Synthesis of (3R,6R)- and (3S,6R)-piperidinone PNA

Article abstract of DOI:10.1039/b103901f

Two new conformationally restricted piperidinone PNA adenine monomers 12 and 13 have been synthesised using a stereoselective synthesis strategy analogous to a previously published strategy for pyrrolidinone analogues. In contrast to the pyrrolidinone case, epimerisation occurred during the final hydrolysis step. However, the diastereomeric mixture could be separated by RP-HPLC to give small amounts of pure 12 and 13. These were built into a PNA dodecamer (once in a central position), and the thermal stability (T_m) of the modified oligomers hybridised to complementary DNA, RNA and PNA were measured. PNA modified with either 12 or 13 resulted in a decrease of the T_m compared to unmodified PNA and to pyrrolidinone modified PNA. Thus, any preorganisation for duplex formation of PNA with a six-membered piperidinone ring seems to be inferior to preorganisation with a five-membered ring in the pyrrolidinone PNA analogues studied earlier.

Full text of DOI:10.1039/b103901f

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Synthesis of (3R,6R)- and (3S,6R)-piperidinone PNA
Ask Püschl,a,b Thomas Boesen,b Tullia Tedeschi,a,b Otto Dahlb and Peter E. Nielsen*a
a Center for Biomolecular Recognition, Department for Biochemistry and Genetics,
Biochemistry Laboratory B, The Panum Institute, Blegdamsvej 3C, DK-2200, Copenhagen,
Denmark
1
b Department of Chemistry, The H. C. Ørsted Institute, University of Copenhagen,
Universitetsparken 5, DK-2100, Copenhagen, Denmark
Received (in Cambridge, UK) 1st May 2001, Accepted 17th August 2001
First published as an Advance Article on the web 10th October 2001
Two new conformationally restricted piperidinone PNA adenine monomers 12 and 13 have been synthesised using a
stereoselective synthesis strategy analogous to a previously published strategy for pyrrolidinone analogues. In con-
trast to the pyrrolidinone case, epimerisation occurred during the final hydrolysis step. However, the diastereomeric
mixture could be separated by RP-HPLC to give small amounts of pure 12 and 13. These were built into a PNA
dodecamer (once in a central position), and the thermal stability (T m) of the modified oligomers hybridised to
complementary DNA, RNA and PNA were measured. PNA modified with either 12 or 13 resulted in a decrease
of the T m compared to unmodified PNA and to pyrrolidinone modified PNA. Thus, any preorganisation for
duplex formation of PNA with a six-membered piperidinone ring seems to be inferior to preorganisation with a
five-membered ring in the pyrrolidinone PNA analogues studied earlier.
duplex formation is accompanied by a decrease in entropy.2e
This entropy loss might be reduced by using a more rigid PNA
Introduction
Peptide nucleic acid (PNA) is an acyclic, pseudopeptide mimic
of natural nucleic acids.1 Only the nucleobases are retained,
linked together by an achiral, uncharged amide backbone
(Fig. 1). PNA is an excellent structural mimic of DNA and
RNA; it binds tightly and with high sequence specificity to com-
plementary oligonucleotides.2a–g PNA–DNA or PNA–RNA
analogue as has been attempted by using a variety of confor-
mationally constrained chiral backbones.3a–f We have recently
described pyrrolidinone PNA (pyr-PNA, Fig. 1) in which atoms
of PNA are constrained by the introduction of a methylene
bridge (see Fig. 1).4 This bridge prevents rotation around the
C–N bond of the amide unit connected to the base residue and
Fig. 1
DOI: 10.1039/b103901f
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This journal is © The Royal Society of Chemistry 2001
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pre-organises PNA in the rotameric conformation prevailing
in PNA–DNA, PNA–RNA and PNA–PNA duplexes as well
as PNA2–DNA triplexes.5a–d The results from the pyr-PNA
study, which involved only adenine monomers, showed that
PNA oligomers containing the (3S,5R) isomer had the highest
affinity towards RNA. However, the affinity was slightly
decreased (∆T m per modification Ϫ1 to Ϫ3 ЊC) compared to
unmodified PNA.4 This decrease in affinity could well be the
result of unfavourable constraints of the PNA in terms of
duplex formation caused by the introduction of the five-
membered pyrrolidinone ring. Indeed, model building seemed
to suggest that a six-membered piperidinone PNA monomer
(pip-PNA, Fig. 1) could better adjust to a duplex structure.
We now present our results on the syntheses and properties of
two such monomers: [(3R,6R)-pip-PNA and (3S,6R)-pip-PNA
(Fig. 1, B = adenin-9-yl)].
(3S)-2 (trans) isomer by analogy with the outcome for the
corresponding 5-membered analogue4 and the known pre-
dominance of formation of the trans-isomer (trans : cis ratio
14 : 1) when the enolate of 1 is alkylated with Me3SnCH2I.7
Protection of the hydroxy group as its benzyloxymethyl (Bom)
ether was followed by removal of the Boc group to give 3. The
lactam nitrogen was alkylated and the silyl protecting group
was removed with Et3Nؒ3HF in THF to produce reasonably
pure 5. This hydroxy ester was immediately mesylated to 6 as
it is unstable due to lactone formation. Conversion into the
azide 7 was followed by hydrogenation to the amine which was
Boc protected in a one-pot procedure using 10% Pd/C as the
catalyst. The Bom protecting group was stable to this treatment
but was removed by hydrogenation using Pearlman’s catalyst to
give 8. Under Mitsunobu conditions, the configuration at C-3
was inverted to yield the pure diastereomer 9. The content of 8
in 9 was less than 1% according to 13C NMR where the region
of the strong Boc CH3 signal of 9 (δC 27.9) was devoid of the
corresponding signal of 8 (δC 27.5). Apparently, the minor
diastereomer carried over from 2 had been removed by one or
several of the column chromatography steps employed in the
transformation of 5 to 9. The hydroxy groups in 8 and 9 were
substituted with adenine under Mitsunobu conditions. 13C
NMR of these intermediates strongly indicated that the correct
N9 isomers had formed. The exocyclic amine in the nucleobases
were Z-protected using Rapoport’s reagent8 to give 10 and 11,
respectively. According to RP-HPLC, 10 was pure, but some
Results and discussion
Synthesis of the (3S,6R)- and (3R,6R)-monomer esters 10 and 11
The adenine monomers were prepared as outlined in Scheme 1.
