Synthesis of alkylcarbonate analogs of O-acetyl-ADP-ribose

Article abstract of DOI:10.1039/c3ob41016a

The non-hydrolyzable alkylcarbonate analogs of O-acetyl-ADP-ribose have been synthesized from the phosphorylated ribose derivatives after coupling with AMP morpholidate promoted by mechanical grinding. The analogs were assessed for their ability to inhibi

Full text of DOI:10.1039/c3ob41016a

Organic &
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Synthesis of alkylcarbonate analogs of
O-acetyl-ADP-ribose†
Cite this: Org. Biomol. Chem., 2013, 11,
5702
Marcela Dvorakova,a,b Radim Nencka,c Milan Dejmek,c Eva Zbornikova,c
Anna Brezinova,c Marie Pribylova,a Radek Pohl,c Marie E. Migaudd and
Tomas Vanek*a
Received 15th May 2013,
Accepted 28th June 2013
The non-hydrolyzable alkylcarbonate analogs of O-acetyl-ADP-ribose have been synthesized from the
phosphorylated ribose derivatives after coupling with AMP morpholidate promoted by mechanical grind-
ing. The analogs were assessed for their ability to inhibit the human sirtuin homolog SIRT1.
DOI: 10.1039/c3ob41016a
www.rsc.org/obc
H2A1.1 protein, which has been involved in transcriptional
regulation.7
Introduction
O-Acetyl-adenosine diphosphoribose (OAADPR) is the most
Further studies of OAADPR targets are restricted by its
common metabolite generated by nicotinamide adenine di- limited availability and susceptibility to enzymatic hydrolysis.
nucleotide (NAD+)-dependent histone deacetylases called sir- OAADPR is a substrate for NUDIX hydrolases, which hydrolyse
tuins. Sirtuins play a key role in gene silencing and the the pyrophosphate linkage,7 and for a range of esterases, such
regulation of metabolism and lifespan extension.1 Among as ARH hydrolases and macrodomain proteins, hydrolysing
histone deacetylases, they utilize a unique mechanism for the acetyl functionality to generate ADP-riboside.8
protein deacetylation by coupling it with a stoichiometric con-
In order to overcome the extreme instability of the acetyl
sumption of NAD+.2 The product of this enzymatic reaction, moiety, a range of alkylcarbonate analogs of OAADPR were syn-
2′-O-acetyl-ADP-ribose, isomerizes rapidly to form a 1 : 1 mixture thesized. The alkylcarbonate functionality was selected due to
of 2′-O-acetyl and 3′-O-acetyl-ADP-ribose regioisomers.3
its enhanced stability when compared to esters and its lower
The role of OAADPR in biological processes remains largely susceptibility to acyl migrations, which have been reported for
unknown. It is hypothesized that OAADPR may serve as a acetate esters on carbohydrates.9 Methyl, ethyl and isobutylcar-
second messenger, in a similar manner to structurally related bonates were chosen as structural mimics not only of the
molecules such as ADPR. From
a few studies already acetyl moiety of OAADPR, but also of the more recently con-
implemented, OAADPR appears to positively regulate the firmed products of protein deacylations, which are the propio-
assembly of the Sir complex4 and the opening of TRPM2 (tran- nyl and butyryl derivatives of ADP-riboses.10 The synthesized
sient receptor potential channel 2) calcium channel.5 In derivatives were then assessed for their ability to inhibit the
addition, it caused a delay in oocyte and blastomere matu- SIRT1 sirtuin homolog.
ration.6 OAADPR is also known to bind the macro domain of
Results and discussion
Synthetic strategy
aInstitute of Experimental Botany, Academy of Sciences of Czech Republic, v.v.i.,
Rozvojova 263, Prague 6, 165 02, Czech Republic. E-mail: vanek@ueb.cas.cz,
The synthesis of alkylcarbonate OAADPR analogs started from
1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranoside (1) which was
reacted with benzyl alcohol under Lewis acid catalysis to afford
1-O-benzyl-2,3,5-tri-O-benzoyl-β-D-ribofuranoside (2).
1-O-Benzyl-β-D-ribofuranoside (3) was obtained after depro-
tection of benzoyl groups in 2 under Zemplén conditions. The
free primary position on this ribofuranoside 3 was then regio-
dvorakova@ueb.cas.cz, pribylova@ueb.cas.cz; Fax: +420 225106832;
Tel: +420 225106804
bFaculty of Natural Sciences, Charles University in Prague, Albertov 6, Prague 2,
Czech Republic
cInstitute of Organic Chemistry and Biochemistry, Academy of Sciences of Czech
Republic, v.v.i., Flemingovo nam. 2, Prague 6, 166 10, Czech Republic.
E-mail: radim.nencka@uochb.cas.cz, dejmek@uochb.cas.cz,
zbornikova@uochb.cas.cz, anna.brezinova@uochb.cas.cz, pohl@uochb.cas.cz
dSchool of Pharmacy, Queen’s University Belfast, Medical Biology Centre, 97 Lisburn
selectively protected with
a tert-butyldimethylsilyl group
Road, Belfast, Northern Ireland, UK BT9 7BL. Tel: +44 (0)2890 9726 89;
E-mail: m.migaud@qub.ac.uk
(TBDMS) to give the ribofuranoside 4 (Scheme 1).
Bis(alkylcarbonates) 5a–c were prepared from the corres-
ponding chloroformates under basic catalysis utilizing DMAP
†Electronic supplementary information (ESI) available: Copies of 1H-NMR and
13C-NMR spectra of all new compounds. See DOI: 10.1039/c3ob41016a
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Scheme 1 (a) BnOH, SnCl4, DCM, 0 °C, 2 h, quant.; (b) MeONa–MeOH, 12 h,
100%; (c) TBDMSCl, DMAP, pyridine, DCM, 0 °C, 18 h, 75%.
Table 1 Synthesis of alkylcarbonate derivatives 5a–d
Scheme 2 (a) CAN, MeCN–H2O (9/1), 3 h, 86–96%; (b) (i) (BnO)2PN(iPr)2, 2,4-
DNP, DCM, 12 h, (ii) tBuOOH, 2 h, 68–96%; (c) TFA–H2O (4/1), 0 °C, 15 min,
67%.
N,N-diisopropyl phosphoramidite reagent prepared from phos-
phorus trichloride.13 This benzyl protected phosphorus
reagent was selected due to the possibility of concomitant
removal of all benzyls from the carbohydrate derivatives 9a–c
in one step, affording the free phosphate derivatives for sub-
sequent coupling with AMP morpholidate.
Entry
1
Reagent
Product (product yield)a
MeOCOCl
The phosphorylation step proceeded smoothly to give all
the phosphorylated derivatives 9a–c and 10a–c in high yields
(Scheme 2). However, the removal of the protecting groups
proved to be challenging. Firstly, the isopropylidene group
deprotection in derivatives 10a–c was accomplished using
TFA–H2O (4/1). This optimized method prevented the cleavage
of the alkylcarbonate moiety that occurs under acidic
conditions; however, it did not forestall its migration with
concomitant alkoxy group cleavage. Therefore, from all the
derivatives 10a–c, only the 2,3-O-cyclic carbonate product 9d
was formed. Such a migration could be explained due to
similar electronic and steric properties of 2-OH and 3-OH
groups in ribosides and because a more structurally rigid con-
formation of riboside derivatives is generated.
2
3
EtOCOCl
(CH3)2CHCH2COCl
4
(Bu)2SnO
TBABr
MeOCOCl
Secondly, the removal of benzyl groups in derivatives 9a–d
was carried out. The reaction was performed in an autoclave
under 10 bars hydrogen pressure in various solvents, as using
only balloon pressure was found to be insufficient (Table 2).
When THF was utilized to hydrogenolyse derivatives 9a–b, the
a Yield after column chromatography.
and pyridine (Table 1, entries 1–3).11 Monosubstituted deriva- anomeric benzyl group was not deprotected even after pro-
tives could not be prepared in this way as an excess of chloro- longed reaction times and products 12a–b were formed. The
formate reagent was necessary to ensure any product use of methanol to hydrogenate derivative 9c, on the other
formation. Other attempts to obtain monosubstituted products hand, deprotected the anomeric benzyl group, but a partial
through either the stannylene acetal methodology (Table 1, methylation of this position occurred to give a 1 : 1 mixture of
entry 4)12 or using 3-O-alkylcarbonate-1,2,5-O-protected ribo- 13c : 14c as revealed by HPLC-MS (Table 2). The use of a THF–
sides 6a–c (Scheme 2)11 were unsuccessful due to rapid car- water mixture finally afforded the desired products 11d and
bonate migration with a concomitant methoxy group cleavage 13a–c. However, the dialkylcarbonate products 13a–c were
which readily afforded the 2,3-O-cyclic carbonate derivatives 5d highly unstable and a spontaneous migration of 2-O-alkylcar-
and 9d. The deprotection of the TBDMS group in 5a–c and bonate moiety occurred to form 1,2-O-cyclic carbonate deriva-
6a–c was accomplished using a rather unusual reagent, cerium tives 15a–c (Table 2). This is probably caused by the high
ammonium nitrite (CAN), which has been found to avoid reactivity of the anomeric position under either acidic or basic
acetyl migration in furanosides (Scheme 2).9 Similarly, this conditions; therefore once this position is deprotected, it is
reagent prevented the tendency of alkylcarbonates to migrate susceptible to the attack of the adjacent alkylcarbonate moiety
from secondary to primary positions within the ribofuranoside to form the stable cyclic carbonate.
scaffold.11
For the final step of the pyrophosphate formation, the
The resulting carbohydrate derivatives 7a–c and 8a–c were phosphomorpholidate methodology developed by Moffatt and
suitable for their direct phosphorylation with bis(benzyloxy)- Khorana (1959) was employed.14 This methodology for the
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Table 2 Hydrogenation of disubstituted phosphate derivatives 9a–d on 10% Pd/C at 10 bars hydrogen pressure
Substrate
Solvent
THF
Reaction time
24 or 48 h
Product (product yield)a, product ratiob
THF
24 or 48 h
MeOH
48 h
THF–H2O
THF–H2O
48 h
48 h
THF–H2O
THF–H2O
48 h
48 h
a Isolated product yield without purification. b According to HPLC-MS.
synthesis of unsymmetrical pyrophosphates, based on the acti- This novel, environmentally friendly procedure significantly
vation of one of the two coupling substrates, prevents the for- improved the yield of the reaction.
mation of symmetrical diphosphate side-products, even
though the strictly anhydrous conditions and long reaction coupled with AMP morpholidate in a ball mill in the presence
times negatively influence the yield. of 1H-tetrazole and hexahydrate of magnesium chloride
Thus the phosphate derivatives 11d, 12b, and 15a–c were
Recently, a further improvement of the phosphomorpholi- according to the reported procedure.15 The corresponding pyro-
date methodology was reported by Ravalico et al. (2011),15 phosphate analogs of OAADPR 13d and 16a–c and 1′′-O-
who performed the reaction in a ball mill without the use benzyl-2′′,3′′-O-diethylcarbonate pyrophosphate 17b were gen-
of a solvent, but in the presence of 6 equivalents of water. erated in 45–68% yields (Table 3).
