Tricyclanos: Conformationally constrained nucleoside analogues with a new heterotricycle obtained from a d-ribofuranose unit

Article abstract of DOI:10.1039/c7ob02296d

A novel type of nucleoside analogue in which the sugar part is replaced by a new tricycle, 3,7,10-trioxa-11-azatricyclo[5.3.1.0^5,11]undecane has been prepared by substrate-controlled asymmetric synthesis. 1,5-Dialdehydes obtained from properly protected or unprotected uridine, ribothymidine, cytidine, inosine, adenosine and guanosine by metaperiodate oxidation reacted readily with tris(hydroxymethyl)aminomethane to provide the corresponding tricyclic derivatives with three new stereogenic centers. Through a double cyclisation cascade process the tricyclic compounds were obtained in good to high yields, with very high diastereoselectivity. Formation of one stereoisomer, out of the eight possible, was observed in all cases. The absolute configuration of the new stereotriad-containing tricyclic systems was aided by conventional NMR experiments followed by chemical shift calculations using an X-ray crystal structure as reference that was in good agreement with H-H distances obtained from a new ROESY NMR method. The synthesis was compatible with silyl, trityl and dimethoxytrityl protecting groups. A new reagent mixture containing ZnCl₂, Et₃SiH and hexafluoroisopropanol was developed for detritylation of the acid-sensitive tricyclano nucleosides.

Full text of DOI:10.1039/c7ob02296d

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DOI: 10.1039/C7OB02296D.
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Tricyclanos: conformationally constrained nucleoside analogues
with a new heterotricycle obtained from the D-ribofuranose unit
Received 00th January 20xx,
Accepted 00th January 20xx
Máté Kicsák,a Attila Mándi,b Szabolcs Varga,a Mihály Herczeg,a Gyula Batta,b Attila Bényei,c Anikó
DOI: 10.1039/x0xx00000x
Borbás,*a and Pál Herczegh*a
www.rsc.org/
A novel type of nucleoside analogues in which the sugar part is replaced by a new tricycle, 3,7,10-trioxa-11-
azatricyclo[5.3.1.05,11]undecane has been prepared by substrate-controlled asymmetric synthesis. 1,5-Dialdehydes
obtained from properly protected or unprotected uridine, ribothymidine, cytidine, inosine, adenosine and guanosine by
metaperiodate oxidation reacted readily with tris(hydroxymethyl)aminomethane to provide the corresponding tricyclic
derivatives with three new stereogenic centers. Through a double cyclisation cascade process the tricyclic compounds
were obtained in good to high yields, with very high diastereoselectivity. Formation of one stereoisomer, out of the eight
possible, was observed in all cases. The absolute configuration of the new stereotriad-containing tricyclic systems was
aided by conventional NMR experiments followed by chemical shift calculations using an X-ray crystal structure as
reference that was in good agreement with H-H distances obtained from a new ROESY NMR method. The synthesis was
compatible with silyl, trityl and dimethoxytrityl protecting groups. A new reagent mixture containing ZnCl2, Et3SiH and
hexafluoroisopropanol was developed for detritylation of the acid-sensitive tricyclano nucleosides.
monomers can be prepared from simple ribonucleosides by
periodate oxidation and a subsequent reductive amination (Scheme
1a). In these compounds the morpholine ring is connected to
Introduction
Nucleoside analogues are able to interfere with the replication of
viruses or with the proliferation of cancer cells by competing with
their natural counterparts. Therefore, chemically modified
nucleosides and nucleotides have attracted considerable attention
and numerous therapeutically important derivatives have been
developed over the last decades.1–3
heterocyclic base recognition moieties of RNA (adenine, cytosine,
uracil and guanine). Oligonucleotide analogues were prepared from
these morpholino derivatives by coupling the morpholine nitrogen
to the phosphoramidite moiety derived from the primary hydroxyl
of another monomer.6 The phosphorodiamidate connected
morpholino oligomers (PMOs) inhibit the translation of target
mRNA by steric blocking and they are resistant to enzymatic
degradation. PMOs proved to be effective antivirals against RNA
viruses.7 They exhibited also antibacterial and anticancer activity as
well as inhibited in-stent restenosis after implantation of coronary
stents and proved to be promising therapeutic agents in the
treatment of Duchenne muscular dystrophy.8,9 Modified
oligonucleotides containing a single morpholino unit show also
potent gene-silencing activity and prolonged action time.10
Azanucleosides are nucleoside analogues in which the furanose unit
has been replaced by
pyrrolidine, piperidine, morpholine, etc. Some azanucleosides are in
advanced clinical trials as anticancer drugs, and others have
promising antimalarial, antitrypanosomal and antibacterial
properties.4
a nitrogen-containing ring including
In the 1990s, Summerton devised and began developing
morpholinos, an important group of azanucleosides having
morpholine ring in the place of ribose moieties.5 The morpholino
The 2‘-amino-locked nucleosides, bearing an -CH2-NH-bridge
between the C2‘ and C4‘ atoms, also represent notable members of
the azanucleoside family.11,12 These nucleoside derivatives are also
a kind of bicyclic morpholine analogues containing a bridged
morpholine ring connected to the nucleobase. Incorporation of
amino-locked nucleosides and other conformationally restricted bi-,
tri- or tetracyclic monomers into oligonucleotides has been a widely
used approach to produce nucleic acid analogues with increased
duplex stability and high enzymatic stabilty.13–16 Given the potential
of morpholinos and other azanucleosides as drug leads,
a.Department of Pharmaceutical Chemistry, University of Debrecen, Egyetem tér 1,
H-4032 Debrecen, Hungary. E-mail: borbas.aniko@pharm.unideb.hu,
herczeghp@gmail.com; Tel: +36 52512900
b.Department of Organic Chemistry, University of Debrecen,
c. Department of Physical Chemistry, University of Debrecen
† Footnotes relating to the title and/or authors should appear here.
