Unusual radical 6-endo cyclization to carbocyclic-ENA and elucidation of its solution conformation by 600 MHz NMR and ab initio calculations

Article abstract of DOI:10.1039/b813870b

In our previous paper (J. Am. Chem. Soc., 2007, 129, 8362), we reported the synthesis of 7′-Me-Carba-LNA and 8′-Me-Carba-ENA thymidine through 5-hexenyl or 6-heptenyl radical cyclization. Both 5-hexenyl and 6-heptenyl radical cyclized exclusively in the exo form, giving unwanted exocyclic C7′-methyl group. In the present study, we showed that the regioselectivity of the 5-hexenyl radical cyclization could be favorably tuned by introduction of a hydroxyl group β to the olefinic double bond, yielding about 9% of the 6-endo cyclization product. Possible pathways to give 6-endo cyclization product 9 compared to the intermediates responsible to give the 5-exo cyclization product 5 has been discussed. Based on this unique 6-endo cyclization strategy, a carbocyclic ENA modified thymidine (carba-ENA) has been successfully synthesized, which also enabled us to perform its full solution conformation analysis by using NMR (¹H at 600 MHz) observables for the first time.

Full text of DOI:10.1039/b813870b

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Unusual radical 6-endo cyclization to carbocyclic-ENA and elucidation of its
solution conformation by 600 MHz NMR and ab initio calculations†
Chuanzheng Zhou, Oleksandr Plashkevych and Jyoti Chattopadhyaya*
Received 8th August 2008, Accepted 28th August 2008
First published as an Advance Article on the web 27th October 2008
DOI: 10.1039/b813870b
In our previous paper (J. Am. Chem. Soc., 2007, 129, 8362), we reported the synthesis of
7¢-Me-Carba-LNA and 8¢-Me-Carba-ENA thymidine through 5-hexenyl or 6-heptenyl radical
cyclization. Both 5-hexenyl and 6-heptenyl radical cyclized exclusively in the exo form, giving unwanted
exocyclic C7¢-methyl group. In the present study, we showed that the regioselectivity of the 5-hexenyl
radical cyclization could be favorably tuned by introduction of a hydroxyl group b to the olefinic double
bond, yielding about 9% of the 6-endo cyclization product. Possible pathways to give 6-endo cyclization
product 9 compared to the intermediates responsible to give the 5-exo cyclization product 5 has been
discussed. Based on this unique 6-endo cyclization strategy, a carbocyclic ENA modified thymidine
(carba-ENA) has been successfully synthesized, which also enabled us to perform its full solution
conformation analysis by using NMR (1H at 600 MHz) observables for the first time.
containing LNA or ENA are preorganized in the A-type
Introduction
canonical structure, and thus typically have high affinity and
The conformationally constrained oligonucleotides have been
specificity toward complementary RNA strand. Subsequently,
attracting much interest in the field of antisense1,2 and siRNA
other locked nucleotides with 2¢,4¢-linkage such as PrNA8 and
technology3 in the past two decades. The development of the 2¢,4¢-
fused five-membered Locked Nucleic Acid (LNA, Fig. 1)4–6 and
six-membered Ethylene-bridged Nucleic Acid (ENA)7 modified
nucleotides have considerably boosted research in this field.
These ribonucleotide analogues consist of a rigid bicyclic sys-
tems constraining sugar conformation to 3¢-endo form by a
methylene (for LNA) or ethylene linkage (for ENA) between
the 2¢-oxygen and 4¢-carbon of the ribose ring. Oligonucleotides
aza-ENA9 have been incorporated into oligonucleotides, and all
of them, just like LNA or ENA, have shown typically high
affinity toward complementary RNA sequence. They also have
shown relatively greater nuclease resistance.8 This presumably led
Nielsen et al.10 to design and synthesize the 6-membered locked
2¢,4¢-carbocyclic-ENA uridine (carba-ENA-U) through the ring-
closing methathesis.10 This work10 however did not include any
studies on the nuclease stability of carba-ENA-U containing
oligonucleotides. Independently, we have reported11 the synthesis
of five- as well as six-membered 2¢,4¢-carbocyclic analogues
(7¢-Me-carba-LNA and 8¢-Me-carba-ENA) of thymidine via 5-
hexenyl or 6-heptenyl radical cyclization, respectively. In both 5-
hexenyl (for carba-LNA analog) and 6-heptenyl (for the carba-
ENA analog) radical, the radical cyclization occurred exclusively
in the exo form giving exocyclic equatorial methyl group (Fig. 2).
It has also been found11 that these 7¢-Me-carba-LNA and
Fig. 1 Locked Nucleic Acid (LNA) and Ethylene-bridged Nucleic Acid
(ENA) modifications and their carbocyclic analogues carba-LNA and
carba-ENA.
