Synthesis and Deprotection of Biodegradably and Thermally Protected Dinucleoside-2′,5′-Monophosphate Prodrug Model of 2-5A

Article abstract of DOI:10.1002/cbdv.201700220

Protected dinucleoside-2′,5′-monophosphate has been prepared to develop a prodrug strategy for 2-5A. The removal of enzymatically and thermally labile 4-(acetylthio)-2-(ethoxycarbonyl)-3-oxo-2-methylbutyl phosphate protecting group and enzymatically labile 3′-O-pivaloyloxymethyl group was followed at pH 7.5 and 37?°C by HPLC from the fully protected dimeric adenosine-2′,5′-monophosphate 1 used as a model compound for 2-5A. The desired unprotected 2′,3′-O-isopropylideneadenosine-2′,5′-monophosphate (9) was observed to accumulate as a major product. Neither the competitive isomerization of 2′,5′- to a 3′,5′-linkage nor the P–O5′ bond cleavage was detected. The phosphate protecting group was removed faster than the 3′-O-protection and, hence, the attack of the neighbouring 3′-OH on phosphotriester moiety did not take place.

Full text of DOI:10.1002/cbdv.201700220

Article Type: Full Paper
Synthesis and Deprotection of Biodegradably and Thermally
Protected Dinucleoside-2´,5´-Monophosphate
Prodrug Model of 2-5A
Emilia Kiuru,a Suvi Malmikare a and Mikko Ora*, a
a Department of Chemistry, University of Turku, FI-20014 Turku,
(e-mail: mikora@utu.fi)
Protected dinucleoside-2´,5´-monophosphate has been prepared to develop a prodrug strategy for 2-5A. The removal of
enzymatically and thermally labile 4-acetylthio-2-ethoxycarbonyl-3-oxo-2-methylbutyl phosphate protecting group and
enzymatically labile 3´-O-pivaloyloxymethyl group was followed at pH 7.5 and 37oC by HPLC from the fully protected dimeric
adenosine-2´,5´-monophosphate
1
used as
a
model compound for 2-5A. The desired unprotected 2´,3´-O-
isopropylideneadenosine-2´,5´-monophosphate (9) was observed to accumulate as a major product. Neither the competative
isomerization of 2´,5´- to a 3´,5´-linkage nor the P-O5´ bond cleavage was detected. The phosphate protecting group removed
faster than the 3´-O-protection and, hence, the attack of the neighbouring 3´-OH on phosphotriester moiety did not take place.
Keywords: Prodrugs • Nucleotides • 2-5A • Protecting group • Deprotection
Introduction
The 2-5A/RNase L system is one of the most important immunity pathway that acts against viral infections. As a result of
infectious agents cells secrete interferons which activates enzyme, called 2´,5´-adenylate synthetase, to produce 2´,5´-
oligoadenylates (2-5A; [px5´A(2´p5´A)n; x = 1-3; n ≥ 2]) from adenosine triphosphate. The 2-5A molecules, in turn, activates 2-
5A-dependent RNase (RNase L) that cleaves viral RNA resulting in apoptosis.[1-4] Accordingly, the synthetic structurally modified
2´,5´-oligoadenylates which activate RNase L constitute a potential class of therapeutic agents to compact against viral
diseases and cancer.[5] To be applicable as a drug, the 2-5A must exhibit a good cellular uptake and resistance against
biodegradation. Numerous modified 2-5A pro-nucleotides, such as phosphorothioate,[6-9] alanyltyrosine,[10] 2´-O-
phosphoglyceryl,[11] 5´-capped,[12] aminofuctionalized[13] and phosphoramidate derivatives[14] as well as 3´-deoxyadenosine,[15] 3´-
O,4-C-bridged adenosine,[16] 3´-fluoro-3-deoxyadenosine,[17] 3´-O-methyladenosine[18] and 3´-amino-3´-deoxyadenosine[19]
analogues have been synthesized to increase the stability in serum and cytoplasm. Several techniques have also been tested to
[20]
enhance the cell delivery of 2-5A and their modified analogues, as an example, encapsulation in liposomes
and conjugation
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to a lipophilic groups,[21-23] but no satisfactory prodrug strategy for the enhancement of cellular uptake has been developed.
