Design, synthesis, and duplex-stabilizing properties of conformationally constrained tricyclic analogues of LNA

Article abstract of DOI:10.1039/c5ob02576a

The design, synthesis and biophysical evaluation of two highly-constrained tricyclic analogues of locked nucleic acid (LNA), which restrict rotation around the C4′-C5′-exocyclic bond (torsion angle γ) and enhance hydrophobicity in the minor groove and along the major groove, are reported. A structural model that provides insights into the sugar-phosphate backbone conformations required for efficient hybridization to complementary nucleic acids is also presented.

Full text of DOI:10.1039/c5ob02576a

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Design, synthesis, and duplex-stabilizing
properties of conformationally constrained
Cite this: DOI: 10.1039/c5ob02576a
tricyclic analogues of LNA†
Robert D. Giacometti,a Juan C. Salinas,a Michael E. Østergaard,b Eric E. Swayze,b
Punit P. Seth*b and Stephen Hanessian*a
The design, synthesis and biophysical evaluation of two highly-constrained tricyclic analogues of locked
nucleic acid (LNA), which restrict rotation around the C4’–C5’-exocyclic bond (torsion angle γ) and
enhance hydrophobicity in the minor groove and along the major groove, are reported. A structural
model that provides insights into the sugar–phosphate backbone conformations required for efficient
hybridization to complementary nucleic acids is also presented.
Received 5th November 2015,
Accepted 16th December 2015
DOI: 10.1039/c5ob02576a
www.rsc.org/obc
Introduction
The synthesis of conformationally restricted nucleic acid ana-
logues, which enhance RNA-binding affinity, nuclease stability,
cell-uptake, and other desirable properties of therapeutic
oligonucleotides, has witnessed a resurgence in recent years.1–5
Recent examples include C2′,C4′-conformationally restricted
furanose ring analogues,6–9 restricting rotation around the
C4′–C5′-exocyclic bond in DNA,10–14 replacing the furanose
ring with other ring systems,15–18 and conformational restric-
tion of the sugar-phosphate backbone.19–22 We recently
showed that dual conformational restriction of α-L-LNA,23 by
restricting the conformational equilibrium of the furanose
ring, as well as rotational freedom about the C4′–C5′ exocyclic
bond (torsion angle γ), can enhance RNA-binding affinity
beyond that obtained by restricting the conformation of the
furanose ring alone.24,25 To extend these observations, we
hypothesized that dual conformational restriction of β-D-LNA, by
locking the conformation of the furanose ring in the C3′-endo
sugar pucker and locking γ in the +sc range could further
enhance the RNA-binding affinity of oligonucleotides that
incorporate this motif (Fig. 1).
Fig. 1 Strategy for dual constraint of nucleic acid analogues.
spread use in oligonucleotide-based diagnostics and thera-
peutics, and a large number of analogues have been prepared
in an effort to further enhance its hybridization and thera-
peutic properties.1,28,29 We have recently shown that (S)- or (R)-
configured methyl groups introduced at the C6′-position of
LNA (2′-O-constrained ethyl-2′,4′-bridged nucleosides; abbrev.
S-cEt (4) and R-cEt (5)) are well tolerated and the corres-
ponding modified oligonucleotides show LNA-like affinity for
complementary RNA.30–32 In contrast, the ability of alkyl sub-
stituents introduced at the C5′-position to influence duplex
stability was highly dependent on their configuration.33 An
analogue with a (S)-5′-Me group (3) showed LNA-like binding
affinity while the epimeric (R)-5′-Me analogue (2) was signifi-
cantly less stabilizing.