Electronic Supplementary Information (ESI) available: Experimental details,
characterization of all reported compounds, DFT and computational data. CCDC
1571164. For ESI and crystallographic data in CIF or other electronic format see
DOI: 10.1039/x0xx00000x
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development of new analogues is an important area in medicinal diastereoisomers, in 58% yield after column chromatographic
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DOI: 10.1039/C7OB02296D
and synthetic chemistry. Inspired by both the gene silencing activity purification. No other product could be isolated from the reaction
and the special structure of morpholinos, we envisioned the mixture. The moderate yield was caused by the incomplete
synthesis of a novel type of nucleoside analogues having structure conversion of 1 and the difficulty of purification of the product. In
F, in which the ribose unit is substituted with a morpholine- order to discriminate the two primary hydroxyl groups of the
containing tricyclic ring system (Scheme 1b). Due to the two resulting tricyclano and in hope of higher yield, we decided to block
primary hydroxyl groups, these analogues can be coupled to each the 5’-hydroxyl function of uridine. Therefore,
1
was
other and can be incorporated into DNA or RNA oligonucleotides at regioselectively reacted with tert-butyldimethylsilyl (TBDMS)
any desired position using standard phosphoramidite chemistry. chloride and the partially blocked 2a21 was subjected to the two-
Moreover, the rigidity of the tricyclic sugar mimic could endow the step transformation including oxidation and condensation with Tris.
synthetic oligonucleotides with an increased stability against To our delight, the cyclocondensation reaction between E and the
nucleases.
dialdehyde22 took place with both high efficacy and
stereoselectivity to give 3a in 72% yield over two steps, in
completely stereo pure form. Removal of the TBDMS protecting
group by TBAF afforded uracil-tricyclano 4 that was identical to the
compound obtained via dialdehyde without protecting the 5’-
hydroxyl group.
Our literature survey revealed that the heterotricyclic ring system
formed by condensation of the ribose-derived dialdehyde and
tris(hydroxymethyl)aminomethane has not been described until
now. The new tricycle has been named as 3,7,10-trioxa-11-
azatricyclo[5.3.1.05,11]undecane (Fig. 1). The absolute configuration
of the new stereogenic centers C2, C5 and C8 could be determined
by means of X-ray crystallography, experimental and theoretical
NMR methods (vide infra).
Scheme 1. Synthesis of (a) morpholinos and (b) proposed synthesis
of morpholine-containing tricyclanos.
Cyclocondensation of aminoalcohols and monoaldehydes is a long-
known reaction17 which has been used extensively for the synthesis
of bis-oxazolidine bicyclic systems.18 It was demonstrated for the
first time by Broadbent et al.19 that condensation of 2-amino-1,3-
propanediols and 1,4-diketones led to the formation of pyrrolidino-
bis-oxazolidine tricycles. However, the reaction required harsh
conditions, use of p-toluenesulfonic acid in refluxing toluene, and
only moderate yields were achieved. Giovenzana et al.20 described
that a completely stereoselective 2:2 condensation of glyoxal and
tris(hydroxymethyl)aminomethane (Tris) took place in aqueous
medium without acid catalysis to give a pentacyclic derivative in
high yield. We assumed that upon such mild conditions
cyclocondensation between the 1,5-dialdehyde derivative of
nucleosides (B) and Tris (E) could proceed in a stereoselective
manner providing the desired heterotricyclic derivatives (F).
Figure 1. Numbering of tricyclano nucleosides having the 3,7,10-
trioxa-11-azatricyclo[5.3.1.05,11]undecane core.
As our aim was to prepare nucleoside analogues which can be
incorporated into short oligonucleotides, we tested the
compatibility of tricyclano synthesis with 4,4’-dimethoxytrityl
(DMTr), the standard protecting group in solid phase nucleic acid
synthesis. Thus, the known compound 2b10 was prepared and
converted to the DMTr-protected uracil-tricyclano 3b in 76% yield
over two steps (Scheme 2). The condensation of the intermediate
Herein, we present the synthesis and structural determination of
uridine-, ribothymidine-, inosine-, cytidine-, adenosine- and
guanosine-derived members of the new nucleoside family which we
suggest to call tricyclanos, by analogy of Summerton’s morpholinos.
dialdehyde with
E
proceeded, again, with the same
diastereoselectivity as observed with the unprotected and 5’-silyl
protected dialdehydes, revealing that the protecting group has no
influence on the stereochemical outcome of the cyclisation.