Generally, most of the prodrug approaches of nucleoside 5´-phosphomonoesters and 3´,5´-phosphodiesters are based on
enzymatic transformation that triggers a chemical removal of the remnants of phosphate protecting group.[24,25] In case of 2-5A,
both the negatively charged phosphate functions and 3´-OH groups must be protected, otherwise 3´-OH attacks readily on
adjacent phosphotriester moiety.[26,27]
We have previously studied on removal of esterase labile protecting groups from dimeric adenylyl-2´,5´-adenosines[28] (2´,5´-
ApA) (2a and b) and from 2-5A trimers[29] (pA2´p5´A2´p5´A) (3a and b) using 3-acetyloxy-2,2-bis(ethoxycarbonyl)propyl
protection[30,31] for the phosphate moieties and pivaloyloxymethyl (PivOCH2) or acetyloxymethyl (AcOCH2) groups for the 3´-OH.
In all cases, the exposed 3´-OH function attacks on the still protected phosphodiester linkage resulting in a complex product
distribution. With trimers, a dramatic retardation of the enzymatic removal of 3-acetyloxy-2,2-bis(ethoxycarbonyl)propyl
protecting group was also observed upon accumulation of the negative charge on the phosphodiester linkages. To develop an
improved prodrug strategy, we now report on an approach based on the enzymatically labile 4-acetylthio-2-ethoxycarbonyl-3-
oxo-2-methylbutyl group[32,33] of the internucleosidic phosphodiester linkage that undergoes also thermolytic removal. 3´-
Hydroxyl function was protected with the enzymatically removable pivaloyloxymethyl group. For this reason, the dimeric model
o
compound 1 was synthesized and the removal of the protecting groups was followed by HPLC at 37 C and pH = 7.5 in the
presence and absence of carboxyesterase.
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Figure 1. Structures of dimeric adenylyl-2´,5´-adenosines and trimeric 2-5A.
Results and Discussion
Preparation of Fully Protected Adenosine-2´,5´-monophosphate (1). 2´,3´-O-Isopropylidene-N6-(4-methoxytrityl)adenosine (4)
was prepared as describer previously.[34,35] 5´-Hydroxyl group of commercially available 2´,3´-O-isopropylideneadenosine was
temporarily trimethylsilylated followed by the tritylation of N6 and the removal of silyl group by treating with ammonia (Scheme 1).
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Scheme 1. Conditions: i) 1.TMSCl, Py, 2. MMTrCl, Py, 3. NH4OH.
The adenosine building block 6a was synthesized analogously as reported earlier.[28] Accordingly, 5´-OH and N6-amino functions
of adenosine were first 4-methoxytritylated to afford 5 (Scheme 2). Alkylation of 5 with pivaloyloxymethyl chloride in the
presence of NaH and sodium iodine as a catalyst gave a mixture of 3´-O- (6a) and 2´-O-(pivaloyloxymethyl)-5´-O,N6-bis(4-
methoxytrityl)adenosine (6b). The separation of the isomers proved difficult and, hence, a mixture of 6a and 6b was used in a
next step. Attempts with 2´-O-tert-butyldimethylsilylated adenosine failed, since TBDMS group migrated between 2´-and 3´-OH
groups upon pivaloyloxymethylation.
Scheme 2. Conditions: i) MMTrCl, pyridine; ii) PivOCH2Cl, NaH, NaI, THF.
Compound 1 was prepared by phosphitylation method using the appropriately protected nucleosides (4 and 6a) and ethyl 4-
(acetylthio)-2-(hydroxymethyl)-2-methyl-3-oxobutanoate. A mixture of 3´-O- (6a; see Scheme 3) and 2´-O-(pivaloyloxymethyl)-
5´-O,N6-bis(4-methoxytrityl)-3´-O-(pivaloyloxymethyl)adenosine
(6b)
was
first
phosphitylated
with
bis(diethylamino)phosphorochloridite in DCM in the presence of triethylamine. The subsequent tetrazole-promoted displacement
of the diethylamino ligands of the diethylaminophosphoramidite by 2´,3´-O-isopropylidene-N6-(4-methoxytrityl)adenosine and
ethyl 4-(acetylthio)-2-(hydroxymethyl)-2-methyl-3-oxo-butanoate in acetonitrile gave the phosphite ester that was oxidized with I2
in a mixture of THF, H2O and 2,6-lutidine. The phosphate ester 7 obtained was passed through a short silica gel column and
used without further purification. Finally, the 4-methoxytrityl groups were removed with 80% aqueous acetic acid to give the
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fully protected diadenosine monophosphate as a mixture of 2´,5´- (1) and 3´,5´-isomers (10) separated by a reverse phase
HPLC. Assignment of the isomers 1 and 10 as 2´,5´- and 3´,5´-phosphotriesters is based on the couplings between C2´ or C3´
and CH2 of POM obtained from HMBC spectra.