The starting material, (6R)-6-(tert-butyldiphenylsilyloxy-
methyl)piperidin-2-one,6 was Boc protected at nitrogen to give
1. Diastereoselective hydroxylation of 1 was carried out as
described for the 5-membered analogue,4 giving a 95 : 5 mixture
of diastereoisomers. The major isomer was assigned as the
Scheme 1 Synthesis of (3S,6R)-12 and the (3R,6R)-13 pip-PNA A monomers. a) Boc2O, DMAP, CH3CN; b) HMDS, BuLi, THF; c) MoOPH;
d) Bom chloride, DIEA, CH2Cl2; e) TFA–CH2Cl2 (1 : 1); f ) NaH, BrCH2CO2CH3, THF; g) (C2H5)3Nؒ3HF, THF; h) MsCl, pyridine; i) NaN3, DMF;
j) H2, 10% Pd/C, Boc2O, AcOEt; k) H2, 10% Pd(OH)2/C, MeOH; l) adenine, PPh3, DEAD, dioxane; m) N-benzyloxycarbonyl-NЈ-methylimidazolium
triflate, CH2Cl2; n) LiOH, THF, then HCl; o) PhCO2H, PPh3, DEAD, dioxane; p) NaOMe, MeOH.
2758
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Table 1 Melting temperatures (T m/ЊC) of PNA–RNA, PNA–DNA
and PNA–PNA duplexesa, b
impurities from the Mitsunobu reagents were present in 11.
Both compounds were diastereomerically pure according to
1H NMR.
T m
Entry
Monomer incorporated
RNA
DNA
PNA
Synthesis of the (3S,6R)- and (3R,6R) monomer acids 12 and 13
The methyl esters 10 and 11 were hydrolysed using standard
conditions for the hydrolysis of PNA monomer methyl esters:
LiOH in aq. THF at 0 ЊC. After 2 min, TLC revealed one new
spot in each case corresponding to the hydrolysis product, but
unexpectedly two spots after 60 min when the hydrolyses were
complete. The two spots had identical Rf values whether 10 or
11 was hydrolysed, and each corresponded to one of the single
spots seen after 2 min. Apparently the esters or the acid salts
epimerise at C-3 under the hydrolysis conditions, although
epimerisation did not occur during hydrolysis of the five-
membered ring pyr-PNA analogues.4 Attemps to purify the
diastereomers by repeated column chromatography on silica
failed to give pure products, probably because they epimerised
slowly on the column, but small amounts of pure 12 (6 mg) and
13 (2 mg) were finally obtained after preparative RP-HPLC.
The proof of 12 being the (3S,6R)- and 13 the (3R,6R)-isomer
rests on TLC evidence. Thus, 12 had the same Rf value as the
new spot which appeared after 2 min hydrolysis of 10, and 13
corresponded to the new spot appearing after 2 min hydrolysis
of 11.
1
2
3
4
5
Unmodified PNA
(3S,6R)-pip-PNA 12
(3S,6R)-pip-PNA 13
(3S,5R)-pyr-PNA
(3S,5R)-pyr-PNA
59.0
47.5
49.0
44.0
54.0
(23)c ϩ 49.5
24.5 ϩ (36.5)
30.5
24 ϩ (38)
(25) ϩ 41
75.5
69.0
68.5
65.5
69.5
a Measured in aqueous buffer containing 100 mM NaCl, 10 mM phos-
phate, 0.1 mM EDTA, pH 7.0; heating rate: 1 K minϪ1. UV absorbance
measured at 260 nm. b The PNA sequence was H-TACTCATACTCT-
LysNH2, with the monomer at the italic position. The complementary
sequences were 5Ј-d(AGAGTATGAGTA), 5Ј-AGAGUAUGAGUA, or
H-AGAGTATGAGTA-NH2 for DNA, RNA and PNA, respectively.
c Transition with low hyperchromicity in brackets.
thus seems to be inferior to the preorganisation by the cyclic
pyr-PNA analogues studied earlier in terms of producing a
hybridisation-competent conformation. However, it cannot be
excluded that PNA oligomers composed exclusively or pre-
dominantly of pip-PNA hybridise better than the mixed back-
bone oligomers 12 and 13 studied here. Therefore, more data in
terms of sequence context and backbone context (number and
position of pip-PNA modifications in an otherwise unmodified
PNA “background”) are required to fully evaluate the proper-
ties of pip-PNA. Nonetheless, the present results stress that
only a narrow ensemble of hybridisation competent PNA
backbone conformations are available for potent RNA and/or
DNA binding.
Oligomer synthesis
The monomers (3S,6R)-pip-PNA 12 and (3R,6R)-pip-PNA 13
were incorporated into the middle position (italic) of the
12-mer PNA sequence H-TAC-TCA-TAC-TCT-LysNH2 using
standard PNA coupling procedures,9 and the oligomers were
purified to high homogeneity by RP-HPLC. Some epimeris-
ation may have occurred during solid phase synthesis, but
the CD spectra of the PNA–PNA duplexes were markedly
different, so extensive epimerisation can be ruled out. The
same sequence with the five-membered analogues (3R,5R)-pyr-
PNA and (3S,5R)-pyr-PNA incorporated were prepared for
comparison.
Experimental
General
Benzyl chloromethyl ether,10 (6R)-6-[(tert-butyldiphenylsilyl)-
oxymethyl]piperidin-2-one,6
oxodiperoxymolybdenum(pyr-
idine)hexamethylphosphoric triamide (MoOPH),11 and N-
benzyloxycarbonyl-NЈ-methylimidazolium triflate 12 were pre-
pared according to literature procedures. All other reagents
were purchased from Sigma-Aldrich and used without purifi-
cation. Solvents were HPLC-grade from LAB-SCAN. Aceto-
nitrile, N,N-dimethylformamide, benzene, toluene, dioxane,
methylene chloride and pyridine were dried over 4 Å molecular
sieves. Tetrahydrofuran was dried by distillation from
sodium–benzophenone. TLC was run on Merck 5554 silica
60 aluminium sheets. Column chromatography was performed
as flash chromatography on Merck 9385 silica gel 60 (0.040–
0.063 mm). Reactions were carried out under nitrogen except
in the case of hydrogenations. FABMS were recorded in the
positive ion mode. Elemental analyses were performed at
the Microanalytical Laboratory, Department of Chemistry,
University of Copenhagen. NMR spectra were obtained on a
300 or 400 MHz spectrometer. δ-Values are in ppm relative
to DMSO-d6 (2.50 for proton and 39.5 for carbon) or CDCl3
(7.29 for proton and 76.9 for carbon) or CD3OD (3.35 for
proton and 49.1 for carbon). RP-HPLC was run on a Waters
486 HPLC system with diode array detector, abs. 260 nm,
Waters 19 × 300 mm C-18 column, eluents 0.1% aq. TFA
(buffer A) and CH3CN–H2O 9 : 1 v/v (buffer B).
