Alpha-L-RNA (alpha-L-ribo configured RNA): synthesis and RNA-selective hybridization of alpha-L-RNA/alpha-L-LNA chimera.

Article abstract of DOI:10.1016/S0960-894X(01)00807-1

Synthesis of the novel alpha-L-ribofuranosyl phosphoramidite derivative was accomplished via the alpha-L-ribofuranosyl thymine nucleoside. Amidite was used in automated syntheses of chimeric oligonucleotides composed of mixtures of the novel alpha-L-RNA nucleotide monomer ((alphaL)T, alpha-L-ribo configured RNA), and DNA, LNA (T(L), locked nucleic acid) or alpha-L-LNA ((alphaL)T(L), alpha-L-ribo configured locked nucleic acid) nucleotide monomers. For alpha-L-RNA/DNA and alpha-L-RNA/alpha-L-LNA chimeras, RNA-selective hybridization was obtained, for alpha-L-RNA/alpha-L-LNA chimera we found increased binding affinity compared to the corresponding DNA:RNA reference duplex. In addition, alpha-L-RNA/alpha-L-LNA chimera displayed significant stabilization towards 3'-exonucleolytic degradation. These results indicate that alpha-L-RNA/alpha-L-LNA chimeras deserve further evaluation as antisense molecules.

Full text of DOI:10.1016/S0960-894X(01)00807-1

Bioorganic & Medicinal Chemistry Letters 12 (2002) 593–596
ꢀ-L-RNA (ꢀ-L-ribo Configured RNA): Synthesis and
RNA-Selective Hybridization of ꢀ-L-RNA/ꢀ-L-LNA Chimera
Lise Keinicke,a Mads D. Sørensena and Jesper Wengelb,*
aDepartment of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen, Denmark
bNucleic AcidCenter, y Department of Chemistry, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark
Received 18 October 2001; revised 22 November 2001; accepted 26 November 2001
Abstract—Synthesis of the novel a-l-ribofuranosyl phosphoramidite derivative 7 was accomplished via the a-l-ribofuranosyl
thymine nucleoside 4. Amidite 7 was used in automated syntheses of chimeric oligonucleotides composed of mixtures of the novel
a-l-RNA nucleotide monomer (ꢀLT, a-l-ribo configured RNA), and DNA, LNA (TL, locked nucleic acid) or a-l-LNA (ꢀLTL, a-l-
ribo configured locked nucleic acid) nucleotide monomers. For a-l-RNA/DNA and a-l-RNA/a-l-LNA chimeras, RNA-selective
hybridization was obtained, for a-l-RNA/a-l-LNA chimera we found increased binding affinity compared to the corresponding
DNA:RNA reference duplex. In addition, a-l-RNA/a-l-LNA chimera displayed significant stabilization towards 30-exonucleolytic
degradation. These results indicate that a-l-RNA/a-l-LNA chimeras deserve further evaluation as antisense molecules. # 2002
Elsevier Science Ltd. All rights reserved.
In the antisense therapeutic strategy, chemically mod-
ified oligonucleotides are administered in order to
specifically inhibit the translation of disease-related
mRNA sequences by duplex formation. Essential
requirements for therapeutic efficiency of antisense oli-
gonucleotides include good aqueous solubility, resis-
tance toward enzymatic degradation, and high binding
affinity and specificity towards the RNA target strand.
Accordingly, synthesis of a large number of chemically
modified oligonucleotides has been accomplished.1 The
unprecedented thermal stability of duplexes involving
LNA (locked nucleic acid, b-d-ribo isomer, Fig. 1)1b,2,3
has prompted us to study the properties of various LNA
stereoisomers, including a-l-LNA (a-l-ribo configured
LNA, Fig. 1).1b,3ꢀ5 Very efficient recognition of single-
stranded DNA and RNA has been demonstrated not
only for LNA but also for a-l-LNA.2ꢀ5
fully modified 20-O-alkyl-RNA and fully modified LNA
to RNase H independent antisense applications, for
example, as steric blockers, as agents interfering with
double-stranded RNA targets, or as end-gaps in gap-
mer9 antisense oligonucleotides. However, high binding
affinity and efficient mis-match discrimination are of
utmost importance for any antisense application, as is
Although significantly less stabilizing than LNA or a-l-
LNA, 20-O-alkyl-RNAs6ꢀ8 are presently among the
preferred nucleotide modifications in antisense oligo-
nucleotides. The fact that 20-O-alkyl-RNA/RNA
duplexes9 and LNA/RNA duplexes2f are not substrates
for the RNA-cleaving enzyme RNase H limits the use of
Figure 1. Structures of LNA and a-l-LNA nucleotide monomers and
sketches of their locked N-type (C30-endo, 3E) furanose conforma-
tions.5 Also shown are structures of RNA and a-l-RNA nucleotide
monomers. In Table 1, the notations ꢀLT (a-l-ribo configured RNA),
TL (LNA) and ꢀLTL (a-l-LNA) are used for the monomers shown
above (base=thymin-1-yl).