Scheme 3. Conditions: i) (Et2N)2PCl, Et3N, DCM; ii) TetH, MeCN; iii) TetH, MeCN; iv) I2, THF, H2O, 2,6-lutidine; v) 80% AcOH.
Deprotection of Fully Protected Adenosine-2´,5´-monophosphate (1). The decomposition of 1 (tR = 29.4 min) was followed in the
presence of porcine liver carboxyesterase (PLE; 2.6 units/ml) in a HEPES buffer at pH 7.5 and at 37 oC. The composition of the
aliquot withdrawn at appropriate intervals from the reaction solution was determined by RP HPLC. According to the MS-HPLC
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analyses, the 4-acetylthio-2-ethoxycarbonyl-3-oxo-2-methylbutyl protecting group was completely removed before the
pivaloyloxymethyl group started to release. Accordingly, 3´-O-pivaloyloxymethyladenosin-2´-yl 2´,3´-O-isopropylideneadenosin-
5´-yl phosphate (8; tR = 20.5 min; [M-H-] at m/z 749.2) was observed to accumulate as an intermediate and was further
decomposed nearly quantitatively to the desired unprotected adenosin-2´-yl 2´,3´-O-isopropylideneadenosin-5´-yl phosphate (9;
tR = 15.2 min; [M-H-] at m/z 635.1) as shown in Scheme 4 and Figure 2. The half-lives for the disappearance of 1 and 8 were 6.3
min (k = 1.84 x 10-3 s-1) and 612 min (k = 1.89 x 10-5 s-1), respectively. The small intermediate detected at the retention time 24.9
min accumulated to such an extent (< 2%) that it could have not been reliably quantified but, most likely, it represents the
deacetylated starting material. No sign of isomerization of 2´,5´-to 3´,5´-isomer or P-O5´-bond cleavage was detected. Only
traces of unknown side products could be observed. We have recently described that the esterase- and thermolabile 2,2-
disubstituted 4-acylthio-3-oxobutyl protecting groups are removable also in a cell extract. For example, treatment of 4-acetylthio-
2,2-dimethyl-3-oxobutyl protected bis(2´-C-methyluridin-5´-yl)phosphate in a whole cell extract of human prostate carcinoma
released the desired 5´,5´-phosphodiester as a main product.[32]
Scheme 4. PLE-triggered deprotection of 1.
The data clearly shows that the now reported protection group strategy is superior compared to the earlier reported one for the
dimers 2a and b.[28] During 1 day, approximately 80% of 1 was converted to the desired unprotected phosphodiester 9 the
amount of which was finally about 90% (Figure 2). With 2a and b, 2´,5´ApA, in turn, accumulated only as a minor product during
1 day (20% and 5%, respectively), since the isomerization to 3´,5´-isomer and cleavage of P-O5´ bond markedly competed with
the deprotection (Figure 3 and 4).[28] The removal of 4-acetylthio-2-ethoxycarbonyl-3-oxo-2-methylbutyl from 1 proceeds more
than one order of magnitude faster (t1/2
bis(ethoxycarbonyl)propyl from 2a (t1/2 = 395 min; k = 2.9×10-5 s-1).
= 6.3 min; k = 1.84 x
10-3 s-1) than that of 3-acetyloxymethoxy-2,2-
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Figure 2. RP-HPLC traces for the carboxyesterase-catalyzed deprotection of 1 at pH 7.5 and 37.0 oC (I = 0.1 M with NaCl). The traces from top to
bottom refer to aliquots withdrawn at 15 s, 7 h and 48 h. For the chromatographic conditions, see the experimental section.
Figure 3. RP-HPLC traces for the HLE-catalyzed hydrolysis of 2b at pH 7.5 and 37.0 oC (I = 0.1 mol L-1 with NaCl).[28]
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Figure 4. RP-HPLC traces for the HLE-catalyzed hydrolysis of 2a at pH 7.5 and 37.0 oC (I = 0.1 M with NaCl).[29]
As mentioned above, it is known that enzyme triggered deprotection is markedly retarded upon accumulation of a negative
charge on the neighboring phosphodiester linkages as a result of reduced affinity of the enzyme to charges substrates.[29] Our
previous studies with thermolabile 4-acetylthio-2,2-dimethyl-3-oxobutyl protected oligomeric 3´,5´-phosphodiesters have been
shown that the non-enzymatic removal of the second protecting group from internucleosidic phosphodiester linkages was only
slightly retarded (by a factor of 1.15-1.50) compared to the first one.[33] Moreover, now presented 4-acetylthio-2-ethoxycarbonyl-
3-oxo-2-methylbutyl protecting group is known to be even 19.6 and 1.6 times more labile than 4-acetylthio-2,2-dimethyl-3-
oxobutyl group thermolytically and enzymatically, respectively.[32] For these reason it can be assumed that our prodrug strategy,
in all likelihood, offers a potential method also for trimeric oligoadenylates (2-5A). To measure the non-enzymatic departure rate
of the 4-acetylthio-2-ethoxycarbonyl-3-oxo-2-methylbutyl from phosphotriester linkage, the decomposition of 1 was also followed
in the absence of enzyme at pH 7.5 and 37.0 oC. Starting material 1 was observed to undergo quantitative conversion into 8 five
times slower than in the presence of enzyme, the half-live being 34.4 min (k = 3.36 x 10-4 s-1). Accordingly, in the presence of
enzyme, intermediate 8 is most likely formed by two competative routes, i.e., a minor non-enzymatic one and a major enzymatic
pathway.
