixture become dark coloured, in order to oxidize the tryphenylphosphine in excess.
Then, iodine in excess was reduced adding a saturated solution of Na2S2O4 (about 50 mL). The
mixture was extracted 3 times with toluen; the organic phase was dried with Na2SO4, filtered and
concentrated under reduced pressure. The product was purified by flash column chromatography
(from 9.5:0.5 to 8:2, PE:EtOAc) giving 11 (784 mg, 94 % yield). 1H NMR (400 MHz, CDCl3) δ
7.79 – 7.27 (m, 5H, H Ph), 5.98 (d, J1,2 = 3.7 Hz, 1H, H1), 4.69 – 4.60 (m, 3H, H2, CH2 Bn), 4.38
(t, J = 7.5 Hz, 1H, H4), 4.15 (s, 1H, H3), 3.40 (d, J = 2.7 Hz, 1H, H5'), 3.38 (s, 1H, H5''), 1.54 (s,

3H, Me), 1.33 (s, 3H, Me). 13C NMR (101 MHz, CDCl3) δ 137.08 (1C, Cquat Ph), 128.55 (1C, C
Ph), 128.47 (1C, C Ph), 128.05 (1C, C Ph), 127.90 (1C, C Ph), 112.70 (1C, Cquat), 106.52 (1C, C1),
85.68 (1C, C4), 84.66 (1C, C2), 83.82 (1C, C3), 71.59 (1C, CH2 Bn), 27.06 (1C, Me), 25.95 (1C,
Me), 6.32 (1C, C5). MS: m/z = 413.2 [M+Na]+, 803.2 [2M+Na]+. C15H19IO4 (390,21): calcd C,
46.17; H, 4.91; found C, 46.09; H, 4.92;
1.10. S-(((3aS,5S,6S,6aS)-6-(benzyloxy)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-
yl)methyl) ethanethioate (12). Compound 11 (530 mg, 1.359 mmol, 1 equiv) was dissolved in dry
DMF (4 mL). Potassium thioacetate (388 mg, 3.398 mmol, 2.5 equiv) was added and the reaction
was stirred at 80 °C for 1 hour. Then the solvent was evaporated under reduced pressure and the
product was purified by flash column chromatography (9:1, PE:EtOAc) giving 12 (450 mg, 98 %
yield) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ 7.45 – 7.09 (m, 5H, H Ph), 5.89 (d, J1,2 = 3.9
Hz, 1H, H1), 4.63 (d, J2,1 = 3.9 Hz, 1H, H2), 4.60 – 4.52 (m, 2H, CH2 Bn), 4.17 (td, J = 7.0, 2.1 Hz,
1H, H4), 3.89 (d, J = 2.1 Hz, 1H, H3), 3.23 (ddd, J = 33.3, 13.7, 7.1 Hz, 2H, H5), 2.33 (s, 3H,
OAc), 1.55 (s, 3H, Me), 1.32 (s, 3H, Me). 13C NMR (101 MHz, CDCl3) δ 192.29 (1C, Cquat CO),
134.51 (1C, Cquat Ph), 125.94 (1C, C Ph), 125.42 (1C, C Ph), 125.28 (1C, C Ph), 110.20 (1C, Cquat),
103.38 (1C, C1), 82.30 (1C, C2), 81.58 (1C, C3), 81.02 (1C, C4), 69.07 (1C, CH2 Bn), 29.38 (1C,
C5), 27.96 (1C, CH3 OAc), 24.41 (1C, Me), 23.51 (1C, Me). MS: m/z = 361.2 [M+Na]+. C17H22O5S
(338,42): calcd: C, 60.33; H, 6.55; found C, 60.25; H, 6.54;
1.11. ((3aS,5S,6S,6aS)-6-(benzyloxy)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-
yl)methanethiol (13). Compound 12 (357 mg, 1.056 mmol, 1 equiv) was dissolved in dry MeOH
(10 mL). Na (2,5 mg, 0.106 mmol, 0.1 equiv) was added and the reaction was stirred for 1 hour.