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Inhibitory activity of the prepared pyrophosphates
The inhibitory activity of pyrophosphates 13d + 13e, 16a–c and
17b towards the human sirtuin homolog, SIRT1, was tested
using a commercially available assay kit. The prepared pyro-
phosphates as structural mimics of the sirtuin enzymatic
product, OAADPR, were expected to act as nonselective com-
petitive inhibitors of sirtuins. Therefore, the SIRT1 assay has
been chosen to test their inhibitory activity as it is the most
commonly used assay and offers a broad spectrum of com-
pounds for the comparison of activities. The best inhibitory
activity was displayed by compounds 16b, 16c and 17b, which
was quite surprising as these compounds possess a longer
alkyl chain and in the case of compound 17b also the benzyl
protecting group on 1′′-OH. The IC50 values of these three com-
pounds were 53 μM for compound 17b, 56 μM for compound
16b and 65 μM for compound 16c (Fig. 1). Even though the
inhibitory activity of these compounds was in the μM range, it
is still about one order of magnitude higher than the potency
of the known sirtuin inhibitor, Suramin, which was used as a
control in these experiments and shown to have an IC50 value
of 5.8 μM under these conditions. From these results, we can
conclude that the rigidity of the northern ribose moiety caused
by the presence of cyclic carbonate prevents the compounds
from better binding to the binding site of the enzyme, and
that the best alkyl group fitting into the binding pocket is
ethyl.
Fig. 1 Inhibitory activity of compounds 13d + 13e, 16a–c and 17b.
Table 3 Ball mill coupling: AMP morpholidate, MgCl2·6H2O, 1H-tetrazole,
water
Reactant
Product (product yield)a
Conclusions
3′′-O-Methyl, ethyl and isobutylcarbonate-1′′,2′′-O-cyclic carbo-
nate-ADP-ribose derivatives 16a–c and 2′′,3′′-O-cyclic carbonate-
ADP-ribose derivative 13d were synthesized and their inhibi-
tory activity against the human sirtuin homolog, SIRT1, was
assessed and compared with the inhibitory activity of a pre-
pared 1′′-O-benzyl-2′′,3′′-O-ethylcarbonate-ADP-ribose derivative
17b and a known SIRT1 inhibitor, Suramin. The best inhibi-
tory activity was displayed by compound 17b followed by 16b
and 16c. The IC50 values of these three compounds were
53 μM (17b), 56 μM (16b) and 65 μM (16c). Even though the
inhibitory activity of these compounds was in the μM range, it
was still one order of magnitude higher than the activity of
Suramin (IC50 = 5.8 μM). From these results, it seems that the
rigidity of the northern ribose moiety caused by the presence
of cyclic carbonate prevents the compounds from better
binding to the binding site of the enzyme.
Experimental
General
All reactions requiring anhydrous or inert conditions were
carried out under a positive atmosphere of argon in oven-dried
glassware. Solutions or liquids were introduced in round
bottom flasks using oven dried syringes through rubber septa.
All reactions were stirred magnetically using Teflon-coated
a Yield after preparative HPLC.
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stirring bars. If needed, reactions were warmed up using an solution of NaHCO3 (80 mL), the aqueous layer was extracted
electrically heated silicon oil bath, and the stated temperature with DCM (3 × 50 mL), and the combined organic layers were
corresponds to the temperature of the bath. When reactions washed with water and brine, dried, filtered and evaporated.
required cooling to −78 °C, a dry ice/acetone bath was used.
Purification by chromatography (7/3, v/v, hexane–EtOAc)
Organic solutions obtained after aqueous work-up were dried gave 1-O-benzyl-2,3,5-tri-O-benzoyl-β-D-ribofuranoside 2 (1.10 g,
over MgSO4. The removal of solvents was accomplished using quant.) as a white solid.
a rotary evaporator at water aspirator pressure.
m.p.: 68–69 °C; [α]2D0 = −16.1° (c 1.27, CHCl3); Anal. calcd
1
Chemicals were purchased from Sigma-Aldrich Chemical for C20H21O9P: C 71.73, H 5.11; found: C 71.64, H 5.50%; H
Company. Technical grade solvents for extractions and chrom- NMR (CDCl3): δ 8.03 (4H, tddd, J = 15.5, 8.4, 3.2, 1.2 Hz, Ar),
atography were purchased from Penta Chemicals s.r.o., Czech 7.88 (2H, tdd, J = 8.4, 3.2, 1.2 Hz, Ar), 7.57 (1H, tt, J = 7.9, 1.3 Hz,
Republic. Solvents used in reactions were distilled from appro- Ar), 7.50 (2H, tt, J = 7.8, 1.3 Hz, Ar), 7.42 (2H, t, J = 7.8 Hz,
priate drying agents and stored under argon over activated Ar), 7.33–7.30 (9H, m, Ar), 5.93 (1H, dd, J = 6.8, 4.7 Hz, H-3),
Linde 4 Å molecular sieves. Column and flash chromatography 5.77 (1H, d, J = 4.7 Hz, H-2), 5.34 (1H, s, H-1), 4.82 (1H, d, J =
was carried out using Merck Silica (60–200 μm) or Merck 11.7 Hz, CH2Ph), 4.77–4.71 (2H, m, H-4, H-5), 4.60 (1H, d, J =
neutral Alumina 90 (63–200 μm). Analytical TLC was per- 11.7 Hz, CH2Ph), 4.56 (1H, dd, J = 11.8, 5.0 Hz, H-5); 13C NMR
formed with Merck Silica gel 60 F254 plates. Visualisation was (CDCl3): δ 166.2 (CO), 165.3 (CO), 165.2 (CO), 136.8 (Ar), 133.5
accomplished by UV-light (254 nm) and staining with a vanil- (Ar), 133.4 (Ar), 133.1 (Ar), 129.8 (Ar), 129.8 (Ar), 129.7 (Ar),
lin solution, followed by heating. IR spectra were recorded 128.5 (Ar), 128.3 (Ar), 128.0 (Ar), 127.9 (Ar), 104.5 (C-1), 79.1
using a Nicolet 6700 FT-IR spectrometer. 1H, 13C and 2D (C-4), 75.6 (C-2), 72.4 (C-3), 69.7 (CH2Ph), 64.7 (C-5); HRMS m/z
(H-COSY, HMQC) NMR spectra were all recorded using Bruker 575.1691 (calc. for C33H28O8Na ([M + Na]+): 575.1682).
DPX 400 MHz. Trimethylsilane (0 ppm, 1H NMR) and CDCl3
1-O-Benzyl-β-D-ribofuranoside
3. 2,3,5-Tri-O-benzoyl-1-O-
(77 ppm, 13C NMR) were used as internal references. The benzyl-β-D-ribofuranoside 2 (400 mg, 0.75 mmol) was dissolved
chemical shifts (δ) are reported in ppm (parts per millions) in MeOH and a catalytic amount of a MeONa–MeOH solution
and the coupling constants (J values) are recorded in Hz (25% w.t., 0.1 mL) was added. The reaction mixture was stirred
(hertz). The hydrogen and carbon assignments were done overnight. The reaction was quenched with DOWEX H+ resin
according to 2D experiments (1H–1H COSY and 1H–13C until neutral pH, filtered and evaporated.
HMQC). Mass spectra were recorded using an LTQ Orbitrap XL
The residue was dissolved in water (20 mL) and washed
spectrometer. Optical rotations were measured using a Perkin- with hexane (3 × 10 mL) and the aqueous layer was freeze-
Elmer 341 polarimeter at 589 nm. Melting points were dried to give pure 1-O-benzyl-β-D-ribofuranoside 3 (184 mg,
recorded using a Büchi Melting Point apparatus B-540. The quant.) as a white solid.
elementary analyses were performed using a Perkin-Elmer
2400 Series II CHNS/O analyzer.
m.p.: 95–96 °C; [α]2D0 = −62.8° (c 1.34, H2O); Anal. calcd for
C20H21O9P: C 59.99, H 6.71; found: C 59.26, H 6.41%; 1H NMR
(D2O): δ 7.42–7.35 (5H, m, Ar), 5.08 (1H, s, H-1), 4.78–4.75 (1H,
m, CH2Ph), 4.53 (1H, d, J = 11.2 Hz, CH2Ph), 4.16 (1H, dd, J =
SIRT1 enzyme inhibitory assays
For the evaluation of the inhibitory activity on SIRT1 enzyme, 6.7, 4.6 Hz, H-3), 4.03 (1H, br d, J = 4.6 Hz, H-2), 4.00 (1H, dd,
a commercially available assay kit from Enzo Life Sciences was J = 6.7, 3.3 Hz, H-4), 3.77 (1H, dd, J = 12.3, 3.3 Hz, H-5a), 3.59
used. For the reaction were used: 1 U per well of SIRT1 enzyme (1H, dd, J = 12.3, 6.7 Hz, H-5b); 13C NMR (D2O): δ 137.0 (Ar),
(1 U = 1 pmol min−1 at 37 °C), 25 μM NAD+ (Km = 558 μM, 129.2 (Ar), 129.0 (Ar), 128.8 (Ar), 106.6 (C-1), 83.2 (C-4), 74.7
Vmax = 1863 AFU min−1), 25 μM Fluor de Lys® peptide substrate (C-2), 71.3 (C-3), 70.2 (CH2Ph), 63.3 (C-5); HRMS m/z 239.0879
(Km = 64 μM, Vmax = 1107 AFU min−1), reaction time 45 min at (calc. for C12H17O5 ([M + H]+): 239.0919).
37 °C, then 200 μM Fluor de Lys® developer and 40 μM NAD
were added. The results were measured after 15 min of incu-
1-O-Benzyl-5-O-tert-butyldimethylsilyl-β-D-ribofuranoside 4.
solution of 1-O-benzyl-β-D-ribofuranoside (300 mg,
A
3
bation at 37 °C using a Tecan microplate fluorimeter at an exci- 1.24 mmol) and DMAP (31.0 mg, 0.025 mmol) in dry pyridine
tation wavelength of 360 nm and detection of emitted light at (20 mL) was cooled down to 0 °C under an argon atmosphere.
460 nm. The test was repeated twice, and the compounds were To this solution was added a solution of TBDMSCl in dry DCM
assessed in triplicate. No slow-onset inhibition was observed (10 mL) in three portions by a syringe over 6 h at 0 °C. The
for any of the tested compounds.
reaction mixture was stirred overnight and allowed to warm up
to room temperature. The reaction mixture was then evapor-
ated and the residue was dissolved in DCM, washed with a
Synthesis
2,3,5-Tri-O-benzoyl-1-O-benzyl-β-D-ribofuranoside 2.16 Dry solution of CuSO4, NaHCO3 and brine, dried and evaporated.
BnOH (266 μL, 2.57 mmol) was added to a solution of 1-O-
Purification by flash chromatography (gradient from 7/1 to
acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranoside 1 (1.00 g, 1.98 mmol) 4/1, v/v, hexane–EtOAc) gave 1-O-benzyl-5-O-tert-butyldimethyl-
in dry DCM at 0 °C, followed by addition of SnCl4 (232 μL, silyl-β-D-ribofuranoside 4 (307 mg, 85%) as a colorless oil.
1.98 mmol). The reaction mixture was stirred at 0 °C for 2 hours By column chromatography using a gradient from 100/0 to
and then it was allowed to warm up to room temperature while 95/5, v/v, CHCl3–EtOH, α- and β-anomers could be separated:
stirring overnight. The reaction was quenched with a saturated α-anomer: 18 mg (5%), β-anomer: 271 mg (75%).