Results and discussion
Synthesis
Removal of the DMTr group from 3b having the acid-sensitive
double aminal structure was a critical part of the reaction path.
Attempted experiments upon standard conditions23 using
dichloroacetic acid led to partial decomposition of the tricycle of 3b.
Hence, our attention was turned to find a new method for DMTr
Our initial experiments were carried out with unprotected uridine
(1), which was oxidized with metaperiodate form of anion exchange
resin, and the obtained crude dialdehyde was reacted directly with
Tris (E) in methanol at room temperature (Scheme 2). The reaction
proceeded cleanly providing compound 4, one of the eight possible
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cleavage which the acid sensitive tricyclano could survive. For this synthesized and converted to 7 in two steps including oxidation
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DOI: 10.1039/C7OB02296D
purpose we have recently elaborated a complex system of reagents with metaperiodate followed by condensation reaction with E.
including a Lewis acid (boron trifluoride diethyl etherate), a Finally, the trityl protecting group was successfully cleaved by our
reducing quenching agent (triethylsilane) and the mild protic acid detritylating reagent combination to result in 8 that was also
1,1,1,3,3,3-hexafluoroisopropanol. This reagent cocktail, due to prepared from unprotected 5. Acetylation of 8 furnished 9 which
synergistic effect of the components, proved to be efficient for was used for structural determination studies.
rapid and clean deprotection of a large variety of trityl- and
dimethoxytrityl-protected compounds.24 However, applying this
method for removal of the DMTr group of 3b, partial decomposition
has been observed. Since the Lewis acid is a variable component of
.
the reagent combination, by exchanging BF3 Et2O into the milder
Lewis acid zinc chloride fine tuning of the reactivity was achieved.
Treating 3b with the reagent cocktail containing zinc chloride the
DMTr deprotection went to completion within half an hour without
noticeable decomposition providing 4 in 71% yield.
The synthesis of the uracil-tricyclano was also carried out using the
cheap trityl (Tr) protecting group which resulted in the
corresponding tricyclano 3c with 76% overall yield from 2c. To our
delight, the ZnCl2-containing reagent cocktail cleaved also the Tr
protecting group of tricyclano 3c without damaging the acid-
sensitive tricyclic system (Scheme 2). This result suggested that the
O-DMTr protecting group can be changed to the less expensive Tr
group in our further experiments.
Scheme 3. Synthesis of thymine-tricyclano 8 without protecting
groups and using 5’-Tr protecting groups. Reagents and conditions:
(i) TrCl, dry C5H5N, rt, overnight, 75%; (ii) IO4- resin, MeOH, rt, dark,
overnight; (iii) E, 3 Å MS, dry MeOH, rt, overnight, 7: 64% (2 steps
from 6), 8: 53% (2 steps from 5); (iv) ZnCl2, (F3C)2CHOH, H3CNO2,
Et3SiH, rt, 1 h, 8: 55% (from 7); (v) Ac2O, dry C5H5N, rt, overnight,
73%. 6, 7: R = Tr; 8: R’ = H; 9: R’ = Ac.
We envisioned that the synthesis of the cytosine-tricyclano can be
easily accomplished by using O,N-bistrityl protection. Unexpectedly,
simultaneous tritylation of the 5’ hydroxyl and the amino group of
10 proved to be very sluggish and inefficient providing the
bisprotected 11a in only 30% yield after a ten-day reaction (Scheme
4). The usual oxidation and tricyclization procedure showed similar
efficacy as in the cases of uridine and thymidine congeners to
provide cytosine-tricyclano 13a in 65% isolated yield.
Unfortunately, our new detritylation method was not completely
successful. Despite the high excess of ZnCl2 (8 equiv.) only the O-Tr
group could be cleaved in a 5-hour reaction to give 14a in 56%
Scheme 2. Synthesis of uracil-tricyclano 4 without protecting groups
and using 5’-TBDMS, DMTr and Tr protecting groups. Reagents and
conditions: (i) TBDMSCl, imidazole, dry DMF, rt, overnight, 2a: 64%;
(ii) DMTrCl, dry Et3N, dry C5H5N, rt, overnight, 2b: 81%; (iii) TrCl, dry
yield. Longer exposure of 13a to the detritylation conditions led to
-
C5H5N, rt, overnight, 2c: 83%; (iv) IO4 resin, MeOH, rt, dark,
decomposition of the tricyclic system rather than N-deprotection.