Then silica gel was added, the solvent was evaporated under reduced pressure and the product was
purified by flash column chromatography (8:2, PE:EtOAc) giving 13 (270 mg, 86 % yield) as a
yellow oil. The corresponding disulfide compound was formed as secondary product (78 mg, 13 %
yield). 1H NMR (400 MHz, CDCl3) δ 7.60 – 6.94 (m, 5H, H Ph), 5.92 (d, J1,2 = 3.9 Hz, 1H, H1),

4.63 (d, J2,1 = 3.9 Hz, 1H, H2), 4.58 (d, J = 3.9 Hz, 2H, CH2 Bn), 4.46 – 4.38 (m, 1H, H4), 4.05 (d, J
= 1.1 Hz, 1H, H3), 3.00 (ddd, J = 22.4, 13.6, 7.3 Hz, 2H, H5), 1.50 (s, 3H, Me), 1.30 (s, 3H, Me).
13C NMR (101 MHz, CDCl3) δ 137.17 (1C, Cquat Ph), 128.54 (1C, C Ph), 128.02 (1C, C Ph), 127.85
(1C, C Ph), 112.72 (1C, Cquat), 105.87 (1C, C1), 86.46 (1C, C4), 84.87 (1C, C2), 83.55 (1C, C3),
71.61 (1C, CH2 Bn), 27.28 (1C, C5), 27.07 (1C, Me), 26.14 (1C, Me). MS: m/z = 319.2 [M+Na]+.
C15H20O4S (296,38): calcd. C, 60.79; H, 6.80, found C, 60.68; H, 6.79.
1.12. Sodium ((3aS,5S,6S,6aS)-6-(benzyloxy)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-
yl)methanesulfonate (14). Compound 13 (128 mg, 0.432 mmol 1 equiv) was suspended in
NaHCO3 saturated solution (1 mL). A 0.1 M TEMPO solution in ACN (864 µL, 0.086 mmol, 0.2
equiv) and a 0.5 M KBr solution (173 µL, 0.086 mmol, 0.2 equiv) were added; then NaOCl 0.35 M
solution (2.5 mL, 0.864 mmol, 2 equiv) was added and the reaction was stirred for 10 minutes.
Then EtOH was added and the solvent was evaporated under reduced pressure; the product was
purified by flash column chromatography (from 100 % EtOAc to 8:2, EtOAc:EtOH) giving 14 (135
mg, 85 % yield). 1H NMR (400 MHz, CD3OD) δ 7.81 – 6.63 (m, 5H, H Ph), 5.89 (d, J1,2 = 3.8 Hz,
1H, H1), 4.69 – 4.57 (m, 4H, H2, H4, CH2 Bn), 4.34 (s, 1H, H3), 3.35 (dd, J5’,5’’ = 13.8, J5’,4 = 8.9
Hz, 1H, H5'), 3.17 (dd, J5’’,5’ = 13.8, J5’’,4 = 4.6 Hz, 1H, H5''), 1.49 (s, 3H, Me), 1.29 (s, 3H, Me).
13C NMR (101 MHz, CD3OD) δ 137.82 (1C, Cquat Ph), 127.96 (1C, C Ph), 127.59 (1C, C Ph),
127.37 (1C, C Ph), 111.92 (1C, Cquat), 106.11 (1C, C1), 84.68 (1C, C3), 84.30 (1C, C2), 81.92 (1C,
C4), 71.25 (1C, CH2 Bn), 54.65 (1C, C5), 25.49 (1C, Me), 24.50 (1C, Me). MS: m/z = 343.3 [M-
H]-. C15H19NaO7S (366,36): calcd C, 49.18; H, 5.23; found C, 49.10; H, 5.22.
1.13. Sodium ((3aS,5S,6S,6aS)-6-hydroxy-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-
yl)methanesulfonate (15). Compound 14 (88 mg, 0.240 mmol) was dissolved in EtOH (3 mL).