Department of Bioorganic Chemistry, Box 581, Biomedical Center, Uppsala
University, SE-751 23, Uppsala, Sweden. E-mail: jyoti@boc.uu.se
1
† Electronic supplementary information (ESI) available: H, 13C, COSY,
HMQC, HMBC, DEPT, 1D-NOE NMR spectra of compounds 1, 7–
14. Comparison of sugar conformation parameters of carba-ENA-T (1)
with that of carba-ENA-U, 8¢-Me-carbo-ENA-T, ENA-T and aza-ENA
T. Coordinates of structure of compound 1 in pdb format.. See DOI:
10.1039/b813870b
Fig. 2 Radical cyclization of 5-hexenyl and 6-heptenyl radical nucleosides
following exclusively exo-cyclization pathway.11
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8¢Me-carba-ENA containing oligonucleotides at the 3¢-end are
fully resistant for over 48 h in the human blood serum.11
Through single LNA substitution at identical site as that with the
carbocyclic counterpart it has been shown that the 7¢-Me-carba-
LNA and 8¢-Me-carba-ENA containing oligonucleotides are
considerably more stable to nucleases in the human serum11 than
that of the identically LNA-substituted oligo-DNA sequences
which in average survive for less then 9 h.
During our efforts to functionalize the five membered 2¢,4¢-
carba-LNA-type thymidine,12 we found that introducing a 6¢-
hydroxyl to the 5-hexenyl carbon radical, as in the intermediate
Ts:6¢ in Scheme 1, lead to 6-endo hexenyl cyclization product
9 (~9%) in conjunction with the competing 5-exo cyclized
product 7 and 8 (Scheme 1). Here, we report synthesis of
carbocyclic ENA thymidine (carba-ENA-T, Fig. 1) through 6-endo
hexenyl cyclization and its structure by NMR and ab initio
calculations.
7 by NMR (see ESI), which is one of the isomers (7¢S) formed
by the 5-exo cyclization reaction. The lower Rf spot was also
separated and found it to be a mixture of two components: the 2nd
isomer (7¢R) of 5-exo cyclization product 8 and 6-endo cyclization
product 9.
The characterization and conformational analysis of 7, 8 and 9
have been performed using NMR data obtained by 1H, 13C, DEPT
as well as COSY, 1H-13C HMQC and long range 1H-13C correlation
3
(HMBC) experiment. JHC HMBC correlations between H1¢ and
C8¢ in compound 9 and between H1¢ and C7¢ in compound 8
(Fig. 3B) unequivocally proves that the oxa-bicyclo [2.2.1] heptane
ring system and oxa-bicyclo [3.2.1] octane ring system have been
formed for compounds 8 and 9 respectively, which was further
3
confirmed by observation of JHH correlation between H7¢ and
3
H2¢ in compound 8, JHH correlation between H8¢¢ and H2¢ in
compound 9 in COSY spectrum (Figure S6 in ESI). The endocyclic
nature of 7¢- and 8¢-methylene groups in compound 9 was verified
by DEPT and HMQC experiment. In the DEPT spectrum, both
C7¢ and C8¢ appeared as the secondary carbon (Figure S3 in ESI)
and in the HMQC spectrum, each of them have two protons
attached (Fig. 3A).
The configuration of C6¢ and C7¢ in compounds 7, 8, 9 was
determined by 1D NOE experiments. For compound 7, irradiation
of H1¢ leads to NOE enhancement for H2¢ (3.1%) and 7¢-Me
(6.0%), and irradiation of H6¢ leads to strong NOE enhancement
(6.8%) for H7¢, but none for 7¢-Me, suggesting both C6¢ and C7¢ are
in S-configuration (Figure S14 in ESI). Since radical cyclization
Results and discussion
1. Free radical cyclization
The key intermediate 6 was synthesized according to our previous
procedure.12 The free radical cyclization was carried out in toluene
in presence of n-Bu3SnH and a catalytic amount of AIBN by
reflux to give two spots on the TLC. The product with higher Rf
was separated by short chromatography and identified as product
Scheme 1 Reagents and conditions: (i) Bu4SnH, AIBN, toluene, reflux 4h; (ii) Dess–Martin periodinane, dichloromethane, r.t. 3 h; (iii) NaBH4, ethanol,
r.t. 2 h; (iv) phenyl chlorothionoformate, pyridine, r.t. 3 h; (v) Bu4SnH, AIBN, toluene, reflux 1 h; (vi) 20% Pd(OH)2/C, ammonium formate, methanol,
reflux 2 h.
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Fig. 3 1H-13C HMQC (panel A) and HMBC (panel B) spectra of mixture of compounds 8 (dashed line, labeled in normal form) and 9 (dotted line,
labeled in italic form).
does not change the configuration of distal C6¢, compounds 6,
8 and 9 should have the same C6¢-S configuration as that in
compound 7. For compound 8, irradiation of H1¢ leads to NOE
enhancement for H2¢ (1.8%) and H7¢ (5.2%), but none for 7¢-Me.