Deprotection of Fully Protected Adenosine-3´,5´-monophosphate (10). For comparison, removal of the 4-acetylthio-2-
ethoxycarbonyl-3-oxo-2-methylbutyl was also followed from 10. The observed rate constant for the PLE-catalyzed deacetylation
of the phosphotriester 10 was 3.36 x 10-4 s-1 (t1/2 = 34.4 min). Only product observed to accumulate was 2´-O-
pivaloyloxymethyladenosin-2´-yl 2´,3´-O-isopropylideneadenosin-5´-yl phosphate (8). Unexpectedly, the pivaloyloxymethyl
group was released much slower. Even after 3 d, we could not reliable verify the release of POM.
Mechanisms. As discussed previously, the mercapto intermediate formed by the esterase-catalyzed deacetylation undergoes an
intramolecular cyclization with a concomitant release of the remnants of the protecting group (Scheme 5A).[32] Also the non-
enzymatic departure of the protecting group takes place by cyclization after the hydration of the keto group at C3 and by the
subsequent migration of the acetyl group from sulfur into the oxyanion of the C3 of geminal diol (Scheme 5B).[32] Earlier studies
has also been shown that the 4,4-disubstituted dihydrothiophen-3(2H)-one released is not markedly alkylating and was not
observed to form glutathione adduct.[32] The removal of the pivaloyloxymethyl group takes place by an esterase-catalyzed
deacylation and a consequtive half-acetal hydrolysis with a loss of formaldehyde.
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Scheme 5. A) Mechanism of the enzymatic removal of the protecting group. B) Mechanism of the non-enzymatic removal of the protecting
groups.
Conclusions
In summary, the data suggest that decreasing the stability of the phosphate protecting group by replacement of the earlier used
3-acetyloxymethoxy-2,2-bis(ethoxycarbonyl)propyl group with the esterase and thermolabile 4-acetylthio-2-ethoxycarbonyl-3-
oxo-2-methylbutyl group allows a feasible prodrug strategy for 2-5A. The fully protected dimeric adenylyl-2´,5´-adenosine
prodrug model 1 was observed to undergo the deprotection nearly quantitatively into the desired unprotected product (9). We
also showed that the deprotection of 1 produced exclusively 8 in the absence of enzyme. An additional advantage of the now
used phosphate protecting group is that it is removable even if the enzymatic deprotection is retarded.
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Experimental Section
General
4-(Acetylthio)-2-(hydroxymethyl)-2-methyl-3-oxo-butanoate was synthesized as described previously.[32] THF, DCM, MeCN and
Py were dried over 4Å molecular sieves. TEA was dried by refluxing over CaH2 and distilled. The assignment of the NMR
signals is based on 2D COSY and HSQC spectra. 1H, 13C, 31P and 2D-NMR spectra were recorded on a Bruker Avance 400 or
500 NMR spectrometer. High-resolution mass spectra were acquired using a Bruker Daltonics micrOTOF-Q and electrospray
ionization technique. LC-ESI-MS: Agilent 6120 quadrupole LC-ESI-MS. The kinetic samples were analyzed on a Merck Hitachi
LaChrom D7000 HPLC using Hypersil C18 column (4 x 250 mm x 5 µm).