*Corresponding author. Tel.: +45-6650-2510; fax: +45-6615-8780;
e-mail: jwe@chem.sdu.dk
yA research center funded by The Danish National Research Foun-
dation for studies on nucleic acid chemical biology.
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00807-1

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L. Keinicke et al. / Bioorg. Med. Chem. Lett. 12 (2002) 593–596
TBDMSCl in pyridine in the presence of imidazole
afforded a mixture of the 20-O- and 30-O-silylated pro-
ducts. Separation of these by column chromatography
(2.0–6.0% acetone/0.5% pyridine/97.5–93.5% dichloro-
methane; v/v/v) furnished nucleoside 6 in 45% yield.
Nucleoside 6 was dissolved in anhydrous dichloro-
methane in the presence of N,N-diisopropylethylamine
and
chloridite to give phosphoramidite 7 in 73% yield
2-cyanoethyl
N,N-diisopropylphophoramido-
(Scheme 1).
The oligomers (Table 1) used in this study were synthe-
sized on an automated DNA synthesizer using the
phosphoramidite approach.12 The a-l-ribofuranosyl
phosphoramidite building block 7 was used for the
synthesis of the a-l-RNA oligomers 11, 12 and 16–20.
The stepwise coupling yield for amidite 7 was approx-
imately 98% (20 min coupling time; 1H-tetrazole as
activator) using procedures described previously.4a
After detritylation, cleavage from the solid support and
deacylation were effected using 40% aqueous methyl-
amine (10 min, 55 ꢁC). After cooling to ꢀ18 ꢁC, the solid
support was removed (centrifugation), washed [2 ꢂ 0.25
cm3; EtOH/CH3CN/H2O (3:1:1, v/v/v)], and the com-
bined liquid phase evaporated to dryness under reduced
pressure. The residue was desilylated using a method
described previously13 during 20 h at 55 ꢁC. Desilylation
of the oligomers was incomplete when using milder
desilylation conditions as revealed by MALDI-MS
analysis. Standard conditions of the synthesizer were
used for incorporation of DNA monomers whereas the
incorporation of LNA or a-l-LNA monomers followed
procedures described previously.2a,2b,4a Analysis by
capillary gel electrophoresis verified the purity of the
novel a-l-RNA oligomers 11, 12 and 16–20 as being
>90%, whereas MALDI-MS analysis confirmed their
compositions.14 The reference DNA oligomers 8 and
13,2a,4b the LNA oligomers 9 and 14,2a,2b and the a-l-
LNA oligomers 10 and 154b,4c have been prepared and
studied previously.
Scheme 1. Synthesis of the a-l-ribofuranosyl thymine nucleoside 4 and
the phosphoramidite derivative 7: (i) MsCl, pyridine (84%); (ii) aq
NaOH, ethanol; (iii) aq NaOH, ethanol, 65 ꢁC (59%, two steps); (iv)
DMTCl, pyridine (93%); (v) TBDMSCl, imidazole, pyridine (45%); (vi)
NC(CH2)2OP(Cl)N(i-Pr)2, EtN(i-Pr)2, CH2Cl2 (73%). [Si]=1,1,3,3-
tetraisopropyldisiloxane-1,3-diyl. DMT = 4,40-dimethoxytrityl.
the opportunity to fine-tune these parameters, for exam-
ple, by combining nucleotide monomers of different che-
mical structures. This report is focused on the potential of
modulating RNA-recognition by use of the novel a-l-
RNA (a-l-ribo configured RNA, Fig. 1) with special focus
on a-l-RNA/DNA and a-l-RNA/a-l-LNA chimeras.