(Figure S15 in ESI). Hence, R-configuration was assigned for C7¢
of compound 8.
The relative ratio of compounds 7/8/9 was 80:11:9. Thus the
radical cyclization of Ts: 6¢ gives 9% of 6-endo cyclization product
9 compared to 4 → 5 (Fig. 2), in which compound 5occurs as an
exclusive product owing to the participation of 5-exo cyclization
pathway. This difference suggested that the 6¢-hydroxyl group
affects the outcome of the regioselectivity of free radical cyclization
to some extent. It is well known that cyclization of 5-hexenyl-1-
radical is a kinetically controlled process and the exo cyclization
product, cyclopentane, is preferred (exo/endo > 98/2).13 The
effect of substituents especially alkyl group on regioselectivity
of 5-hexenyl-1-radical cyclization has been studied.14 Generally,
alkylation at C1 and C5 increase the selectivity on 6-endo
cylization15 and methylation at C2, C3, C4 and C6 enhance
the 5-exo cylization.16,17 In some cases, the 6-endo product can
however be found as the major product.15 But unfortunately, the
mechanism behind the regioselectivity upon alkyl substitutation
is still not clear, and the role of 4-hydroxyl substitution has never
been illustrated heretofore.
It is however likely two possible effectors could contribute to
the enhanced 6-endo hexenyl cyclization by the 6¢-OH in Ts: 6’
(Scheme 1). First, it is known that b-hydroxyl or alkyloxyl has a
marked stabilizing effect on a carbon radical (b-Oxygen Effect):18
Thus, in the intermediate 15 of 6-endo cyclization, the 6¢-OH
located at the b position to radical at C7¢ can have a stabilizing
effect by inductive effect and/or by resonance delocalization of
the charge into the s*(O6¢-C6¢) orbital19,20 since the single occupied p
orbital and the empty s*(O6¢-C6¢) orbital orientate periplanar with
respect to each other (Fig. 4).
Fig. 4 Graphical representations of the molecular structures of 6-endo cyclization intermediate (15), 5-exo cyclization transition state 16 and 6-endo
cyclization transition state 17.
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Second possibility is that the 6¢-hydroxyl could stabilize tran-
sition state of 6-endo radical cyclization. During the attack of
carbon radical to olefin, the C2¢ radical behaves as a nucleophile
and becomes slightly positively charged,14 whereas the terminal
carbon in the olefinic moiety becomes fractionally negative to give
5-exo (16) and 6-endo (17) cyclization transition states (Fig. 4).
When R = H, QM calculations21 reveal that strain engendered
in accommodating the required disposition of reactive centers
is greater for the 6-endo transition state 17 than for the 5-
exo transition state 16, and as a result the 5-exo cyclization is
kinetically predominant. While, when R = OH, the 6¢-hydroxyl can
stabilize inductively the developing negative charge on the olefin
moiety in the 6-endo transition structure 17, and thus lowering the
activation energy of 6-endo cyclization, as a result, more 6-endo
cyclization product (9%) was obtained. Due to the discovery of the
6-endo cyclization through the stabilizing effect of the 6¢-hydroxy
group, we are now attempting to prepare other derivatives with
electron-withdrawing groups at C6¢ in order to increase the yield
of the cyclization step.
COSY, HMQC and HMBC experiments performed using 500
and 600 MHz NMR (see Figure S26 to S31 in ESI). In order
to reconstruct the solution structure of carba-ENA-T, we have
utilized vicinal proton couplings analysis using data from homod-
ecoupling 1H NMR experiments and the results of the theoretical
simulations. Theoretical vicinal proton coupling constants have
been back-calculated using Haasnoot-de Leeuw-Altona general-
ized Karplus equation23,24 from the corresponding torsional angles
of the ab initio optimized molecular structures obtained utilizing
HF/6–31G* or B3LYP/6–31++G* geometry optimization by
GAUSSIAN 98.25 As shown in Table 1, the experimental vicinal
coupling constants of compound are well reproduced by this
theoretical approach (Table 1). This indicates that the modified
nucleoside 1 is indeed in rigid locked conformation and its average
molecular structure observed experimentally is close to that of the
minimized theoretical structure.
The molecular structure obtained from ab initio geometry
optimization is shown in Fig. 5. The six-membered carbocyclic
moiety adopts a perfect chair conformation. The furanose ring
of carba-ENA-T (Table S1) is found to be locked in the North-
type conformation characterized by pseudorotational phase angle
P = 19.6◦ and the puckering amplitude Wm = 45.9◦. The sugar
pucker parameters are very similar to that of carba-ENA-U,10 8¢-
Me-carba-ENA-T,11 ENA-T26 and aza-ENA-T,9 which suggests
the 2¢,4¢-linkage induced locking of the sugar is so strong that the
sugar pucker becomes rigid and not sensitive to a large extent to
substitutions at the base or/and at the 2¢,4¢-linkage.