overnight; (v) E, 3 Å MS, dry MeOH, rt, 3a, 3b: overnight, 3c: 2 days,
Therefore, we decided to change the N-trityl to the more acid-
3a: 72% (2 steps from 2a), 3b: 76% (2 steps from 2b), 3c: 76% (2
sensitive N-DMTr protecting group. First, the amino group of 10 was
steps from 2c), 4: 58% (2 steps from 1); (vi) TBAF, dry THF, rt,
selectively protected with DMTr, then the obtained 11b26 was
overnight, 4: 48% (from 3a); (vii) ZnCl2, (F3C)2CHOH, H3CNO2, Et3SiH,
selectively O-tritylated at the primary position to result in 12b in
rt, 2 h, 4: 71% from 3b, 66% from 3c. 2a, 3a: R = TBDMS; 2b, 3b: R =
56% overall yield from 10. After periodate oxidation, the resulting
DMTr; 2c, 3c: R = Tr.
dialdehyde was reacted with E to produce 13b in a good 72% yield
over two steps. Finally, 13b was successfully treated with our
The tricyclano derivative of ribothymidine was also prepared using
detritylation cocktail to result in 14b. Although TLC monitoring of
Tr protecting group at position 5’ (Scheme 3). First, 625 was
the reaction showed clean and efficient deprotection, 14b could
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only be isolated in 26% yield because the purification process was
difficult due to the very poor solubility of the product.
View Article Online
DOI: 10.1039/C7OB02296D
Scheme 5. Synthesis of hypoxanthine-tricyclano 19 via unprotected,
TBDMS- and Tr-protected dialdehyde intermediates. Reagents and
conditions: (i) TBDMSCl, imidazole, dry DMF, rt, overnight, 16a:
o
54%; (ii) TrCl, dry C5H5N, dry DMF, dry DMSO, 60 C, 4 days, 16b:
Scheme 4. Synthesis of cytosine-tricyclano using O- and N-bistrityl
-
39%; (iii) IO4 resin, MeOH, rt, dark, overnight; (iv) E, 3 Å MS, dry
(including Tr and DMTr) protecting groups. Reagents and
MeOH, rt, overnight, 19: 38% (2 steps from 15), 17a: 52% (2 steps
from 16a), 17b: 42% (2 steps from 16b); (v) Ac2O, dry C5H5N, rt,
overnight, 18b: 86% (from 17b); (vi) TBAF, dry THF, rt, overnight,
19: 56% (from 17a); (vii) ZnCl2, (F3C)2CHOH, H3CNO2, Et3SiH, rt, 2 h,
19: 58% (from 17b). 16a: R = TBDMS; 16b: R = Tr; 17a: R = TBDMS,
R’ = H; 17b: R = Tr, R’ = H; 18b: R = Tr, R’ = Ac.
o
conditions: (i) TrCl, DMAP, Et3N, dry DMF, 50 C, 10 days, 11a: 30%;
(ii) TMSCl, DMTrCl, dry C5H5N, rt, 4 days, 11b: 82%; (iii) TrCl, dry
-
C5H5N, rt, 7 days, 12b: 68%; (iv) IO4 resin, MeOH:CH2Cl2 (13a: 3:1,
13b: 1:1), rt, dark, 13a: overnight, 13b: 3 days; (v) E, 3 Å MS, dry
MeOH, rt, 13a: 2 days, 13b: overnight, 13a: 65% (2 steps from 11a),
13b: 72% (2 steps from 12b); (vi) ZnCl2, (F3C)2CHOH, H3CNO2, Et3SiH,
rt, 5 h, 14a: 56% (from 13a), 7h, 14b: 26% (from 13b); 11a, 13a: R =
R’ = Tr, 11b: R = H, R’ = DMTr. 12b, 13b: R = Tr, R’ = DMTr; 14a: R =
H, R’ = Tr; 14b: R = R’ = H.
To get access to adenine-tricyclano, 20 was N-benzoylated (21),29
silylated (22a)30 and subsequently transformed into 23a (Scheme 6).
The cyclocondensation led to the formation of
a single
stereoisomer, however, partial loss of the benzoyl group was
observed, owing to the nucleophilicity of the Tris reactant.
Desilylation of 23a with TBAF and subsequent debenzoylation of
the resulting 24a in methanolic ammonia took place smoothly to
provide the desired free tricyclano 25 in 86% overall yield. In order
to test the applicability of our new detritylation method in a purine-
containing tricyclano, the 6-N,5’-O-bistritylated adenosine
derivative 22b31 was prepared. Next, 22b was converted to 23b
using the usual two-step reaction including oxidation followed by
cyclocondensation of the obtained dialdehyde with Tris. Complete
deprotection of 23b required an overnight treatment with the
detritylating cocktail containing 8 equiv. of ZnCl2. Fortunately,
despite the long reaction time and high excess of Lewis acid, neither
depurination nor decomposition of the tricyclic core occurred in a
noticeable amount and compound 25 was isolated in 63% yield.