Thermal stability
The T m’s of the PNA–RNA, PNA–DNA and PNA–PNA
duplexes are given in Table 1. Both pip-PNA modified 12-mers
(entry 2 and 3) hybridised to RNA, but with a large decrease in
T m compared to the unmodified PNA (entry 1). Contrary to
the (3S,5R) pyr-PNA modified sequence (entry 5), which was
bound better to RNA than its diastereomers both in the present
(compare with entry 4) and in the previously published cases,4
the (3S,6R) pip-PNA 12 modified sequence (entry 2) did not
bind significantly better to RNA than its diastereomer (entry
3). The data for binding to DNA are difficult to interpret
because two transitions occurred in most cases, but the modifi-
cation clearly decreased the binding to DNA. Binding to PNA
is compromised as well, albeit somewhat less pronounced for
both pip-PNA modified sequences.
Conclusion
Two new conformationally restricted piperidinone PNA
adenine monomers 12 and 13 have been synthesised and incor-
porated into a PNA dodecamer (once in a central position).
Modifying PNA with either 12 or 13 resulted in a large decrease
in duplex stability with RNA (∆T m 10–11.5 ЊC) as well as with
DNA, and a smaller decrease with PNA. In particular, 12
with the similar (3S,6R) configuration to that of the best of the
pyr-PNA [(3S,5R) configuration] bound much less efficiently
to RNA. Therefore, any expected preorganisation of the
PNA single strand induced by these cyclic pip-PNA analogues
(6R)-N-tert-Butoxycarbonyl-6-[(tert-butyldiphenylsilyl)-
oxymethyl]piperidin-2-one (1)
Boc2O (3.3 g, 15.0 mmol) and then DMAP (61 mg, 0.5 mmol)
were added to
a stirred solution of (6R)-6-[(tert-butyl-
diphenylsilyl)oxymethyl]piperidin-2-one6 (3.68 g, 10.0 mmol)
in CH3CN (14 ml) at rt. The solution was stirred at rt overnight.
J. Chem. Soc., Perkin Trans. 1, 2001, 2757–2763
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More Boc2O (3.3 g, 15.0 mmol) was added and the solution
again stirred overnight. The reaction mixture was quenched by
the addition of 10% aq. citric acid (25 ml) and CH2Cl2 (50 ml).
The aqueous phase was extracted with CH2Cl2 (2 × 50 ml). The
organic phases were combined, washed with brine (25 ml),
dried (Na2SO4) and evaporated in vacuo. The crude product was
purified by chromatography (a stepwise gradient of AcOEt in
hexane from 1 : 4 to 1 : 0 v/v). Yield: 4.02 g of 1 as a white solid
(9H, s, Boc), 0.97 (9H, s, ButSi). 13C-NMR (100.6 MHz,
DMSO-d6): δ 170.7, 152.4, 138.0, 135.1, 132.4, 132.3, 130.0,
128.3, 128.2, 128.0, 127.9, 127.7, 127.5, 93.5, 82.2, 72.7, 69.2,
64.9, 55.8, 27.5, 26.6, 25.2, 21.2, 18.7. FABMS m/z 603.9
(M ϩ H). This purified intermediate (1.76 g, 2.92 mmol) was
dissolved in CH2Cl2 (2.9 ml). TFA (2.9 ml) was added dropwise
at 0 ЊC during 1 min and the solution was stirred for 8 min. The
reaction was quenched by the slow addition of saturated aq.
NaHCO3 (40 ml). The aqueous phase was extracted with
CH2Cl2 (50 ml) and AcOEt (2 × 50 ml). The combined organic
phases were dried (MgSO4) and evaporated in vacuo. Purifi-
cation by chromatography (AcOEt–hexane 2 : 1 v/v) afforded
0.87 g (57%) of 3 a clear oil which was pure on TLC, Rf 0.41
1
(86%), mp 74–75 ЊC, Rf 0.80 (AcOEt). H-NMR (400 MHz,
DMSO-d6): δ 7.66–7.39 (10H, m, Ph), 4.18 (1H, m, H-6), 3.78
(1H, m, SiOCHA), 3.62 (1H, m, SiOCHB), 2.48–2.29 (2H, m,
H-3), 2.02–1.89 (3H, m, H-4 ϩ H-5), 1.65 (1H, m, H-4 or H-5),
1.38 (9H, s, Boc), 0.97 (9H, s, ButSi). 13C-NMR (100.6 MHz,
DMSO-d6): δ 171.2, 152.5, 135.1, 135.0, 132.5, 132.3, 129.9,
127.9, 127.8, 81.7, 64.5, 55.7, 34.4, 27.5, 26.5, 24,5. 18.7, 17.6
(Found: C, 69.4; H, 8.1; N, 2.9. Calc. for C27H37NO4Si: C, 69.3;
H, 8.0; N, 3.0%).
1
(AcOEt–hexane 2 : 1 v/v). H-NMR (400 MHz, DMSO-d6):
δ 7.63–7.60 (4H, m, Ph), 7.49–7.41 (6H, m, Ph), 7.36–7.27 (6H,
m, Ph and NH), 4.91 (1H, d, J 6.8, OCHAO), 4.83 (1H, d, J 6.8,
OCHBO), 4.59 (2H, s, CH2-Ph), 3.95 (1H, m, H-3), 3.62 (1H, m,
SiOCHA), 3.53–3.46 (2H, m, SiOCHB and H-6), 2.02–1.98 (2H,
m, H-4 or H-5), 1.68–1.65 (2H, m, H-4 or H-5), 1.00 (9H,
s, ButSi). 13C-NMR (100.6 MHz, DMSO-d6): δ 170.5, 138.2,
135.1, 132.9, 132.8, 129.9, 128.3, 128.0, 127.7, 127.5, 94.0,
71.2, 68.9, 66.1, 53.1, 26.7, 26.3, 22.4, 18.9. FABMS m/z 503.8
(M ϩ H).