Palladium hydroxide (80 mg) was added, the mixture was stirred under vacuum in order to remove
the residual gas and then H2 was added. The reaction was stirred at room temperature for 24 hours
and then the catalyst was filtered on a syringe filter and washed with EtOH. The solvent was
evaporated giving 15 (64 mg, 97 % yield). 1H NMR (400 MHz, CD3OD) δ 5.89 (d, J1,2 = 3.8 Hz,

1H, H1), 4.53 (d, J2,1 = 3.8 Hz, 1H, H2), 4.46 – 4.39 (m, 2H, H3, H4), 3.34 (dd, J5’,5’’ = 14.0, J5’,4
5.7 Hz, 1H, H5'), 3.14 (dd, J5’’,5’ = 14.0, J5’’,4 = 4.9 Hz, 1H, H5''), 1.50 (s, 3H, Me), 1.30 (s, 3H,
Me). 13C NMR (101 MHz, CD3OD) δ 111.89 (1C, Cquat), 105.95 (1C, C1), 86.36 (1C, C2), 84.37
(1C, C4), 77.23 (1C, C3), 54.64 (1C, C5), 25.48 (1C, Me), 24.51 (1C, Me). MS: m/z = 253.1 [M-
H]-. C8H13NaO7S (276,24): calcd C, 34.78; H, 4.74; found. C, 34.83; H, 4.75.
=
1.14. Sodium ((2S,3S,4S)-3,4,5-trihydroxytetrahydrofuran-2-yl)methanesulfonate (2).
Compound 15 (40 mg, 0.145 mmol) was dissolved in D2O (0.7 mL) and the solution at pH 4 was
transferred into an NMR tube and heated at 90 °C for 1 day. The reaction was followed by 1H-NMR
until completion, then the pH was neutralized adding NaOH 5 % and the solvent was lyophilised to
give 2 (33 mg, quantitative yields). 1H NMR (400 MHz, D2O) δ 5.10 (d, J = 3.2 Hz, 1H, H1β), 5.08
(d, J1,2 = 2.2 Hz, 1H, H1α), 4.22 (ddd, J = 7.9, 5.7, 4.6 Hz, 1H, H4α), 3.96 – 3.88 (m, 3H, H4β,
H3β, H2β), 3.85 (dd, J = 4.0, J2,1 = 2.2 Hz, 1H, H2α), 3.83 – 3.77 (m, 1H, H3α), 3.15 – 2.97 (m, 2H,
H5). 13C NMR (101 MHz, D2O) δ 101.09 (1C, C1α), 95.33 (1C, C1β), 80.78 (1C, C2α), 79.08 (1C,
C3α), 78.80 (1C, C4α), 77.44, 76.79, 75.34 (3C, C2β, C3β, C4β), 55.27 (1C, C5β), 53.79 (1C,
C5α). MS: m/z = 213.1 [M-H]-. C5H9NaO7S (236,18): calcd. C 25.43, H 3.84, found C 25.39, H
3.85.