To further explore the scope of the novel synthetic pathway parallel
to the synthesis of pyrimidine-based tricyclano, similar experiments
with inosine, adenosine, and guanosine towards purine tricyclanos
were also carried out. First, the impact of the purine base on the
cyclocondensation was studied in the case of inosine (Scheme 5).
Reaction between Tris and the 5’-TBDMS- or Tr-protected
dialdehyde derivatives obtained from inosine via 16a27 or 16b28
afforded 17a or 17b, respectively, as the only products. Formation
of other stereoisomers was not visible, and the moderate yields
(52% for 17a and 42% for 17b over two steps) can be explained
mainly by the difficulty of purification of the tricyclic products and,
to a lesser extent, by the incomplete conversions of dialdehydes
Usual deprotection of both 17a and 17b yielded efficiently the
unprotected hypoxanthine-tricyclano 19, which can also be
obtained directly from 15 without protecting the 5’ position. 17b
was acetylated to afford the crystalline 18b which was recrystallized
from i-PrOH. Fortunately, the obtained crystal was good for X-ray
diffraction thereby gave us the key for determination of the
absolute configuration of the new tricyclic ring system. The crystal
structure of 18b is shown in Fig. 2.
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Scheme 6. Synthesis of adenine-tricyclano 25 using O-TBDMS and
N-benzoyl or O- and N-bistrityl protection. Reagents and conditions:
(i) TMSCl, Bz2O, dry C5H5N, rt, overnight, then H2O, cc. NH3, 21: 96%;
(ii) TBDMSCl, DMAP, 4 Å MS, dry C5H5N, rt, overnight, 22a: 83%
(from 21); (iii) TrCl, dry C5H5N, 60 oC, overnight, 22b: 63% (from 20);
Scheme 7. Synthesis of guanine-tricyclano 29 with O- and N-bistrityl
and O- and N-bis(dimethoxytrityl) protecting groups. Reagents and
conditions: (i) TrCl, DMAP, Et3N, dry DMF, 50 C, 10 days, 27a: 49%;
o
(ii) TMSCl, DMTrCl, dry DMF, dry C5H5N, 4 days, 27b: 47%, 27c: 30%;
-
(iv) IO4 resin, MeOH, rt, dark, overnight; (v) E, 3 Å MS, dry MeOH,
-
(iii) NaIO4, MeOH:H2O (3:1) or IO4 resin, MeOH:CH2Cl2 (2:3), rt,
rt, overnight, 23a: 24% (2 steps from 22a), 23b: 70% (2 steps from
dark, overnight; (iv) E, 3 Å MS, dry MeOH, rt, 28a: 2 days, 28b:
overnight, 28a: 73% (2 steps from 27a), 28b: 80% (2 steps from
27b); (v) ZnCl2, (F3C)2CHOH, H3CNO2, Et3SiH, rt, 5 h, 20% (from 28b).
27a, 28a: R = R’ = Tr; 27b, 28b: R = R’ = DMTr; 27c: R =H, R’ = DMTr;
29: R = R' = H.
-
22b); (vi) IO4 resin, MeOH:CH2Cl2 (3:2), rt, dark, overnight; (vii)
TBAF, dry THF, rt, overnight, 24a: 92% (from 23a); (viii) cc. NH3,
MeOH, rt, overnight, 25: 95% (from 24a); (ix) ZnCl2, (F3C)2CHOH,
H3CNO2, Et3SiH, rt, 24 h, 63% (from 23b); 20: R = H; 21: R = Bz; 22a,
23a: R = Bz, R’ = TBDMS; 22b, 23b R = R’ = Tr; 24a: R = Bz, R’ = H.
Determination of the absolute configuration
Finally, in order to extend our synthetic method to all
ribonucleosides, guanine-tricyclano 29 was also prepared (Scheme
7). First, guanosine 26 was selectively tritylated at the primary
hydroxyl and the amino group of the heterocyclic base to obtain
27a,32 which was transformed into the tricyclano 28a in high
efficacy (73% yield) by the usual two-step procedure. Unexpectedly,
the complete deprotection required a three-day reaction with the
detritylating cocktail, upon which decomposition also occurred, and
the desired product could not be isolated. To avoid decomposition
upon deprotection, the above synthetic route from 26 to 28 was
carried out by the use of the more acid-labile DMTr protecting
group. For this purpose, 26 was treated with TMSCl and DMTrCl to
produce the required O- and N-bis(dimethoxytrityl) derivative 27b33
in 47% yield, together with the mono-N-protected byproduct 27c.
Transformation of 27b into the tricyclano derivative was carried out
in the usual manner to obtain 28 in 80% yield. To our great delight,
treatment of 28b with our mild reagent combination led to a
satisfactory level of O-and N-deprotection within 5 hours and the
required fully deprotected guanosine derivative 29, the last
member of the tricyclano family, could successfully be isolated and
characterised. We assume that the moderate yield of 29, which was
mainly caused by the incomplete deprotection, can be increased by
further fine tuning of the detritylation conditions.