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α-Anomer: [α]2D0 = +68.5° (c 0.36, CHCl3); 1H NMR (CDCl3): δ
1-O-Benzyl-2,3-O-bis(ethylcarbonate)-5-O-tert-butyldimethyl-
7.38–7.30 (5H, m, Ar), 5.12 (1H, d, J = 4.4 Hz, H-1), 4.85 (1H, d, silyl-β-D-ribofuranoside 5b. For the reaction, 270 mg
J = 11.8 Hz, CH2Ph), 4.58 (1H, d, J = 11.8 Hz, CH2Ph), 4.15–4.10 (0.76 mmol) of ribofuranoside 4 and ethyl chloroformate
(2H, m, H-2, H-4), 4.00 (1H, ddd, J = 8.6, 6.2, 2.4 Hz, H-3), 3.77 reagent was used.
(1H, dd, J = 11.2, 3.2 Hz, H-5a), 3.72 (1H, dd, J = 11.2, 3.2 Hz,
Purification by flash chromatography (isocratic 9/1, v/v,
H-5b), 2.94 (1H, d, J = 9.8 Hz, OH), 2.61 (1H, d, J = 8.7 Hz, OH), hexane–EtOAc) gave 1-O-benzyl-2,3-O-bis(ethylcarbonate)-5-O-
0.89 (9H, s, SiC(CH3)3), 0.06 (3H, s, Si(CH3)2), 0.05 (3H, s, Si- tert-butyldimethylsilyl-β-D-ribofuranoside 5b (287 mg, 76%) as
(CH3)2); 13C NMR (CDCl3): δ 137.2 (Ar), 128.5 (Ar), 127.9 (Ar), a colorless oil.
101.1 (C-1), 85.9 (C-4), 72.1 (C-2), 71.3 (C-3), 69.6 (CH2Ph), 63.4
[α]2D0 = −34.5° (c 0.67, CHCl3); IR: νmax(CHCl3): 3029, 2956,
(C-5), 25.9 (SiC(CH3)3), 18.3 (SiC(CH3)3), −5.4 (Si(CH3)2), −5.5 2929, 2858, 1756 (CO), 1464, 1373, 1282, 1094, 1007, 950, 838,
(Si(CH3)2); HRMS m/z 377.17543 (calc. for C18H30O5NaSi 699; 1H NMR (CDCl3): δ 7.40–7.27 (5H, m, Ar), 5.33 (1H, dd, J =
([M + Na]+): 377.17547).
6.2, 4.8 Hz, H-3), 5.19 (1H, dd, J = 4.8, 1.5 Hz, H-2), 5.15 (1H,
β-Anomer: [α]D20 = −64.2° (c 0.34, CHCl3); 1H NMR (CDCl3): δ d, J = 1.5 Hz, H-1), 4.77 (1H, d, J = 11.9 Hz, CH2Ph), 4.53 (1H,
7.36–7.28 (5H, m, Ar), 5.03 (1H, s, H-1), 4.73 (1H, d, J = d, J = 11.9 Hz, CH2Ph), 4.25–4.17 (5H, m, H-4, CH2CH3), 3.78
11.8 Hz, CH2Ph), 4.48 (1H, d, J = 11.8 Hz, CH2Ph), 4.31 (1H, td, (2H, dd, J = 4.4, 3.9 Hz, H-5), 1.30 (6H, t, J = 7.1 Hz, CH2CH3),
J = 5.5, 4.7 Hz, H-3), 4.12 (1H, br d, J = 4.0 Hz, H-2), 4.00 (1H, dt, 0.88 (9H, s, SiC(CH3)3), 0.06 (3H, s, Si(CH3)2), 0.05 (3H, s, Si-
J = 6.5, 5.2 Hz, H-4), 3.85 (1H, dd, J = 10.2, 5.2 Hz, H-5a), 3.69 (CH3)2); 13C NMR (CDCl3): δ 154.1 (CO), 154.0 (CO), 137.0 (Ar),
(1H, dd, J = 10.2, 6.5 Hz, H-5b), 2.71 (1H, br s, OH), 2.45 (1H, 128.4 (Ar), 127.9 (Ar), 127.8 (Ar), 103.7 (C-1), 80.9 (C-4), 77.8
d, J = 4.3 Hz, OH), 0.90 (9H, s, SiC(CH3)3), 0.09 (6H, s, Si- (C-2), 75.1 (C-3), 69.4 (CH2Ph), 64.5 (CH2CH3), 63.8 (C-5), 25.8
(CH3)2); 13C NMR (CDCl3): δ 137.5 (Ar), 128.4 (Ar), 127.9 (Ar), (SiC(CH3)3), 18.2 (SiC(CH3)3), 14.3 (CH2CH3), 14.2 (CH2CH3),
127.7 (Ar), 106.3 (C-1), 83.1 (C-4), 75.4 (C-2), 73.3 (C-3), 69.3 −5.5 (Si(CH3)2), −5.5 (Si(CH3)2); HRMS m/z 521.21766 (calc. for
(CH2Ph), 65.1 (C-5), 25.9 (SiC(CH3)3), 18.4 (SiC(CH3)3), −5.4 (Si- C24H38O9NaSi ([M + Na]+): 521.21773).
(CH3)2), −5.4 (Si(CH3)2); HRMS m/z 377.17543 (calc. for
1-O-Benzyl-2,3-O-bis(isobutylcarbonate)-5-O-tert-butyl-
dimethylsilyl-β-D-ribofuranoside 5c. For the reaction, 1.04 g
(2.93 mmol) of ribofuranoside 4 and isobutyl chloroformate
reagent was used.
Purification by flash chromatography (isocratic 95/5, v/v,
hexane–EtOAc) gave 1-O-benzyl-2,3-O-bis(isobutylcarbonate)-5-
C18H30O5NaSi ([M + Na]+): 377.17547).
General procedure for the synthesis of bis(alkylcarbonate)
ribofuranosides 5a–c
To a solution of 1-O-benzyl-5-O-tert-butyldimethylsilyl-β-D-ribo- O-tert-butyldimethylsilyl-β-D-ribofuranoside 5c (1.57 g, 97%) as
furanoside 4 (1 × n), DMAP (0.2 × n) and pyridine (2.2 × n) in a white solid.
DCM was added alkyl chloroformate (2.2 × n) at 0 °C. The reac-
m.p.: 65–66 °C; [α]D20 = −30.8° (c 0.35, CHCl3); IR: νmax/cm−1
tion mixture was stirred overnight and allowed to warm up to (CHCl3): 3035, 2958, 2929, 2857, 1754 (CO), 1471, 1381, 1346,
room temperature, and then it was diluted with DCM, washed 1283, 1132, 1101, 1050, 998, 839, 698; 1H NMR (CDCl3): δ
with a solution of CuSO4, NaHCO3, water and brine, dried and 7.37–7.27 (5H, m, Ar), 5.33 (1H, dd, J = 6.2, 4.9 Hz, H-3), 5.20
evaporated.
(1H, dd, J = 4.9, 1.6 Hz, H-2), 5.16 (1H, d, J = 1.6 Hz, H-1), 4.77
1-O-Benzyl-2,3-O-bis(methylcarbonate)-5-O-tert-butyl- (1H, d, J = 11.9 Hz, CH2Ph), 4.53 (1H, d, J = 11.9 Hz, CH2Ph),
dimethylsilyl-β-D-ribofuranoside 5a. For the reaction, 378 mg 4.25 (1H, td, J = 6.2, 4.9 Hz, H-4), 3.95 (2H, ddd, J = 10.3, 6.7,
(1.07 mmol) of ribofuranoside 4 and methyl chloroformate 4.8 Hz, CH2CH(CH3)2), 3.89 (2H, ddd, J = 10.3, 6.7, 1.9 Hz,
reagent was used.
CH2CH(CH3)2), 3.79 (1H, dd, J = 10.8, 4.7 Hz, H-5a), 3.75 (1H,
Purification by flash chromatography (gradient from 99/1 to dd, J = 10.8, 4.9 Hz, H-5b), 1.95 (2H, doublet of heptuplet, J =
9/1, v/v, hexane–EtOAc) gave 1-O-benzyl-2,3-O-bis(methylcarbo- 6.7, 1.1 Hz, CH2CH(CH3)2), 0.95–0.92 (12H, m, CH2CH(CH3)2),
nate)-5-O-tert-butyldimethylsilyl-β-D-ribofuranoside 5a (372 mg, 0.88 (9H, s, SiC(CH3)3), 0.06 (3H, s, Si(CH3)2), 0.05 (3H, s, Si-
74%) as a colorless oil.
(CH3)2); 13C NMR (CDCl3): δ 154.3 (CO), 154.2 (CO), 137.0 (Ar),
[α]2D0 = −41.3° (c 0.31, CHCl3); IR: νmax/cm−1 (CHCl3): 3034, 128.4 (Ar), 127.9 (Ar), 103.7 (C-1), 80.9 (C-4), 77.8 (C-2), 75.2
2957, 2929, 2857, 1760 (CO), 1444, 1290, 1134, 1099, 999, 838, (C-3), 74.6 (CH2CH(CH3)2), 74.5 (CH2CH(CH3)2), 69.4 (CH2Ph),
699; 1H NMR (CDCl3): δ 7.37–7.29 (5H, m, Ar), 5.34 (1H, dd, J = 63.8 (C-5), 27.7 (CH2CH(CH3)2), 27.7 (CH2CH(CH3)2), 25.8
5.9, 4.9 Hz, H-3), 5.20 (1H, dd, J = 4.6, 1.1 Hz, H-2), 5.15 (1H, (SiC(CH3)3), 18.8 (CH2CH(CH3)2), 18.8 (CH2CH(CH3)2),
d, J = 1.1 Hz, H-1), 4.77 (1H, d, J = 12.0 Hz, CH2Ph), 4.53 (1H, 18.8 (CH2CH(CH3)2), 18.2 (SiC(CH3)3), −5.5 (Si(CH3)2), −5.5
d, J = 12.0 Hz, CH2Ph), 4.22 (1H, J = 10.7, 4.9 Hz, H-4), (Si(CH3)2); HRMS m/z 577.28017 (calc. for C28H46O9NaSi
3.79–3.76 (8H, m, H-5, CH3), 0.88 (9H, s, SiC(CH3)3), 0.06 (3H, ([M + Na]+): 577.28033).
s, Si(CH3)2), 0.06 (3H, s, Si(CH3)2); 13C NMR (CDCl3): δ 154.7
1-O-Benzyl-2,3-O-carbonate-5-O-tert-butyldimethylsilyl-β-D-
(CO), 137.0 (Ar), 128.4 (Ar), 127.9 (Ar), 127.8 (Ar), 103.7 (C-1), ribofuranoside 5d. A solution of 1-O-benzyl-5-O-tert-butyl-
80.9 (C-4), 78.0 (C-2), 75.4 (C-3), 69.5 (CH2Ph), 63.8 (C-5), 55.2 dimethylsilyl-β-D-ribofuranoside 4 (1.00 g, 2.80 mmol) and
(CH3), 55.1 (CH3), 25.8 (SiC(CH3)3), 18.2 (SiC(CH3)3), −5.5 dibutyltin oxide (0.77 g, 3.10 mmol) in toluene (50 mL) was
(Si(CH3)2), −5.5 (Si(CH3)2); HRMS m/z 493.18634 (calc. for refluxed under Dean Stark overnight. The reaction mixture
C22H34O9NaSi ([M + Na]+): 493.18643).
was then cooled down to room temperature and tetra-
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n-butylammonium bromide (1.00 g, 3.10 mmol) was added.