Locked nucleosides enhance the thermal stability of modi-
fied oligonucleotide duplexes by locking the conformation of
the furanose ring into a C3′-endo (RNA-like) sugar pucker
(Fig. 1).26,27 LNA-modified oligonucleotides have found wide-
aDepartment of Chemistry, Université de Montréal, P.O. Box 6128, Downtown
Station, Montréal, QC H3C 3J7, Canada. E-mail: stephen.hanessian@umontreal.ca
bDepartment of Medicinal Chemistry, Ionis Pharmaceuticals, 2855 Gazelle Court,
Carlsbad, CA 92010, USA. E-mail: PSeth@ionisph.com
†Electronic supplementary information (ESI) available: Experimental details and
crystallographic data. CCDC 1434799 and 1434800. For ESI and crystallographic
data in CIF or other electronic format see DOI: 10.1039/c5ob02576a
NMR studies with (R)- and (S)-5′-Me-modified LNA nucleo-
sides (2 and 3) suggest the possibility of conformational
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mobility around the C4′–C5′ exocyclic bond in (R)-5′-Me LNA
(2b, torsion angle γ in +ap range), presumably owing to a repul-
sive 1,3-eclipsing interaction between the (R)-configured C5′-
Me and the C3′-hydroxy group (2a). This interaction is absent
in the (S)-5′-Me LNA analogue (3), where torsion angle γ is in
the canonical +sc range. This hypothesis was further strength-
ened by crystal structures of A-form DNA–DNA decamer
duplexes containing (R)- and (S)-6′-Me-substituted 3′-fluoro-
hexitol nucleic acids (FHNA), which exhibited tight intramolecular
contacts between the (R)-6′-Me and the 3′-phosphodiester func-
tionalities.34 The FHNA scaffold represents a mimic of the C3′-
endo sugar pucker35 and the duplex stabilizing properties
observed for the (R)- and (S)-6′-Me FHNA analogues mirrored
those obtained with (R)- and (S)-5′-Me LNA (i.e., the (R)-5′-Me
isomer was destabilizing and the (S)-5′-Me epimer was
stabilizing).
To determine if the strategy of dual conformational
constraint is a valid approach for further enhancing the
RNA-binding properties of LNA (1), we designed TriNA 1 (6)
and TriNA 2 (7) as highly conformationally restricted tri-
cyclic analogues of LNA (Fig. 2). In essence, TriNA 1 also
represents a conformationally restricted hybrid of S-cEt (4)
and (R)-5′-Me LNA (2a), while TriNA 2 represents the confor-
mationally restricted hybrid of R-cEt (5) and (S)-5′-Me LNA
(3). Although both TriNA 1 and TriNA 2 constrict the confor-
mation of the furanose ring to a C3′-endo sugar pucker,
while further restricting γ to the +sc range, the steric bulk
of the appended six-membered ring is expected to project
into different regions of duplexes modified with each
monomer (Fig. 3).
Fig. 3 Overlays of TriNA 1, TriNA 2, and LNA nucleosides, along with an
overlay of TriNA 1 and TriNA 2 and an S-cEt-modified A-form DNA–DNA
duplex. The six-membered ring of TriNA 2 projects into the minor
groove while that of TriNA 1 extends toward the edge of the major
groove.
In this report, we describe the synthesis of nucleoside phos-
phoramidite building blocks of TriNA 1 and 2, as well as the
oligonucleotides that incorporate each of them, to demon-
strate that that the strategy of dual constraint is also effective
within other classes of conformationally restricted nucleic acid
analogues.
Results and discussion
Synthesis of TriNA 1
The synthesis of nucleoside TriNA 1 was envisaged to originate
from sugar-derived synthetic precursor 8, which was previously
synthesized from diacetone-D-allofuranose in five steps.30 Oxi-
dation of alcohol 8 in the presence of pyridinium chlorochro-
mate (PCC), followed by a Hosomi–Sakurai allylation reaction
afforded (S)-configured homoallylic alcohol 9. Previous work
Fig. 2 Design, strategy, and rationale for TriNA 1 and TriNA 2.
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has shown that addition of nucleophiles to the intermediate the freshly-generated alkoxide (12). This furnished the key oxa-
aldehyde occurs exclusively from the Si face.25,30,36 It was cyclic bridge, which is present in LNA, appropriately substi-
planned that activation of this secondary hydroxy group would tuted to allow for further elaboration to the corresponding six-
provide a suitable route for establishing the correct stereo- membered ring embedded in the tricyclic scaffold of TriNA
chemistry at C6′ of TriNA 1, by way of an SN2 displacement 1. To avoid undesirable side-reactions and improve its solubi-
reaction. Consequently, it was necessary to invert the configur- lity during the subsequent synthetic sequence, the nucleobase
ation of the secondary hydroxy group in 9 for the purpose of was first protected as its N-benzyloxymethyl derivative, before
preparing TriNA 1. To this end, alcohol 9 was oxidized with TBAF-mediated deprotection was used to access primary
Dess–Martin periodinane and then reduced with lithium alcohol 14. Initially we hoped to add vinyl-containing organo-
aluminum hydride to provide the corresponding (R)-config- metallic reagents to the corresponding aldehyde of 14, to
ured alcohol, which was subsequently mesylated to give 10. stereoselectively furnish the C5′ hydroxy group in one step
The thymine nucleobase was installed using a three-step while providing a synthetic handle for the planned ring-
sequence that comprised removing the acetonide protective closing metathesis reaction.