3
Simple NMR methods (e.g. JHH coupling constants) were
insufficient to unambiguously disclose the stereochemistry around
the three new stereocenters which might be due to the
conformational diversity predicted theoretically (see Fig. S4 in the
Supporting Information). Finally, combined use of X-ray
crystallography, a new NMR ROESY method for H-H distance
determination and chemical shift calculations based on theoretical
conformational distribution and comparison with NMR data gave
satisfactory results.
X-ray studies
X-ray quality crystals of 18b were grown from i-propanol. Details of
data collection and summary of parameters are given in the ESI,
Table S1. In the structure, hydrogen atom positions were placed
into geometric positions except N-H and O-H protons which could
be located in the difference electron density maps but the N-H and
O-H distances were restrained. The O-H hydrogen atoms of the
solvent i-propanol were shifting between two orientations and the
small disorder of the solvent molecule was not modelled as
chemical evidence indicated the presence of i-propanol molecules.
However this did not affect the overall correctness of the structure
determination. The isotropic displacement parameters of the
hydrogen atoms were approximated from the U(eq) value of the
atom they were bonded to. Refinement of non-hydrogen atoms
was carried out with anisotropic temperature factors. Selected
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bond lengths and angles of compound 18b are shown at Tables S2 organic compounds. The histogram for the angle of the said planes
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DOI: 10.1039/C7OB02296D
and S3, respectively. The space group was non centrosymmetric is shown at Fig. S165 clearly demonstrating, that our system with
and the results unambiguously support that the sample is 52-58 degree angle is rather unique.
enantiopure. The high sensitivity of the detector made it possible to
DFT NMR calculations
determine the absolute configuration reliably on the basis of
anomalous dispersion effect. The Flack parameter for 4034
quotients was found to be 0.03(12).34
For the DFT NMR studies all possible 8 : 8 stereoisomers of 3c and
9, the proven one of 18b and the presumed one of 13a were
utilized. For each stereoisomer an OPLS (Optimized Potentials for
Liquid Simulations)38 conformational search was carried out for
CHCl3 and conformers were saved in a 21 kJ mol-1 energy window.
These conformers were reoptimized at the B3LYP/6-31+G(d,p) level
and NMR shift values were computed for conformers over 1%
Boltzmann population at the mPW1PW91/6-311+G(2d,p) level.39
Corrected C and H-NMR data40 were compared to the experimental
shift values. While for 9 the C-NMR data (Table S7) alone were
rather persuasive, for 3c a combination of C (Table S5) and H-NMR
(Table S6) was necessary to distinguish between the 8 stereoisomes
resulting in the same diastereomer elucidated by X-ray
crystallography for 18b. Corrected mean absolute error (CMAE)
values41 and largest deviations from the experimental data of 18b
and 13a were similar for the single investigated stereoisomers as
for the best stereoisomers of 3c and 9 (Tables S9–S12) Therefore
upon the computational NMR results for all tricyclanos the products
are homochiral with the X-ray-determined compound 18b.
Newly developed distance-based experimental NMR method to
elucidate stereochemistry
A recently introduced zero-quantum suppressed off-resonance
ROESY method42 (an easy-ROESY variant) proved to be especially
useful to corroborate stereochemical conclusions. Since the method
is less sensitive to zero-quantum artifacts present between scalar-
coupled nuclei (e.g. CH2 groups or aromatic ortho protons used for
distance calibration), good distance agreement was found between
NMR and X-ray (Figs. S167-S169 and Table S4) where independent
X-ray structure was available (18b). Interestingly, theoretical
calculations showed conformational diversity of the tricyclic ring
and the side chains that prevents the possibility of direct
comparison with ad hoc selected conformers. Conformational
diversity was recently shown to persist in "rigid" molecules like
strychnine.43 For the 500 MHz off-resonance ROESY spectra
typically theta = 54.7o magic tilt angle was used, with a spin lock
field strength of 8.33 kHz (p90 = 30 us) at ± 5.9 kHz offsets applied in
the two halves of mixing the time of 200 ms. For zero quantum
filtering adiabatic chirp pulses of 30 and 50 ms with simultaneous
spoil gradients were used as described in ref. 42.
Figure 2. ORTEP view of the structure of compound 18b, at 40%
probability level with partial numbering scheme. One of the
molecules from the asymmetric unit is shown and solvent
molecules are omitted for clarity.
The absolute configuration for both 18b molecules is
(1R,2S,5R,8R,9R). The structure is stabilized by weak C-H∙∙O and C-
H∙∙N hydrogen bonds. There are also hydrogen bonds between the
i-propanol molecules and O-H of one of the i-propanol molecules
and carbonyl oxygen of the hypoxanthine moiety (O96). Altogether
bond length and bond distance data correspond to the expected
values35 and distance of N11 from the C2-C5-C8 plane is 0.497 and
0.515 Å for the two molecules, respectively. Ring puckering
analysis36 of the structure revealed that the 5-membered rings of
N11-C2-O3-C4-C5
and
N11-C8-O7-C6-C5
have
envelope
conformation on O3 and O7, respectively (Fig. S165). However, the
six membered ring of N11-C2-C1-O10-C9-C8 is very strained as C1-
C2-N11-C8 atoms are in one plane (Fig. S166) while C9 and O10 are
at the same side of the plane resembling a half chair conformation.