2´,3´-O-Isopropylidene-N6-(4-methoxytrityl)adenosine (4). 2´,3´-O-Isopropylideneadenosine (13.0 mmol, 4.00 g) was
dissolved into dry pyridine (117 ml) and trimethylsilylchloride (26.0 mmol, 3.31 ml) was added. After 1 h 45 min, 4-
methoxytritylchloride (13.0 mmol, 4.00 g) was added and the reaction was allowed to proceed for overnight. Aqueous 28%
NH4OH solution (29.0 ml) was added and the reaction mixture was stirred for 45 min. The volatiles were evaporated to dryness
and the residue was dissolved in DCM and washed with 5% aq. NaHCO3. The organic phase was dried on Na2SO4, filtered and
evaporated to dryness. Crude product was purified by silica gel chromatography using a 1:1 (v/v) mixture of EtOAc and
1
petroleum ether as an eluent to yield 4 (1.68 g, 22%). H-NMR (400 MHz, (CD3)2SO): 8.43 (s, H-C(8)); 7.93 (s, H-C(2)); 7.39-
7.19 (m, 12H of MMTr); 6.84 (d, J = 8.79, 2H of MMTr); 6.12 (d, J = 3.20, H-C(1´)); 5.34 (dd, J = 6.40 and 3.2, H-C(2´)); 4.94 (dd,
J = 6.40 and 2.80, H-C(3´)); 4.20 (m, H-C(4´)); 3.71 (s, MeO of MMTr); 3.54 (dd, J = 11.59 and 4.80, H-C(5´)); 3.49 (dd, J =
11.59 and 5.20, H-C(5´´)); 1.50 (s, CH3); 1.30 (s, CH3). 13C-NMR (101 MHz, (CD3)2SO): 158.21 (MMTr); 154.12 (C(6)); 151.80
(C(2)); 148.48 (C4); 145.54 (MMTr); 140.85 (C(8)); 137.47, 130.32, 128.94, 128.17, 127.02 (MMTr); 121.30 and 121.29 (spiro C
of isopropylidene and C(5)); 113.49 (MMTr); 90.18 (C(1´)); 87.03 (C(4´)); 83.73 (C(2´)); 81.76 (C(3´)); 70.47 (MMTr); 61.99
+
(C(5´)); 55.47 (OMe); 27.50 (Me); 25.62 (Me). HR-MS: 602.2351 ([M+Na]+, C56H55N5NaO8 ; calc. 602.2374).
5´-O,N6-Bis(4-methoxytrityl)adenosine (5). Adenosine (18.7 mmol, 5.00 g) was coevaporated from dry pyridine and dissolved
in the same solvent (70 ml). A solution of 4-methoxytritylchloride (39.3 mmol, 12.1 g) in dry pyridine (70 ml) was added. The
o
reaction mixture was stirred at 40 C overnight. The reaction was quenched by adding MeOH (18 ml) and the volatiles were
removed under reduced pressure. The residue was dissolved in DCM and washed with sat. aq. NaHCO3 and brine. The organic
phase was dried on Na2SO4, filtered and evaporated to dryness. Crude product was purified by silica gel chromatography using
1
DCM containing 1% MeOH and 0.1% pyridine as an eluent to yield 5 (9.4 g, 62%). H-NMR (400 MHz, (CD3)2SO): 8.34 (s, H-
C(2)); 7.84 (s, H-C(8)); 7.39-7.17 (24H of MMTr); 6.86-6.82 (4H of MMTr); 5.91 (d, J = 4.80, H-C(1´)); 5.52 (s, 2´-OH); 5.21 (s,
3’-OH); 4.72 (t, H-C(2’)); 4.28 (t, H-C(3’)); 4.05 (m, H-C(4’)); 3.71 (MeO); 3.20-3.19 (m, H-C(5´ and 5´´)). 13C-NMR (101 MHz,
(CD3)2SO): 158.64, 158.21 (MMTr); 154.08 (C(6)); 151.72 (C(2)); 149.0, 145.54, 144.83, 144.57 (C(4) and MMTr); 141.00
(C(8)); 137.53, 135.38, 130.51, 130.32, 128.96, 128.44, 128.35, 128.26, 128.16, 127.27, 127.01, 124.37 (MMTr); 121.43 (C(5));
113.65 and 113.48 (MMTr); 88.65 (C(1´)); 86.21 (spiro C of MMTr); 83.60 (C(4´)); 73.24 (C(2´)); 70.79 (C(3´)); 70.47 (spiro C of
+
MMTr); 64.22 (C(5´)); 55.47 (OMe). HR-MS: 812.3420 ([M+H]+, C50H46N5O6 ; calc. 812.3443).
