Amino acids bearing nucleobases for the synthesis of novel peptide nucleic acids

Article abstract of DOI:10.1039/a603696a

All of the four nucleobases found in DNA have been incorporated in their protected form into the 4-position of N-tert-butoxycarbonyl-L-proline methyl ester with cis-stereochemistry. An efficient route for the synthesis of N-tert-butoxycarbonyl-trans-4-hydroxy-D-proline methyl ester has been developed from which the enantiomers may be synthesized. In addition an efficient synthesis of N-tert-butoxycarbonyl-N-(2-hydroxyethyl)glycine methyl ester has been achieved and its hydroxy group replaced with protected nucleobases using the Mitsunobu reaction.

Full text of DOI:10.1039/a603696a

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Amino acids bearing nucleobases for the synthesis of novel peptide
nucleic acids
Gordon Lowe* and Tirayut Vilaivan
Dyson Perrins Laboratory, Oxford University, South Parks Road, Oxford OX1 3QY, UK
All of the four nucleobases found in D NA have been incorporated in their protected form into the 4-
position of N-tert-butoxycarbonyl-L-proline methyl ester with cis-stereochemistry. An efficient route for the
synthesis of N-tert-butoxycarbonyl-trans-4-hydroxy-D -proline methyl ester has been developed from which
the enantiomers may be synthesized. In addition an efficient synthesis of N-tert-butoxycarbonyl-N-(2-
hydroxyethyl)glycine methyl ester has been achieved and its hydroxy group replaced with protected
nucleobases using the M itsunobu reaction
Introduction
Oligonucleotides are potentially useful for the regulation of
genetic expression by binding with DNA or mRNA. The
principle, which is known as the antisense principle,1 provides
a way to control protein synthesis at the nucleic acid level
which should be much more effective than inhibition of
enzymes. Furthermore, a highly specific inhibition can be
achieved by a relatively short antisense oligonucleotide, whose
sequence can be derived directly from the sequence of the
target nucleic acids. However, for the antisense principle to be
put into practice, the antisense oligonucleotides must be suf-
ficiently stable under physiological conditions, able to pass
through the cell membrane, and bind specifically and tightly
Fig. 1 Comparison of structure of the target peptide nucleic acids and
to the target nucleic acids.1 As natural oligonucleotides are
readily degraded by nucleases in vivo, there is considerable
interest in synthetic oligonucleotide analogues which are
stable under physiological conditions. Recently, there has
been interest in oligonucleotide analogues in which the sugar
phosphate backbone is replaced by a peptide chain 2 after the
success of the so-called peptide nucleic acids (PNA),3,4
although such analogues were made much earlier.5 PNAs
were shown not only to retain the ability to recognise their
complementary oligonucleotides, but also with a remarkably
high affinity which is believed to be due to the uncharged
nature of the peptide backbone.4 These properties, in add-
ition to their stability towards nucleases and their ease of
synthesis make PNAs very attractive candidates for the anti-
sense technology as well as other areas of research.6 We
report here the design and synthesis of amino acids bearing
nucleobases with a view to their use for the synthesis of novel
peptide nucleic acids.
DNA
its greater conformational flexibility.9 However, the lack of
negative charge on the peptide backbone of both the ‘rigid’ and
the ‘flexible’ peptide nucleic acids would be expected to allow a
higher affinity for nucleic acids if the correct conformation were
to be easily adopted. Moreover, these novel peptide nucleic
acids can also be modified easily by replacing the glycine spacer
with a range of amino acids to affect physical and biological
properties such as solubility or cell permeability, both of which
are important for therapeutic applications.
The initial target molecules were N-Boc amino acids 1 and 2.
The sugar phosphate backbone of a nucleic acid consists of a
repeating unit of six atoms, configurationally and conforma-
tionally constrained by the -ribose or 2Ј-deoxy--ribose ring.
If this could be replaced by an isostructural dipeptide unit, the
new backbone would be amenable to preparation by solid-
phase peptide synthesis. Several molecular modelling investi-
gations2a,2b,7 indicated that the peptide backbone can adopt the
helical B-form of DNA with a relatively low energy. Our pre-
liminary model8 suggested that a peptide chain consisting of an
alternating sequence of a ‘nucleo-amino acid’ derived from pro-
line and a ‘spacer amino acid’, which could be any amino acid,
should be a suitable structural analogue of the ribose phos-
phate backbone of nucleic acids as shown in Fig. 1. The
(2R,4R) (‘cis-’) stereochemistry of proline was the obvious
choice since this is analogous to the stereochemistry of deoxy-
ribonucleotides. If the 3Ј-methylene group in the pyrrolidine
ring is removed, the resulting analogue of DNA would not be
expected to form as stable a duplex as the cyclic analogue, due to
Although only the -proline series would be able to mimic
natural nucleotides, initially the more readily accessible and
inexpensive -proline series was investigated. Kaspersen and
Pandit had reported the preparation of various pyrrolidine
nucleosides by construction of the pyrimidine and purine rings
from protected 4-aminoproline and 4-aminoprolinol, because
reactions of 4-tosyl- or 4-chloro-proline derivatives with
sodium salts of nucleobases or 2,4-dimethoxypyrimidines failed
to give the desired products in acceptable yield.10 The ring-
construction method, although well documented for both pyr-
imidine11 and purine12 bases, requires a number of steps,
uncommon reagents and includes harsh conditions. Our plan
was to convert the hydroxy group of N-Boc-trans-4-hydroxy-
proline methyl ester into a good leaving group so that nucleo-
philic displacement with appropriately protected nucleobases
would result in nucleo-amino acids with inverted configuration
at the 4-position.
J. Chem. Soc., Perkin Trans. 1, 1997
539

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with compound 4b attempted. However, the protecting groups
used appeared to be labile under the reaction conditions and
substantial cleavage was observed.
Results and discussion
The reaction of N-Boc-trans-4-hydroxy--proline 3 with diazo-
methane gave the methyl ester 4a in near-quantitative yield. The
1H NMR spectrum of this product showed two sets of broad
signals for each proton, notably the two singlets of the But
group at δ ~1.5. The effect is quite common in amides and
urethanes and was previously observed in N-acetylproline and
N-Boc-proline13 and was attributed to the presence of two
relatively stable conformers caused by restricted rotation
around the C–N bond.14 The methyl ester 4a was then treated
with trifluoromethanesulfonic anhydride in pyridine–dichloro-
methane or with toluene-p-sulfonyl chloride in anhydrous
pyridine to give crystalline trifluoromethanesulfonate 4bЈ or
toluene-p-sulfonate 4b in 79 and 87% yield, respectively.
The trifluoromethanesulfonate 4bЈ was too unstable under
the conditions required for the displacement reactions, but
the toluene-p-sulfonate 4b reacted smoothly with N 6-benzoyl-
adenine15 (N 6-BzA)–K2CO3 in dimethylformamide (DMF) in
the presence of 18-crown-6 to give the product 5a in 68% yield,
and which was confirmed to be the N 9-isomer by 13C NMR
spectroscopy16 after the N-Boc protecting group was removed
in order to simplify the spectrum. A positive nuclear Over-
hauser effect (NOE) of H4Ј (1.5%) on irradiation at H2Ј was
observed, indicating that inversion at C4 had taken place, as
expected, giving the cis-product 5a (Scheme 1).