The C1-C2 and C51-C52 distances are both 1.516(7) Å indicating sp3
carbons. The angle of C1-O10 and C1-C2-N11-C8 planes is 58 degree
(52 degree for the other molecule in the asymmetric unit). Search
of the Cambridge Structural Database (Ver. 5.38, update May,
2017)37 resulted around 2600 hits for morpholine ring moiety in
Mechanism
We suppose that the formation of the new tricycle can be described
as a series of consecutive reactions (Scheme 8.). First a Schiff base
(i2) is formed through
a hemiaminal (i1) followed by an
intramolecular addition of one of the primary alcohols resulting in
the oxazolidine i3. A second hemiaminal (i4) is formed by the
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addition of NH group to the second aldehyde. The final step is the for the N-trityl group of cytosine and guanine – and this mild
View Article Online
DOI: 10.1039/C7OB02296D
addition of the second hydroxyl group onto the iminium deprotection method neither affected the acid-sensitive
intermediate i5. The stereoselectivity of the reaction is controlled double aminal system nor caused depurination.
by the two chiral centers of the dialdehyde. Since the water side
Studies towards the synthesis of tricyclano-containing
oligonucleotide derivatives and utilisation of the tricyclisation
method for producing new N-containing heterotricyclic
systems are under way.
product is continously removed with molecular sieves there is no
equilibration in the particular reaction steps. Therefore, we
postulate that formation of the kinetic product is preferred
resulting in the high stereoselectivity of the reaction. The observed
stereochemistry at C2 and C8 is in accordance with the polar Felkin-
Anh modell generally applied for addition reactions of aldehydes or
ketones with an electronegative group.44 Chiral centers at C2 and C5
(or at C8 and C5) form simultaneously in the final cyclisation step.
Experimental
1. Typical procedure for the cyclocondensation reaction between
the nucleoside dialdehyde and Tris: 12’-O-trityl-uracil-tricyclano
(3c)
-
To a solution of 2c (2.00 g, 4.13 mmol) in MeOH (100 mL) IO4 -form
of anion exchange resin (8.0 g) was added and stirred overnight in
dark. Next day the resin was filtered off through a short pad of
Celite® and washed successively with MeOH and CH2Cl2. The
solvent was evaporated in vacuum. The residue was dissolved in dry
MeOH (100 mL) and 3 Å molecular sieves (2 g) were added to the
reaction. After stirring for half an hour Tris (747 mg, 6.17 mmol, 1.5
equiv.) was added to the mixture. Next day another portion of Tris
(249 mg, 2.06 mmol, 0.5 equiv.) was added to the stirred reaction
mixture. After a total reaction time of 2 days the molecular sieves
were filtered off through a short pad of Celite® and washed
successively with MeOH and CH2Cl2. The solvent was evaporated in
vacuum and the crude product was purified by flash column
chromatography (CH2Cl2/MeOH 98:2 → 98:3 → 97:3) to yield
compound 3c (1.78 g, 76%, over two steps) as a white foam. Rf=
0.42 (CH2Cl2/MeOH 95:5); 1H NMR (400 MHz, CDCl3) δ = 9.77 (s, 1H,
NH), 7.48 (d, J = 8.2 Hz, 1H, uracil CH-6), 7.43 (d, J = 7.3 Hz, 6H, 6 x
Tr Ar-H), 7.29 (t, J = 7.4 Hz, 6H, 6 x Tr Ar-H), 7.23 (t, J = 7.2 Hz, 3H, 3
x Tr Ar-H), 6.14 (d, J = 4.7 Hz, 1H, H-1’), 5.67 (d, J = 8.2 Hz, 1H, uracil
CH-5), 4.80 (d, J = 4.6 Hz, 1H, H-8’), 4.68 (d, J = 4.7 Hz, 1H, H-2’),
4.21 (q, J = 4.4 Hz, 1H, H-9’), 3.87 (d, J = 9.0 Hz, 2H, H-6’a, H-4’a),
3.78 (d, J = 8.9 Hz, 1H, H-6’b), 3.77 (d, J = 8.8 Hz, 1H, H-4’b), 3.64 (d,
J = 5.5 Hz, 2H, H-13’a,b), 3.38 (d, J = 4.3 Hz, 2H, H-12’a,b), 2.90 (t, J =
5.8 Hz, 1H, OH); 13C NMR (100 MHz, CDCl3) δ = 163.4 (1C, uracil CO-
4), 150.5 (1C, uracil CO-2), 143.5 (3C, 3 x Tr Ar-C), 139.9 (1C, uracil
CH-6), 128.7, 128.0, 127.3 (15C, 15 x Tr Ar-CH), 103.0 (1C, uracil CH-
5), 90.3 (1C, C-2’), 88.4 (1C, C-8’), 87.1 (1C, Tr Cq), 77.7 (1C, C-1’),
75.3 (1C, C-5’), 73.9 (1C, C-9’), 72.0 (1C, C-4’), 71.3 (1C, C-6’), 63.7
(1C, C-13’), 63.6 (1C, C-12’); ESI-TOF-MS: m/z calcd for
C32H31N3NaO7 [M+Na]+ 592.206, found 592.205.