The spectroscopic data corresponded to those previously
The reaction mixture was cooled down to 0 °C and stirred for reported.11
10 min before methyl chloroformate was added (the same
5-O-tert-Butyldimethylsilyl-1,2-O-isopropylidene-3-O-isobu-
product was also obtained when ethyl chloroformate or iso- tylcarbonate-α-D-ribofuranoside 6c. For the reaction was
butyl chloroformate was used as the reagent). The reaction used 330 mg (1.08 mmol) of 5-O-tert-butyldimethylsilyl-1,2-O-
mixture was stirred for 2 h, and then it was diluted with isopropylidene-α-D-ribofuranoside and isobutyl chloroformate
chloroform, washed with a solution of NaHCO3, water and reagent.
brine, dried and evaporated.
Purification by silica gel column chromatography (gradient
Purification by flash chromatography (gradient 97/3 to 93/7, from 95/5 to 9/1, v/v, hexane–EtOAc) afforded 5-O-tert-butyldi-
v/v, hexane–EtOAc) gave 1-O-benzyl-2,3-O-carbonate-5-O-tert- methylsilyl-1,2-O-isopropylidene-3-O-isobutylcarbonate-α-D-
butyldimethylsilyl-β-D-ribofuranoside 5d (840 mg, 78%) as a ribofuranoside 6c (0.41 g, 94%) as a colorless oil.
colorless oil.
The spectroscopic data corresponded to those previously
[α]2D0 = −98.1° (c 0.32, CHCl3); IR: νmax/cm−1 (CHCl3): 3033, reported.11
2956, 2931, 2859, 1809 (CO), 1472, 1372, 1259, 1161, 1089,
1012, 839, 699; H NMR (CDCl3): δ 7.39–7.27 (5H, m, Ar), 5.30
1
General procedure for TBDMS deprotection with CAN9
(1H, s, H-1), 5.16 (1H, d, J = 6.8 Hz, H-3), 5.04 (1H, d, J = Ribofuranosides 5a–c and 6a–c (1 × n) were dissolved in AcCN–
6.8 Hz, H-2), 4.68 (1H, d, J = 11.5 Hz, CH2Ph), 4.49 (1H, d, J = H2O (9/1, v/v) and CAN (1.1 × n) was added. The reaction
11.5 Hz, CH2Ph), 4.41 (1H, dd, J = 10.0, 5.5 Hz, H-4), 3.72 (1H, dd, mixture was stirred for 3 h. Then it was diluted with chloro-
J = 10.2, 5.5 Hz, H-5a), 3.61 (1H, t, J = 10.2 Hz, H-5b), 0.91 (9H, form and washed with a solution of NaHCO3. The aqueous
s, SiC(CH3)3), 0.06 (3H, s, Si(CH3)2), 0.05 (3H, s, Si(CH3)2); 13C layer was extracted with CHCl3 (3×) and the combined organic
NMR (CDCl3): δ 155.3 (CO), 136.1 (Ar), 128.6 (Ar), 128.3 (Ar), fractions were dried, filtered and evaporated.
128.2 (Ar), 105.7 (C-1), 86.2 (C-4), 83.6 (C-2), 81.0 (C-3), 69.8
1-O-Benzyl-2,3-O-bis(methylcarbonate)-β-D-ribofuranoside
(CH2Ph), 62.4 (C-5), 25.7 (SiC(CH3)3), 18.1 (SiC(CH3)3), −5.4 7a. For the reaction, 246 mg (0.52 mmol) of ribofuranoside 5a
(Si(CH3)2), −5.5 (Si(CH3)2); HRMS m/z 403.15466 (calc. for was used.
C19H28O6NaSi ([M + Na]+): 403.15474).
Purification by flash chromatography (isocratic 98/2, v/v,
CHCl3–EtOH) gave 1-O-benzyl-2,3-O-bis(methylcarbonate)-β-D-
ribofuranoside 7a (172 mg, 91%) as a colorless oil.
[α]2D0 = −37.4° (c 0.61, CHCl3); IR: νmax/cm−1 (CHCl3): 3029,
2959, 2929, 2857, 1761 (CO), 1444, 1289, 1150, 1092, 950, 699;
General procedure for the synthesis of alkylcarbonate
ribofuranosides 6a–c11
To a solution of 1,2-O-isopropylidene-5-O-tert-butyldimethyl- 1H NMR (CDCl3): δ 7.40–7.30 (5H, m, Ar), 5.35 (1H, dd, J = 6.5,
silyl-β-D-ribofuranoside (1 × n; prepared as reported pre- 4.9 Hz, H-3), 5.21 (1H, d, J = 4.9 Hz, H-2), 5.19 (1H, s, H-1),
viously10), DMAP (0.2 × n) and pyridine (1.2 × n) in DCM was 4.77 (1H, d, J = 11.8 Hz, CH2Ph), 4.60 (1H, d, J = 11.8 Hz,
added alkyl chloroformate (1.2 × n) at 0 °C. The reaction CH2Ph), 4.30 (1H, td, J = 6.5, 3.4 Hz, H-4), 3.86–3.80 (7H, m,
mixture was stirred overnight and allowed to warm up to room H-5a, CH3), 3.67 (1H, ddd, J = 12.3, 8.7, 3.8 Hz, H-5b), 2.04
temperature, and then it was diluted with DCM, washed with a (1H, dd, J = 8.7, 4.5 Hz, OH); 13C NMR (CDCl3): δ 154.8 (CO),
solution of CuSO4, NaHCO3, water and brine, dried and 154.7 (CO), 136.6 (Ar), 128.6 (Ar), 128.2 (Ar), 104.3 (C-1), 81.7
evaporated.
(C-4), 78.3 (C-2), 74.2 (C-3), 70.7 (CH2Ph), 62.4 (C-5), 55.3
5-O-tert-Butyldimethylsilyl-1,2-O-isopropylidene-3-O-methyl- (CH3), 55.3 (CH3); HRMS m/z 379.09991 (calc. for C16H20O9Na
carbonate-α-D-ribofuranoside 6a. For the reaction was used ([M + Na]+): 379.09995).
1.00
isopropylidene-α-D-ribofuranoside and methyl chloroformate For the reaction, 287 mg (0.58 mmol) of ribofuranoside 5b was
reagent. used.
Purification by silica gel column chromatography (gradient Purification by flash chromatography (isocratic 98/2, v/v,
g
(3.30 mmol) of 5-O-tert-butyldimethylsilyl-1,2-O-
1-O-Benzyl-2,3-O-bis(ethylcarbonate)-β-D-ribofuranoside 7b.
from 85/15 to 65/45, v/v, hexane–EtOAc) afforded 5-O-tert-butyl- CHCl3–EtOH) gave 1-O-benzyl-2,3-O-bis(ethylcarbonate)-β-D-
dimethylsilyl-1,2-O-isopropylidene-3-O-methylcarbonate-α-D- ribofuranoside 7b (190 mg, 86%) as a colorless oil.
ribofuranoside 6a (1.00 g, 83%) as a white solid.
[α]2D0 = −32.0° (c 0.34, CHCl3); IR: νmax/cm−1 (CHCl3): 3029,
The spectroscopic data corresponded to those previously 2955, 2928, 2856, 1756 (CO), 1374, 1281, 1093, 942, 880, 699;
reported.11
1H NMR (CDCl3): δ 7.38–7.29 (5H, m, Ar), 5.34 (1H, t, J =
5-O-tert-Butyldimethylsilyl-1,2-O-isopropylidene-3-O-ethyl- 5.7 Hz, H-3), 5.22 (1H, d, J = 4.8 Hz, H-2), 5.19 (1H, s, H-1), 4.76
carbonate-α-D-ribofuranoside 6b. For the reaction was used (1H, d, J = 11.8 Hz, CH2Ph), 4.60 (1H, d, J = 11.8 Hz, CH2Ph),
510 mg (1.70 mmol) of 5-O-tert-butyldimethylsilyl-1,2-O-isopro- 4.30 (1H, pentuplet, J = 3.7 Hz, H-4), 4.24–4.20 (4H, m,
pylidene-α-D-ribofuranoside and ethyl chloroformate reagent.
CH2CH3), 3.83 (1H, td, J = 12.2, 3.7 Hz, H-5a), 3.67 (1H,
Purification by silica gel column chromatography (gradient ddd, J = 12.2, 8.6, 3.7 Hz, H-5b), 2.04 (1H, dd, J = 8.6, 4.6 Hz,
from 95/5 to 9/1, v/v, hexane–EtOAc) afforded 5-O-tert-butyldi- OH), 1.31 (6H, m, CH2CH3); 13C NMR (CDCl3): δ 154.2
methylsilyl-1,2-O-isopropylidene-3-O-ethylcarbonate-α-D-ribo- (CO), 154.1 (CO), 136.6 (Ar), 128.6 (Ar), 128.1 (Ar), 128.0
furanoside 6b (460 mg, 75%) as a colorless oil.
(Ar), 104.3 (C-1), 81.7 (C-4), 78.1 (C-2), 74.0 (C-3), 70.4 (CH2Ph),
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64.7 (CH2CH3), 64.7 (CH2CH3), 62.5 (C-5), 14.1 (CH2CH3);
Bisbenzyloxy-diisopropylamino phosphine.13 A solution of
HRMS m/z 407.13121 (calc. for C18H24O9Na ([M + Na]+): freshly distilled diisopropylamine (16 mL, 114.6 mmol) in dry
407.13125).
hexane (40 mL) was added dropwise to a solution of PCl3
1-O-Benzyl-2,3-O-bis(isobutylcarbonate)-β-D-ribofuranoside (5 mL, 57.3 mmol) in dry hexane (40 mL) at 0 °C. The reaction
7c. For the reaction, 1.57 g (2.83 mmol) of ribofuranoside 5c mixture was allowed to warm up to room temperature
was used.
while stirring overnight. A solution of dry BnOH (12 mL,
Purification by flash chromatography (isocratic 98/2, v/v, 114.6 mmol) and dry Et3N (16 mL, 114.6 mmol) in dry Et2O
CHCl3–EtOH) gave 1-O-benzyl-2,3-O-bis(isobutylcarbonate)-β-D- (50 mL) was then added and stirring was continued for 1 hour.
ribofuranoside 7c (1.07 g, 86%) as a colorless oil.
The reaction mixture was then filtered and washed with Et2O.
[α]2D0 = −35.4° (c 0.53, CHCl3); IR: νmax/cm−1 (CHCl3): 3034, The filtrate was washed with a solution of NaHCO3, water and
2962, 2930, 2877, 1755 (CO), 1456, 1381, 1282, 1091, 1045, brine, dried and evaporated. The residue obtained was dis-
1
1011, 971, 699; H NMR (CDCl3): δ 7.38–7.29 (5H, m, Ar), 5.34 solved in hexane (100 mL), washed with AcCN (3 × 80 mL), and
(1H, dd, J = 6.4, 4.9 Hz, H-3), 5.22 (1H, dd, J = 4.9, 0.8 Hz, H-2), the hexane layer was dried and evaporated to afford bisbenzyl-
5.19 (1H, d, J = 0.8 Hz, H-1), 4.77 (1H, d, J = 11.8 Hz, CH2Ph), oxy-diisopropylamino phosphine (2.9 g, 15%) as a colourless
4.61 (1H, d, J = 11.8 Hz, CH2Ph), 4.32 (1H, ddd, J = 6.8, 3.6, 3.2 oil.
Hz, H-4), 3.96 (2H, dd, J = 10.4, 6.8 Hz, CH2CH(CH3)2), 3.89
The spectroscopic data corresponded to those previously
(2H, ddd, J = 10.4, 6.8, 1.9 Hz, CH2CH(CH3)2), 3.83 (1H, ddd, reported.