(3S,6R)-N-tert-Butoxycarbonyl-6-[(tert-butyldiphenylsilyl)-
oxymethyl]-3-hydroxypiperidin-2-one (2)
BuLi (10.2 ml, 25.5 mmol, 2.5 M in hexane) was added drop-
wise to a solution of hexamethyldisilazane (5.39 ml, 25.5 mmol)
in THF (20 ml) at Ϫ78 ЊC. The solution was stirred at Ϫ78 ЊC
for 30 min. A solution of 1 (3.99 g, 8.51 mmol) in THF (20 ml)
was added over a period of 5 min with stirring. The tem-
perature of the reaction mixture was slowly raised from Ϫ78
to Ϫ40 ЊC during the next 60 min, before MoOPH (7.39 g,
17.0 mmol) was added in two portions. The green solution was
stirred between Ϫ40 and Ϫ30 ЊC for 30 min and the reaction
was then quenched by the addition of half-saturated aq. NH4Cl
(40 ml). The THF was evaporated off and the aqueous phase
was extracted with AcOEt (3 × 80 ml). The organic phases
were combined, washed with brine (80 ml), dried (Na2SO4)
and evaporated in vacuo to give 6.7 g of a crude product which
was purified by chromatography (a stepwise gradient of
AcOEt–hexane from 2 : 3 to 1 : 0 v/v). Yield 1.46 g (36%) of 2
as a slightly yellow solid, mp 80 ЊC, Rf 0.44 (AcOEt–heptane
2 : 3 v/v). The product was contaminated with 5% of the
(3S,6R)-3-Benzyloxymethoxy-6-[(tert-butyldiphenylsilyl)-
oxymethyl]-N-[methoxycarbonylmethyl]piperidin-2-one (4)
Compound 3 (2.40 g, 4.81 mmol) was dissolved in THF (25 ml).
NaH (60% suspension in mineral oil, 0.39 g, 9.62 mmol)
and then methyl bromoacetate (0.93 ml, 9.62 mmol) were added
at 0 ЊC and the reaction mixture stirred overnight at rt. The
reaction mixture was quenched by the slow addition of half-
saturated aq. NH4Cl (50 ml) at 0 ЊC and extracted twice with
AcOEt (2 × 50 ml). The combined organic phases were washed
with brine (50 ml), dried (Na2SO4) and evaporated in vacuo to
give the crude product as an oil which was purified by chroma-
tography (AcOEt–hexane 2 : 1 v/v). Yield: 2.50 g (90%) of 4 as a
clear oil, pure on TLC, Rf 0.67 (AcOEt). 1H-NMR (400 MHz,
DMSO-d6): δ 7.61–7.57 (4 H, m, Ph), 7.48–7.41 (6H, m, Ph),
7.34–7.28 (5H, m, Ph), 4.93 (1H, d, J 6.6, OCHAO), 4.80 (1H, d,
J 6.6, OCHBO), 4.58 (2H, m, CH2-Ph), 4.11 (1H, d, J 17.0,
NCHACO), 4.05 (1H, m, H-3), 3.84 (1H, d, J 17.0, NCHBCO),
3.65 (3H, m, H-6 and SiOCH2), 3.58 (3H, s, CH3), 2.00 (2H, m,
H-4 or H-5), 1.82 (1H, m, H-4 or H-5), 1.71 (1H, m, H-4 or
H-5), 0.98 (9H, s, ButSi). 13C-NMR (100.6 MHz, DMSO-d6):
δ 169.9, 169.4, 138.2, 135.1, 132.6, 132.4, 130.1, 130.0, 128.2,
128.0, 128.0, 127.7, 127.4, 93.7, 70.6, 68.9, 64.5, 58.4, 51.7, 46.7,
26.6, 25.2, 21.2, 18.7. FABMS m/z 576.0 (M ϩ H).
1
1
(3R,6R)-isomer as judged by H-NMR. H-NMR (300 MHz,
DMSO-d6) [data for the minor isomer are given in brackets]:
δ 7.61–7.39 (10 H, m, Ph), 5.57 (1H, d, J 4.4, OH), [5.43 (1H, d,
J 3.3, OH)], 4.19 (1H, m, H-3 or H-6), 4.00 (1H, m, H-3 or
H-6), 3.73 (1H, m, SiOCHA), 3.57 (1H, m, SiOCHB), 2.12 (2H,
m, H-4 or H-5), 1.98 (1H, m, H-4 or H-5), 1.63 (1H, m, H-4 or
H-5), 1.37 (9H, s, Boc), 0.98 (9H, s, ButSi). 13C-NMR (75.5
MHz, DMSO-d6): δ 173.4, 152.6, 135.0, 132.4, 132.2, 129.9,
127.9, 82.0, 68.4, 64.6, 56.0, 27.5, 27.0, 26.5, 21.2, 18.7 (Found:
C, 66.6; H, 7.8; N, 2.8. Calc. for C27H37NO5Si: C, 67.05; H, 7.7;
N, 2.9%).
(3S,6R)-3-Benzyloxymethoxy-6-hydroxymethyl-N-methoxy-
carbonylmethylpiperidin-2-one (5)
(3S,6R)-3-Benzyloxymethoxy-6-[(tert-butyldiphenylsilyl)-
oxymethyl]piperidin-2-one (3)
Compound 4 (2.50 g, 4.34 mmol) was dried by co-evaporation
from CH3CN–CH2Cl2 1 : 1 v/v and then redissolved in THF
(22 ml). Et3Nؒ3HF (2.75 ml, 17 mmol) was slowly added
and the reaction was stirred at 50 ЊC for 3 h. The solvent was
evaporated off and the crude product purified by chroma-
tography (a stepwise gradient of 5–10% MeOH in CH2Cl2).