Scheme 8. Proposed mechanism of the imine-oxazolidine and
iminium-oxazolidine cascade reactions. (The first attack of the
amine on one of the two aldehyde moieties is interchangeable.)
Conclusions
In conclusion, we developed a new type of nucleoside
analogues by stereoselective transformation of the ribose unit
of nucleosides to a novel, specific heterotricyclic system
comprising a morpholine and two 1,3-oxazolidine rings. The
implemented synthesis is very simple including an oxidation
step followed by
a cyclocondensation of the resulting
dialdehyde with the commercially available and cheap reagent
Tris. The highly efficient and stereoselective formation of one
isomeric form of the newly generated stereotriade observed in
the condensation reactions can be explained by the induced
fitting of the reactants. The reaction proceeds through two
consecutive hemiaminal-oxazolidine cascade reactions and the
stereoselectivity is governed by the two stereogenic centers of
the nucleoside-derived dialdehydes. The stereochemistry was
unambiguously proven by single crystal X-ray diffraction for
18b in agreement with NMR ROESY and computational
methods that allowed to assign absolute configuration in the
absence of X-ray structures. Combined approach was
necessary because of the theoretically predicted flexibility of
the tricycle. The synthetic method is compatible with both
purine and pyrimidine bases as well as bulky protecting groups
like TBDMS, Tr or DMTr. Using a ZnCl2-Et3SiH-(F3C)2CHOH-
containing reagent system, due to the beneficial synergism of
the components, the Tr and DMTr protecting groups of
tricyclanos could efficiently be cleaved in most cases – except
2. Typical deprotection procedure using the three-component
detritylating cocktail: Uracil-tricyclano (4)
3c (342 mg, 0.60 mmol) was added to the mixture of ZnCl2 (342 mg,
2.51 mmol, 4.2 equiv.), hexafluoroisopropanol (6 mL), MeNO2 (3
mL) and Et3SiH (600 L, 3.76 mmol, 6.3 equiv.). After 2 hours
saturated NaHCO3-solution was added and the solvents were
evaporated. The crude product was purified by flash column
chromatography (CH2Cl2/MeOH 9:1 → 8:1 → 85:15 → 8:2) to yield 4
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Organic & Biomolecular Chemistry
1
(129 mg, 66%) as a white foam. Rf= 0.45 (CH2Cl2/MeOH 85:15); H 12 C. Rosenbohm, S. M. Christensen, M. D. Sřrensen, D. S.
View Article Online
DOI: 10.1039/C7OB02296D
NMR (400 MHz, D2O + CD3OD) δ = 7.78 (d, J = 8.1 Hz, 1H, uracil CH-
6), 6.04 (d, J = 5.0 Hz, 1H, H-1’), 5.88 (d, J = 8.1 Hz, 1H, uracil CH-5),
Pedersen, L.-E. Larsen, J. Wengel and T. Koch, Org. Biomol.
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= 3.9, 1.3 Hz, 1H, H-9’), 4.00 (d, J = 9.1 Hz, 1H, H-6’a), 3.93 – 3.86 (m, 1994, 77, 716–744.
2H, H-4’a,b), 3.85 – 3.71 (m, 3H, H-6’b, H-12’a,b), 3.69 (s, 2H, H- 14 D. Renneberg and C. J. Leumann, J. Am. Chem. Soc., 2002,
13’a,b); 13C NMR (100 MHz, D2O + CD3OD) δ = 166.6 (1C, uracil CO-
124, 5993–6002.
4), 152.3 (1C, uracil CO-2), 142.6 (1C, uracil CH-6), 103.8 (1C, uracil 15 T. S. Kumar, A. S. Madsen, J. Wengel and P. J. Hrdlicka, J. Org.
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243.
350.096, found 350.093.
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Conflicts of interest
There are no conflicts of interest to declare.
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The authors gratefully acknowledge financial support from the
National Research, Development and Innovation Office (OTKA K
109208 and 105459, NKFI PD 121020 and ANN 110821). The
research was also supported by the EU and co-financed by the
European Regional Development Fund under the project GINOP-
2.3.2-15-2016-00008 as well as in the framework of TÁMOP-4.2.2.B-
15/1/KONV-2015-0001 and TÁMOP-4.2.1.C-14/1/KONV-2015-0004.
CPU time was granted by the Governmental Information-
Technology Development Agency (KIFÜ). The authors thank Dr.
Dyanne Cruickshank (Rigaku Oxford Diffraction) for collecting X-ray
data and Erzsébet Rőth for excellent technical assistance.
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