Starting from l-arabinose, the known nucleoside 110
was synthesized in five steps. Mesylation of compound 1
in pyridine gave compound 2 in an acceptable yield
(84%). Subsequent treatment with aq NaOH in ethanol
afforded the ribo-configured nucleoside 3, presumably
via an O20,C2-anhydronucleoside intermediate. Sub-
sequent heating of the reaction mixture for 18 h effected
removal of the 1,1,3,3-tetraisopropyldisiloxane-1,3-diyl
protecting group and 1-(a-l-ribofuranosyl)thymine (4)
was isolated in 59% yield (from 2). The NMR data of 4
were identical to those of its enantiomer, 1-(a-d-ribo-
furanosyl)thymine.11 Nucleoside triol 4 was selectively
DMT-protected in 93% yield at the primary hydroxy
group by reaction with 1.5 equiv DMTCl in pyridine to
give derivative 5, which after reaction with 3.0 equiv
Table 1. Melting temperatures (Tm values) towards complementary single-stranded DNA and RNA targets obtained as the maximum of the first
derivative of the melting curve (A260 vs temperature) in medium salt buffer (10 mM sodium phosphate, 100 mM sodium chloride, 0.1 mM EDTA,
pH 7.0) using 1.5 mM concentrations of the two complementsa
Entry
Description of oligomers
Sequence of oligomers
DNA complement
Tm (ꢁC)
RNA complement
Tm (ꢁC)
1
2LNA/DNA
3
4
5
DNA reference
8: GTGATATGC
9: GTLGATLATLGC
30
44
37
26
28
50
45
28
12
a-l-LNA/DNA
ꢀ-L-RNA/DNA
ꢀ-L-RNA/DNA
10: G(ꢀLTL)GA(ꢀLTL)A(ꢀLTL)GC
11: GTGA(ꢀLT)ATGC
12: G(ꢀLT)GA(ꢀLT)A(ꢀLT)GC
<5
6
7
8
9
10
11
12
13
DNA reference
LNA
a-l-LNA
ꢀ-L-RNA
ꢀ-L-RNA/LNA
ꢀ-L-RNA/LNA
ꢀ-L-RNA/ꢀ-L-LNA
ꢀ-L-RNA/ꢀ-L-LNA
13: T10
14: (TL)9T
2
80
63
<5
<5
<5
<5
<5
0
71
66
<5
2
<5
2
15: (ꢀLTL)9T
16: (ꢀLT)9T
17: (ꢀLT)4(TL)4(ꢀLT)T
18: [(ꢀLT)(TL)]4(ꢀLT)T
19: (ꢀLT)4(ꢀLTL)4(ꢀLT)T
20: [(ꢀLT)(ꢀLTL)]4(ꢀLT)T
2
aAll oligomers are depicted with the 50-end positioned to the left. A=adenine monomer, C=cytosine monomer, G=guanine monomer, T=thymine
monomer; A, C, G and T are DNA monomers, that is, 20-deoxy-b-D-ribofuranosyl derivatives. See Fig. 1 for structures of the TL, ꢀLTL, and ꢀLT
monomers (Base=thymin-1-yl).

L. Keinicke et al. / Bioorg. Med. Chem. Lett. 12 (2002) 593–596
595
containing four consecutive a-l-LNA monomers and
alternating a-l-RNA and a-l-LNA monomers, respec-
tively, display very similar binding properties. Thus, Tm
values of 29 ꢁC and 27 ꢁC for 19 and 20, respectively,
were observed toward the RNA target but no Tm values
toward the DNA target. Of utmost importance for
antisense applications are not only binding affinity but
also base-pairing selectivity. It is therefore encouraging
that oligomers 19 and 20 both display satisfactory dis-
criminatory behavior towards an RNA target with one
mis-matched base [r(A7CA6) target: Tm <5 ꢁC]. How-
ever, more experiments are needed in order to fully
understand the binding interactions between RNA and
a-l-RNA/a-l-LNA chimera.