J = 12.2, 3.8, 3.6 Hz, H-5a), 3.67 (1H, ddd, J = 12.2, 8.6, 3.8 Hz,
General procedure for phosphorylation17
H-5b), 2.07 (1H, dd, J = 8.6, 3.6 Hz, 5-OH), 1.98 (2H, doublet of
pentuplet, J = 6.7, 4.2 Hz, CH2CH(CH3)2), 0.94 (12H, d, J = A solution of 2,4-DNP (1.1 × n) in dry DCM was added dropwise
6.7 Hz, CH2CH(CH3)2); 13C NMR (CDCl3): δ 154.4 (CO), 154.3 (CO), to a solution of furanoside (1 × n) and dibenzylphosphorami-
136.7 (Ar), 128.6 (Ar), 128.1 (Ar), 128.0 (Ar), 104.4 (C-1), 81.8 date (1.1 × n) in dry DCM. The resulting reaction mixture was
(C-4), 78.1 (C-2), 74.7 (CH2CH(CH3)2), 74.7 (CH2CH(CH3)2), stirred overnight at room temperature, and then tBuOOH (2 ×
74.1 (C-3), 70.4 (CH2Ph), 62.5 (C-5), 27.7 (CH2CH(CH3)2), 18.8 n, 5.5 M in decane) was added and stirring was continued for
(CH2CH(CH3)2); HRMS m/z 463.19380 (calc. for C22H32O9Na another 20 minutes. Afterwards, the reaction mixture was
([M + Na]+): 463.19385).
1,2-O-Isopropylidene-3-O-methylcarbonate-α-D-ribofurano- Na2S2O3 and water (3×), dried, filtered and evaporated.
side 8a. For the reaction was used 210 mg (0.58 mmol) of 5-O- 5-O-Dibenzylphosphate-1-O-benzyl-2,3-O-bis(methylcarbo-
tert-butyldimethylsilyl-1,2-O-isopropylidene-3-O-methylcarbo- nate)-β-D-ribofuranoside 9a. For the reaction, 132 mg
diluted with DCM, washed with a saturated solution of
nate-α-D-ribofuranoside 6a.
(0.41 mmol) of ribofuranoside 7a was used.
Purification by silica gel column chromatography (gradient
Purification by flash chromatography (gradient, from 3/1
from 6/4 to 1/1, v/v, hexane–EtOAc) gave 1,2-O-isopropylidene- to 2/1, v/v, hexane–EtOAc) gave 5-O-dibenzylphosphate-1-O-
3-O-methylcarbonate-α-D-ribofuranoside 8a (137 mg, 95%) as a benzyl-2,3-O-bis(methylcarbonate)-β-D-ribofuranoside 9a (200 mg,
white solid.
The spectroscopic data corresponded to those previously
reported.11
88%) as a colorless oil.
[α]2D0 = −27.4° (c 0.29, CHCl3); IR: νmax/cm−1 (CHCl3): 3035,
2959, 2927, 2856, 1762 (CO), 1498, 1456, 1378, 1289, 1097,
1,2-O-Isopropylidene-3-O-ethylcarbonate-α-D-ribofuranoside 1001, 951, 882, 698; 1H NMR (CDCl3): δ 7.36–7.28 (15H, m, Ar),
8b. For the reaction was used 148 mg (0.39 mmol) of 5-O-tert- 5.30 (1H, dd, J = 6.7, 5.0 Hz, H-3), 5.19 (1H, d, J = 4.6 Hz, H-2),
butyldimethylsilyl-1,2-O-isopropylidene-3-O-ethylcarbonate-α-D- 5.12 (1H, s, H-1), 5.03 (4H, d, J = 7.6, 5.8 Hz, CH2Ph), 4.74 (1H,
ribofuranoside 6b.
d, J = 11.8 Hz, CH2Ph), 4.47 (1H, d, J = 11.8 Hz, CH2Ph), 4.33
Purification by silica gel column chromatography (gradient (1H, dd, J = 10.8, 5.0 Hz, H-4), 4.23–4.13 (2H, m, H-5), 3.79
from 100/0 to 99/1, v/v, DCM–EtOH) afforded 1,2-O-isopropyli- (1H, s, CH3), 3.77 (1H, s, CH3); 13C NMR (CDCl3): δ 154.7 (CO),
dene-3-O-ethylcarbonate-α-D-ribofuranoside 8b (99.0 mg, 96%) 154.5 (CO), 128.5 (Ar), 128.4 (Ar), 128.0 (Ar), 127.9 (Ar), 103.7
as a colorless oil.
(C-1), 78.6 (d, J 8.3 Hz, C-4), 77.6 (C-2), 74.5 (C-3), 69.5
The spectroscopic data corresponded to those previously (CH2Ph), 69.5 (CH2Ph), 69.4 (CH2Ph), 67.2 (d, J 5.5 Hz, C-5),
reported.11
55.3 (CH3), 55.3 (CH3); 31P NMR (CDCl3): δ −1.10; HRMS m/z
1,2-O-Isopropylidene-3-O-isobutylcarbonate-α-D-ribofurano- 639.16026 (calc. for C30H33O12NaP ([M + Na]+): 639.16018).
side 8c. For the reaction was used 70.0 mg (0.17 mmol) of 5-O-
5-O-Dibenzylphosphate-1-O-benzyl-2,3-O-bis(ethylcarbonate)-
tert-butyldimethylsilyl-1,2-O-isopropylidene-3-O-isobutylcarbo- β-D-ribofuranoside 9b. For the reaction, 190 mg (0.49 mmol)
nate-α-D-ribofuranoside 6c.
of ribofuranoside 7b was used.
The purification by silica gel column chromatography
Purification by flash chromatography (gradient, from 3/1
(gradient from 100/0 to 99/1, v/v, DCM–EtOH) afforded to 7/3, v/v, hexane–EtOAc) gave 5-O-dibenzylphosphate-1-O-
1,2-O-isopropylidene-3-O-ethylcarbonate-α-D-ribofuranoside 8c benzyl-2,3-O-bis(ethylcarbonate)-β-D-ribofuranoside 9b (306 mg,
(48.0 mg, 96%) as a colorless oil.
The spectroscopic data corresponded to those previously
reported.11
96%) as a colorless oil.
[α]2D0 = −22.2° (c 0.33, CHCl3); IR: νmax/cm−1 (CHCl3): 3035,
2955, 2927, 2855, 1757 (CO), 1498, 1456, 1374, 1280, 1094,
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1001, 882, 697; 1H NMR (CDCl3): δ 7.36–7.28 (15H, m, Ar), 5.30 J = 5.4, 3.0 Hz, CH2Ph), 65.0 (d, J = 5.2 Hz, C-5), 55.2 (CH3),
(1H, dd, J = 7.0, 4.7 Hz, H-3), 5.20 (1H, d, J = 4.7 Hz, H-2), 5.13 26.6 (C(CH3)2); 31P NMR (CDCl3): δ 0.26; HRMS m/z 493.1178
(1H, s, H-1), 5.03 (4H, d, J = 7.8, 5.6 Hz, CH2Ph), 4.73 (1H, d, J (calc. for C23H26O10P ([M − CH3]+): 493.1264).
= 11.8 Hz, CH2Ph), 4.46 (1H, d, J = 11.8 Hz, CH2Ph), 4.35 (1H,
5-O-Dibenzylphosphate-1,2-O-isopropylidene-3-O-ethyl-
bdd, J = 11.2, 4.7 Hz, H-4), 4.20–4.13 (4H, m, H-5, CH2CH3), carbonate-α-D-ribofuranoside 10b. For the reaction, 65.0 mg
4.13–4.06 (2H, m, CH2CH3), 1.34–1.27 (6H, m, CH2CH3); 13C (0.25 mmol) of ribofuranoside 8b was used.
NMR (CDCl3): δ 154.0 (CO), 153.9 (CO), 136.6 (Ar), 128.7 (Ar),
Purification by flash chromatography (gradient 100/0 to
128.5 (Ar), 128.4 (Ar), 128.0 (Ar), 127.9 (Ar), 103.7 (C-1), 78.7 (d, 98/2, v/v, DCM–MeOH) yielded 5-O-dibenzylphosphate-1,2-O-
J = 8.0 Hz, C-4), 77.4 (C-2), 74.3 (C-3), 69.5 (CH2Ph), 69.4 isopropylidene-3-O-ethylcarbonate-α-D-ribofuranoside 10b
(CH2Ph), 67.3 (dd, J = 5.4, 5.2 Hz, C-5), 64.7 (CH2CH3), 64.7 (88.0 mg, 68%) as a yellow oil.
(CH2CH3), 14.1 (CH2CH3); 31P NMR (CDCl3): δ –1.11; HRMS m/z
[α]2D0 = +56.0° (c 0.43, CHCl3); IR: νmax/cm−1 (CHCl3): 3034,
2928 2856, 1751 (CO), 1456, 1376, 1268, 1115, 1015, 873, 697;
639.16026 (calc. for C30H33O12NaP ([M + Na]+): 639.16018).
5-O-Dibenzylphosphate-1-O-benzyl-2,3-O-bis(isobutylcarbo- 1H NMR (CDCl3): δ 7.35–7.33 (10H, m, Ar), 5.74 (1H, d, J = 3.7
nate)-β-D-ribofuranoside 9c. For the reaction, 1.07 g (2.43 mmol) Hz, H-1), 5.07–5.04 (4H, m, CH2Ph), 4.77 (1H, dd, J = 4.4, 4.0
of ribofuranoside 7c was used.
Hz, H-2), 4.68 (1H, dd, J = 8.8, 4.8 Hz, H-3), 4.30 (1H, ddd, J =
Purification by flash chromatography (gradient, from 3/1 11.6, 6.2, 2.4 Hz, H-5a), 4.28–4.25 (1H, m, H-4), 4.21 (2H, q, J =
to 7/3, v/v, hexane–EtOAc) gave 5-O-dibenzylphosphate-1-O- 7.2 Hz, CH2CH3), 4.09 (1H, ddd, J = 11.6, 6.6, 3.6 Hz, H-5b),
benzyl-2,3-O-bis(isobutylcarbonate)-β-D-ribofuranoside 9c (1.51 g, 1.54 (3H, s, C(CH3)2), 1.34 (3H, s, C(CH3)2), 1.32 (3H, t, J = 7.2
89%) as a colorless oil.
Hz, CH2CH3); 13C NMR (CDCl3): δ 154.0 (CO), 135.7 (Ar), 128.6
[α]2D0 = −21.4° (c 0.72, CHCl3); IR: νmax/cm−1 (CHCl3): 3035, (Ar), 128.5 (Ar), 128.0 (Ar), 127.9 (Ar), 113.5 (C(CH3)2), 104.2
2929, 2856, 1756 (CO), 1498, 1471, 1456, 1381, 1280, 1034, (C-1), 77.2 (C-2), 76.1 (d, J = 6.2 Hz, C-4), 74.0 (C-3), 69.4 (dd,
1001, 971, 919, 697; 1H NMR (CDCl3): δ 7.37–7.28 (15H, m, Ar), J = 5.4, 3.0 Hz, CH2Ph), 65.0 (d, J = 5.4 Hz, C-5), 64.7 (CH2CH3),
5.29 (1H, dd, J = 7.0, 4.8 Hz, H-3), 5.20 (1H, d, J = 4.8 Hz, H-2), 26.6 (C(CH3)2), 26.6 (C(CH3)2), 14.1 (CH2CH3); 31P NMR
5.14 (1H, s, H-1), 5.03 (4H, d, J = 8.0, 5.3 Hz, CH2Ph), 4.74 (1H, (CDCl3): δ –1.02; HRMS m/z 545.15443 (calc. for C25H31O10NaP
d, J = 11.8 Hz, CH2Ph), 4.47 (1H, d, J = 11.8 Hz, CH2Ph), ([M + Na]+): 545.15470).