group in the presence of iron(III) chloride hexahydrate, acetylat-
Unfortunately, attempts to use several alkynyl- and vinyl-
ing the intermediate 1,2-diol, and a Vorbrüggen glycosylation based organometallic reagents under a variety of conditions
reaction to give 11. Notably, the use of high-quality FeCl3·6H2O were largely unsuccessful and resulted in low yields and
was crucial for ensuring that the yield of the deprotection reac- diastereoselectivities. Consequently, we elected to pursue a
tion was consistently high and the number of side-products longer synthetic sequence that involved preparing a Weinreb–
minimized.25
Nahm amide instead.37 To this end, alcohol 14 was oxidized to
At this point, potassium carbonate was used to effect the the corresponding carboxylic acid, which could readily be con-
crucial one-pot deprotection–cyclization sequence, in which verted to Weinreb amide 15 in the presence of N,O-dimethyl-
the C2′ acetate ester was hydrolyzed, with concomitant intra- hydroxylamine and HBTU. Fortunately, alkynyl-based Grignard
molecular displacement of the homoallylic mesylate group by reagents, such as 1-propynylmagnesium bromide, could suc-
Scheme 1 Synthesis of TriNA 1. Reagents and conditions: (a) PCC, NaOAc, 4 Å MS, CH2Cl2, r.t., 3 h; (b) BF3·OEt2, allyltrimethylsilane, CH2Cl2,
−40 °C, 1 h, 91% over two steps; (c) DMP, CH2Cl2, r.t., 2 h, 91%; (d) LiAlH4, THF, −78 °C, 2 h; (e) MsCl, DMAP, pyridine, r.t., 14 h, 80% over two steps;
(f) FeCl3·6H2O, CH2Cl2, 0 °C to r.t., 45 min.; (g) Ac2O, pyridine, DMAP, CH2Cl2, 1 h; (h) thymine, BSA, 1,2-DCA, reflux, 1 h; then TMSOTf, 0 to 90 °C,
4 h, 60% over three steps; (i) K2CO3, MeOH, 60 °C, 6 h, 83%; ( j) BOMCl, DBU, DMF, 0 °C, 1 h; (k) TBAF, THF, r.t., 9 h, 83% over two steps; (l) CrO3,
H2SO4, H2O, acetone, 0 °C to r.t., 2 h; (m) MeNH(OMe)·HCl, HATU, i-Pr2NEt, CH2Cl2, r.t., 8 h, 62% over two steps; (n) 1-propynylMgBr, THF, 0 °C to
r.t., 1 h, 80%; (o) H2, Pd/CaCO3/Pb, 1,10-phenanthronline, DMF, r.t., 2 d; (p) Grubbs II, CH2Cl2, r.t., 1 h, 80% over two steps; (q) NaBH4, CeCl3·7H2O,
MeOH, 0 °C, 30 min., 93%; (r) p-nitrobenzoyl chloride, DMAP, i-Pr2NEt, CH2Cl2, r.t., 2 h, 93%; (s) LevOH, EDC·HCl, DMAP, i-Pr2NEt, CH2Cl2, r.t., 10 h,
86%; (t) H2, Pd(OH)2/C, MeOH/EtOH/EtOAc, r.t., 2 d; then i-Pr2NEt, r.t., 30 min., 79%; (u) NC(CH2)2OP[N(i-Pr2)]2, N-methylimidazole, 1H-tetrazole,
DMF, r.t., 6 h, 85%.