Figure 2. Time course of snake venom phosphodiesterase digestion of
the DNA reference 13 and the a-l-RNA/a-l-LNA chimeras 19 and
20. A solution of the oligomers (ꢃ0.2OD) in 2mL of a buffer (0.1 M
Tris–HCl; pH=8.6; 0.1 M NaCl; 14 mM MgCl2) was digested with 1.2
U snake venom phosphodiesterase [30 mL of a solution in the follow-
ing buffer: 5 mM Tris–HCl; pH=7.5; 50% glycerol (v/v)] at 25 ꢁC.
The stability of a-l-RNA/a-l-LNA chimera toward 30-
exonucleolytic degradation in vitro was evaluated using
snake venom phosphodiesterase (SVPDE).16 During
SVPDE digestion of unmodified oligonucleotides, for
example the DNA reference 13 (see Fig. 2), the absor-
bance at 260 nm rapidly increases due to conversion
into the nucleoside constituents.16 In contrast, the a-l-
RNA/a-l-LNA chimeras 19 and 20 are both very sig-
nificantly stabilized toward degradation by SVPDE
(Fig. 2; no significant hyperchromicity observed). These
qualitative experiments indicate that a-l-RNA/a-l-
LNA chimera, like a-l-DNA,17 a-l-LNA,18 and DNA/
a-l-LNA chimera,18 are significantly protected toward
30-exonucleolytic degradation.
Results from hybridization experiments (Tm values)
toward single-stranded DNA and RNA complements
are depicted in Table 1. In entries 1–5, different variations
of a 9-mer mixed-base sequence are studied. Introduction
of three thymine LNA monomers (9)2a,2b or three a-l-
LNA monomers (10)4c significantly improves the ther-
mal stability towards both DNA and RNA when com-
pared to the results obtained with the corresponding
DNA reference 8. Both 9 and 10 display a weak RNA
selectivity as witnessed by the slightly lower thermal
stabilities of the duplexes involving the DNA comple-
ment. Results for the two a-l-RNA/DNA chimeras 11
and 12 are despicted in entries 4 and 5. Incorporation of
one a-l-RNA nucleotide leads to unchanged (toward
RNA) or reduced (toward DNA, ÁTm=ꢀ4 ꢁC) thermal
stability when compared to the DNA reference 8. When
three a-l-RNA monomers are incorporated (oligomer
12), hybridization towards both DNA and RNA is
adversely influenced, most so, however, toward DNA.
The results reported herein suggest that further studies
should be performed in order to evaluate the full
potential of a-l-RNA/a-l-LNA chimeras as antisense
molecules. If the pronounced RNA selectivity obtained
for 20 (composed of alternating a-l-RNA and a-l-LNA
monomers) turns out to be a general feature of a-l-RNA/
a-l-LNA chimeras, one may envision reduced toxicity
and improved specificity compared to the current anti-
sense molecules which are known to be able also to
hybridize toward DNA targets. A similarily pronounced
RNA selectivity has been reported for a few other
oligonucleotide analogues, for example, b-l-DNA,19
arabinonucleic acids,20 20-O,30-C-linked bicyclic oligo-
nucleotides,21 and a-d-LNA.22 However, their usefulness
as antisense molecules is hampered either by compara-
tively low binding affinity toward RNA19,20 or the
necessity of using fully modified oligomers in order to
obtain efficient RNA binding.21,22 It is therefore impor-
tant that the a-l-RNA/DNA chimera 11 retains the
ability to hybridize to RNA and that the a-l-RNA/a-l-
LNA chimera 20, with alternating a-l-RNA and a-l-
LNA monomers, displays increased binding affinity
towards RNA (ÁTm value=+8 ꢁC compared to the
DNA reference 13). It is striking that both DNA/a-l-
LNA and a-l-RNA/a-l-LNA chimeras display increased
binding affinity toward RNA which stresses the flexibility
and power of these LNA-type chimeric oligonucleotides.
Various combinations of the different monomers in a
homothymine 10-mer context are evaluated in the sec-
ond series of oligomers (Table 1, entries 6–13). As
reported earlier, the (almost) fully modified LNA and a-
l-LNA oligomers 14 and 15, respectively, indeed dis-
play very efficient hybridization towards both DNA and
RNA.2a,3b,3c In contrast, a melting point was not
obtained for the corresponding a-l-RNA 16 neither
toward DNA nor RNA. Exchange of four a-l-RNA
monomers of 16 with LNA monomers gave the a-l-
RNA/LNA chimeras 17 and 18. With four consecutive
LNA monomers (17), no hybridization toward the
DNA complement was detected. However, a Tm value
of 27 ꢁC toward the RNA complement was observed.