2. Radical deoxygenation to give carba-ENA-T (1)
Compound 8 and 9 appeared to have nearly the same polarity and
efforts to separate them using column chromatography have failed.
Therefore, we treated the mixture with Dess–Martin periodinane
to obtain ketones 10 and 11, which can be separated easily by silica
gel column chromatography. Compound 11 was then reduced to
alcohol again with NaBH4. This reduction was highly stereos-
elective to give compound 12 with C6¢(R)-OH stereochemistry
as the only product with high yield (82%). Thus the oxidation
followed by reduction constitutes an inversion of the configuration
of C6¢(S)-OH in 9 to C6¢(R)-OH 12 (in Scheme 1) efficiently.
Compound 12 was converted to its 6¢-O-phenoxythiocarbonyl
derivative 13 (63%) followed by standard radical deoxygenation22
to give intermediate 14 (72%), which was debenzylated with 20%
Pd(OH)2/C, ammonium formate in methanol to give the title
compound 1 in 90% yield.
3. Molecular structure of carba-ENA-T based on NMR and
ab initio calculations
Fig. 5 Molecular structure of carba-ENA-T optimized ab initio [only
The product 1, carba-ENA-T, has been characterized using NMR
and ab initio optimized molecular modeling. All the peaks in
1H and 13C NMR spectra have been assigned through DEPT,
B3LYP geometry is shown since it is identical to the HF optimized
˚
geometry (RMSD < 0.05 A)]. The PDB coordinates are presented in the
Electronic Supplementary information (ESI).
Table 1 Experimental and calculated vicinal 3JHH coupling constants and torsion angles of carbo-ENA-T (compound 1, Scheme 1)
UH.H , (cal.3J, Hz),
HF/6–31G**
UH.H (cal.3J, Hz),
B3LYP/6–31++G**
Torsion
vicinal proton coupling
3JH,H exp. (Hz)
UH.H (◦) exp.
H1¢-C1¢-C2¢-H2¢
H2¢-C2¢-C3¢-H3¢
H2¢-C2¢-C7¢-H8¢e
H2¢-C2¢-C7¢-H8¢a
H6¢e-C6¢-C7¢-H7¢a
H6¢e-C6¢-C7¢-H7¢e
H6¢a-C6¢-C7¢-H7¢a
H6¢a-C6¢-C7¢-H7¢e
H7¢a-C7¢-C8¢-H8¢e
H7¢a-C7¢-C8¢-H8¢a
H7¢-C7¢-C8¢-H8¢e
H7¢e-C7¢-C8¢-H8¢a
91.89◦(1.0)
47.55◦(5.7)
91.75◦(1.0)
47.22◦(5.8)
3JH-1¢,H-2¢
3JH-2¢,H-3¢
3JH-2¢,H-8¢¢
3JH-2¢,H-8¢
3JH-6¢¢,H-7¢¢
3JH-6¢¢,H-7¢
3JH-6¢,H-7¢¢
3JH-6¢,H-7¢
3JH-7¢¢,H-8¢¢
3JH-7¢¢,H-8¢
3JH-7¢,H-8¢¢
3JH-7¢,H-8¢
1.0
5.2
3.7
2.0
5.0
1.8
11.0
7.0
5.5
12.0
1.8
7.0
84.3 to 92.6
47.2 to 55.2
-61.1 to -52.5
61.0 to 72.0
-51.24◦(4.6)
65.58◦(2.1)
-51.26◦(4.6)
65.52◦(2.1)
41.29◦(6.7)
42.83◦(6.6)
46.1 to 54.0
-74.24◦(0.9)
159.54◦(12.0)
44.01◦(6.3)
-72.73◦(1.1)
161.29◦(12.2)
45.74◦(5.9)
-87.5 to -67.1
157.5 to 172.1
48.0 to 56.0
-53.1 to -45.3
-155.2 to -146.7
61.6 to 72.9
-42.39◦(6.4)
-160.40◦(11.9)
73.66◦(1.0)
-43.67◦(6.2)
-161.91◦(12.1)
72.44◦(1.1)
-44.34◦(6.1)
-45.80◦(5.8)
-48.2 to -40.5
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(s, 1H, H1¢), 7.22–7.34 (m, 10H), 7.75 (s, 1H, H6), 8.71 (s, 1H,
N3H).13C NMR (600 MHz, CDCl3): d 12.0 (5-CH3), 18.1 (7¢CH3),
42.1 (C7¢), 45.3 (C2¢), 65.8 (C5¢), 72.4(Bn CH2), 73.9(Bn CH2),
79.0 (C3¢), 81.0 (C6¢), 88.6 (C1¢), 88.9 (C4¢), 109.5 (C5), 127.5,
127.9, 128.0, 128.1, 128.5, 128.6, 135.9, 136.8, 137.5, 149.9, 163.8.