Yield 1.11 g (76%) of 5 as a clear oil, TLC, Rf 0.60 (minor),
Rf 0.54 (major) (CH2Cl2–MeOH 9 : 1 v/v). The product was
contaminated with approx. 10% of the lactone of 5 as judged
by 1H-NMR. 1H-NMR (400 MHz, DMSO-d6): δ 7.37–7.28
(5H, m, Ph), 4.95 (1H, d, J 6.6, OCHAO), 4.86–4.80 (2H, m,
OCHBO and OH), 4.60 (2H, m, CH2-Ph), 4.21 (1H, d, J 14.1,
NCHACO), 4.04–3.98 (2H, m, NCHBCO and H-3), 3.63 (3H,
s, CH3), 3.50–3.42 (3H, m, OCH2 and H-6), 2.06–1.96 (2H, m,
H-4 or H-5), 1.77–1.68 (2H, m, H-4 or H-5). 13C-NMR (100.6
MHz, DMSO-d6): δ 169.9, 169.8, 138.2, 128.7, 127.7, 127.5,
93.7, 70.8, 68.9, 62.2, 58.8, 51.7, 46.8, 25.3, 21.3. FABMS
m/z 338.1 (M ϩ H).
Compound 2 (1.45 g, 3.01 mmol) was dried by co-evaporation
from CH3CN–CH2Cl2 1 : 1 v/v and then redissolved in CH2Cl2
(7 ml). Bom-Cl (1.25 ml, 9.0 mmol) and then diisopropylethyl-
amine (DIEA) (1.6 ml, 9.0 mmol) were added at 0 ЊC. The
solution was stirred at rt overnight. More CH2Cl2 (50 ml) was
added and the solution was extracted with half-saturated aq.
NH4Cl (2 × 25 ml). The organic phase was washed with brine
(25 ml) and evaporated in vacuo. Purification by chroma-
tography (AcOEt–hexane 2 : 3 v/v) afforded the Bom-protected
intermediate as a clear oil. Yield 1.78 g (98%), pure on TLC, Rf
1
0.71 (AcOEt–hexane 2 : 1 v/v). H-NMR (400 MHz, DMSO-
d6): δ 7.63–7.56 (4H, m, Ph), 7.49–7.38 (6H, m, Ph), 7.34–7.27
(5H, m, Ph), 4.84 (2H, m, OCH2O), 4.58 (2H, s, CH2-Ph), 4.18
(1H, m, H-3 or H-6), 4.14 (1H, m, H-3 or H-6), 3.76 (1H, m,
SiOCHA), 3.59 (1H, m, SiOCHB), 2.21–2.07 (2H, m, H-4 or
H-5), 1.91 (1H, m, H-4 or H-5), 1.75 (1H, m, H-4 or H-5), 1.37
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(3S,6R)-3-Benzyloxymethoxy-N-methoxycarbonylmethyl-6-
(methylsulfonyloxymethyl)piperidin-2-one (6)
1H-NMR (400 MHz, DMSO-d6): δ 6.88 (1H, t, J 5.6, BocNH),
5.08 (1H, d, J 3.7, OH), 4.15 (1H, d, J 17.3, NCHACO), 3.91
(1H, d, J 17.3, NCHBCO), 3.83 (1H, m, H-3), 3.64 (3H, s, CH3),
3.42 (1H, m, H-6), 3.14–3.07 (2H, m, BocN-CH2), 2.02–1.94
(2H, m, H-4 or H-5), 1.68–1.42 (2H, m, H-4 or H-5), 1.37 (9H,
s, Boc). 13C-NMR (75.5 MHz, CDCl3): δ 173.7, 169.4, 155.6,
78.5, 66.7, 57.9, 51.5, 46.3, 41.7, 27.5, 26.1, 22.0. FABMS
m/z 317.1 (M ϩ H).
Compound 5 (1.68 g, 4.98 mmol) was dried by evaporation
from CH3CN–CH2Cl2 1 : 1 v/v and then redissolved in CH2Cl2
(23 ml). Et3N (0.96 ml, 6.9 mmol) and methanesulfonyl chloride
(0.97 g, 8.5 mmol) were added at 0 ЊC. The reaction mixture
was stirred at 0 ЊC for 45 min and then quenched by addition of
half-saturated aq. NaHCO3 (50 ml) and CH2Cl2 (75 ml). The
aqueous phase was extracted with CH2Cl2 (75 ml) and the
combined organic phases evaporated. The crude product was
purified by chromatography (2% MeOH in CH2Cl2) to give
1.74 g (85%) of 6 as a clear oil, pure on TLC, Rf 0.26 (MeOH-
(3R,6R)-6-tert-Butoxycarbonylaminomethyl-3-hydroxy-N-
methoxycarbonylmethylpiperidin-2-one (9)
Compound 8 (315 mg, 1.0 mmol) was dried by evaporation
from CH3CN (5 ml) and then redissolved in THF (5 ml). A
solution of PPh3 (786 mg, 3.0 mmol) in THF (2 ml) and a
solution of benzoic acid (610 mg, 5.0 mmol) in toluene (10 ml)
were successively added at 0 ЊC, and the mixture stirred for
2 min, before DEAD (0.79 ml, 5.0 mmol) was added dropwise.
The clear yellow solution was allowed to warm to rt and stirred
overnight. AcOEt (100 ml) was added and the mixture was
extracted with 10% aq. citric acid (25 ml), brine (25 ml), half-
saturated aq. NaHCO3 (25 ml), and brine (25 ml). The organic
phase was dried (MgSO4) and evaporated. The crude product
was purified by chromatography (AcOEt–hexane 2 : 1 v/v,
then pure AcOEt). Yield 305 mg (73%) of the intermediate as
a white foam, pure on TLC, Rf 0.25 (AcOEt–hexane 2 : 1 v/v).
1H-NMR (300 MHz, CDCl3): δ 7.98 (2H, d, J 6.9, Ph), 7.47
(1H, m, Ph), 7.35 (2H, m, Ph), 5.65 (1H, t, J 5.7, BocNH), 5.36
(1H, m, H-3), 4.04 (2H, m, NCH2CO), 3.64 (3H, s, CH3), 3.44
(2H, m, BocN-CH2), 3.16 (1H, m, H-6), 2.30–1.90 (4H, m, H-4
and H-5), 1.36 (9H, s, Boc). 13C-NMR (75.5 MHz, CDCl3):
δ 169.3, 167.6, 165.2, 155.9, 132.8, 129.4, 129.2, 127.9, 79.0,
69.0, 57.6, 51.8, 48.0, 41.6, 27.9, 22.8, 22.1. This intermediate
(305 mg, 0.73 mmol) was dissolved in MeOH (5.3 ml) and
cooled to 0 ЊC. NaOMe in methanol (1.0 M, 1.4 ml, 1.4 mmol)
was added dropwise and the solution was stirred at 0 ЊC for 3 h.