1.15. Sodium (3aS,5S,6S,6aS)-6-(benzyloxy)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxole-5-
carboxylate (16). Compound 8 (133 mg, 0.475 mmol, 1 equiv) was suspended in NaHCO3
saturated solution (1 mL). A 0.1 M TEMPO solution in ACN (950 µL, 0.095 mmol, 0.2 equiv) and
a 0.5 M KBr solution (190 µL, 0.095 mmol, 0.2 equiv) were added; then NaOCl 0.35 M solution
(2.7 mL, 0.949 mmol, 2 equiv) was added and the reaction was stirred for 40 minutes. EtOH was
added, the solvent was evaporated under reduced pressure, and the product was purified by flash
column chromatography (100 % EtOAc) giving 16 (143 mg, 95 % yield). 1H NMR (400 MHz,
CD3OD) δ 7.39 – 7.24 (m, 5H, H Ph), 5.97 (d, J1,2 = 3.6 Hz, 1H, H1), 4.68 (d, J2,1 = 3.6 Hz, 1H,
H2), 4.65 – 4.61 (m, 3H, H4, CH2 Bn), 4.48 (s, 1H, H3), 1.45 (s, 3H, Me), 1.27 (s, 3H, Me). 13C
NMR (101 MHz, CD3OD) δ 177.49 (1 C, C5), 141.38 (1 C, Cquat Ph), 132.03 (1 C, C Ph), 131.59 (1

C, C Ph), 131.51 (1 C, C Ph), 116.63 (1 C, Cquat), 110.54 (1 C, C1), 88.48 (1 C, C3), 87.44 (1 C,
C4), 86.62 (1 C, C2), 75.20 (1C, CH2 Bn), 28.60 (1C, Me), 28.45 (1C, Me). MS: m/z = 293.2 [M-
H]-. C15H17NaO6 (316,28): calcd. C, 56.96; H, 5.42; found. C, 56.88; H, 5.43.
1.16. Sodium (3aS,5S,6S,6aS)-6-hydroxy-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxole-5-
carboxylate (17). Compound 16 (125 mg, 0.395 mmol) was dissolved in MeOH (5 mL). Palladium
hydroxide (100 mg) was added, the mixture was stirred under vacuum in order to remove the
residual gas and then H2 was added. The reaction was stirred at room temperature for 24 hours and
then the catalyst was filtered on a syringe filter and washed with MeOH. The solvent was
evaporated giving 17 (88 mg, quantitative yields). 1H NMR (400 MHz, D2O) δ 5.89 (d, J1,2 = 3.6
Hz, 1H, H1), 4.53 (d, J = 0.5 Hz, 1H, H4), 4.51 (d, J2,1 = 3.6 Hz, 1H, H2), 4.32 (s, 1H, H3), 1.30 (s,
1H, Me), 1.16 (s, 1H, Me). 13C NMR (101 MHz, D2O) δ 174.79 (1 C, C5), 113.15 (1 C, Cquat),
105.74 (1C, C1), 85.87 (1C, C4), 83.97(1C, C2), 76.42 (1C, C3), 24.71 (1C, Me), 24.39 (1C, Me).
MS: m/z = 203.2 [M-H]-. C8H11NaO6 (226,16): calcd. C, 42.49; H, 4.90; found C, 42.52; H, 4.89.
1.17. Sodium (2S,3S,4S)-3,4,5-trihydroxytetrahydrofuran-2-carboxylate (3). Compound 17 (40
mg, 0.177 mmol) was dissolved in D2O (0.7 mL) and the pH 4 solution was heated at 90 °C
overnight into an NMR tube. After reaction’s completion the pH was neutralized adding NaOH 5 %
and the solvent was lyophilised to give 3 (32 mg, quantitative yields). 1H NMR (400 MHz, D2O) δ
5.38 (d, J = 4.3 Hz, 1H, H1β), 5.34 (d, J = 1.3 Hz, 1H, H1α), 4.48 (d, J = 4.2 Hz, 1H, H4), 4.28 (t, J
= 5.8 Hz, 1H, H3β), 4.23 – 4.17 (m, 2H, H3α, H4β), 4.03 (dd, J = 5.8, 4.3 Hz, 1H, H2β), 4.00 (dd, J
= 2.7, 1.8 Hz, 1H, H2α). 13C NMR (101 MHz, D2O) δ 176.25 (1C, C5), 102.08 (1C, C1α), 96.55
(1C, C1β), 82.91 (1C, C4α), 80.12 (1C, C4β), 80.04 (1C, C2α), 78.82 (1C, C3α), 77.30 (1C, C3β),
75.49 (1C, C2β). MS: m/z = 163.1 [M-H]-. C5H7NaO6 (186,10): calcd. C, 32.27; H, 3.79; found. C,
32.22; H, 3.80.