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cessfully be added to 15, which led to the alkynyl ketone. Che- (Scheme 1). However, recent work in our laboratory32 on the
moselective semi-hydrogenation of the alkynyl ketone to synthesis of S-cEt analogue 4 highlighted the complementary
access cis-alkene 16 was successfully achieved using Lindlar’s advantages of using a nucleoside, rather than sugar-based
catalyst and 1,10-phenanthroline, with DMF as the solvent.38
precursor.41 Accordingly, beginning with nucleoside 21
The key cyclohexenone moiety was established through a (Scheme 2), the homoallylic alcohol was introduced through a
ring-closing metathesis reaction, catalyzed by Grubbs’ second- two-step sequence comprising oxidation with Dess–Martin per-
generation catalyst.39 Chemo- and stereoselective reduction of iodinane42 followed by a Brown allylation reaction43 with
the enone to the corresponding (R)-configured allylic alcohol (−)-Ipc2B(allyl).
(17) was accomplished under Luche’s conditions. The stereo-
With 22 in hand, the secondary alcohol was converted to its
chemistry and arrangement of the tricyclic core was confirmed corresponding mesylate and the cyclohexylidene acetal moiety
by X-ray crystallographic analysis, using a single crystal of was removed under acidic conditions to furnish diol 23.
para-nitrobenzoate ester derivative 18.40 Conversion of alcohol Although mono-protection of the C3′–OH was unselective, the
17 to the levulinate ester was necessary because of difficulties desired C3′-O-naphthyl ether was successfully installed using a
encountered during protection of the highly hindered two-step sequence that involved protection of the diol moiety
5′-hydroxyl group as the 4,4′-dimethoxytrityl ether. Subsequent as a diastereomeric mixture of naphthylidene acetals, followed
hydrogenolysis/hydrogenation of levulinate ester 17 afforded by selective deprotection of the C2′-hydroxy group in the pres-
19, the key TriNA 1 monomer that was phosphitylated (20) and ence of TiCl4/NaBH3CN44 to furnish alcohol 24. Base-promoted
incorporated into oligonucleotides for evaluation in thermal intramolecular displacement of the mesylate provided access
stability studies.
to the bicyclic framework of 25 in 45% yield over three steps. It
is worth noting that this protected bicyclic nucleoside is the
C6′-epimer of 13, which was a key intermediate in the syn-
thesis of TriNA 1.
At this point, the synthetic sequence converged with the
sequence used to prepare TriNA 1. Accordingly, the N3-imide
nitrogen atom of the nucleobase was protected with BOMCl
Synthesis of TriNA 2
As a consequence of a difference of only two stereocenters
between TriNA 1 and 2 at C5′ and C6′, preliminary studies indi-
cated that TriNA 2 could be synthesized using the same syn-
thetic precursor (1) and sequence established for TriNA 1
Scheme 2 Synthesis of TriNA 2. Reagents and conditions: (a) DMP, NaHCO3, CH2Cl2, r.t., 2 h; (b) (−)-Ipc2B(allyl), Et2O, r.t., overnight, then 1 M
NaOH, 30% H2O2, 67% over two steps; (c) MsCl, DMAP, pyridine, 0 °C to r.t., 12 h; (d) HCl/THF/MeOH, r.t., 6 h, 60% over two steps; (e) 2-naphthalde-
hyde dimethylacetal, CSA, 1,2-DCA, 70 °C, 6 h; (f) TiCl4, NaBH3CN, MeCN, 0 °C, 8 h; (g) K2CO3, MeOH, r.t., 12 h, 45% over three steps; (h) BOMCl,
DBU, DMF, 0 °C, 1.5 h; (i) TBAF, THF, r.t., 3 h, 75% over two steps; ( j) PDC, DMF, 40 °C, 12 h; (k) MeNH(OMe)·HCl, HATU, i-Pr2NEt, CH2Cl2, r.t., 8 h,
65% over two steps; (l) 1-propynylMgBr, THF, 0 °C, 1 h, 88%; (m) H2, Pd/CaCO3/Pb, 1,10-phenanthroline, DMF, r.t., 3 d; (n) Grubbs II, toluene, 70 °C,
6 h, 36% over two steps; (o) NaBH4 CeCl3·7H2O, MeOH, 0 °C, 30 min., 60%; (p) LevOH, EDC·HCl, DMAP, i-Pr2NEt, CH2Cl2, r.t., 1 h, 86%; (q) H2,
Pd(OH)2/C, MeOH/EtOH/EtOAc, r.t., 2 d, 45%; (r) NC(CH2)2OPCl[N(i-Pr2)], CH2Cl2, r.t., 25 min., 75%.