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3´-O-(Pivaloyloxymethyl)-5´-O,N6-bis(4-methoxytrityl)adenosine (6a). Compound 5 (3.08 mmol, 2.50 g) was dissolved in
dry THF (26 ml) and 1 equivalent of 60% dispersion NaH (123 mg, 3.08 mmol) was added. After stirring for 1 h at room
temperature, the mixture was added into a mixture of pivaloyloxymethyl chloride (3.39 mmol, 0.53 ml) and NaI (0.31
mmol, 46 mg). The reaction was allowed to proceed for 5 h and quenched by adding water (70 ml). The mixture was
extracted three times with Et2O, the ether layer was dried over Na2SO4 and evaporated to dryness. The crude products
was purfied by silica gel chromatography, eluting with a 1:1 (v/v) mixture of EtOAc and petroleum ether to give a mixture
1
of 6a and 6b (0.32 g, 11% yield). The isomers were not separated from each other. H-NMR of 6a (500 MHz, (CDCl3): 8.00
(s, H-C(2)); 7.99 (s, H-C(8));. 7.45-7.19 (24H of MMTr); 7.00 (NH); 6.83-6.80 (4H of MMTr); 5.91 (d, J = 7.55, H-C(1´)); 5.47-
5.30 (CH2); 4,90 (t, J = 6.85, H-C(2’)); 4.65 (s, 2´-OH); 4,50 (t, H-C(3’)); 4.22 (m, H-C(4’)); 3.79 (MeO); 3.50 (dd, J = 13.25 and
1
4.70, H-C(5´)); 3.38 (dd, J = 13.20 and 5.50, H-C(5´´)); 1.16 (Me of POM). H-NMR of 6b (500 MHz, (CDCl3): 7.97 (s, H-C(2));
7,94 (s, H-C(8));. 7.45-7.19 (24H of MMTr); 6.94 (NH); 6.83-6.80 (4H of MMTr); 6.10 (d, J = 6.15 Hz, H-C(1´)); 5.47-5.30 (CH2);
5.11 (t, J = 6.20 Hz, H-C(2’)); 4,50 (t, H-C(3’)); 4,35 (m, H-C(4’)); 3.79 (MeO); 3.47 (dd, J = 13.10 and 4.95 Hz, H-C(5´)); 3.27
(dd, J = 13.05 and 4.70 Hz, H-C(5´´)); 2.67 (s, 3´-OH); 1.14 (Me of POM). 13C-NMR (126 MHz, (CDCl3): 177.74 (C=O); 158.70,
158.35 (MMTr); 154.25 (C(6)); 154.25 and 154.10 (C(2)); 148.59 and 148.30 (C4); 145.20, 145.12, 144.07, 144.00, 143.96,
143.88 (MMTr); 138.86 and 138.61 (C(8)); 137.24, 137.14, 135.19, 134.94, 130.38, 130.33, 130.21, 129.24, 128.90, 128.43,
128.37, 128.27, 128.23, 127.88, 127.06, 126.89 (MMTr); 121.46 and 121.28 (C(5)); 113.23 and 113.19 (MMTr); 89.67 (C(1´));
89.05 and 88.69 (CH2); 87.10 (C(1´)); 86.86 and 86.81 (spiro C of MMTr); 83.87 (C(4´)); 83.58 (C(4´)); 81.38 (C(2´)); 79.24
(C(3´)); 74.67 (C(2´)); 71.08 (C(3´)); 70.99 and 70.63 (spiro C of MMTr); 63.18 (C(5´)); 55.21 (OMe); 38.77 and 38.74 (spiro C of
+
POM); 26.96 and 26.87 (Me of POM). HR-MS: 948.3902 ([M+Na]+, C56H55N5NaO8 ; calc. 948,3943).