1.18. ((3aS,5R,6R,6aS)-6-(benzyloxy)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-
yl)methyl trifluoromethanesulfonate (18). To a stirred solution of 8 (110 mg, 0.389 mmol, 1
equiv) in dry DCM (4 mL), DTBMP (200 mg, 0.975 mmol, 2.5 equiv) was added. The solution was

cooled at -75 °C and triflic anhydride (132 µL, 0.785 mmol, 2 equiv) was added by syringe; the
temperature was increased to 0 °C and after 30 minutes the reaction mixture was directly loaded on
a silica gel column and quickly filtrated with (6:4, PE:EtOAc, 0.1 % TEA) giving 18. The
compound was used immediately for the next step because of its instability.
1.19. Dibenzyl 2-(((3aS,5R,6R,6aS)-6-(benzyloxy)-2,2-dimethyltetrahydrofuro[2,3-
d][1,3]dioxol-5-yl)methyl)malonate (19). To an -80 °C solution of LiHMDS (2.05 mL, 2.037
mmol, 3 equiv) in dry THF (3.6 mL), dibenzylmalonate (509 µl, 2.037 mmol, 3 equiv) was added
and then the mixture was stirred at -80 °C for 30 minutes. The freshly prepared triflate 18 (239 mg,
0.853 mmol, 1 equiv) was dissolved in dry THF (3.6 mL) and slowly added by syringe to the cold
dibenzylmalonate solution. The temperature was slowly increased to 0 °C for 1 hour, then the
reaction was stirred at 0°C for other 20 minutes, and finally at room temperature for 1.5 hours. The
reaction was quenched adding NH4Cl saturated solution and extracted with EtOAc; the organic
phase was dried with Na2SO4, filtered and concentrated under reduced pressure. The product was
purified by flash column chromatography (8.5:1.5, PE:EtOAc) giving 19 (183 mg, 50 % yield over
two steps) as a light yellow oil. 1H NMR (400 MHz, CDCl3) δ 7.40 – 7.24 (m, 15H, H Ph), 5.87 (d,
J1,2 = 3.9 Hz, 1H, H1), 5.21 (d, J = 12.3 Hz, 1H, H Bn), 5.16 – 5.04 (m, 3H, H Bn), 4.61 (d, J2,1
=
3.9 Hz, 1H, H2), 4.57 – 4.47 (m, 2H, H Bn), 4.21 – 4.13 (m, 1H, H4), 3.88 – 3.76 (m, 2H, H3, H6),
2.46 – 2.30 (m, 1H, H5’), 2.28 – 2.19 (m, 1H, H5”),, 1.43 (s, 3H, Me), 1.30 (s, 3H, Me). 13C NMR
(101 MHz, CDCl3) δ 169.22 (1C, Cquat CO), 168.85 (1C, Cquat CO), 137.15 (1C, Cquat Ph), 135.42
(1C, Cquat Ph), 135.34 (1C, Cquat Ph), 128.65 (1C, C Ph), 128.62 (1C, C Ph), 128.40 (1C, C Ph),
128.39 (1C, C Ph), 128.29 (1C, C Ph), 128.25 (1C, C Ph), 128.06 (1C, C Ph), 127.86 (1C, C Ph),
127.73 (1C, C Ph), 127.08 (1C, C Ph), 112.70 (1C, Cquat), 105.95 (1C, C1), 85.53 (1C, C3), 84.85
(1C, C2), 82.10 (1C, C4), 71.64 (1C, CH2 Bn), 67.33 (2C, CH2 Bn), 48.74 (1C, C6), 32.98 (1C,
C5), 26.74 (1C, Me), 26.18 (1C, Me). MS: m/z = 569.4 [M+Na]+. C32H34O8 (546,61): calcd. C,
70.31; H, 6.27; found. C, 70.25; H, 6.26.