The reaction was quenched by addition of half-saturated aq.
NH4Cl (20 ml). The aqueous phase was extracted with AcOEt
(4 × 40 ml). The organic phases were combined, dried over
MgSO4 and evaporated in vacuo. Chromatography (AcOEt,
then CH2Cl2–MeOH 9 : 1 v/v) afforded 9 as a hygroscopic foam,
yield: 195 mg (85%), pure on TLC, Rf 0.30 (CH2Cl2–MeOH
9 : 1 v/v). 1H-NMR (400 MHz, CDCl3): δ 5.52 (1H, br s,
BocNH), 4.04 (1H, d, J 17.2, NCHACO), 3.94–3.90 (2H, m,
NCHBCO and H-3), 3.61 (3H, s, CH3), 3.37 (1H, m, BocN-CH2
or H-6), 3.29 (1H, m, BocN-CH2 or H-6), 2.98 (1H, m, BocN-
CH2 or H-6), 1.97 (1H, m, H-4 or H-5), 1.88 (2H, m, H-4 or
H-5), 1.81 (1H, m, H-4 or H-5), 1.30 (9H, s, Boc). 13C-NMR
(100.6 MHz, CDCl3): δ 172.9, 169.3, 155.8, 79.2, 67.7, 57.6,
51.9, 47.9, 42.1, 27.9, 24.1, 22.2. FABMS m/z 317.2 (M ϩ H).
1
CH2Cl2 2 : 98 v/v). H-NMR (300 MHz, CDCl3): δ 7.38–7.28
(5H, m, Ph), 5.11 (1H, d, J 7.0, OCHAO), 4.90 (1H, d, J 7.0,
OCHBO), 4.69 (2H, s, CH2-Ph), 4.32–4.09 (5H, m, H-3, N-CH2
and MsO-CH2), 3.82 (1H, m, H-6), 3.77 (3H, s, O-CH3), 3.06
(3H, s, S-CH3), 2.27 (1H, m, H-4 or H-5), 2.30 (1H, m, H-4
or H-5), 1.95 (1H, m, H-4 or H-5), 1.85 (1H, m, H-4 or H-5).
13C-NMR (75.5 MHz, CDCl3): δ 170.2, 169.4, 137.6, 128.3,
127.8, 127.6, 94.2, 70.4, 69.8, 68.9, 56.9, 52.2, 47.8, 37.5, 25.1,
21.6. FABMS m/z 416.0 (M ϩ H).
(3S,6R)-6-Azidomethyl-3-benzyloxymethoxy-N-methoxy-
carbonylmethylpiperidin-2-one (7)
Compound 6 (1.70 g, 4.10 mmol) was dissolved in DMF (21 ml)
and NaN3 (1.33 g, 20.5 mmol) was added. The solution
was stirred at 80 ЊC overnight. The solvent was evaporated off
and the resulting oil partitioned between half-saturated aq.
NaHCO3 (50 ml) and AcOEt (75 ml). The aqueous phase was
extracted with AcOEt (2 × 75 ml). The combined organic
phases were washed with brine, dried (MgSO4) and evaporated
in vacuo. The crude product was purified by chromatography
(a stepwise gradient of 2–10% MeOH in CH2Cl2) to give 1.45 g
(98%) of 7 as an oil, pure on TLC, Rf 0.47 (AcOEt–hexane
4 : 1 v/v). 1H-NMR (400 MHz, DMSO-d6): δ 7.37–7.28 (5H, m,
Ph), 4.93 (1H, d, J 6.6, OCHAO), 4.80 (1H, d, J 6.6, OCHBO),
4.60 (2H, m, CH2-Ph), 4.16 (1H, d, J 17.2, NCHA), 4.06 (1H, m,
H-3), 4.02 (1H, d, J 17.2, NCHB), 3.64 (3H, s, CH3), 3.63–3.54
(3H, m, H-6 and N3CH2), 2.07–2.05 (2H, m, H-4 or H-5), 1.74–
1.71 (2H, m, H-4 or H-5). 13C-NMR (100.6 MHz, DMSO-d6):
δ 169.9, 169.5, 138.2, 128.3, 127.7, 127.5, 93.7, 70.6, 69.0, 56.4,
52.0, 51.8, 46.8, 24.9, 21.9. FABMS m/z 363.2 (M ϩ H).
(3S,6R)-6-tert-Butoxycarbonylaminomethyl-3-hydroxy-N-
methoxycarbonylmethylpiperidin-2-one (8)
10% Pd/C (0.27 g) was added to a stirred solution of 7 (1.45 g,
4.0 mmol) and Boc2O (1.74 g, 8.0 mmol) in AcOEt (40 ml) at
0 ЊC. The mixture was hydrogenated at 1 atm for 90 min at rt,
and then passed through Celite. The solvent was evaporated off
and the crude product (2.64 g) was purified by chromatography
(AcOEt) to give 1.67 g (96%) of the Bom protected inter-
(3S,6R)-3-[N6-(Benzyloxycarbonyl)adenin-9-yl]-6-tert-butoxy-
carbonylaminomethyl-N-methoxycarbonylmethylpiperidin-2-one
(10)
1
mediate, pure on TLC, Rf 0.44 (AcOEt). H-NMR (400 MHz,
DMSO-d6): δ 7.37–7.27 (5H, m, Ph), 6.92 (1H, t, J 6.0, NH),
4.94 (1H, d, J 6.6, OCHAO), 4.80 (1H, d, J 6.6, OCHBO), 4.59
(2H, m, CH2-Ph), 4.14 (1H, d, J 17.0, NCHACO), 4.02 (1H, m,
H3), 3.90 (1H, d, J 17.0, NCHBCO), 3.64 (3H, s, CH3), 3.41
(1H, m, H-6), 3.10 (2H, m, BocN-CH2), 2.00 (2H, m, H-4 or
H-5), 1.68 (2H, m, H-4 or H-5), 1.37 (9H, s, Boc). 13C-NMR
(100.6 MHz, DMSO-d6): δ 169.7, 169.6, 155.9, 138.1, 128.2,
127.7, 127.5, 93.6, 78.0, 70.4, 68.9, 57.3, 51.8, 46.9, 41.5, 28.2,
24.8, 21.4. FABMS m/z 437.2 (M ϩ H). This purified inter-
mediate (1.67 g, 3.82 mmol) was dissolved in MeOH (57 ml),
10% Pd(OH)2/C (0.22 g) was added and the mixture was hydro-
genated overnight at 1 atm and rt and then passed through
Celite. The solution was evaporated and the crude product
(1.0 g) was purified by chromatography (a stepwise gradient of
5–10% MeOH in CH2Cl2). Yield 0.83 g of 8 as a white oil
(68%), pure on TLC, Rf 0.32 (CH2Cl2–MeOH 9 : 1 v/v).