4.39–4.34 (1H, m, H–4), 4.21 (1H, dd, J = 10.3, 6.2, 2.7 Hz,
5-O-Dibenzylphosphate-1,2-O-isopropylidene-3-O-isobutyl-
H-5a), 4.14 (1H, dd, J = 10.3, 6.2 Hz, H-5b), 3.95 (2H, ddd, J = carbonate-α-D-ribofuranoside 10c. For the reaction, 48.0 mg
10.3, 6.7, 2.7 Hz, CH2CH(CH3)2), 3.87 (2H, dt, J = 10.3, 6.7 Hz, (165 μmol) of ribofuranoside 8c was used.
CH2CH(CH3)2), 1.96 (2H, doublet of hexuplet, J = 6.7, 2.3 Hz,
Purification by flash chromatography (gradient 8/2 to
CH2CH(CH3)2), 0.94 (6H, dd, J = 6.7, 0.7 Hz, CH2CH(CH3)2), 7/3, v/v, hexane–EtOAc) yielded 5-O-dibenzylphosphate-1,2-O-
0.93 (6H, d, J = 6.7 Hz, CH2CH(CH3)2); 13C NMR (CDCl3): δ isopropylidene-3-O-isobutylcarbonate-α-D-ribofuranoside 10c
154.2 (CO), 154.1 (CO), 136.6 (Ar), 128.5 (Ar), 128.4 (Ar), 128.0 (70.0 mg, 77%) as a yellow oil.
(Ar), 127.9 (Ar), 103.8 (C-1), 78.7 (d, J = 8.2 Hz, C-4), 77.4 (C-2),
[α]2D0 = +43.0° (c 1.25, CHCl3); IR: νmax/cm−1 (CHCl3): 3032,
74.7 (CH2CH(CH3)2), 74.7 (CH2CH(CH3)2), 74.4 (C-3), 69.5 2964, 2877, 1747 (CO), 1498, 1456, 1385, 1377, 1277, 1168,
1
(CH2Ph), 69.4 (CH2Ph), 67.2 (d, J = 5.5 Hz, C-5), 27.7 (CH2CH 1134, 1096, 1014, 918, 697; H NMR (CDCl3): δ 7.37–7.35 (10H,
(CH3)2), 27.7 (CH2CH(CH3)2), 18.8 (CH2CH(CH3)2), 18.8 m, Ar), 5.74 (1H, d, J = 3.6 Hz, H-1), 5.11–5.08 (4H, m, CH2Ph),
(CH2CH(CH3)2); 31P NMR (CDCl3):
δ
–1.11; HRMS m/z 4.64 (1H, t, J = 4.1 Hz, H-2), 4.46 (1H, ddd, J = 9.0, 6.8, 4.6 Hz,
723.25411 (calc. for C36H45O12NaP ([M + Na]+): 723.25408).
H-3), 4.41 (1H, dd, J = 12.1, 2.2 Hz, H-5a), 4.23 (1H, ddd, J =
5-O-Dibenzylphosphate-1,2-O-isopropylidene-3-O-methylcar- 9.0, 4.5, 2.3 Hz, H-4), 4.16 (1H, dd, J = 12.0, 4.5 Hz, H-5b),
bonate-α-D-ribofuranoside 10a. For the reaction, 1.20
(4.83 mmol) of ribofuranoside 8a was used.
g
3.93–3.85 (2H, m, CH2CH(CH3)2), 1.96 (1H, d, J = 6.7 Hz,
CH2CH(CH3)2), 1.55 (3H, s, C(CH3)2), 1.30 (3H, s, C(CH3)2),
Purification by chromatography (gradient from 6/4 to 4/6, 0.92 (6H, d, J = 6.7 Hz, CH2CH(CH3)2); 13C NMR (CDCl3):
v/v, hexane–EtOAc) yielded 1,2-O-isopropylidene-5-O-dibenzyl- δ155.6 (CO), 128.7 (Ar), 128.6 (Ar), 128.0 (Ar), 127.9 (Ar), 110.0
phosphate-3-O-methylcarbonate-α-D-ribofuranoside 10a (2.0 g, (C(CH3)2), 103.9 (C-1), 77.7 (C-2), 75.8 (d, J = 10.3 Hz, C-4), 74.5
81.0%) as a yellow oil.
(C-3), 74.3 (CH2CH(CH3)2), 69.7 (m, CH2Ph), 64.9 (m, C-5), 27.7
[α]2D0 = +48.1° (c 0.09, CHCl3); IR: νmax/cm−1 (CHCl3): 3030, (CH2CH(CH3)2), 26.7 (C(CH3)2), 26.5 (C(CH3)2), 18.9 (CH2CH-
2959, 1756 (CO), 1498, 1456, 1444, 1385, 1376, 1277, 1114, (CH3)2); 31P NMR (CDCl3): δ –1.97; HRMS m/z 573.18585 (calc.
1014, 874, 697; 1H NMR (CDCl3): δ 7.41–7.30 (10H, m, Ar), 5.73 for C27H35O10NaP ([M + Na]+): 573.18600).
(1H, d, J = 3.7 Hz, H-1), 5.05 (1H, d, J = 8.1 Hz, CH2Ph), 5.04
Deprotection of the isopropylidene group12
(1H, dd, J = 8.1, 4.2 Hz, CH2Ph), 4.77 (1H, t, J = 4.2 Hz, H-2),
4.67 (1H, dd, J = 8.8, 4.8 Hz, H-3), 4.30 (1H, ddd, J = 11.6, 6.1, Furanoside (1 × n) was treated with a mixture of TFA–H2O (4/1,
2.4 Hz, H-5), 4.27–4.23 (1H, m, H-4), 4.14–4.06 (1H, m, H-5), 10 mL) at 0 °C for 2.5 hours. The reaction mixture was then
3.80 (3H, s, CH3), 1.53 (3H, s, C(CH3)2), 1.33 (3H, s, C(CH3)2); slowly poured into a saturated solution of Na2CO3. The
13C NMR (CDCl3): δ 154.6 (CO), 135.7 (d, J = 6.8 Hz, Ar), 128.6 aqueous layer was extracted with DCM (3 × 40 mL) and
(Ar), 128.5 (Ar), 128.0 (Ar), 127.9 (Ar), 113.5 (C(CH3)2), 104.2 the combined organic layers were washed with brine, dried,
(C-1), 77.2 (C-2), 76.1 (d, J = 7.6 Hz, C-4), 74.2 (C-3), 69.4 (dd, filtered and evaporated.
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5-O-Dibenzylphosphate-2,3-O-carbonate-β-D-ribofuranose 9d. 4.13–4.03 (5H, m, CH2CH3, H-5a), 4.00–3.95 (1H, m, H-5b),
For the reaction, 500 mg (0.98 mmol) of ribofuranoside 10a 1.17 (6H, t, J = 7.1 Hz, CH2CH3); 13C NMR (MeOD): δ 155.7
was used.
(CO), 155.6 (CO), 138.5 (Ar), 129.5 (Ar), 129.4 (Ar), 129.0 (Ar),
Purification by flash chromatography (gradient from 1/0 to 105.3 (C-1), 80.6 (d, J = 8.5 Hz, C-4), 78.9 (C-2), 76.2 (C-3), 70.7
98/2, v/v, CHCl3–EtOH) gave 5-O-dibenzylphosphate-2,3-O-car- (CH2Ph), 67.6 (d, J = 4.2 Hz, C-5), 65.9 (CH2CH3), 14.6
bonate-β-D-ribofuranose 9d (275 mg, 67%) as a white solid. (CH2CH3); 31P NMR (D2O): δ 1.17; HRMS m/z 463.10117 (calc.
When ribofuranosides 10b and 10c were used as the starting for C18H24O12P ([M − H]+): 463.10109).
material, the same product was obtained.
5-O-Phosphate-2,3-O-bis(methylcarbonate)-β-D-ribofuranose
[α]2D0 = −12.7° (c 0.14, CHCl3); Anal. calcd for C20H21O9P: C 13a. For the reaction, 250 mg (0.41 mmol) of ribofuranoside
55.05, H 4.85; found: C 55.08, H 4.82%; IR: νmax/cm−1 (CHCl3): 9a in a THF–mQ H2O (1/2, v/v, 15 mL) mixture was used.
3068, 3035, 2959, 2856, 1762 (CO), 1498, 1456, 1378, 1289,
1150, 1097, 951, 882, 698; H NMR (CDCl3): δ 7.39–7.32 (10H, phosphate-1,2-O-carbonate-3-O-methylcarbonate-β-D-ribofura-
The spectroscopic analyses revealed the product to be 5-O-
1
m, Ar), 5.60 (1H, d, J = 2.2 Hz, H-1), 5.10 (2H, d, J = 8.6 Hz, noside 15a (105 mg, 83%) as a colorless oil.
1
CH2Ph), 5.03 (2H, dd, J = 8.6, 5.7 Hz, CH2Ph), 4.94 (2H, s, H-2,
[α]2D0 = +46.8° (c 0.21, MeOH); H NMR (D2O): δ 6.38 (1H, d,
H-3), 4.49 (1H, dd, J = 8.5, 5.5 Hz, H-4), 4.26 (1H, dd, J = 11.3, J = 4.8 Hz, H-1), 5.51 (1H, t, J = 5.2 Hz, H-2), 5.13 (1H, dd, J =
8.5 Hz, H-5a), 3.98 (1H, dt, J = 11.3, 5.5 Hz, H-5b); 13C NMR 7.8, 5.2 Hz, H-3), 4.52 (1H, d, J = 7.8 Hz, H-4), 4.18 (1H, dd, J =
(CDCl3): δ 153.5 (CO), 135.3 (Ar), 128.9 (Ar), 128.7 (Ar), 128.1 12.0, 5.1 Hz, H-5a), 4.07–4.00 (1H, m, H-5b), 3.85 (3H, s, CH3);
(Ar), 101.8 (C-1), 84.0 (C-2), 83.9 (d, J 6.0 Hz, C-4), 80.0 (C-3), 13C NMR (D2O): δ 155.1 (CO), 154.8 (CO), 103.7 (C-1), 78.7 (d,
70.0 (m, CH2Ph), 67.2 (d, J = 5.4 Hz, C-5); 31P NMR (CDCl3): δ J = 7.5 Hz, C-4), 77.8 (C-2), 73.0 (C-3), 62.7 (d, J = 2.2 Hz, C-5),
1.19; HRMS m/z 436.0961 (calc. for C20H21O9P ([M + H]+): 56.0 (CH3); 31P NMR (D2O): δ –0.22; HRMS m/z 312.99668 (calc.
436.0923).
for C8H10O11P ([M − H]+): 312.99662).