Recent studies21,22 suggested that a direct displacement of
hydroxy group with a nucleobase by the Mitsunobu reaction 23
is a very versatile method for the preparation of carbocyclic
nucleosides. Reactions of compound 4a with N 3-benzoyl-
thymine and N 2-isobutyryl-O 6-(4-nitrophenylethyl)guanine in
the presence of triphenylphosphine–diethyl azodicarboxylate
(DEAD) were attempted and were found to give the desired
products 5c and 5d {after removal of the O 6-nitrophenylethyl
group by treatment with 1,8-diazabicyclo[5.4.0]undec-7-ene
(DBU) in pyridine}20 in 42 and 34% yield, respectively. The
analogous reaction with N 6-benzoyladenine (N 6-BzA) was
also attempted but the product 5a was obtained in a lower
yield (34%) than by the displacement reaction of the toluene-
p-sulfonate. However, no product could be obtained from
the reaction of compound 4a and N 4-benzoylcytosine under
similar conditions, probably because of the insolubility of the
protected nucleobase in the reaction medium.
Having solved the chemistry in the -hydroxyproline series
we moved to the -series. The required protected trans-4-
hydroxy--proline was not commercially available but its syn-
thesis starting from cis-4-hydroxy--proline by inversion of the
4-hydroxy group is well documented.21,24 The cis-4-hydroxy--
proline was synthesized by epimerisation of trans-4-hydroxy--
proline by heating it with acetic anhydride followed by acid
hydrolysis and fractional crystallisation of the epimeric
hydroxyprolines (Scheme 2). The procedure used was essentially
Scheme 1 Reagents: Boc = tert-butoxycarbonyl, Ts = p-tolylsulfonyl
i, CH2N2; ii, TsCl, Py; iii, 4b; N6-BzA, K2CO3, 18-crown-6; iv, 4a;
N6-BzA, Ph3P, DEAD; v, 4b; N4-BzC, K2CO3, 18-crown-6; vi, 4a;
N3-BzT, Ph3P, DEAD; vii, 4a; N2-IbuG(ONpe), Ph3P, DEAD; followed
by DBU, Py
Scheme 2 Reagents and conditions: i, 1:2 Ac2O–HOAc, reflux, 5.5 h;
ii, 2  HCl, reflux, 3 h (82%); iii, Et3N–EtOH–water followed by
recrystallisation (57%); iv, Boc2O–ButOH–aq. NaOH, room temp.,
overnight (quant.); v, CH2N2, 0 ЊC (quant.); vi, HCO2H–DEAD–Ph3P,
THF, room temp., 5 h; vii, NH3–MeOH, room temp., 2 h (78% from
4b); viii, N6-BzA, Ph3P, DEAD (6a; 23%); ix, N3-BzT, Ph3P, DEAD
(6b; 42%)
The analogous reaction with N 4-benzoylcytosine17 (N 4-BzC)
gave a mixture of two products—the N1-isomer 5b and the O2-
isomer in 31 and 32% yield, respectively. The two isomers could
be separated readily by chromatography on silica gel. The iden-
tity of the O2-isomer was shown by a characteristic downfield
shift of the C4Ј resonance compared with that for the N1-
isomer.18 A subsequent NOE experiment further confirmed the
assignment as the NOE effect between H6 and H4 was observed
only in the N1-isomer. Furthermore, the cis-configuration in
both compounds was confirmed by the presence of positive
NOE between H4Ј–H2Ј resonances.
that developed by Greenstein and Winitz24a except that
triethylamine was used for neutralising the epimeric hydro-
chloride salt instead of silver carbonate which considerably
simplified the work-up and gave a comparable yield of the
product. Protection of cis-4-hydroxy--proline as its N-Boc and
methyl ester derivative to give compound 4c was achieved in
a similar fashion to the trans--isomer.21 An early method
of inversion of the 4-OH group involved conversion of the
N,C-protected amino acid into the corresponding toluene-p-
sulfonate followed by SN 2 displacement with acetate ion and
hydrolysis.24 We achieved considerable improvement following
the method of Peterson and Vince21 in which the alcohol 4c
was converted into the acetate with inverted configuration by
Mitsunobu reaction in the presence of acetic acid. Subsequent
Reaction of the toluene-p-sulfonate 4b with N 2-isobutyryl-
guanine (N 2-IbuG) under similar conditions gave the undesired
N7-isomer as the major product, and reaction with thymine
gave a mixture of elimination product and mono- and di-
substituted thymines. These results suggested that protection
of thymine at N3 and guanine at O6 might be necessary. As a
result, N 3-benzoylthymine19 and N 2-isobutyryl-O 6-(4-nitro-
phenylethyl)guanine20 were synthesized and their reactions
540
J. Chem. Soc., Perkin Trans. 1, 1997

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methanolysis with sodium methoxide–methanol gave the pro-
tected trans-4-hydroxy--proline 4d in 68% overall yield. A
modification using formic acid instead of acetic acid in the
Mitsunobu reaction 25 gave a better yield (78%) of compound
4d after removal of the formyl group by treatment with dilute
ammonia in aq. methanol.26 Mitsunobu reactions of compound
4d with N 6-BzA and N 3-BzT gave the desired products 6a and
6b which were identical in all respects with the cis--isomers
except for the sign of optical rotation.
Synthesis of the amino acids 2 required for the flexible PNAs
(Fig. 1) was also investigated. The analogous route to the rigid
analogue, starting from N-(2-hydroxyethyl)glycine, was studied.
The amino acid is known in the literature,27 but was not com-
mercially available. The existing methods of synthesis of this
simple compound include hydrolysis of the condensation prod-
uct of hydroxyacetonitrile and ethanolamine,28 oxidative
dealkylation of N-(2-hydroxyethyl)iminodiacetic acid,29 and
oxirane ring opening with glycine and its derivatives.30 All are
either lengthy or inefficient. In fact, this hydroxy amino acid
could be prepared easily from the reaction between chloroacetic
acid and excess of ethanolamine in aqueous solution. Sub-
sequent precipitation with ethanol followed by recrystallisation
from ethanol–water gave the pure crystalline amino acid in 43%
yield.
Scheme 4 Reagents and conditions: i, A–Ph3P–DEAD in THF, room
temp., overnight (27%); ii, BzCl, Py (quant.); iii, N3-BzT–Ph3P–DEAD
in THF; iv, aq. NaOH–acetone 60% for 10, 78% for 11 (from 7)
tion between C2/C6–H1Ј and C1Ј–H6 and the lack of correlation
between C4/H1Ј were consistent with the proposed N1-substitu-
tion and ruled out the possibilities of substitution at N3, O2 or O4.
Since the acyclic thymidine analogue 9 could be synthesized
from readily available starting materials and could be purified
easily on a large scale, it was selected for the first solid phase
peptide synthesis. The purpose was to establish suitable reac-
tion conditions and to synthesize a model PNA for preliminary
biological tests. Saponification of the methyl ester 9 by an
equivalent excess of aq. NaOH–acetone led to substantial
debenzoylation, giving mainly the free thymine acid 11. Since
protection of thymine was not necessary for the oligomer syn-
thesis, the reaction was repeated with an excess of base and the
reaction time was increased to give the debenzoylated derivative
11 as the sole product in very good yield (78% overall yield from
hydroxy ester 7 if recrystallisation of the intermediate 9 was
omitted) (Scheme 4). Recrystallisation once from ethanol–water
gave pure material suitable for the peptide synthesis. Details of
the synthesis of peptides and their biophysical properties will be
discussed elsewhere.