Compound 9 (369 mg, 1.17 mmol) was dried by co-evaporation
from dry CH3CN and redissolved in dry dioxane (24 ml). PPh3
(0.660 g, 2.92 mmol) was added followed by adenine (0.789 g,
5.84 mmol). To this stirred suspension was slowly (during
20 min) added DEAD (0.36 ml, 2.29 mmol) at rt, and the
suspension stirred at rt overnight. The solvent was evaporated
off and the residue purified by chromatography (AcOEt then
CH2Cl2–MeOH 9 : 1 v/v) to give an intermediate, yield 267 mg
(53%), pure on TLC, Rf 0.33 (CH2Cl2–MeOH 9 : 1 v/v).
13C-NMR (75.5 MHz, CDCl3): δ 169.7, 167.9, 156.2, 155.5,
152.3, 149.4, 139.7, 118.5, 79.4, 58.1, 53.9, 52.1, 49.6, 47.3, 42.1,
28.0, 25.6, 23.4. FABMS m/z 434.2 (M ϩ H). The intermediate
(245 mg, 0.57 mmol) was dissolved in dry CH2Cl2 (3.8 ml) and
N-benzyloxycarbonyl-NЈ-methylimidazolium triflate (0.621 g,
1.70 mmol) was added, followed by stirring at rt overnight.
Half-saturated aq. NaHCO3 (25 ml) and CH2Cl2 (50 ml) were
J. Chem. Soc., Perkin Trans. 1, 2001, 2757–2763
2761

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added and the aqueous phase extracted with CH2Cl2 (50 ml)
and AcOEt (50 ml).The combined organic phases were dried
(MgSO4) and evaporated in vacuo. The crude product was puri-
fied by chromatography (AcOEt then AcOEt–MeOH 9 : 1 v/v)
to give 191 mg (60%) of 10 as a white solid, pure on TLC,
Rf 0.52 (CH2Cl2–MeOH 9 : 1 v/v), pure on RP-HPLC (RT 21.2
(3R,6R)-3-[N6-(Benzyloxycarbonyl)adenin-9-yl]-6-tert-butoxy-
carbonylaminomethyl-N-carboxymethylpiperidin-2-one (13)
Compound 13 was prepared in a similar way to 12. From 11
(66 mg, 0.12 mmol) the crude product (50 mg) showed on TLC
(CHCl3–MeOH–HOAc 85 : 10 : 5 v/v/v) two spots of similar
intensity, Rf 0.36 and 0.27. Preparative RP-HPLC gave 13 as a
colourless solid, pure on TLC, Rf 0.27 (CHCl3–MeOH–HOAc
85 : 10 : 5), Rf 0.36 (CHCl3–EtOH–HOAc 80 : 15 : 5 v/v/v).
Compound 13 was dried under high vacuum for two days, yield
2 mg (3%). HR FABMS m/z 554.2363 (M ϩ H calc. 554.2363).
1H-NMR (300 MHz, CD3OD): δ 8.71 (1H, s, H-adenine),
8.61 (1H, s, H-adenine), 7.52 (2H, m, Ph), 7.42 (3H, m, Ph),
5.48 (1H, m, H-3), 5.40 (2H, s, CH2-Ph), 4.40 (1H, d, J 17,
NCHACO), 4.08 (1H, d, J 17, NCHBCO), 3.69 (1H, m, H-6),
3.58 (2H, m, BocN-CH2), 2.95 (1H, m, H-4 or H-5), 2.32 (2H,
m, H-4 or H-5), 2.23 (1H, m, H-4 or H-5), 1.49 (9H, s, Boc).
1
min). H-NMR (400 MHz, CDCl3): δ 10.0 (1H, br s, ZNH),
8.62 (1H, s, H-8-adenine), 7.91 (1H, s, H-2-adenine), 7.30–7.19
(5H, m, Ph), 5.71 (1H, br s, BocNH), 5.18 (2H, m, OCH2-Ph),
5.00 (1H, m, H-3), 4.13 (1H, d, J 17.2, NCHACO), 3.98 (1H, d,
J 17.2, NCHBCO), 3.61 (3H, s, CH3), 3.58 (1H, m, BocN-CH2
or H-6), 3.41 (1H, m, BocN-CH2 or H-6), 3.11 (1H, m, BocN-
CH2 or H-6), 2.30 (1H, m, H-4 or H-5), 2.07 (2H, m, H-4 or
H-5), 1.88 (1H, m, H-4 or H-5), 1.38 (9H, s, Boc). 13C-NMR
(100.6 MHz, CDCl3): δ 169.6, 167.4, 156.1, 152.0, 151.2, 149.2,
142.6, 135.2, 128.1, 128.0, 127.9, 121.4, 78.4, 67.1, 58.3, 54.2,
52.1, 47.3, 42.0, 28.0, 25.4, 23.4. FABMS m/z 568.0 (M ϩ H).
Oligomer synthesis
(3R,6R)-3-[N6-(Benzyloxycarbonyl)adenin-9-yl]-6-tert-butoxy-
carbonylaminomethyl-N-methoxycarbonylmethylpiperidin-2-one
(11)
H-TAC-TC(12)-TAC-TCT-LysNH2.