5-O-Phosphate-2,3-O-bis(ethylcarbonate)-β-D-ribofuranose
13b. For the reaction, 300 mg (0.47 mmol) of furanoside 9b in
Removal of benzyl groups
Furanoside (1 × n) was dissolved in an appropriate solvent, a THF–mQ H2O (1/1, v/v, 15 mL) mixture was used.
poured into a hydrogenation tube to which 10% Pd/C (20 mg) The HPLC-MS analysis revealed the product to be a
was added. The hydrogenation was carried out in an autoclave 9 : 1 mixture of 5-O-phosphate-2,3-O-bis(ethylcarbonate)-D-ribo-
under 10 bars of hydrogen pressure for 24 h. The reaction furanose 13b in α-(A) and β-(B) form, and 5-O-phosphate-1,2-O-
mixture was then filtered through celite, washed with MeOH carbonate-3-O-ethylcarbonate-β-D-ribofuranoside
15b
(C)
and evaporated. (150 mg, 86%), which upon storage underwent a migration
5-O-Phosphate-1-O-benzyl-2,3-O-bis(methylcarbonate)-β-D- towards product 15b. When the NMR was measured, the
ribofuranoside 12a. For the reaction, 387 mg (0.63 mmol) of mixture ratio was 4 : 1 (A + B : C).
ribofuranoside 9a in THF was used.
1H NMR (D2O): δ 6.40 (0.5H, d, J = 4.9 Hz, H-1C), 5.66 (1H,
The spectroscopic analyses revealed the product to be 5-O- d, J = 4.5 Hz, H-1B), 5.53 (0.5H, t, J = 4.9 Hz, H-2C), 5.46 (1H,
phosphate-1-O-benzyl-2,3-O-bis(methylcarbonate)-β-D-ribofura- d, J = 2.7 Hz, H-1A), 5.30 (1H, t, J = 4.7 Hz, H-3A), 5.29 (1H, q,
noside 12a (217 mg, 79%).
J = 3.0 Hz, H-3B), 5.15–5.10 (2.5H, m, H-2A, H-2B, H-3C), 4.56
[α]D20 = −18.5° (c 0.71, MeOH); IR: νmax/cm−1 (KBr): 3436 (0.5H, br s, H-4C), 4.51 (1H, br s, H-4B), 4.35 (1H, dd, J = 8.6,
(NH), 2958, 2926, 1812, 1760 (CO), 1632 (CN), 1445, 1289, 4.7 Hz, H-4A), 4.30–4.22 (10H, m, CH2CH3, H-5Aa), 4.10–4.00
1
1265, 1081, 946, 788; H NMR (D2O): δ 7.34–7.25 (5H, m, Ar), (4H, m, H-5Ab, H-5B, H-5C), 1.31–1.28 (13.5H, m, CH2CH3);
5.27–5.24 (1H, m, H-3), 5.11–5.10 (2H, m, H-1, H-2), 4.77 (1H, 13C NMR (D2O): δ 155.1 (CO), 154.8 (CO), 154.5 (CO), 154.4
d, J = 11.7 Hz, CH2Ph), 4.51 (1H, d, J = 11.7 Hz, CH2Ph), 4.28 (CO), 154.1 (CO), 103.8 (C-1C), 98.9 (C-1A), 94.5 (H-1B), 80.4 (d,
(1H, br s, H-4), 4.20–3.97 (2H, m, H-5), 3.73 (6H, s, CH3); 13C J = 8.3 Hz, C-4B), 79.2 (m, H-4A, H-4C), 78.1 (C-3C), 77.9
NMR (D2O): δ 156.4 (CO), 156.3 (CO), 138.6 (Ar), 129.6 (Ar), (C-2C), 74.4 (C-3A), 73.8 (C-3B), 73.7 (C-2A), 72.9 (C-2B), 66.2
129.5 (Ar), 129.1 (Ar), 105.2 (C-1), 80.8 (d, J = 6.9 Hz, C-4), 79.2 (CH2CH3), 66.0 (CH2CH3), 66.0 (CH2CH3), 65.9 (CH2CH3), 65.8
(C-2), 76.5 (C-3), 70.7 (CH2Ph), 67.5 (m, C-5), 56.0 (CH3); 31P (CH2CH3), 64.8 (d, J = 4.9 Hz, C-5A), 64.5 (d, J = 4.3 Hz, C-5B),
NMR (D2O): δ 1.00; HRMS m/z 435.06981 (calc. for C16H20O12P 62.7 (d, J = 4.3 Hz, C-5C), 13.4 (CH2CH3), 13.4 (CH2CH3), 13.3
([M − H]+): 435.06979).
(CH2CH3); 31P NMR (D2O): δ 0.21; 15b: HRMS m/z 327.01233
5-O-Phosphate-1-O-benzyl-2,3-O-bis(ethylcarbonate)-β-D-ribo- (calc. for C9H12O11P ([M − H]+): 327.01227).
furanoside 12b. For the reaction, 245 mg (0.38 mmol) of ribo-
furanoside 9b in THF was used.
5-O-Phosphate-1-O-methyl-2,3-O-bis(isobutylcarbonate)-β-D-
ribofuranose 13c. For the reaction, 225 mg (0.32 mmol) of
The spectroscopic analyses revealed the product to be 5-O- ribofuranoside 9c in MeOH was used.
phosphate-1-O-benzyl-2,3-O-bis(ethylcarbonate)-β-D-ribofurano-
side 12b (150 mg, 85%).
The HPLC-MS analysis revealed the product to be a
1 : 1 mixture of 5-O-phosphate-1-O-methyl-2,3-O-bis(isobutyl-
[α]2D0 = −103.4° (c 0.19, MeOH); 1H NMR (MeOD): δ carbonate)-β-D-ribofuranose 13c and 5-O-phosphate-2,3-O-bis-
7.27–7.17 (5H, m, Ar), 5.18 (1H, dd, J = 6.6, 4.5 Hz, H-3), (isobutylcarbonate)-β-D-ribofuranoside 14c (120 mg, 87%).
5.05–5.04 (2H, m, H-1, H-2), 4.71 (1H, d, J = 11.6 Hz, CH2Ph),
13c: HRMS m/z 429.11678 (calc. for C15H26O12P ([M − H]+):
4.43 (1H, d, J = 11.6 Hz, CH2Ph), 4.22 (1H, q, J = 5.2 Hz, H-4), 429.11674).
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14c: HRMS m/z 443.13227 (calc. for C16H28O12P ([M − H]+): gradient 0–13% B in 40 min) revealed pure diphosphate deriva-
433.13239).
tive 16a eluted at 25 min in 61% yield (34 mg).
1H NMR (D2O): δ 8.54 (1H, s, H-8), 8.16 (1H, s, H-2), 6.28
(1H, d, J = 4.9 Hz, H-1′′), 6.13 (1H, d, J = 5.7 Hz, H-1′), 5.44 (1H,
t, J = 5.3 Hz, H-2′′), 5.03 (1H, dd, J = 7.4, 5.6 Hz, H-3′′), 4.75
(1H, dd, J = 5.7, 5.0 Hz, H-2′), 4.52 (1H, dd, J = 5.0, 3.8 Hz,
H-3′), 4.43–4.37 (2H, m, H-4′, H-4′′), 4.21 (3H, br s, H-5′, H-5′′
a), 4.06 (1H, ddd, J = 12.1, 5.8, 3.4 Hz, H-5′′b); 13C NMR (D2O):
δ 154.7 (CO), 151.7 (CO), 148.1 (C-2), 140.3 (C-6), 135.1 (C-4),
131.7 (C-8), 103.8 (C-1′′), 87.1 (C-1′), 83.9 (d, J = 2.4 Hz, C-4′),
79.1 (m, C-4′′), 77.8 (C-2′′), 74.4 (C-2′), 73.2 (C-3′′), 70.3 (C-3′),
65.2 (m, C-5′′), 63.7 (m, C-5′), 56.0 (CH3); 31P NMR (D2O):
δ −11.40; HRMS m/z 642.04870 (calc. for C18H22O17N5P2
([M − H]+): 642.04914).
5-O-Phosphate-2,3-O-bis(isobutylcarbonate)-β-D-ribofuranose
13c
For the reaction, 470 mg (0.67 mmol) of ribofuranoside 9c in a
THF–mQ H2O (3/2, v/v, 15 mL) mixture was used.
The HPLC-MS analysis revealed the product to be a
9 : 1 mixture of 5-O-phosphate-2,3-O-bis(isobutylcarbonate)-β-D-
ribofuranose 13c and 5-O-phosphate-1,2-O-carbonate-3-O-iso-
butylcarbonate-β-D-ribofuranoside 15c (280 mg, 97%), which
underwent a migration towards product 15c.
13c: HRMS m/z 429.11678 (calc. for C15H26O12P ([M − H]+):
429.11674).
1′,2′-O-Carbonate-3′-O-ethylcarbonate-ADP-ribofuranoside
16b. For the reaction, 16.0 mg (47.9 μmol) of ribofuranoside
15b was used.
The purification by reversed phase HPLC on a C18 column
(10 × 250 mm, A: ammonium formate 10 mM, B: AcCN, gradi-
ent 0–13% B in 20 min, 13–30% B in 10 min, 30–50% B in
10 min, run time 40 min) revealed pure diphosphate derivative
16b eluted at 15 min in 52% yield (16 mg).
15c: [α]2D0 = +30.1° (c 0.33, MeOH); IR: νmax/cm−1 (KBr):
3437, 2925, 2855, 1820, 1755 (CO), 1470, 1383, 1258, 1178,
1
1095, 1008, 962, 787; H NMR (D2O): δ 6.35 (1H, d, J = 4.9 Hz,
H-1), 5.49 (1H, t, J = 5.3 Hz, H-2), 5.10 (1H, dd, J = 7.5, 5.4 Hz,
H-3), 4.51 (1H, d, J = 7.5 Hz, H-4), 4.16 (1H, ddd, J = 12.2, 5.6,
2.2 Hz, H-5a), 4.05–3.90 (3H, m, H-5b, CH2CH(CH3)2), 1.95
(1H, m, CH2CH(CH3)2), 0.83 (6H, d, J = 6.7 Hz, CH2CH(CH3)2);
13C NMR (D2O): δ 155.1 (CO), 154.3 (CO), 103.8 (C-1), 79.1 (d,
J = 7.9 Hz, C-4), 77.8 (C-2), 75.8 (CH2CH(CH3)2), 72.3 (C-3), 62.8
(d, J = 4.0 Hz, C-5), 27.2 (CH2CH(CH3)2), 17.9 (CH2CH(CH3)2);
31P NMR (D2O): δ 0.01; HRMS m/z 355.04372 (calc. for
C11H16O11P ([M − H]+): 355.04357).
1H NMR (D2O): δ 8.43 (1H, s, H-8), 8.18 (1H, s, H-2), 6.19
(1H, d, J = 4.9 Hz, H–1′′), 6.04 (1H, d, J = 5.4 Hz, H-1′), 5.35
(1H, t, J = 5.4 Hz, H-2′′), 4.93 (1H, dd, J = 6.9, 5.8 Hz, H-3′′),
H-2′under the water signal, 4.42 (1H, dd, J = 4.9, 3.3 Hz, H-3′),
4.35–4.31 (1H, m, H-4′′), 4.30–4.26 (1H, m, H-4′), 4.15–4.06
(5H, m, H-5′, H-5′′a, CH2CH3), 4.00–3.93 (1H, m, H-5′′b), 1.15
(3H, t, J = 7.2 Hz, CH2CH3); 13C NMR (D2O): δ 154.0 (CO), 150.3
(CO), 145.5 (C-2), 145.1 (C-6), 143.9 (C-4), 140.6 (C-8), 103.8
(C-1′′), 87.2 (C-1′), 83.9 (d, J = 4.3 Hz, C-4′), 79.1 (d, J = 5.1 Hz,
C-4′′), 77.8 (C-2′′), 74.4 (C-2′), 73.0 (C-3′′), 70.2 (C-3′), 66.1
(CH2CH3), 65.1 (d, J = 2.5 Hz, C-5′), 63.7 (d, J = 4.5 Hz, C-5′′),
13.2 (CH2CH3); 31P NMR (D2O): δ −11.48; HRMS m/z 656.06396
(calc. for C19H24O17N5P2 ([M − H]+): 656.06479).