The N- and C-termini of the amino acid were protected in
the same way as hydroxyproline to give the N-Boc-amino acid
methyl ester Boc-Eg(OH)-OMe 7 in good overall yield (Scheme
3). Reactions of the alcohol 7 and BzA under Mitsunobu con-
Scheme 3 Reagents and conditions: i, NH2CH2CH2OH (2.5 mol equiv.)
in water, room temp., overnight (43%); ii, Boc2O, ButOH, aq. NaOH,
room temp., overnight (90%); iii, CH2N2, 0 ЊC (quant.)
ditions or via the toluene-p-sulfonate gave only a mixture of the
N7- and N9-isomers, which could not be separated by column
chromatography or crystallisation. Unprotected adenine, how-
ever, reacted with the alcohol 7 under Mitsunobu reaction
conditions to give a single isomer in 27% yield, which was
shown to be the desired N9-isomer 8 by 13C NMR spec-
troscopy.16 The yield was not good but isolation of the product
was far more convenient than for the benzoylated derivative due
to the difference in polarity of the product and triphenylphos-
phine oxide. Protection of the exocyclic amino group by treat-
ment with benzoyl chloride gave the dibenzoylated adenine
derivative in quantitative yield. Treatment with aq. sodium
hydroxide–acetone resulted in cleavage of the methyl ester and
of one benzoate group to give the Boc-protected N 6-benzoyl-
adenine derivative 10 in 60% yield (Scheme 4).
Experimental
Mps were recorded on a Kofler block apparatus and are quoted
uncorrected. Specific rotations were measured on a Perkin-
Elmer 241 polarimeter, and [α]D -values are given in 10Ϫ1 deg
cm2 gϪ1. IR spectra were recorded on a Perkin-Elmer 1750 Fou-
rier Transform Infrared spectrometer. Elemental analyses were
performed by Ms V. Lamburn on a Carlo Erba CHN analyser
model 1106. Routine H and 13C NMR spectra were recorded
1
on a Varian Gemini 200 spectrometer operating at 200 MHz
(1H) and 50.28 MHz (13C). 13C spectra were recorded in broad-
band-decoupled mode and the chemical-shift assignment was
assisted by a distortionless enhancement by polarisation trans-
fer (DEPT) experiment performed on the Varian Gemini 200
spectrometer. 19F NMR spectra were recorded on a Bruker AM
The alcohol 7 reacted with N 3-benzoylthymine under stand-
ard Mitsunobu conditions to give the product 9, which could be
purified by column chromatography followed by crystallisation
from ethanol in 56% yield. Unlike the adenine analogue 8, the
protected thymine derivative 9 was relatively non-polar and
eluted rapidly from the column. This property allowed a 20
mmol preparation of compound 9 to be carried out easily. The
position of substitution in product 9 was confirmed to be N1 of
thymine by a 13C–1H heteronuclear multiple-bond correlation
(HMBC) experiment, whereby long-range 13C–1H couplings
over 2 and 3 bonds were detected and the connectivity of the
molecule could be deduced from this information. The correla-
250 spectrometer at 235.35 MHz. High-field H NMR spectra
1
were recorded on a Bruker AMX 500 spectrometer (500 MHz).
The H/13C HMBC experiment was performed on a Bruker
1
1
AMX 500 spectrometer. H and 13C chemical shifts are quoted
in ppm relative to tetramethylsilane and were internally refer-
enced to the residual protonated solvent signal. 19F chemical
shifts were externally referenced to CFCl3 in CHCl3. J-Values
are given in Hz. Chemical ionisation and fast-atom bombard-
ment mass spectra were recorded on a VG 20-250 masslab and
a VG Micromass ZAB-1F mass spectrometer. Electrospray
mass spectra were recorded on a VG Biotech BioQ or VG Bio-
tech Platform. Masses are quoted as m/z-values unless other-
J. Chem. Soc., Perkin Trans. 1, 1997
541

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wise stated, only the molecular ions and major fragments being
quoted.
(Boc CH3), 37.1 and 35.9 [CH2(3) rotamers], 51.8 and 52.3
[CH2(5) rotamers], 51.2 (CO2CH3), 57.0 and 57.3 [CH(2)
rotamers], 78.5 and 79.1 [CH(4) rotamers], 80.7 (Boc C), 127.9
and 130.3 (tosyl CH), 133.5 [tosyl C(4Ј)], 145.6 [tosyl C(1Ј)],
153.5 and 154.0 (Boc CO rotamers) and 172.9 and 173.1 (ester
CO rotamers); m/z [CI(NH3)] 400 (M + H+, 1%), 361
(M Ϫ C4H8 + NH4+, 22), 300 ([M Ϫ Boc + 2 H]+, 77), 128
{[C4H6N(CO2Me) + H]+, 48} and 68 (C4H5N + H+, 100);
νmax(KBr)/cmϪ1 1751s (C᎐O ester), 1695s (C᎐O Boc), 1407s
Distilled water was used for all chemical experiments. Chem-
icals and solvents were obtained from Aldrich Chemical Com-
pany Ltd., Avocado Research Chemicals Ltd. and Lancaster
Synthesis Ltd. and were purified according to the literature,31 if
necessary. N-Boc-trans-4-hydroxy--proline was obtained from
Calbiochem-Novabiochem Ltd. Toluene-p-sulfonyl chloride
was purified by recrystallisation from light petroleum (distil-
lation range 60–80 ЊC). DMF (peptide synthesis grade) was
obtained from Rathburn Chemical Ltd. and was used without
further purification except when strictly anhydrous conditions
were required, when it was re-distilled from calcium hydride
under reduced pressure. Tetrahydrofuran (THF) and 1,4-
dioxane were distilled from sodium wire/benzophenone under
argon and stored over 4 Å molecular sieves. Pyridine was dis-
tilled from calcium hydride and stored over 4 Å molecular
sieves. Moisture-sensitive reactions were performed under
argon in flame-dried glassware.
᎐
᎐
(᎐SO2O᎐), 1369s and 1180s (᎐SO2O᎐); λmax(CHCl3)/nm 242
(ε/dm3 molϪ1 cmϪ1, 568), 258 (466), 264 (568), 268 (530) and
274 (470); [α]D25 Ϫ37.4 (c 0.70, CHCl3).
cis-4-Hydroxy-D-proline
A suspension of trans-4-hydroxy--proline (1.31 g, 10.0 mmol)
in a mixture of acetic anhydride (10 ml) and glacial acetic acid
(20 ml) was heated under reflux for 5.5 h. The dark solution was
cooled, and evaporated under reduced pressure to give a thick
oil. The oil was dissolved in 2  HCl (25 ml) and the solution
was refluxed for 3 h, then was decolourised with charcoal and
filtered. The filtrate was concentrated under reduced pressure to
give a light yellow oil. Trituration with diethyl ether gave a
precipitate which was a mixture of epimeric hydrochloride salts.
Recrystallisation from ethanol gave a crystalline solid (1.37 g,
82%), mp 145–149 ЊC. A portion of the hydrochloride salt (0.50
g, 3.0 mmol) was dissolved in water (2 ml), and triethylamine (1
ml) and absolute ethanol (40 ml) were added. The solution was
stored at room temperature until crystallisation was complete.
The crystals were collected by suction filtration and were
recrystallised twice from water–ethanol to give pure cis-4-
hydroxy--proline as needles (0.225 g, 57%), mp 250–254 ЊC
(decomp.) [lit.,24 mp 252–257 ЊC (decomp.)] (Found: C, 46.1;
H, 7.2; N, 10.8. Calc. for C5H9NO3: C, 45.8; H, 6.9; N, 10.7%);
δH (200 MHz; D2O) 2.00 (1 H, m) and 2.25 (1 H, ddd, J 14.3,
10.4 and 4.5) [CH2(3)], 3.10 (1 H, ddd, J 12.4, 1.8 and 1.8) and
3.22 (1 H, dd, J 12.4 and 3.8) [CH2(5)], 3.96 [1 H, dd, J 10.4
and 3.9, CH(2)] and 4.33 [1 H, m, CH(4)]; [α]D23 +58.6 (c 2.0,
H2O) {lit.,24 [α]D25 +60.3 (c 2.0, H2O)}.