9: 1H NMR (600 MHz, CDCl3): d 1.71 (s, 3H, 5-CH3), 1.45 (m, 1H,
Conclusion
In this study, we have devised effective control switch to tune
the radical cyclization reaction from 5-exo pathway to 6-endo
pathway. It has been achieved by the introduction of one hydroxyl
group at C6¢ to the 5-hexenyl carbon radical which has led
to enhancement of the regioselectivity of the radical center
and ultimately led to the 6-endo radical cyclization. Radical
deoxygenation of this 6-endo cyclization product results in carba-
ENA-T in high yield. The molecular structure of carba-ENA-
T has been studied using experimental NMR observables and
Karplus empirical approaches, as well as theoretical ab initio
calculations. The furanose ring of carba-ENA-T has been shown
H7¢), 1.62 (d,1H, J
= 10.2 Hz, 6¢OH), 1.77 (m, 1H, 8¢H),
6¢H, 6¢OH
1.88 (m, 1H, 8¢¢H), 2.15 (m, 1H, H7¢), 2.56 (s, 1H, H2¢), 3.72 (d,
1H, J = 11.0 Hz, 5¢H), 4.00 (m, 1H, H6¢), 4.15 (d, 1H, J = 11.0 Hz,
5¢H), 4.33 (d, 1H, J = 4.9 Hz, 3¢H), 4.40–4.63 (m, 4H, BnCH2),
5.78 (s, 1H, H1¢), 7.22–7.34 (m, 10H), 8.00 (s, 1H, H6), 8.71 (s, 1H,
N3H). 13C NMR (600 MHz, CDCl3): d 11.8 (5-CH3), 19.1 (C8¢),
29.0(C7¢), 42.1 (C2¢), 68.1 (C5¢), 68.9 (C6¢), 72.0(Bn CH2), 73.6 (Bn
CH2), 73.8 (C3¢), 87.5(C4¢), 87.7(C1¢), 109.4, 127.4, 127.8, 127.9,
128.0, 128.5, 128.6, 136.2, 137.40, 137.47, 150.1, 163.9. MALDI-
TOF MS m/z: [M + H]+ 479.2, calcd 479.2.
to be locked in the North-type conformation (P = 19.6◦, Wm
=
45.9◦) and the six-membered carbocyclic moiety has been found
to adopt a perfect chair conformation. Work is now in progress to
synthesize carba-ENA nucleosides (compound 1 and its analogs)
through the free-radical addition to the terminal -CH N- or to an
aldehyde or an aziridinylimine function as radical acceptors.
Synthesis of (1R, 3R, 4R, 5R, 7S)-7- benzyloxy-1-benzyloxmethyl-
6-one-3-(thymin-1-yl)-2-oxa-bicyclo[2.2.1] heptane (10) and (1R,
5R, 7R, 8S)-8-benzyloxy-5-benzyloxmethyl-4-one-7-(thymin-1-yl)-
6-oxa-bicyclo[3.2.1]octane (11)
Experimental
Mixture of 8 and 9(342 mg, 0.71 mmol) was dissolved in dry DCM,
Dess–Martin periodinane (15% in DCM, 1.8 mL, 0.85 mmol) was
added and stirred at r.t. for 2 h. Then diluting the reaction mixture
with DCM, filtered through celite bar, the filtrate was washed
with aqueous Na2S2O3 twice, saturated NaHCO3 solution once
and NaCl solution once. After drying over MgSO4, it was applied
to silica short column chromatography (EtOAc/cyclohexane 2/8
to 4/6) to give 150 mg of 10 and 112 mg of 11 (overall yield
Synthesis of (1R, 3R, 4R, 5S, 6S, 7S)-7-benzyloxy-1-
benzyloxmethyl-6-hydroxyl-3-(thymin-1-yl)-2-oxa-bicyclo[2.2.1]
heptane(7), (1R, 3R, 4R, 5R, 6S, 7S)-7- benzyloxy-1-
benzyloxmethyl-6-hydroxyl-3-(thymin-1-yl)-2-oxa-bicyclo[2.2.1]
heptane(8) and (1R, 4S, 5R, 7R, 8S)-8-benzyloxy-5-
benzyloxmethyl-4-hydorxyl-7-(thymin-1-yl)-6-oxa-
bicyclo[3.2.1]oct-4-one (9)
4.2 g (5.5 mmol) of 612 was dissolved in 200 mL of dry toluene
which was purged by N2 for ca 30 min. The mixture was heated
under reflux and Bn3SnH (1.85 ml in 20 mL dry toluene), AIBN
(0.55 g in 20 mL dry toluene) was added dropwise in 2h. The
reaction was found to be incomplete after 30 min (TLC). So
another part of Bn3SnH (0.9 mL in 10 mL toluene) and AIBN
(0.25 g in 10 mL toluene) was added dropwise in 1 h and
continued reflux for further 1 h. The solvent was evaporated and
the residue was applied to silica short column chromatography
(EtOAc/cyclohexane, 2/8 to 6/4) to give 1.3 g (49%) of compound
7, 0.32 g (12%) of mixture of 8 and 9 (8/9 = 11:9) and recovered
0.7 g of substrate 6. 7: 1H NMR (500 MHz, CDCl3): d 1.15 (d, 3H,
77%). 10: 1H NMR (500 MHz, CDCl3): d 1.36 (d, 3H, J
=
7¢CH3, 7¢H
7.6 Hz, 7¢CH3), 1.49(s, 3H, 5-CH3), 2.46(m, 1H, H7¢), 2.99 (s, 1H,
H2¢), 3.90 (d, J = 11.7 Hz, 5¢H), 4.0 (d, J = 11.7 Hz, 5¢¢H),
4.18(s, 1H, H3¢), 4.49–4.61(m, 4H, BnCH2), 5.54(s, 1H, H1¢),
7.20–7.33(m, 10H, Bn-Ph), 7.72(s, 1H, H6), 8.76 (s, 1H, N3H).