3´-O-Pivaloyloxymethyladenosin-2´-yl
2´,3´-O-isopropylideneadenosin-5´-yl
4-acetylthio-2-ethoxycarbonyl-3-oxo-2-
methylbutyl phosphate (1). Compound 6a (0.34 mmol, 0.314 g) was dried over P2O5 overnight and dissolved in dry DCM (1.9
ml) under nitrogen atmosphere. TEA (1.7 mmol, 0.24 ml) and bis(diethylamino)phosphorochloridite (0.44 mmol, 0.093 ml) were
added and the mixture was stirred under nitrogen for 2 hours. The product was isolated by passing the mixture through a short
silica gel column with a 7:3 mixture of EtOAc and hexane containing 0.5% TEA. The solvent was removed under reduced
pressure and the residue was coevaporated from dry MeCN to remove the traces of triethylamine. The phosphitylated
nucleoside was dissolved in dry MeCN (0.36 ml) under nitrogen and tetrazole (5.09 mmol, 0.754 ml of 0.45 M solution in MeCN)
and 2´,3´-O-isopropylidene-N6-(4-methoxytrityl)adenosine (5, 0.238 mmol; 0.14 g) in dry MeCN (0.36 ml) were added. The
reaction mixture was stirred for 30 min and then tetrazole (7.64 mmol, 1.13 ml of 0.45 M solution in MeCN) and ethyl 4-
(acetylthio)-2-(hydroxymethyl)-2-methyl-3-oxo-butanoate (0.52 mmol, 0.129 g) in dry MeCN were added. The phosphite ester
(6b) formed was oxidized with iodine (0.2 g) in a mixture of THF (4.0 ml), H2O (2.0 ml) and 2,6-lutidine (1.0 ml) overnight. A
solution of 5% NaHSO3 (100 ml) was added and the mixture was extracted 3 times with DCM. The organic phase was dried on
Na2SO4 and evaporated to dryness. The crude product was passed through a short silica column eluting first with a 1:1 (v/v)
mixture of EtOAc and DCM and then with DCM containing 3-5 % MeOH. The product 7 obtained was used without further
purification and 80% (v/v) aq AcOH (7.0 ml) was added to carry out the detritylation. After stirring overnight at room temperature,
the reaction mixture was evaporated to dryness. The residue was coevaporated twice with water. The product was purified first
by silica gel chromatography eluting with DCM containing 2-20 % MeOH, and then by HPLC on a SunFire C18 column (250 x
10 mm, 5 µm, flow rate 3.0 ml min-1), using isogratic elution with water and MeCN (45%) to yield 1 (14 mg, 3.9%). 1H-NMR (500
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MHz, CD3CN): 8.23, 8.22, 8.20 and 8.19 (s, 2H of H-C(2)); 8.10, 8.09 and 8.01 (s, 2H of H-C(8)); 6.23 (br. s, NH2); 6.13 (d, J =
2.00, H-C(1’)); 6.11 (br. s, NH2); 6.043 and 6.041 (d, J = 7.00, H-C(1’)); 5.48 (CH of POM); 5.45 (dd, J = 6.50 and 2.50, H-C(2´));
5,45 (dd, J = 6.50 and 2.50, H-C(2´)); 5,22 (d, J = 6.50, CH of POM); 5.00 (m, H-C(3´)); 4.32 (m, H-C(4´)); 4.24 (m, H-C(4´));
4.15-3.98 (m, H-C(5´), H-C(5´´), CH2 of Et and POCH2); 3.87-3.77 (m, 1H of CH2(5´) and CH2S); 3.65 (br. d, J = 12.5, 1H of
CH2(5´´)); 2.32 and 2.31 (s, SAc); 1.58 and 1.36 (s, Me of isopropylidene); 1.20-1.10 (m, Me, 3 x Me of POM and CH3 of Et).
13C-NMR (126 MHz, CD3CN): 198.73 and 198.70 (C=O); 194.07 (C=O of SAc); 177.58 (C=O of POM); 169.01 (C=OOEt);
156.44 and 156.00 (C(6)); 152.94 and 152.52 (C(2)); 149.28 and 148.61 (C(4)); 141.00, 140.98, 139.86 and 139.83 (C(8));
120.61 and 119.82 (C(5)); 114.09 (spiro C of isopropylidene); 90.42 (C(1´)); 88.74 (CH2 of POM); 88.09 and 88.02 (C(1´)); 85.96,
85.94, 85.09 and 85.03 (C(4´)); 84.25 (C(2´)); 81.15 (C(3´)); 76.46 and 76.42 (C(2´)); 69.27, 69.23 and 69.17 (POCH2); 67.91
and 67.87 (C(5´)); 62.17 (CH2 of Et); 61.85 (C(5´)); 59.72 (spiro C); 38,48 (spiro C of POM); 36.43 and 36.40 (CH2S); 29.30
(SAc); 26.37 (Me of isopropylidene); 26.37 and 26.24 (Me of POM); 24.54 (Me of isopropylidene); 16.41 (Me); 13.18 (CH3 of Et).
31P-NMR (202 MHz, CD3CN): -2.71 and -2.71. (Multiplicity of some signals is due to the presence of Rp and Sp diastereomers.)
HR-MS: 981.3171 ([M+H]+, C39H54N10O16PS+; calc. 980.3172).