N-tert-Butoxycarbonyl-trans-4-trifluoromethylsulfonyloxy-L-
proline methyl ester 4bЈ
The alcohol 4a 32 (0.24 g, 1.0 mmol) was dissolved in dry dichlo-
romethane (10 ml) in a dry, round-bottomed flask fitted with a
septum under argon. This solution was stirred and cooled in an
ice–salt-bath at Ϫ10 ЊC. Anhydrous pyridine (100 µl, 1.25 mmol)
was added via a microsyringe, followed by trifluoromethane-
sulfonic anhydride (210 µl, 1.25 mmol). A white precipitate
immediately formed, while the solution turned yellow. After the
reaction mixture had been stirred for 2 h, it was allowed to
warm to ambient temperature and was stirred for another 1 h.
The reaction mixture was evaporated to dryness, and the resi-
due was extracted several times with 1:1 diethyl ether–hexane.
The resulting light yellow solution was passed through a short
silica gel column and the column was washed with more 1:1
diethyl ether–hexane. The solution obtained was evaporated to
dryness to give the title compound as a solid (0.30 g, 79%), mp
53–55 ЊC (from diethyl ether–hexane); δH (200 MHz; CDCl3)
1.35–1.60 (9 H, 2 × s, Boc rotamers), 2.25–2.70 [2 H, br m,
CH2(3)], 3.75 (3 H, s, Me), 3.80–4.05 [2 H, m, CH2(5)], 4.35–
4.65 [1 H, m, CH(2)] and 5.40–5.55 [1 H, m, CH(4)]; δF (235.35
MHz; CDCl3) Ϫ76.86 and Ϫ76.90 (2 × s, CF3SO2 rotamers);
m/z [CI(NH3)] 395 (M + NH4+, 4%), 378 (M + H+, 1), 339
(M Ϫ C4H8 + NH4+, 16), 278 ([M Ϫ Boc + 2 H]+, 44), 128
{[C4H6N(CO2Me) + H]+, 100} and 68 (C4H5N + H+, 58);
νmax(KBr)/cmϪ1 1742 (C᎐O ester), 1687 (C᎐O urethane), 1410
N-tert-Butoxycarbonyl-trans-4-hydroxy-D-proline methyl ester
4d
DEAD (360 µl, 2.0 mmol ) was added dropwise to a stirred
solution of the cis--alcohol 4c21 (0.418 g, 1.71 mmol), triphe-
nylphosphine (0.538 g, 2.0 mmol) and formic acid (75 µl, 2.0
mmol) in THF (10 ml) at Ϫ15 ЊC. The reaction mixture was
warmed to room temperature and was stirred for 5 h. The clear
solution was evaporated to dryness, the residue was treated with
diethyl ether, and the mixture was filtered to remove triphenyl-
phosphine oxide. The filtrate was evaporated to dryness and the
residue chromatographed on a silica gel column with dichloro-
methane–acetone (20:1) as eluent. N-tert-Butoxycarbonyl-
trans-4-formyloxy--proline methyl ester (Rf 0.52) was obtained
as a clear oil (0.431 g). This was dissolved in methanol (20 ml)
containing conc. aq. ammonia (250 µl) and the reaction mixture
was stirred at room temperature for 2 h. The solvents were
removed under reduced pressure and the residue was purified
by column chromatography (SiO2; ethyl acetate) to give N-tert-
butoxycarbonyl-trans-4-hydroxy--proline methyl ester 4d as
an oil (0.310 g, 78% from 4c) (Found: C, 53.6; H, 8.1; N, 5.7.
Calc. for C11H19NO5: C, 53.9; H, 7.8; N, 5.7%); δH (500 MHz;
CDCl3) 1.42 and 1.47 (9 H, 2 × s, Boc rotamers), 2.08 and 2.29
[2 H, m, CH2(3)], 3.40–3.70 [2 H, m, CH2(5)], 3.75 (3 H, s, Me
ester), 4.42 [1 H, br m, CH(2)] and 4.52 [1 H, br m, CH(4)];
δC(50.28 MHz; CDCl3) 28.1 and 28.2 (Boc CH3 rotamers), 38.3
and 38.9 [CH2(3) rotamers], 52.0 and 52.2 (CO2CH3), 54.6
[CH2(5)], 57.5 and 57.9 [CH(2) rotamers], 69.2 and 69.9 [CH(4)
rotamers], 80.3 and 80.5 (Boc C rotamers), 154.3 (Boc CO) and
174.1 (ester CO); m/z [CI(NH3)] 246 (M + H+, 18%), 207
([M Ϫ C4H8 + NH4]+, 19), 190 ([M Ϫ C4H8 + H]+, 100), 146
([M Ϫ Boc + 2 H]+, 53), 86 (69); νmax(neat)/cmϪ1 3439br (O–H),
᎐
᎐
(᎐SO2O᎐) and 1142 (᎐SO2O᎐).
N-tert-Butoxycarbonyl-trans-4-p-tolylsulfonyloxy-L-proline
methyl ester 4b
A solution of N-Boc-trans-hydroxy--proline methyl ester 4a
(4.90 g, 20.0 mmol) in dry pyridine (10 ml) was cooled in an
ice-bath. Recrystallised toluene-p-sulfonyl (tosyl) chloride
(4.00 g, 21.0 mmol) was added with stirring of the mixture,
which was continued until the reaction mixture became homo-
geneous. This solution was kept in a refrigerator at 0 ЊC for
48 h. The reaction mixture was poured into a solution of citric
acid (40 g) in ice-cold water (250 ml) and extracted with diethyl
ether (4 × 50 ml). The combined organic phase was dried
(MgSO4) and evaporated to give the crude product as a solid
(6.93 g, 87%). Recrystallisation from diethyl ether–light petrol-
eum (distillation range 40–60 ЊC) gave an analytical sample N-
tert-butoxycarbonyl-trans-4-p-tolylsulfonyloxy--proline methyl
ester, mp 78–79 ЊC (Found: C, 54.0; H, 6.1; N, 3.3. C18H25NO7S
requires C, 54.1; H, 6.3; N, 3.5%); δH (200 MHz; CDCl3) 1.39
and 1.42 (9 H, 2 × s, Boc rotamers), 2.00–2.60 [2 H, br m,
CH2(3)], 2.46 (3 H, s, tosyl CH3), 3.50–4.00 [2 H, br m, CH2(5)],
3.71 (3 H, s, Me ester), 4.35 [1 H, m, CH(2)], 5.03 [1 H, br m,
CH(4)], 7.36 [2 H, d, J 7.9, tosyl H(3,5)] and 7.78 [2 H, d, J 7.9,
tosyl H(2,6)]; δC(50.28 MHz; CDCl3) 21.5 (tosyl CH3), 28.0
542
J. Chem. Soc., Perkin Trans. 1, 1997

View Article Online
1748s (C᎐O ester) and 1678s (C᎐O urethane); [α]24 +78.7 (c
68%) which was identical with the material obtained from the
᎐
᎐
D
0.625, MeOH).
Mitsunobu reaction.