13C NMR (500 MHz, CDCl3): d 12.0 (5-CH3), 14.3 (7¢CH3), 43.1
(C7¢), 49.6 (C2¢), 63.3 (C5¢), 72.5 (BnCH2), 74.1 (BnCH2), 86.1
(C4¢), 88.5 (C1¢), 109.9, 127.5, 127.9, 128.21, 128.24, 128.5, 128.62,
128.66, 135.6, 136.3, 137.3, 149.9, 163.7, 208.5(C6). MALDI-TOF
1
MS m/z: [M + H]+ 477.2, calcd 477.2. 11: H NMR (500 MHz,
CDCl3): d 1.38 (s, 3H, 5-CH3), 2.14 (m, 2H, H8¢ and H8¢¢), 2.46
(m, 1H, H7¢), 2.72–2.81 (m, 2H, H2¢ and H7¢¢), 3,93 (d, J =
11.7 Hz, 5¢H), 4.05 (d, J = 11.7 Hz, 5¢¢H), 4.44–4.60 (m, 5H,
H3¢ and BnCH2), 5.98 (s, 1H, H1¢), 7.22–7.35 (m, 10H), 8.01 (s,
1H, H6), 8.60 (s, 1H, N3H). 13C NMR (CDCl3): d11.7 (5-CH3),
20.7(C8¢), 34.5 (C7¢), 43.4(C2¢), 65.4 (C5¢), 72.3 (Bn CH2), 73.9 (Bn
CH2), 76.2 (C3¢), 87.4 (C4¢), 88.9 (C1¢), 109.9 (C5), 127.4, 128.0,
128.2, 128.5, 128.6, 135.8, 136.7, 137.1, 150.1(C2), 163.7(C4),
205.6(C6). MALDI-TOF MS m/z: [M + H]+ 477.2, calcd
477.2.
J
= 7.6 Hz, 7¢CH3), 1.50(s, 3H, 5-CH3), 2.25 (d, 1H,
7¢CH3, 7¢H
J6¢H, 6¢OH = 11.6 Hz, 6¢OH), 2.66 (d, 1H, J = 2.75 Hz, 2¢H), 2.72
(m, 1H, H7¢), 3.81 (d, 1H, J = 11.0 Hz, 5¢H), 3.91(d, 1H, J =
11.0 Hz, 5¢¢H), 4.42–4.61 (m, 4H, BnCH2), 5.75 (s, 1H, H1¢),
7.23–7.34 (m, 10H), 7.72 (s, 1H, H6), 8.87 (broad, 1H, N3H). 13
C
NMR (CDCl3): d 8.4 (7¢CH3), 12.1 (5-CH3), 33.1(C7¢), 47.8(C2¢),
66.2(C5¢), 71.9(C6¢), 72.1 (Bn CH2), 73.9 (Bn CH2), 76.8 (C3¢),
83.8 (C1¢), 89.1 (C4¢), 109.6 (C5), 127.6, 127.9, 128.1, 128.5, 128.6,
136.0 (C6), 137.0, 137.6, 149.9 (C2), 164.0(C4). MALDI-TOF MS
m/z: [M + Na]+ 501.2, calcd 501.2.Though we isolated a mixture
of 8 and 9, their proton and carbon NMR peaks could be assigned
clearly, so here they are given separately. 8: 1H NMR (600 MHz,
Synthesis of (1R, 4R, 5R, 7R, 8S)-8-benzyloxy-5-benzyloxmethyl-
4-hydroxyl-7-(thymin-1-yl)-6-oxa-bicyclo[3.2.1]octane (12)
CDCl3): d 1.31 (d, 3H, J
5-CH3), 1.77 (m, 1H, 7¢H), 2.23 (1H, J
= 7.2 Hz, 7¢CH3), 1.47 (s, 3H,
110 mg (0.23 mmol) of compound 11 was dissolved in 95% ethanol,
NaBH4 (17 mg, 0.46 mmol) was added in portions in 10 min. The
mixture was allowed to stir at r.t. for 2h. The reaction mixture was
diluted with saturated NaHCO3, and extracted with DCM. The
separated organic phase was dried over MgSO4 and applied to
7¢CH3, 7¢H