1.20. 2-(((3aS,5R,6R,6aS)-6-hydroxy-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-
yl)methyl)malonic acid (20). Compound 19 (119 mg, 0.218 mmol) was dissolved in EtOH (4 mL).
Palladium hydroxide (100 mg) was added, the mixture was stirred under vacuum in order to remove
the residual gas and then H2 was added. The reaction was stirred at room temperature for 24 hours
and then the catalyst was filtered on a syringe filter and washed with MeOH. The solvent was
evaporated giving 20 (59 mg, quantitative yields). 1H NMR (400 MHz, CD3OD) δ 5.88 (d, J1,2 = 3.7
Hz, 1H, H1), 4.50 (d, J2,1 = 3.7 Hz, 1H, H2), 4.07 – 3.96 (m, 2H, H3, H4), 3.59 (dd, J6,5 = 10.4, 4.3
Hz, 1H, H6), 2.38 – 2.26 (m, 1H, H5’), 2.20 – 2.07 (m, 1H, H5’’), 1.49 (s, 3H, Me), 1.29 (s, 3H,
Me). 13C NMR (101 MHz, CD3OD) δ 173.18 (1C, Cquat CO), 172.76 (1C, Cquat CO), 113.26 (1C,
Cquat), 107.41 (1C, C1), 88.10 (1C, C2), 86.98 (1C, C4), 79.31 (1C, C3), 49.66 (1C, C6) 33.95 (1C,
C5), 26.74 (1C, Me), 26.00 (1C, Me). MS: m/z = 275.2 [M-H]-. C11H16O8 (276,24): calcd. C, 47.83;
H, 5.84; found C, 47.79; H, 5.85.
1.21. Sodium 2-(((2R,3S,4S)-3,4,5-trihydroxytetrahydrofuran-2-yl)methyl)malonate (4).
Compound 20 (40 mg, 0.157 mmol) was dissolved in D2O (0.7 mL) and the solution was
transferred into an NMR tube. The reaction was checked by 1H-NMR and the pH 2 solution was
reacted for 48 hours at room temperature. Then the pH was neutralized adding NaOH 5 % and the
solvent was lyophilised to give 4 (33 mg, 99 %). 1H NMR (400 MHz, D2O) δ 5.04 (d, J = 4.6 Hz,
1Hβ), 5.01 (d, J = 2.5 Hz, 1Hα), 3.86 – 3.74 (m, 3H, H2α, H2β, H4), 3.64 (dd, J = 6.2, 4.6 Hz, 1H,
H3), 3.52 – 3.47 (m, 1H, H6), 2.00 – 1.91 (m, 1H, H5’), 1.85 – 1.75 (m, 1H, H5’’). 13C NMR (101
MHz, D2O) δ 178.80 (1C, Cquat), 178.66 (1C, Cquat), 100.61 (1C, C1α), 94.84 (1C, C1β), 81.63 (1C,
C2α), 79.58 (1C, C3), 79.54 (1C, C6), 78.00 (1C, C2β), 76.11 (1C, C4), 35.62 (1C, C5β), 33.87
(1C, C5β). MS: m/z = 235.1 [M-H]-, 257.1 [M-2H+Na]-. C8H12O8 (236,18): calcd. C, 40.68; H,
5.12; found. C, 40.60; H, 5.11.
Acknowledgements
This project was funded by Fondazione Cariplo (Grant n° 2010-0653), Regione Lombardia

“Cooperazione scientifica e tecnologica internazionale” (Grant 16876 SAL-18), MIUR-Regione
Lombardia (ID 30190679), Fondazione Banca del Monte di Lombardia, and MIUR under project
PRIN2008/24M2HX
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HIGHLIGHTS
We designed and synthesized a set of new Ara-5P analogues, modified at position 5.
We tested the affinity of the synthesised compounds by STD-NMR spectroscopy studies.
The synthesised compounds were studied in vivo on P. aeruginosa and E. coli strains.
None of the compounds was active, either in vivo or on isolated API.
Best regards,
Luca Gabrielli