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and the TBDPS protective group removed with TBAF to furnish measured as additional controls. Relative to an unmodified
primary alcohol 26. Oxidation to the analogous aldehyde DNA control, incorporation of TriNA 1 produced an average
proved to be rather challenging, yielding several different side- increase in duplex thermal stability of +4.4 °C mod−1, while
products when carried out in the presence of Dess–Martin TriNA 2 was found to be even more stabilizing, producing an
periodinane, under Swern conditions, or with a mixture of average increase in Tm of +6.2 °C mod−1. By comparison, LNA,
TEMPO and PIDA. To avoid isolating the aldehyde, we opted to S-cEt, and R-cEt produced average Tm increases of +6 °C
directly oxidize the primary alcohol to a carboxylic acid, before mod−1. Consequently TriNA 1, which has the 5′-(R),6′-(S) con-
converting it to Weinreb amide 27. The oxidation was success- figuration at the stereogenic centers of constraint on the LNA
fully accomplished under mild conditions in the presence of scaffold was found to be less stabilizing compared with TriNA
PDC and DMF.45 Notably, this reaction occurs under essen- 2, which has the 5′-(S),6′-(R) configuration at the analogous
tially neutral conditions and was found to be significantly stereocenters. In some ways, this outcome mirrors and further
more practical than the acidic conditions used during the syn- confirms our previous observations with (R)-5′-Me LNA (2),
thesis of TriNA 1.
which was found to be significantly less stabilizing as com-
Addition of 1-propynylmagnesium bromide to Weinreb pared to the (S)-5′-Me analogue (3).33
amide 27 furnished propargyl ketone 28, which we hoped to
To further understand the origins of the differential duplex
selectively reduce and then cyclize under ring-closing meta- stabilizing properties of TriNA 1 and TriNA 2, as well as
thesis conditions to form the tricyclic core of TriNA 2. To that those for the analogous α-L-configured tricyclic nucleosides –
end, we proceeded with the partial hydrogenation of 28 using α-L-TriNA 1 and α-L-TriNA 2 (34 and 35, Fig. 4) – we created a
Lindlar’s catalyst, then cyclized the resultant α,β-unsaturated structural model in which TriNA analogues 6 and 7 were over-
ketone to enone 29 using Grubbs’ II generation catalyst. Sur- laid onto the crystal structure of a S-cEt-modified A-form DNA
prisingly, reduction of the enone under Luche conditions duplex (Fig. 5).31 Both TriNA analogues were well accommo-
favoured formation of the desired (S)-alcohol (19) over the dated within the duplex, with the six-membered carbocyclic
corresponding epimer, with a ratio of 2.1 : 1. Formation of the rings projecting into the minor groove for TriNA
1
levulinate ester and subsequent hydrogenolysis/hydrogenation (cyan-coloured) and towards the edge of the major groove for
provided levulinate ester 31, which crystallized to deliver TriNA 2 (magenta-coloured). Visual analysis of the structures
definitive evidence for the structure of TriNA 2 through X-ray suggested that the (R)-5′-methylene group of the carbocyclic
crystallographic analysis.40 Phosphitylation of 31 provided ring in TriNA 1 may experience a tight contact (2.7 Å) with one
access to phosphoramidite 32, which was incorporated into of the non-bridging oxygen atoms of the 5′-phosphodiester
oligonucleotides for evaluation in duplex thermal stability linkage. In contrast, the analogous distance for TriNA 2 is
studies.