= 11.0 Hz, 6¢OH),
6¢H, 6¢OH
2.55 (s, 1H, 2¢H), 3.83 (d, 1H, J = 11.3 Hz, 5¢H), 3.93 (d, 1H,
J = 11.3 Hz, 5¢H), 4.00 (dd, 1H, J = 11.0 Hz, J
=
6¢H, 6¢OH
6¢H, 7¢H
3.5 Hz, 6¢H), 4.04 (s, 1H, 3¢H), 4.40–4.62 (m, 4H, BnCH2), 5.50
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short column chromatography (EtOAc/cyclohexane 2/8 to 4/6)
to give 90 mg of compound 12 (81%). 1H NMR (500 MHz, CDCl3):
d 1.44(s, 3H, 5-CH3), 1.79–2.11 (m, 4H, H7¢, H7¢¢, H8¢, H8¢¢),
Synthesis of (1R, 5R, 7R, 8S)-8-hydroxy-5-hydroxymethyl-7-
(thymin-1-yl)-6-oxa-bicyclo[3.2.1]octane (1)
20 mg (0.043 mmol) of compound 14 was dissolved in methanol
(2 mL), to which ammonium formate (168 mg) and 20%
Pd(OH)2/C (68 mg) was added. The reaction mixture was heated
under reflux for 1 h and filtered through celit bar. Evaporation
2.67 (m, 1H, H2¢), 3.67(dd, 1H, J
= 11.7 Hz, J
=
6¢H, 6¢OH
6¢H, 7¢¢
3.2 Hz), 3.81 (d, 1H, J
= 11.0 Hz, H5¢), 3.90 (d, 1H,
5¢H, 5¢¢H
J
= 11.9 Hz, H6-OH), 4.14 (d, 1H, J
= 11.3 Hz,
6¢OH, 6¢H
5¢¢H, 5¢H
H5¢¢), 4.40 (d, 1H, J3¢H, 2¢H = 11.7 Hz, H3¢), 4.45–4.63 (m, 4H,
BnCH2), 5.75 (s, 1H, H1¢), 7.26–7.79 (m, 10H), 7.97(s, 1H, H6),
8.76(s, 1H, N3H).). 13C NMR (500 MHz, CDCl3): d11.8 (5-CH3),
17.3 (C8¢), 27.2 (C7¢), 42.4 (C2¢), 69.1 (C5¢), 71.7 (C6¢), 73.2 (Bn
CH2), 73.7 (Bn CH2), 75.1 (C3¢), 82.2 (C4¢), 87.2 (C1¢), 109.5 (C5),
127.8, 127.9, 128.1, 128.5, 128.6, 128.7, 136.1, 136.2, 137.3, 150.2
(C2), 163.9 (C4).MALDI-TOF MS m/z: [M + H]+ 479.6, calcd
479.2.
1
of the solvent gave 11 mg of pure product 1 (90%). H NMR
(500 MHz, DMSO-d6): d 1.12 (dd, 1H, J
= 4.0 Hz,
6¢He, 7¢Ha
J
= 13.1 Hz, 6¢He), 1.54 (m, 1H, 8¢He), 1.55–1.64 (m,
6¢He, 6¢Ha
3H, 7¢He, 7¢Ha and 6¢Ha), 1.87 (m, 1H, 8¢Ha), 2.16 (broad, 1H,
H2¢), 3.41 (dd, 1H, J 5¢H, 5¢¢ = 12.3 Hz, J 5¢H, 5¢OH = 4.4 Hz), 3.48 (dd,
1H, J
5.24 (d, 1H, J
= 12.3 Hz, J
= 4.4 Hz), 4.1 (broad, 1H, H3¢),
5¢H, 5¢¢
5¢¢H, 5¢OH
= 4.0 Hz, 3¢OH), 5.31 (broad, 1H, 5¢OH),
3¢H, 3¢OH
5.61 (s, 1H, H1¢), 8.30 (s, 1H, H6), 11.22 (broad, 1H, N3H). 13C
NMR (DMSO-d6): d12.3 (C5-CH3), 17.4 (C7¢), 20.1 (C8¢), 25.4
(C6¢), 44.9 (C2¢), 61.4 (C5¢), 64.0 (C3¢), 85.8 (C4¢), 86.1 (C1¢), 107.0
(C5), 136.3 (C6), 150.1 (C2), 163.9 (C4).MALDI-TOF MS m/z:
[M + H]+, found 282.9, calcd 283.1.