N-tert-Butoxycarbonyl-cis-4-(N 6-benzoyladenin-9-yl)-L-proline
methyl ester 5a and N-tert-butoxycarbonyl-cis-4-(N6-benzoyl-
adenin-9-yl)-D-proline methyl ester 6a (by Mitsunobu reaction)
DEAD (2.0 ml, 12 mmol ) was added dropwise to a cooled (ice-
bath), stirred THF suspension (25 ml) of benzoyladenine (2.60
g, 11.0 mmol), the alcohol 4a (2.45 g, 10.0 mmol) and tri-
phenylphosphine (2.94 g, 11.0 mmol). The reaction mixture was
warmed to room temperature and stirred for 48 h. Since the
benzoyladenine was still incompletely dissolved, a further 0.5
mole equivalents of DEAD (1.0 ml, 6.0 mmol) and triphenyl-
phosphine (1.47 g, 5.5 mmol) were added and stirring was
continued for another 24 h. The clear orange solution was
evaporated to dryness and the residue was chromatographed
on silica gel with dichloromethane–acetone (3:1) as eluent. N-
tert-Butoxycarbonyl-cis-4-(N 6-benzoyladenin-9-yl)--proline
methyl ester 5a (RF 0.17) was obtained as a foam (1.58 g, 34%)
(Found: C, 59.1; H, 5.7; N, 18.3. C23H26N6O5 requires C, 59.2;
H, 5.6; N, 18.0%); δH (500 MHz; CDCl3) 1.45 (9 H, br s, Boc),
2.58 and 3.00 [2 H, br m, CH2(3Ј)], 3.72 (3 H, 2 × br s, CO2CH3
rotamers), 3.60–4.55 [3 H, br m, CH(2Ј) and CH2(5Ј)], 5.27 [1
H, br m, CH(4Ј)], 7.53 (2 H, t, J 7.4, benzoyl m-CH ), 7.65 (1 H,
t, J 7.4, benzoyl p-CH), 8.03 (2 H, d, J 7.5, benzoyl o-CH),
8.21 [1 H, s, CH(8)], 8.81 [1 H, s, CH(2)] and 9.01 (1 H, br s,
benzamide NH); δC(50.28 MHz; CDCl3) 28.0 (Boc CH3), 34.5
and 35.5 [CH2(3Ј) rotamer], 50.0 and 50.5 [CH2(5Ј) rotamer],
52.0 [CH(4Ј)], 52.5 (CH3 ester), 57.5 [CH(2Ј)], 81.0 [Boc
(CH3)3C], 123.5 [C(5)], 127.0 and 127.5 (benzoyl CH), 132.8
(benzoyl CH), 133.7 (benzoyl C), 141.5 [CH(8)], 150.0 [C(4)],
152.0 [C(6)], 152.5 [CH(2)], 153.5 (Boc CO), 165.5 (benz-
amide CO) and 172.5 (ester CO); m/z (FAB+) 467
N-tert-Butoxycarbonyl-cis-4-(N 4-benzoylcytosin-1-yl)-L-proline
methyl ester 5b and N-tert-butoxycarbonyl-cis-4-[4-(benzoyl-
amino)pyrimidin-2-yloxy]-L-proline methyl ester
A suspension of the toluene-p-sulfonate 4b (0.40 g, 1.0 mmol),
N 4-benzoylcytosine (0.54 g, 2.5 mmol), anhydrous K2CO3 (0.70
g, 5.0 mmol) and 18-crown-6 (100 mg, cat.) in DMF (5 ml) was
stirred at 70–80 ЊC under argon for 6.5 h. The reaction mixture
was then diluted with dichloromethane (50 ml) and washed
with water (4 × 50 ml). The organic layer was dried (MgSO4),
and filtered through Celite. The filtrate was evaporated to
give an oil, which was chromatographed on silica gel with
5% methanol in ethyl acetate as eluent. The more polar frac-
tions (RF 0.39) were combined and evaporated to give N-
tert-butoxycarbonyl-cis-4-(N 4-benzoylcytosin-1-yl)--proline
methyl ester 5b as a foam (134 mg, 31%). Recrystallisation from
ethanol–water gave a crystalline solid mp 159–161 ЊC (Found:
C, 59.5; H, 5.9; N, 12.6. C22H26N4O6 requires C, 59.7; H, 5.9; N,
12.7%); δH (200 MHz; CDCl3) 1.39 (9 H, br m, Boc), 2.20 and
2.80 [2 H, 2 × m, CH2(3Ј)], 3.69 and 3.96 [2 H, 2 × m, CH2(5Ј)],
3.70 (3 H, br s, Me ester), 4.31–4.35 [1 H, 2 × m, CH(2Ј)], 5.23
[1 H, br m, CH(4Ј)], 7.35–7.58 [4 H, m, benzoyl CH and
CH(5)], 7.86–7.94 [3 H, m, benzoyl CH and CH(6)] and 9.11 (1
H, br s, amide NH); δC(50.28 MHz; CDCl3) 28.1 (Boc CH3),
35.8 [CH2(3Ј)], 49.5 and 50.2 [CH2(5Ј) rotamers], 52.3 (ester
CH3), 54.4 and 54.9 [CH(4Ј) rotamers], 57.4 [CH(2Ј)], 81.2 (Boc
C), 96.9 [CH(5)], 127.9 and 129.1 (benzoyl CH), 133.3 (benzoyl
C), 145.8 and 146.0 [CH(6)], 153.6 (Boc CO), 155.8 [C(2)],
162.4 [C(4)], 167.1 (benzamide CO) and 172.9 (ester CO); m/z
[CI(NH3)] 443 (M + H+, 100%), 387 ([M Ϫ C4H8 + H]+, 10),
343 ([M Ϫ Boc + 2 H]+, 12), 227 (14), 216 ([BzC + H]+, 29), 127
([M Ϫ Boc Ϫ BzC + H]+, 95), 105 (PhCO+, 51) and 68
(M + H+, 14%), 411 ([M Ϫ C4H8 + H]+, 34), 240 ([BzA + H]+,
+
18), 105 (PhCO+, 47), 68 ([C4H5N + H]+, 20) and 57 (C4H9
,
(C4H5N + H+, 52); νmax(KBr)/cmϪ1 1755 and 1683s (C᎐O);
᎐
100); νmax(KBr)/cmϪ1 3416br (N–H), 1750 and 1703s (C᎐O);
λmax(MeOH)/nm 258 (ε/dm3 molϪ1 cmϪ1 2.9 × 104) and 302
(1.3 × 104); [α]D25 +33.8 (c 0.58, MeOH).
᎐
λmax(MeOH)/nm 279 (ε/dm3 molϪ1 cmϪ1 1.6 × 104); [α]D22 +14.2
(c 1.60, CHCl3).