This strong RNA selectivity is thought to be caused by
hybridization of the LNA segment (-TL -) with the
4
RNA but not with the DNA complement, as supported
by hybridization results reported earlier for a TL T oli-
5
gomer.2a A comparable RNA selectivity has not been
observed neither for longer homothymine sequences
(e.g., 14) nor for partly or fully modified LNAs with
mixed base compositions.2b,15 The a-l-RNA/LNA chi-
mera 18 with alternating a-l-RNA and LNA monomers
hybridizes neither with the DNA nor the RNA comple-
ment. The two a-l-RNA/a-l-LNA chimeras 19 and 20
Acknowledgements
The Danish Natural Science Research Council, The
Danish Technical Research Council, The Danish

596
L. Keinicke et al. / Bioorg. Med. Chem. Lett. 12 (2002) 593–596
National Research Foundation, and Exiqon A/S are
acknowledged for financial support. Ms. Britta M.
Dahl, University of Copenhagen, is thanked for oligo-
nucleotide synthesis. Dr. Michael Meldgaard, Exiqon
A/S, is thanked for MALDI-MS analysis.
tion. A similar furanose conformation, although more flexible,
is likely to be preferred for an a-l-RNA monomer as indicated
by calculations, see: Latha, Y. S.; Yathindra, N. Biopolymers
1992, 32, 249.
6. Inoue, H.; Hayase, Y.; Iwai, A.; Miura, K.; Ohtsuka, E.
Nucleic Acids Res. 1987, 15, 6131.
7. Lesnik, E. A.; Guinosso, C. J.; Kawasaki, A. M.; Sasmor,
H.; Zounes, M.; Cummins, L. L.; Ecker, D. J.; Cook, P. D.;
Freier, S. M. Biochemistry 1993, 32, 7832.
References and Notes
8. Martin, P. Helv. Chim. Acta 1995, 78, 486.
9. Manoharan, M. Biochim. Biophys. Acta 1999, 1489, 117.
10. Czernecki, S.; le Diguarher, T. Synthesis 1991, 683.
11. Sawai, H.; Nakamura, A.; Hayashi, H.; Shinozuka, K.
Nucleosides Nucleotides 1994, 13, 1647.
12. Caruthers, M. H. Acc. Chem. Res. 1991, 24, 278.
13. Wincott, F.; DiRenzo, A.; Schaffer, C.; Grimm, S.; Tracz,
D.; Workman, C.; Sweedler, D.; Gonzalez, C.; Scaringe, S.;
Usman, N. Nucleic Acids Res. 1995, 23, 2677.
14. MALDI-MS ([MꢀH]ꢀ measured/[MꢀH]ꢀ calc): 11 (2770/
2769), 12 (2804/2801), 16 (3121/3121), 19 (3170/3169), 20
(3172/3169).
15. Oerum, H.; Jacobsen, M. H.; Koch, T.; Vuust, J.; Borre,
M. B. Clin. Chem. 1999, 45, 1898.
16. (a) Rosemeyer, H.; Seela, F. Helv. Chim. Acta 1991, 74,
748. (b) Svendsen, M. L.; Wengel, J.; Dahl, O.; Kirpekar, F.;
Roepstorff, P. Tetrahedron 1993, 49, 11341.
17. Asseline, U.; Hau, J.-F.; Czernecki, S.; Le Diguarher, T.;
Perlat, M.-C.; Valery, J.-M.; Thuong, N. T. Nucleic Acids Res.
1991, 19, 4067.
18. Sørensen, M. D.; Kværnø, L.; Bryld, T.; Hakansson, A.
E.; Verbeure, B.; Gaubert, G.; Herdewijn, P.; Wengel, J. J.
Am. Chem. Soc. in press.
19. (a) Fujimori, S.; Shudo, K. J. Am. Chem. Soc. 1990, 112,
7436. (b) Ashley, G. W. J. Am. Chem. Soc. 1992, 114, 9731.