3.2 Å, and the tightest contact (2.9 Å) is likely between the (S)-
5′-methylene group and the 3′-oxygen atom of the 3′-adjacent
nucleotide. This suggested that the incorporation of TriNA 1
into the duplex might produce subtle changes about torsion
angles α and/or β – or enhance conformational mobility of the
phosphodiester backbone – in order to relieve this tight
spacing, and consequently lead to a smaller enhancement in
duplex stability. However, locking γ in the canonical +sc range
in TriNA 1 does provide a greater enhancement in duplex stabi-
lity as compared to (R)-5′-Me LNA analogue 2, which is not
stabilizing relative to DNA.33
Duplex thermal stability measurements
The duplex stabilizing properties of TriNA 1 and TriNA 2 versus
complementary RNA were measured using
a previously
described oligonucleotide sequence.46 Modified nucleotides
were incorporated into DNA oligonucleotides at four different
locations to provide a position and sequence context for the
Tm studies (Table 1). Moreover, the duplex–stabilizing properties
of LNA, S-cEt, and R-cEt modified oligonucleotides were
To determine the generality of these observations, we also
inspected the corresponding structural models of α-L-TriNA 124
(magenta-coloured) and α-L-TriNA 225 (cyan-coloured) overlaid
onto the NMR structures of an α-L-LNA-modified DNA–RNA
duplex.47 As was observed for TriNA 1 and 2, the α-L-configured
tricyclic nucleosides also displayed differential increases in
duplex stability, with α-L-TriNA 1 (ΔTm + 8 °C mod−1) being
more stabilizing than α-L-TriNA 2 (ΔTm + 4 °C mod−1). Inspec-
tion of these structural models showed that the 3′-methylene
group of the six-membered carbocycle in α-L-TriNA 2 can
experience a tight contact (2.5 Å) with one of the non-bridging
oxygen atom of the 3′-phosphodiester linkage. In contrast, the
tightest contact in α-L-TriNA 1 (3.4 Å) is likely between the (R)-
5′-methylene group and the non-bridging atom of the 5′-phos-
phodiester linkage. Thus, it appears that in the β-D-TriNA and
the α-L-TriNA series, the analogue that produces a smaller
Table 1 Duplex stabilizing properties of TriNA 1 and TriNA 2
ΔTm/modb (°C)
Sequence (5′ to 3′)a
LNA
S-cEt
R-cEt
TriNA 1
TriNA 2
GGA GTTCTCGA
GGATG TCTCGA
GGATGT CTCGA
GGATGTTC CGA
Average ΔTm
6.3
5.6
7.0
5.0
6.0
6.3
5.1
6.9
4.7
5.8
6.6
5.6
7.0
4.6
6.0
5.3
4.0
4.5
3.6
4.4
6.8
6.1
6.4
5.5
6.2
a Red boldface letters indicate site of modification, base code T =
thymine, U = uracil, C = cytosine, A = adenine and G = guanine b Tm
values were measured in 10 mM sodium phosphate buffer (pH 7.2)
containing 100 mM NaCl and 0.1 mM EDTA. Sequence of RNA
complement is 5′-r(UCGAGAACAUCC)-3′. Tm of unmodified DNA
control = 49.3 0.24 °C (n = 5).
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Conclusions
In conclusion we have reported the design and synthesis of
dual-constrained tricyclic nucleosides of LNA. An analysis of
structural models of TriNA 1 and TriNA 2 duplexes provides
insight into the possible origins of the differential duplex
stabilizing properties of these highly constrained nucleic acid
analogues when they are incorporated into oligonucleotide
sequences. Recent studies have shown that oligonucleotides
modified with tricyclo-DNA (tc-DNA),48 a conformationally
restricted analogue with enhanced hydrophobicity, display excel-
lent splice correction activity in animal disease models.49 The
improved activity was attributed to enhanced cellular uptake
properties that stem from their propensity to spontaneously
self-assemble into nanoparticles in aqueous solutions.50 TriNA
1 and 2 also represent conformationally restricted nucleic acid
analogues with enhanced hydrophobicity in and along the
major and minor grooves, which have the potential to improve
the binding affinity, nuclease stability, cell-uptake, and other
desirable properties of therapeutic antisense oligonucleotides.
Fig. 4 Comparison of the duplex stabilizing properties of dual con-
strained TriNA analogues derived from α-L-LNA and β-D-LNA.
Acknowledgements
R.D.G. is grateful to NSERC (CGS D) for a postgraduate scholar-
ship. Bradley L. Merner is thanked for initial forays toward the
syntheses. We thank Michel Simard and Benoît Deschênes–
Simard for X-ray crystallographic analyses.
Notes and references
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Fig. 5 Structural models of tricyclic nucleosides overlaid on oligo-
nucleotide duplexes containing the corresponding antecedent bicyclic
modifications (S-cEt or α-L-LNA). For clarity, the second strand of the
duplex has been removed and only one region of the oligonucleotide is
shown. In the case of TriNA 1 the (R)-5’-methylene group of the carbo-
cyclic ring may experience a tight contact (2.7 Å) with one of the non-
bridging oxygen atoms of the 5’-phosphodiester linkage. For α-L-TriNA
2, the 3’-methylene group of the six-membered carbocycle may experi-
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