The less polar fractions (RF 0.68) were combined and re-
chromatographed on silica gel with dichloromethane–acetone
10:1 as eluent (RF 0.40). N-tert-Butoxycarbonyl-cis-4-[4-
(benzoylamino)pyrimidin-2-yloxy]--proline methyl ester was
obtained as an oil (138 mg, 32%), δH (200 MHz; CDCl3) 1.38
and 1.43 (9 H, 2 × s, Boc rotamers), 2.30 and 2.55 [2 H, 2 × m,
CH2(3Ј)], 3.63 and 3.88 [2 H, 2 × m, CH2(5Ј)], 3.69 and 3.71 (3
H, 2 × s, Me ester), 4.33–4.53 [1 H, 2 × m, CH(2Ј) rotamers],
5.37 [1 H, m, CH(4Ј)], 7.43–7.60 (3 H, m, benzoyl m- and
p-CH), 7.85–7.95 [3 H, m, benzoyl o-CH and CH(5)], 8.36–8.40
[1 H, m, CH(6)] and 8.78–8.80 (1 H, 2 × s, benzamide NH
rotamers); δC(50.28 MHz; CDCl3) 28.1 and 28.2 (Boc CH3
rotamers), 35.2 and 36.0 [CH2(3Ј) rotamers], 51.5 and 52.2
[CH2(5Ј) rotamers], 52.3 (ester CH3), 57.4 and 57.7 [CH(2Ј)
rotamers], 74.1 and 75.2 [CH2(4Ј) rotamers], 80.3 (Boc C),
104.6 [CH(5)], 127.6 and 129.1 (benzoyl CH), 133.1 (benzoyl
CH), 133.4 (benzoyl C), 153.9 and 154.4 (Boc CO rotamers),
159.7 [C(2)], 160.7 and 160.8 [CH(6) rotamers], 163.9 [C(4)],
166.5 (benzamide CO) and 172.6 and 172.9 (ester CO
rotamers); m/z [CI(NH3)] 443 (M + H+, 16%), 387 ([M Ϫ
C4H8 + H]+,15), 343 ([M Ϫ Boc + 2 H]+, 14), 216 ([BzC + H]+,
100), 127 ([M Ϫ Boc Ϫ BzC + H]+, 47), 105 (PhCO+, 86) and 68
N-tert-Butoxycarbonyl-cis-4-(N6-benzoyladenin-9-yl)--
proline methyl ester 6a was similarly prepared as a foam in 23%
yield starting from the alcohol 4d (1.0 mmol scale reaction)
(Found: C, 58.9; H, 6.1; N, 17.4. C23H26N6O5ؒ0.25H2O requires
C, 58.7; H, 5.7; N, 17.8%); δH (200 MHz; CDCl3) 1.45 (9 H, br s,
Boc), 2.56 and 3.00 [2 H, br m, CH2(3Ј)], 3.72 (3 H, 2 × br s,
CH3 ester), 3.60–4.55 [3 H, br m, CH(2Ј) and CH2(5Ј)], 5.27 [1
H, br m, CH(4Ј)], 7.53 (2 H, t, J 7.4, benzoyl m-CH), 7.65 (1 H,
t, J 7.4, benzoyl p-CH), 8.03 (2 H, d, J 7.5, benzoyl o-CH), 8.21
[1 H, s, CH(8)], 8.81 [1 H, s, CH(2)] and 9.01 (1 H, br s,
benzamide NH); δC(50.28 MHz; CDCl3) 28.1 (Boc CH3), 34.5
and 36.0 [CH2(3Ј) rotamer], 50.1 and 50.8 [CH2(5Ј) rotamer],
52.0 [CH(4Ј)], 52.1 (CH3 ester), 57.5 [CH(2Ј)], 81.2 [Boc
(CH3)3C], 123.1 [C(5)], 128.1 and 128.9 (benzoyl CH), 132.9
(benzoyl CH), 133.8 (benzoyl C), 141.4 [CH(8)], 149.9 [C(4)],
152.3 [C(6)], 152.8 [CH(2)], 153.5 (Boc CO), 165.2 (benzamide
CO) and 172.6 (ester CO); m/z (ES MS) 467 (M + H+, 100%)
and 411 ([M Ϫ C4H8 + H]+, 12); νmax(KBr)/cm-1 3257br (N–H),
1751 (C᎐O), 1701 (C᎐O); λmax(CHCl3)/nm 285 (ε/dm3 molϪ1
᎐
᎐
cmϪ1 1.65 × 104); [α]D22 Ϫ11.6 (c 1.6, CHCl3).
N-tert-Butoxycarbonyl-cis-4-(N 6-benzoyladenin-9-yl)-L-
(C4H5N + H+, 56); νmax(KBr)/cmϪ1 1757 and 1698s (C᎐O);
᎐
proline methyl ester 5a (by displacement of toluene-p-sulfonate 4b)
A mixture of the toluene-p-sulfonate 4b (0.400 g, 1.00 mmol),
N 6-benzoyladenine (0.595 g, 2.50 mmol), anhydrous K2CO3
(0.350 mg, 2.50 mmol) and 18-crown-6 (100 mg) in DMF (5 ml)
was stirred under argon at 80 ЊC overnight. Water (20 ml) was
added to the reaction and the suspension was extracted with
dichloromethane. The organic phase was washed with water,
dried (MgSO4) and evaporated to give the crude pruduct, which
was purified by column chromatography (SiO2; 5% methanol in
dichloromethane) to give compound 5a as a foam (0.316 g,
λmax(MeOH)/nm 238 (ε/dm3 molϪ1 cmϪ1 2.2 × 104) and 280
(2.7 × 104); [α]D25 Ϫ49.5 (c 0.62, MeOH).
N-tert-Butoxycarbonyl-cis-4-(N 3-benzoylthymin-1-yl)-L-proline
methyl ester 5c and N-tert-butoxycarbonyl-cis-4-(N3-benzoyl-
thymin-1-yl)-D-proline methyl ester 6b
To a suspension of the alcohol 4a (0.245 g, 1.0 mmol), N 3-
benzoylthymine (0.230 g, 1.0 mmol) and triphenylphosphine
(294 mg, 1.1 mmol) in dry THF (10 ml) was added DEAD (182
µl, 1.1 mmol) dropwise at Ϫ15 ЊC. The reaction mixture was
J. Chem. Soc., Perkin Trans. 1, 1997
543

View Article Online
stirred at room temperature overnight. The clear solution
was evaporated to dryness and the residue was purified by
column chromatography (SiO2; dichloromethane–acetone 20:1)
to give N-tert-butoxycarbonyl-cis-4-(N 3-benzoylthymin-1-yl)--
proline methyl ester 5c (RF 0.34) as a foam (0.193 g, 42%),
δH (200 MHz; CDCl3) 1.40 (9 H, br s, Boc), 1.90 (3 H, s, thymine
CH3) 2.10 and 2.71 [2 H, 2 × br m, CH2(3Ј)], 3.61 [1 H, m,
CHaHb(5Ј)], 3.72 (3 H, s, Me ester), 3.90 [1 H, dd, J 8.0 and
11.5, CHaHb(5Ј)], 4.26 [1 H, m, CH(2Ј)], 5.18 [1 H, br m,
CH(4Ј)], 7.37 [1 H, s, CH(6)], 7.44 (2 H, t, J 7.7, benzoyl m-H),
7.60 (1 H, t, J 7.4, benzoyl p-H) and 7.86 (2 H, d, J 7.1, benzoyl
o-H); δC(50.28 MHz; CDCl3) 12.4 (thymine CH3), 28.1 (Boc
CH3), 35.1 and 33.8 [CH2(3Ј) rotamers], 49.1 and 49.5
[CH2(5Ј) rotamers], 52.4 [CH(4Ј) and ester CH3], 57.3 [CH2(2Ј)
rotamers], 81.1 (Boc C), 111.6 [C(5)], 129.3 and 130.5 (2 ×
benzoyl CH), 131.6 (benzoyl C), 135.3 (benzoyl p-CH), 136.5
[CH(6)], 150.1 [C(2)], 153.1 (Boc CO), 162.7 [C(4)], 169.2 (CO
amide) and 173.1 (ester CO); m/z [CI(NH3)] 458 (M + H+,
7%), 419 ([M Ϫ C4H8 + NH4]+, 8), 402 ([M Ϫ C4H8 + H]+, 16),
358 ([M Ϫ Boc + 2 H]+, 69), 254 ([M Ϫ Boc Ϫ PhCO + H]+, 53),
127 (T + H+ and [M Ϫ BzT Ϫ Boc + H]+, 100), 105 (PhCO+,
100) and 68 (C4H5N + H+, 72); νmax(KBr)/cmϪ1 1752s, 1701s
[(CH3)2CH], 37.2 and 36.5 [CH2(3Ј) rotamers], 50.5 and 51.4
[CH2(5Ј) rotamers], 52.2 (ester CH3), 54.6 and 55.2 [CH(4Ј)
rotamers], 57.3 and 57.5 [CH2(2Ј) rotamers], 81.1 (Boc C),
111.9 [C(5)], 141.6 [CH(8)], 148.4 [C(2)/C(6)], 153.5 (Boc CO),
157.6 [C(6)/C(2)], 163.1 [C(4)], 172.6 (ester CO) and 180.6
(amide CO); m/z [CI(NH3)] 449 (M + H+, 6%), 349
([M Ϫ Boc + 2 H]+, 5), 222 (IbuG + H+, 100), 152 (G + H+, 14),
127 ([M Ϫ Boc Ϫ IbuG + H]+, 23) and 68 (C4H5N + H+, 29);
νmax(KBr)/cmϪ1 1758s and 1688s (C᎐O); λmax(MeOH)/nm 264sh
᎐
(ε/dm3 molϪ1 cmϪ1 1.5 × 104); [α]D25 +36.2 (c 0.16, MeOH).