H-TAC-TCT-Lys-
MBHA resin (24 mg, loading 0.12 mmol gϪ1, dried in a desic-
cator) was transferred to an Eppendorf tube. Compound
12ؒTFA (3.0 mg, 4.5 µmol), HBTU (2 mg, 5.3 µmol), and DIEA
(4 µl, 23 µmol) were dissolved in DMF (92 µl) and, after preactiv-
ation for 2 min, added to the dry resin. Coupling was allowed
to proceed overnight after which the resin and the coupling
mixture were transferred (in DMF) to a glass reactor and
the oligomerisation continued by the standard procedure.
All Kaiser tests after couplings were yellow (indicating com-
plete reaction). Cleavage and deprotection was carried out
with TFA–trifluoromethanesulfonic acid–thioanisole–m–cresol
300 : 100 : 50 : 50 µl for 2 h at rt. The product was precipitated
several times with ether and finally purified by RP-HPLC.
Yield 1.1 mg, 88% pure on RP-HPLC, MALDI-TOF: 3332.8
(calc. 3333).
Compound 11 was prepared in a similar way to 10. Starting
from 8 (234 mg, 0.74 mmol), the intermediate was obtained in
1
137 mg (43%) yield. H-NMR (300 MHz, CDCl3): δ 8.22 (1H,
s, H-8-adenine), 7.81 (1H, s, H-2-adenine), 6.75 (2H, s, NH2),
6.44 (1H, BocNH), 5.03 (1H, m, H-3), 4.23 (1H, d, J 17.1,
NCHACO), 4.01 (1H, d, J 17.1, NCHBCO), 3.64 (3H, s, CH3),
3.60–3.35 (3H, m, NCH2 and H-6), 2.72 (1H, m, H-4 or H-5),
2.20–2.03 (3H, m, H-4 and H-5), 1.37 (9H, s, Boc). 13C-NMR
(75.5 MHz, CDCl3): δ 169.2, 166.7, 156.1, 155.6, 152.4, 149.2,
140.3, 119.1, 79.4, 57.2, 54.4, 52.0, 48.5, 42.2, 28.1, 24.0, 23.9.
FABMS m/z 434.1 (M ϩ H). This intermediate (130 mg,
0.30 mmol) gave 66 mg (39%) of 11 as a white solid, Rf 0.52
(CH2Cl2–MeOH 9 : 1 v/v), 85% pure on RP-HPLC (RT 21.8
1
min). H-NMR (300 MHz, CDCl3): δ 9.50 (1H, br s, Z-NH),
8.70 (1H, s, H-8-adenine), 7.95 (1H, s, H-2-adenine), 7.40–7.30
(5H, m, Ph), 5.91 (1H, br s, BocNH), 5.26 (2H, s, CH2-Z), 4.95
(1H, m, H-3), 4.20 (1H, d, J 17.1, NCHACO), 4.05 (1H, d,
J 17.1, NCHBCO), 3.68 (3H, s, CH3), 3.62–3.42 (3H, m, N-CH2
and H-6), 2.79 (1H, m, H-4 or H-5), 2.15 (3H, m, H-4 or H-5),
1.41 (9H, s, Boc). 13C-NMR (75.5 MHz, CDCl3): δ 169.2, 166.2,
156.0, 152.2, 151.0, 149.3, 143.0, 135.3, 128.4, 128.3, 128.1,
121.8, 79.6, 67.3, 57.5, 54.8, 54.8, 52.1, 48.7, 42.2, 28.1, 23.9.
FABMS m/z 568.1 (M ϩ H).
H-TAC-TC(13)-TAC-TCT-LysNH2. This was prepared as
above from 13 (1.5 mg). Yield 0.5 mg, pure on RP-HPLC,
MALDI-TOF: 3332.0 (calc. 3333).
H-TAC-TC(3R,5R pyr-PNA)-TAC-TCT-LysNH2. This was
prepared as above from 3.0 mg of modified monomer. Yield:
1.0 mg, 93.4% pure on RP-HPLC. MALDI-TOF: 3321.8
(calc. 3319).
H-TAC-TC(3S,5R pyr-PNA)-TAC-TCT-LysNH2. This was
prepared as above from 3.0 mg of modified monomer. Yield:
1.3 mg, 93.2% pure on RP-HPLC. MALDI-TOF: 3321.8
(calc. 3319).
(3S,6R)-3-[N6-(Benzyloxycarbonyl)adenin-9-yl]-6-tert-butoxy-
carbonylaminomethyl-N-carboxymethylpiperidin-2-one (12)
LiOH (1 M, 0.82 ml, 0.82 mmol) was slowly added to a stirred
solution of 10 (185 mg, 0.326 mmol) in THF (3.4 ml) at 0 ЊC,
and the reaction was stirred at 0 ЊC. A small sample was
removed after 2 min for TLC and acidified. TLC (CHCl3–
MeOH–HOAc 85 : 10 : 5), Rf 0.36 (product) and 0.74 (10).
After 45 min H2O (1 ml) was added and the THF was evapor-
ated off. The product was precipitated by the slow addition of
2 M HCl (0.5 ml) at 0 ЊC. The white precipitate was spun down
in a centrifuge tube and the supernatant removed. The white
pellet was washed twice with H2O (2 × 4 ml) and dried in vacuo
to give the crude product (140 mg). TLC (CHCl3–MeOH–
HOAc 85 : 10 : 5 v/v/v) showed two spots of similar intensity,
Rf 0.36 and 0.27. Attempts to purify the crude product by
chromatography (CH2Cl2–MeOH–HOAc 85 : 10 : 5) failed,
but preparative RP-HPLC purification gave 12 as a colourless
solid, pure on TLC, Rf 0.36 (CHCl3–MeOH–HOAc 85 : 10 : 5),
Rf 0.53 (CHCl3–EtOH–HOAc 80 : 15 : 5 v/v/v). Compound 12
was dried under high vacuum for two days, yield 6.1 mg
(3%). FABMS m/z 554.2 (M ϩ H). Electrospray MS: m/z 554.46
(M ϩ H) (Found: C, 49.9; H, 4.9; N, 14.9. Calc. for C26H31-
N7O7ؒTFA: C, 50.4; H, 4.8; N, 14.7%).
Acknowledgements
Annette W. Jørgensen and Jolanta Ludvigsen are thanked for
their expert technical assistance. The Danish Natural Science
Research Council and The Danish Technical Research Council
are acknowledged for financial support.
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