Synthesis of (1R, 4R, 5R, 7R, 8S)-8-benzyloxy-5-benzyloxmethyl-
4-(O-phenoxythiocarbonyl)-7-(thymin-1-yl)-6-oxa-
bicyclo[3.2.1]octane (13)
80 mg (0.16 mmol) of 12 was coevaporated with dry pyridine twice
and dissolved in the same solvent. Phenyl chlorothionoformate
(45 ul, 0.32 mmol) was added at r.t. and stirring at r.t. 2h.
Then the solvent was evaporated and the residue was diluted
with dichloromethane, washed with NaHCO3, brine in turn,
dried over MgSO4, applied to short column chromatography
(EtOAc/cyclohexane 1/9 to 3/7) to give 61 mg of compound
Theoretical calculations
The geometry optimizations of the carba-ENA-T (1) have been
carried out by GAUSSIAN 98 program package25 at the Hartree–
Fock level using HF/6–31G** and B3LYP/6–31++G**. The
experimental torsion angles have been back-calculated from
experimental vicinal proton JH,H coupling constants employing
Haasnoot-de Leeuw-Altona generalized Karplus equation23,24
3
1
13 (62%). H NMR (500 MHz, CDCl3): d 1.45 (s, 3H, 5-CH3),
taking into account b substituent correction in form:
1.84 (m, 1H, 8¢He), 2.20(m, 2H, H7¢, H7¢¢), 2.30 (m, 1H, 8¢Ha),
2.69 (s, 1H, H2¢), 3.93 (dd, 2H, J = 10.7 Hz, H5¢ and 5¢¢), 4.41
(d, 1H, J = 3.7 Hz, H3¢), 5.44 (s, 1H, H6¢), 5.84 (s, 1H, H1¢),
7.12–7.47 (m, 15H), 7.92 (s, 1H, H6), 8.49 (s, 1H, N3H). 13C NMR
(500 MHz, CDCl3): d 11.8 (5-CH3), 17.7 (C8¢), 23.3 (C7¢), 42.7
(C2¢), 69.1 (C5¢), 72.1 (Bn CH2), 73.1 (C3¢), 73.8 (Bn CH2), 80.1
(C6¢), 82.7 (C4¢), 87.1 (C1¢), 109.6 (C5), 121.9, 126.6, 127.1, 127.9,
128.2, 128.3, 128.6, 129.5, 135.9, 137.1, 137.7, 150.0 (C2), 153.4,
163.7 (C4), 194.8 (PhOC(S)O). MALDI-TOF MS m/z: [M + H]+
615.2, calcd 615.2.
ꢀ
3J = P1 cos2 (f) + P2 cos(f) + P3 +
(Dci
(P4 + P5 cos2(zi f
group
+ P6|Dcigroup|)))
where P1 = 13.70, P2 = -0.73, P3 = 0.00, P4 = 0.56, P5
=
=
group
-2.47, P6 = 16.90, P7 = 0.14 (parameters from.23), and Dci
Dcia- substituent - P7 R Dci
where Dci are taken as Huggins
b- substituent
electronegativities.27
Acknowledgements
Generous financial support from the Swedish Natural Science
Research Council (Vetenskapsra˚det), the Swedish Foundation for
Strategic Research (Stiftelsen fo¨r Strategisk Forskning) and the
EU-FP6 funded RIGHT project (Project no. LSHB-CT-2004-
005276) is gratefully acknowledged.
Synthesis of (1R, 5R, 7R, 8S)-8-benzyloxy-5-benzyloxmethyl-7-
(thymin-1-yl)-6-oxa-bicyclo[3.2.1]octane (14)
40 mg (0.065 mmol) of compound 13 was coevaporated with dry
toluene twice and dissolved in the same solvent (2 ml), to which
was purged dry N2 for 10 min. Bn3SnH (50 ml, 0.18 mmol) and
AIBN (5 mg) was added. The mixture was heated under reflux for
1h. Then evaporating the solvent and the residue was subjected to
short column chromatography (EtOAc/cyclohexane 1/9 to 3/7)
to give 22 mg of compound 14 (72%). 1H NMR (500 MHz, CDCl3):
d 1.34 (dd, 1H, H6¢), 1.43 (s, 3H, 5-CH3), 1.72 (m, 3H, H7¢, H7¢¢,
H8¢), 1.83 (m, 1H, H6¢¢), 1.92 (m, 1H, H8¢¢), 2.58 (s, 1H, H2¢), 3.54
(d, 1H, J = 11.0 Hz, H5¢), 3.67(d, 1H, J = 11.0 Hz, H5¢¢), 4.15
(d, 1H, J = 4.9 Hz, H3¢), 4.44–4.61 (m, 4H, BnCH2), 5.82 (s, 1H,
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Org. Biomol. Chem., 2008, 6, 4627–4633 | 4633
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