N-tert-Butoxycarbonyl-cis-4-(N 2-isobutyrylguanin-9-yl)-L-
proline methyl ester 5d
To a suspension of the alcohol 4a (245 mg, 1.0 mmol), N 2-
isobutyryl-O 6-[(4-nitrophenyl)ethyl]guanine (180 mg, 0.50
mmol) and triphenylphosphine (294 mg, 1.1 mmol) in dry 1,4-
dioxane (10 ml) was added DEAD (182 µl, 1.1 mmol) drop-
wise at room temperature. The mixture was stirred at room
temperature overnight. The clear solution was evaporated to
dryness and the residue was purified by column chromato-
graphy (SiO2; ethyl acetate) to give the N-tert-butoxycarbonyl-
cis-4-(N 2-isobutyryl-O 6-nitrophenylethylguanin-9-yl)--proline
methyl ester (RF 0.39) which was contaminated with triphenyl-
phosphine oxide. The impure material was dissolved in dry
pyridine (5 ml), treated with DBU (150 µl, 1.0 mmol) and
stirred at room temperature overnight. The reaction mixture
was diluted with dichloromethane, and washed successively
with 5% HCl and water. The organic phase was dried
(MgSO4) and evaporated to give the crude product. This was
purified by column chromatography (SiO2; ethyl acetate–
methanol 10:1) to give the product as a foam (76 mg,
34% from 4a). Crystallisation from ethanol–water gave N-
tert-butoxycarbonyl-cis-4-(N2-isobutyrylguanin-9-yl)--proline
methyl ester 5d as needles (dihydrate), mp 132–134 ЊC
(Found: C, 49.6; H, 6.7; N, 17.4. C20H28N6O6ؒ2H2O requires
C, 49.8; H, 6.9; N, 17.3%); δH (200 MHz; CDCl3) 1.22 [6 H, d,
J 6.9, (CH3)2CH], 1.39 (9 H, br s, Boc), 2.44 [1 H, br m,
CHaHb(3Ј)], 2.75–2.88 [2 H, m, CHaHb(3Ј) and (CH3)2CH],
3.69 (3 H, s, Me ester), 3.81 [1 H, dd, J 7.8 and 11.1,
CHaHb(5Ј)], 4.09 [1 H, m, CHaHb(5Ј)], 4.37 [1 H, m, CH(2Ј)],
4.94 [1 H, br m, CH(4Ј)] and 7.77 [1 H, s, CH(8)]; δC(50.28
MHz; CDCl3) 18.9 [(CH3)2CH], 28.1 (Boc CH3), 35.6 and
34.8 [CH2(3Ј) rotamers], 36.0 [(CH3)2CH], 50.0 and 50.3
[CH2(5Ј) rotamers], 52.0 and 52.4 [CH(4Ј) rotamers], 52.5
(CH3 ester), 57.4 and 57.5 [CH2(2Ј) rotamers], 81.1 (Boc C),
121.2 [C(5)], 137.3 [CH(8)], 148.1 and 149.1 [C(2) and
C(6)], 153.8 (Boc CO), 156.0 [C(4)], 172.1 (ester CO), 180.1
(amide CO); m/z [CI(NH3)] 449 (M + H+, 15%), 349
([M Ϫ Boc + 2 H]+, 47), 222 (IbuG + H+, 100), 152 (G + H+,
28), 127 ([M Ϫ IbuG Ϫ Boc + H]+, 18) and 68 (C4H5N + H+,
and 1659s (C᎐O); λmax(MeOH)/nm 253sh (ε/dm3 molϪ1 cmϪ1
᎐
1.8 × 104); [α]D25 +3.85 (c 0.52, MeOH).
N-tert-Butoxycarbonyl-cis-4-(N3-benzoylthymin-1-yl)--
proline methyl ester 6b was similarly obtained as a foam in 42%
yield starting from compound 4d (0.95 mmol scale reaction),
mp 84–86 ЊC (Found: C, 59.8; H, 6.0; N, 8.9. C23H27N3O7ؒ
0.25H2O requires C, 59.8; H, 6.0; N, 9.1%); δH (200 MHz;
CDCl3) 1.43 (9 H, br s, Boc), 1.94 (3 H, s, thymine CH3) 2.13
and 2.71 [2 H, 2 × br m, CH2(3Ј)], 3.58–3.66 [1 H, br m,
CHaHb(5Ј)], 3.75 (3 H, s, Me ester), 3.93 [1 H, dd, J 8.0 and
11.5, CHaHb(5Ј)], 4.29–4.33 [1 H, br m, CH(2Ј)], 5.15–5.18 [1 H,
br m, CH(4Ј)], 7.38 [1 H, s, CH(6)], 7.47 (2 H, t, J 7.7, benzoyl
m-H), 7.63 (1 H, t, J 7.4, benzoyl p-H) and 7.89 (2 H, d, J 7.1,
benzoyl o-H); δC(50.28 MHz; CDCl3) 12.4 (thymine CH3), 28.1
(Boc CH3), 34.0 and 35.3 [CH2(3Ј) rotamers], 49.1 and 49.5
[CH2(5Ј) rotamers], 52.2 and 52.4 [CH(4Ј) and ester CH3],
57.4 [CH2(2Ј) rotamers], 81.2 (Boc C), 111.6 and 111.7 [C(5)
rotamers], 129.4 and 130.6 (2 × benzoyl CH), 131.6 (benzoyl
C), 135.3 (benzoyl p-CH), 136.4 [CH(6)], 150.2 [C(2)], 153.5
(Boc CO), 162.7 [C(4)], 169.2 (CO amide) and 173.2 (ester CO);
m/z (APCI) 458 (M + H+, 20%), 402 ([M Ϫ C4H8 + H]+, 59),
358([M Ϫ Boc + 2H]+,98),254([M Ϫ Boc Ϫ PhCO + H]+,100),
127 (T + H+ and [M Ϫ BzT Ϫ Boc + H]+, 9) and 105 (PhCO+,
25); νmax(KBr)/cmϪ1 1750s, 1700s and 1660s (C᎐O); [α]21 Ϫ1.05
᎐
D
(c 1.05, MeOH).