5-O-Phosphate-2,3-O-carbonate-β-D-ribofuranose
the reaction, 190 mg (0.44 mmol) of ribofuranoside 9d was
used.
11d. For
HPLC-MS of the crude product revealed pure 5-O-phos-
phate-2,3-O-carbonate-β-D-ribofuranose 11d as a mixture of
α- and β-anomers in a 3 : 7 ratio in 84% yield (94 mg).
1H NMR (D2O): δ 6.14 (0.3H, br s, H-1α), 5.59 (0.3H, br s,
H-2α), 5.46 (0.7H, s, H-1β), 5.38 (0.7H, d, J 6.1 Hz, H-2β), 5.17
(0.3H, br s, H-3α), 5.06 (0.7H, d, J = 6.1 Hz, H-3β), 4.44 (0.7H,
br s, H-4β), 4.24–4.17 (0.3H, m, H-4α), 4.08–3.97 (2H, m, H-5);
13C NMR (D2O): δ 102.8 (C-1), 86.1 (C-4, C-2), 82.9 (C-3), 66.10
(C-5); 31P NMR (D2O): δ 0.80; HRMS m/z 254.99111 (calc. for
C6H8O9P ([M − H]+): 254.99114).
1′,2′-O-Carbonate-3′-O-isobutylcarbonate-ADP-ribofuranoside
16c. For the reaction, 32.0 mg (90.0 μmol) of ribofuranoside
15c was used.
The purification by reversed phase HPLC on a C18 column
(10 × 250 mm, A: ammonium formate 10 mM, B: AcCN, gradi-
ent 0–13% B in 20 min, 13–30% B in 10 min, 30–50% B in
10 min, run time 40 min) revealed pure diphosphate derivative
16c eluted at 18 min in 58% yield (36 mg).
Coupling of 5-phosphate-ribofuranoside derivatives with AMP
morpholidate (ball mill technique)15
5-Phosphate ribofuranoside (1 × n), AMP morpholidate (1.1 ×
1H NMR (D2O): δ 8.56 (1H, s, H-8), 8.30 (1H, s, H-2), 6.30
n), 1H-tetrazole (2 × n), MgCl2·6H2O (1.5 × n) and water (6 × n) (1H, d, J = 4.9 Hz, H-1′′), 6.14 (1H, d, J = 5.6 Hz, H-1′), 5.46 (1H,
were put together into a ball milling jar of 25 mL inner t, J = 5.3 Hz, H-2′′), 5.05 (1H, dd, J = 6.7, 5.7 Hz, H-3′′), 4.75
volume. A 1.5 cm steel ball was added inside, the jar was (1H, t, J = 5.0 Hz, H-2′), 4.52 (1H, dd, J = 5.0, 3.9 Hz, H-3′), 4.46
closed and set to a ball milling machine to move at 30 Hz fre- (1H, td, J = 6.7, 3.4 Hz, H-4′′), 4.38 (1H, bd, J = 2.7 Hz, H-4′),
quency for 90 min. Once finished, the jar was left to cool down 4.27–4.20 (3H, m, H-5′, H-5a′′), 4.08 (1H, ddd, J = 11.9, 6.1, 3.4
to room temperature, and then the product was dissolved in Hz, H-5b′′), 3.96 (1H, dd, J = 10.2, 6.7 Hz, CH2CH(CH3)2), 3.89
mQ water and freeze-dried.
(1H, dd, J = 10.2, 6.7 Hz, CH2CH(CH3)2), 1.89 (2H, heptuplet,
1′,2′-O-Carbonate-3′-O-methylcarbonate-ADP-ribofuranoside J = 6.7 Hz, CH2CH(CH3)2), 0.87 (6H, d, J = 6.7 Hz, CH2CH-
16a. For the reaction, 27.0 mg (86.0 μmol) of ribofuranoside (CH3)2); 13C NMR (D2O): δ 156.0 (CO), 154.1 (CO), 150.9 (C-2),
15a was used.
140.5 (C-8), 103.9 (C-1′′), 87.2 (C-1′), 83.9 (d, J 3.4 Hz, C-4′), 79.5
The purification by reversed phase HPLC on a C18 column (d, J 4.2 Hz, C-4′′), 77.8 (C-2′′), 75.7 (CH2CH(CH3)2), 74.4 (C-2′),
(10 × 250 mm, A: ammonium formate 10 mM, B: AcCN, 73.1 (C-3′′), 70.3 (C-3′), 65.2 (d, J 2.2 Hz, C-5′), 63.9 (t, J 3.5 Hz,
5712 | Org. Biomol. Chem., 2013, 11, 5702–5713
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C-5′′), 27.2 (CH2CH(CH3)2), 17.9 (CH2CH(CH3)2), 17.9 (CH2CH- (m, C-5′′), 13.3 (CH2CH3); 31P NMR (D2O): δ −11.28; HRMS m/z
(CH3)2); 31P NMR (D2O): δ −11.38; HRMS m/z 684.09537 (calc. 792.15351 (calc. for C28H36O18N5P2 ([M − H]+): 792.15361).
for C21H28O17N5P2 ([M − H]+): 684.09609).
2′,3′-O-Carbonate-ADP-ribose 13d. For the reaction was used
48.0 mg (185 μmol) of ribofuranoside 11d.
Acknowledgements
The purification by reversed phase HPLC on a C18 column
(10 × 250 mm, A: ammonium formate 10 mM, B: AcCN, gradi-
ent 0–13% B in 40 min) revealed pure diphosphate derivative
13d eluted at 17 min in 45% yield (50.5 mg).
The financial support received from Marie Curie Actions for
early-stage researchers (MEST-CT-2005-020351) is gratefully
acknowledged.
The product 13d exists as an equilibrium mixture of α- and
β-anomeric forms (S2 + S3) together with the 1′,2′-O-carbonate/
ADP/ribofuranoside 13e (S1) form arising from carbonate
migration in a 1 : 1 : 1 ratio.
Notes and references
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1H NMR (D2O): δ 8.53 (1H, d, J = 2.4 Hz, H-8), 8.28 (1H, d,
J = 2.4 Hz, H-2), 6.20 (0.3H, d, J = 4.8 Hz, H-1′′, S1), 6.15 (1H, d,
J = 5.7 Hz, H-1′), 5.71 (0.4H, d, J = 4.0 Hz, H-1′′, S2), 5.57 (0.3H,
s, H-1′′, S3), 5.43 (0.3H, br d, J = 7.3 Hz, H-3′′, S3), 5.32 (0.4H,
dd, J = 7.3, 1.4 Hz, H-3′′, S2), 5.24–5.18 (0.7H, m, H–2′′, S2,
H-2′′, S1), 5.15 (0.4H, d, J = 6.8 Hz, H-2′′, S3), H-2′ and H-3′′, S1
under water signal, 4.56–4.50 (1.6H, m, H-3′, H-4′′, S2, S3),
4.42–4.37 (1H, m, H-4′), 4.28 (0.4H, d, J 4.6 Hz, H-4′′, S1),
4.25–4.21 (2H, m, H-5′), 4.12–4.03 (2H, m, H-5′′, S1, S2, S3); 13
C
NMR (D2O): δ 155.1 (CO), 152.1 (C-2), 149.1 (CO), 140.0 (C-8),
103.2 (C-1′′, S1), 101.1 (C-1′′, S3), 96.5 (C-1′′, S2), 86.9 (C-1′),
84.8 (C-2′′, S3), 83.8 (C-4′), 83.7 (C-4′′, S3), 81.8 (C-3′′, S3), 81.4
(C-3′′, S2), 79.9 (C-4′′, S2), 79.8 (C-2′′, S1), 79.6 (C-4′′, S1), 79.0
(C-2′′, S2), 74.2 (C-2′), 70.3 (C-3′), 68.7 (C-3′′, S1), 66.2 (d, J =
4.7 Hz, C-5′′, S3), 65.4 (d, J = 4.2 Hz, C-5′′, S2), 65.2 (t, J = 2.9 Hz,
C-5′), 63.2 (d, J = 3.3 Hz, C-5′′, S1); 31P NMR (D2O): δ −10.80;
HRMS m/z 586.05823 (calc. for C16H22O15N5P2 ([M + H]+):
586.05821).
1′-O-Benzyl-2′,3′-O-bis(ethylcarbonate)-ADP-ribofuranoside
17b. For the reaction, 40.0 mg (86.1 μmol) of ribofuranoside
12b was used.
The purification by reversed phase HPLC on a C18 column
(10 × 250 mm, A: ammonium formate 10 mM, B: AcCN, gradi-
9 O. P. Chevallier and M. E. Migaud, Beilstein J. Org. Chem.,
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10 min, run time 40 min) revealed pure diphosphate derivative
17b eluted at 35 min in 68% yield (46 mg).
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11 M. Dvorakova, M. Pribylova, R. Pohl, M. E. Migaud and
T. Vanek, Tetrahedron, 2012, 68, 6701.
1H NMR (D2O): δ 8.52 (1H, s, H-8), 8.21 (1H, s, H-2),
7.30–7.23 (5H, m, Ar), 6.06 (1H, d, J = 5.4 Hz, H-1′), 5.24 (1H, 12 H. Dong, Y. X. Zhou, X. L. Pan, F. C. Cui, W. Liu and
dd, J = 6.1, 4.7 Hz, H-3′′), 5.17 (1H, d, J = 1.2 Hz, H-1′′), 5.11 O. Ramstrom, J. Org. Chem., 2012, 77, 1457.
(1H, dd, J = 4.7, 1.2 Hz, H-2′′), 4.67–4.64 (2H, m, H-2′, CH2Ph), 13 S. J. Mills, C. S. Liu and B. V. L. Potter, Carbohydr. Res.,
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CH2Ph), 4.37–4.33 (2H, m, H-4′, H-4′′), 4.27–4.13 (7H, m, H-5′, 14 J. G. Moffatt and H. G. Khorana, J. Am. Chem. Soc., 1959,
H-5′′a, CH2CH3), 4.08–4.00 (1H, m, H-5′′b), 1.23 (6H, m, 81, 1265.
CH2CH3); 13C NMR (D2O): δ 154.2 (CO), 149.4 (CO), 149.1 (C-2), 15 F. Ravalico, I. Messina, M. V. Berberian, S. L. James,
141.9 (C-6), 140.7 (C-4), 140.6 (C-8), 136.0 (Ar), 128.5 (Ar), 128.4 M. E. Migaud and J. S. Vyle, Org. Biomol. Chem., 2011, 9, 6496.
(Ar), 128.2 (Ar), 103.5 (C-1′′), 87.4 (C-1′), 83.8 (d, J = 2.7 Hz, 16 J. D. Trzupek and T. L. Sheppard, Org. Lett., 2005, 7, 1493.
C-4′), 79.3 (d, J = 2.1 Hz, C-4′′), 77.3 (C-2′′), 74.6 (C-2′), 74.5 17 G. Dufner, R. Schworer, B. Muller and R. R. Schmidt,
(C-3′′), 70.2 (C-3′), 69.7 (CH2CH3), 66.0 (m, C-5′, CH2Ph), 65.0
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