2´-O-Pivaloyloxymethyladenosin-3´-yl
2´,3´-O-isopropylideneadenosin-5´-yl
4-acetylthio-2-ethoxycarbonyl-3-oxo-2-
methylbutyl phosphate (10). The 10 was obtained in 4.7% yield (17 mg). 1H-NMR (500 MHz, CD3CN): 8.27, 8.24 and 8.22 (s,
2H of H-C(2)); 8.10, 8.06, 8,05 and 8.01 (s, 2H of H-C(8)); 6.35-6.23 (m, NH2 and 5´-OH); 6.21 (d, J = 9.50, H-(C1´)); 6.20 (d, J
= 9.00, H-(C1´)); 6.13 (br. s, NH2); 6.11 (br. s, NH2); 5.96 (d, J = 8.00, H-C(1’)); 5.89 (d, J = 8.00, H-C(1’)); 5,47-5.41 (m, H-
C(2´)); 5.25-5.07 (CH2 of POM, H-C(2´) and H-C(3´)); 4.53-4.33 (m, H-C(4´), H-C(5´) and H-C(5´´); 4.32-4.25 (m, POCH2); 4.25-
4.19 (m, CH2 of Et); 4.07-3.97 (m, CH2S); 3.81-3.61 (m, H-C(5´) and H-C(5´´)); 2.37 and 2.36 (s, SAc); 1.61 (s, Me of
isopropylidene); 1.51 and 1.50 (s, Me); 1.36 (s, Me of isopropylidene); 1.26 (dt, J = 7.00 and 2.00, CH3 of Et); 0.80 and 0.79 (s, 3
x Me of POM). 13C-NMR (126 MHz, CD3CN): 198.73 and 198.70 (C=O); 194.10 (C=O of SAc); 176.97 (C=O of POM); 169.40
(C=OOEt); 156.58 and 156.03 (C6); 152.99, 152.97 and 152.31 (C2); 148.44 and 148.61 (C4); 141.12, 141.04 and 139.96 (C8);
120.81 and 119.82 (C5); 114.10 and 114.05 (spiro C of isopropylidene); 90.40 and 90.26 (C1´); 87.86 and 87.84 (C1´); 87.38
and 87.35 (CH2 of POM); 85.25 and 85.24 (C4´); 84.22 and 84.18 (C2´); 81.40 and 81.22 (C3´); 77.90 and 77.80 (C2´); 77.00
and 76.90 (C3´); 69.39, 69.33, 69.18 and 69.12 (C5´ and C5´´); 67.64, 67.62 and 67.58 (POCH2); 62.26, 62.25, 62.18 and
62.12 (CH2 of Et, C5´ and C5´´); 60.00 and 59.72 (spiro C); 38.00 (spiro C of POM); 36.58 (CH2S); 29.34 (SAc); 26.38 (Me of
isopropylidene); 25.70 and 26.24 (Me of POM); 24.56 (Me of isopropylidene); 16.79 (Me); 13.23 (CH3 of Et). 31P-NMR (202 MHz,
CD3CN): -2.35, -2.37 and -2.51. (Multiplicity of some signals is due to the presence of Rp and Sp diastereomers.) HR-MS:
981.3171 ([M+H]+, C39H54N10O16PS+; calc. 980.3172).
Kinetic Measurements
The reactions were carried out in sealed tubes immersed in a thermostated water bath at 37 oC. The hydronium ion
concentration of the reaction solution was adjusted with (N-[2-hydroxyethyl]piperazine-N,-[2-ethanesulfonic acid]) (HEPES)
buffer (0.036/0.024 M; 3 ml; pH 7.5) and the ionic strength to 0.1 M with sodium chloride. The initial starting material
concentration was ca. 0.2 mM. The deprotection 1 was followed by reversed-phase HPLC (UV detection at 260 nm, flow rate
0.95 ml min-1) in the absence and presence of porcine liver esterase (PLE; 2.6 units/ml). The samples (200 µl) were withdrawn
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from the reaction solution at appropriate time intervals and were made acidic (pH 3) with 1 M aqueous HCl (15 µl) to inactivate
the enzyme and to quench the hydrolysis. The solution was filtered with Minisart RC 4 filters (0.2 μm). Products were separated
using 5 min isocratic elution with AcOH/AcONa buffer (0.045/0.015 M) containing NH4Cl (0.1 M) and 2% MeCN, followed by 30
min linear gradient up to 70.0% MeCN. The products were identified by HPLC/ESI-MS.
Supplementary Material
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/MS-number.
Acknowledgements
We thank Ms. Tuuli-Maaria Tuominen for the kinetic measurements.
Author Contribution Statement
Dr. Mikko Ora has acted as a supervisor of undergraduate student Ms. Suvi Malmikare who prepared compounds 1, 4-6 and 10.
Mikko Ora has written the article.
Dr. Emilia Kiuru has purified and characterized the compounds 1 and 10.
Dr. Emilia Kiuru and Mikko Ora have acted as supervisors of undergraduate student Ms. Tuuli-Maaria Tuominen mentioned in
acknowledgement who carried out kinetic measurements.
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