20. (a) Damha, M. J.; Wilds, C. J.; Noronha, A.; Brukner, I.;
Borkow, G.; Arion, D.; Parniak, M. A. J. Am. Chem. Soc.
1998, 120, 12976. (b) Noronha, A. M.; Wilds, C. J.; Lok, C.-
N.; Viazovkina, K.; Arion, D.; Parniak, M. A.; Damha, M. J.
Biochemistry 2000, 39, 7050. (c) Wilds, C. J.; Damha, M. J.
Nucleic Acids Res. 2000, 28, 3625.
21. (a) Nielsen, P.; Pfundheller, H. M.; Olsen, C. E.; Wengel,
J. J. Chem. Soc., Perkin Trans. 1 1997, 3423. (b) Raunkjær,
M.; Olsen, C. E.; Wengel, J. J. Chem. Soc., Perkin Trans. 1
1999, 2543.
1. (a) Freier, S. M.; Altmann, K.-H. Nucleic Acids Res. 1997,
25, 4429. (b) Wengel, J. Acc. Chem. Res. 1999, 32, 301. (c)
Herdewijn, P. Biochim. Biophys. Acta 1999, 1489, 167. (d)
Uhlmann, E. Curr. Opin. Drug Discov. Dev. 2000, 3, 203. (e)
Kværnø, L.; Wengel, J. Chem. Commun. 2001, 1419.
2. (a) Singh, S. K.; Nielsen, P.; Koshkin, A. A.; Wengel, J.
Chem. Commun. 1998, 455. (b) Koshkin, A. A.; Singh, S. K.;
Nielsen, P.; Rajwanshi, V. K.; Kumar, R.; Meldgaard, M.;
Olsen, C. E.; Wengel, J. Tetrahedron 1998, 54, 3607. (c) Obika,
S.; Nanbu, D.; Hari, Y.; Andoh, J.; Morio, K.; Doi, T.; Ima-
nishi, T. Tetrahedron Lett. 1998, 39, 5401. (d) Koshkin, A. A.;
Nielsen, P.; Meldgaard, M.; Rajwanshi, V. K.; Singh, S. K.;
Wengel, J. J. Am. Chem. Soc. 1998, 120, 13252. (e) Singh,
S. K.; Wengel, J. Chem. Commun. 1998, 1247. (f) Wahlestedt,
C.; Salmi, P.; Good, L.; Kela, J.; Johnsson, T.; Hokfelt, T.;
Broberger, C.; Porreca, F.; Lai, J.; Ren, K.; Ossipov, M.;
Koshkin, A.; Oerum, H.; Jakobsen, N.; Skouv, J.; Jacobsen,
M. H.; Wengel, J. Proc. Natl. Acad. Sci. U.S.A. 2000, 97,
5633.
3. We have defined LNA as an oligonucleotide containing one
or more 20-O,40-C-methylene-b-d-ribofuranosyl nucleotide
monomer(s) and a-l-LNA as an oligonucleotide containing
one or more 20-O,40-C-methylene-a-l-ribofuranosyl mono-
mer(s).
4. (a) Rajwanshi, V. K.; Hakansson, A. E.; Dahl, B. M.;
Wengel, J. Chem. Commun. 1999, 1395. (b) Rajwanshi,
V. K.; Hakansson, A. E.; Kumar, R.; Wengel, J. Chem. Com-
mun. 1999, 2073. (c) Rajwanshi, V. K.; Hakansson, A. E.;
Sørensen, M. D.; Pitsch, S.; Singh, S. K.; Kumar, R.; Nielsen,
P.; Wengel, J. Angew. Chem. Int. Ed. 2000, 39, 1656. (d)
Hakansson, A. E.; Wengel, J. Bioorg. Med. Chem. Lett. 2001,
11, 935.
5. Despite the apparent differences in the furanose conforma-
tions between an LNA monomer and an a-l-LNA monomer
(Fig. 1), the furanose conformation of an a-l-LNA monomer
22. (a) Nielsen, P.; Dalskov, J. K. Chem. Commun. 2000,
1179. (b) Christensen, N. K.; Dahl, B. M.; Nielsen, P. Bioorg.
Med. Chem. Lett. 2001, 11, 1765.
is also of the N-type (C30-endo, E) because of its l-configura-
3