N-tert-Butoxycarbonyl-cis-4-(N 2-isobutyrylguanin-7-yl)-L-
proline methyl ester
A suspension of the toluene-p-sulfonate 4b (0.40 g, 1.0 mmol),
N 2-isobutyrylguanine (dried by azeotropic distillation with
toluene) (0.55 g, 2.5 mmol), anhydrous K2CO3 (0.70 g, 5.0
mmol) and 18-crown-6 (100 mg) in DMF was stirred at 70 ЊC
under argon for 48 h. The reaction mixture was worked up as
usual to give a foam which was chromatographed on silica gel
eluted first with ethyl acetate and then with 5% methanol
in ethyl acetate. The less polar fractions (RF 0.41, 10% methanol
in ethyl acetate) were combined and evaporated to give the N7-
isomer (121 mg, 27%) as a foam which could be recrystallised
from ethanol–water to give N-tert-butoxycarbonyl-cis-4-(N2-
isobutyrylguanin-7-yl)--proline methyl ester as shiny plates
(monohydrate), mp >200 ЊC (Found: C, 51.3; H, 6.8; N, 17.9.
C20H28N6O6ؒH2O requires C, 51.5; H, 6.5; N, 18.0%); δH (200
MHz; CDCl3) 1.27 [6 H, d, J 7.0, (CH3)2CH], 1.45 (9 H, br s,
Boc), 2.44 and 2.95 [2 H, 2 × m, CH2(3Ј)], 2.75 [1 H, m,
(CH3)2CH], 3.70 (3 H, br s, Me ester), 3.90 and 4.14 [2 H,
2 × m, CH2(5Ј)], 4.44 [1 H, m, CH(2Ј)], 5.46 [1 H, br m,
CH(4Ј)], 8.00 [1 H, s, CH(8)] and 9.10 (1 H, br s, amide NH);
δC(50.28 MHz; CDCl3) 18.9 [(CH3)2CH], 28.1 (Boc CH3), 35.8
55); νmax(KBr)/cmϪ1 1737s, 1708s and 1673s (C᎐O);
᎐
λmax(MeOH)/nm 260sh (ε/dm3 molϪ1 cmϪ1 2.8 × 104) and 278
(2.0 × 104); [α]D25 Ϫ2.43 (c 0.54, MeOH).
N-(2-Hydroxyethyl)glycine
To a swirled aqueous solution of ethanolamine (30.5 g, 0.500
mol in 25 ml) was gradually added solid chloroacetic acid (18.9
g, 0.200 mol). The reaction mixture was stored overnight at
room temperature, then was evaporated to give a thick oil.
Ethanol (95%; 2 l) was added with heating to dissolve the oil
and the solution was kept in a refrigerator until precipitation
was complete. The crude product was collected by filtration and
was recrystallised from water–ethanol to give crystals (10.3 g,
43%), mp 186–188 ЊC [lit.,27 182–184 ЊC (decomp.)] (Found: C,
40.3; H, 8.0; N, 11.8. Calc. for C4H9NO3: C, 40.3; H, 7.6; N,
11.8%); δH (200 MHz; D2O) 2.99 (2 H, dd, J 5.3 and 5.0,
CH2CH2NH), 3.43 (2 H, s, CH2CO2H) and 3.64 (2 H, dd, J 5.3
and 5.0, HOCH2CH2); δC(50.28 MHz; D2O) 48.7 (CH2CH2NH
and CH2CO2H), 56.4 (HOCH2CH2) and 171.4 (CO2H); m/z
[CI(NH3)] 120 (100%, M + H+).
544
J. Chem. Soc., Perkin Trans. 1, 1997

View Article Online
N-tert-Butoxycarbonyl-N-(2-hydroxyethyl)glycine
155.3 and 155.9 [C(6) and Boc CO], 170.6 and 171.0 (ester CO
N-(2-Hydroxyethyl)glycine (1.19 g, 10.0 mmol) was added to
stirred 10% aq. NaOH (6 ml). tert-Butyl alcohol (6 ml) was then
added, followed by di-tert-butyl dicarbonate (2.82 g, 12.5
mmol) dropwise. The solution was stirred at room temperature
overnight. The reaction mixture was evaporated to dryness and
the residue was taken up in water and extracted several times
with chloroform to remove any excess of di-tert-butyl dicarbo-
nate. The solution was then acidified to pH 2 with 10% aq.
KHSO4 and was extracted with ethyl acetate (5 × 40 ml). The
combined organic extracts were dried (MgSO4) and evaporated
to give the crude protected amino acid as a solid. Trituration
with hexane and filtration gave the product as a solid (1.98 g,
90%). A portion of the sample was recrystallised from ethyl
acetate–light petroleum (distillation range 40–60 ЊC) to give
crystals of N-tert-butoxycarbonyl-N-(2-hydroxyethyl)glycine,
mp 78–81 ЊC (Found: C, 49.2; H, 7.9; N, 6.3. C9H17NO5
requires C, 49.3; H, 7.8; N, 6.4%); δH (200 MHz; CDCl3) 1.43
and 1.50 (9 H, 2 × s, Boc rotamers), 3.47 [2 H, br m, CH2CH2N-
(Boc)] and 3.76 (2 H, br m, HOCH2CH2), 3.96 and 4.10 (2 H,
2 × s, CH2CO2H); δC(50.28 MHz; CDCl3) 28.0 and 28.1 (Boc
CH3 rotamers), 50.4–51.0 (CH2CO2H rotamers), 51.5 and 52.0
[CH2CH2N(Boc) rotamers], 60.6 (HOCH2CH2), 81.3 (Boc C),
155.9 and 156.2 (Boc CO rotamers) and 174.6 and 174.9 (CO2H
rotamers); m/z [CI(NH3)] 237 (M + NH4+, 18%), 220 (M +
H+, 100), 181 ([M Ϫ C4H8 + NH4]+, 70), 164 ([M Ϫ
C4H8 + H]+, 96), 120 ([M Ϫ Boc + 2 H]+, 70), 88 ([M Ϫ Boc Ϫ
CH2OH + H]+, 25), 74 ([M Ϫ Boc Ϫ CH2CH2OH + H]+, 20)
rotamers); m/z [CI(NH3)] 351 (M + H+, 100%), 251
([M Ϫ Boc + 2 H]+, 15), 136 (A + H+, 16) and 115
([M Ϫ Boc Ϫ A]+, 8); νmax(KBr)/cmϪ1 1754s (C᎐O ester) and
᎐
1696s (C᎐O urethane).
᎐
N-tert-Butoxycarbonyl-N-[2-(N 6,N 6-dibenzoyladenin-9-yl)-
ethyl]glycine methyl ester and N-tert-butoxycarbonyl-N-[2-(N 6-
benzoyladenin-9-yl)ethyl]glycine 10
The adenine derivative 8 (0.315 g, 0.898 mmol) was dissolved in
dry pyridine (8 ml) and the solution was cooled in an ice-bath.
Benzoyl chloride (520 µl, 4.50 mmol) was then added portion-
wise with stirring of the mixture, which was then stirred at 4 ЊC
overnight and diluted with dichloromethane (50 ml). The solu-
tion was washed successively with 10% aq. HCl, saturated aq.
NaHCO3 and water. Evaporation followed by column chrom-
atography of the residue (SiO2; EtOAc) gave the dibenzoylated
product as an oil (0.533 g, quant.); δH (200 MHz; CDCl3) 1.17
and 1.34 (9 H, 2 × s, Boc rotamers), 3.67 [5 H, m, Me ester
and CH2CH2N(Boc)], 3.72 and 3.86 (2 H, 2 × s, CH2CO2Me
rotamers), 4.31–4.44 (2 H, m, ACH2CH2), 7.29 (4 H, t, J 7.1,
benzoyl m-CH), 7.42 (2 H, t, J 7.1, benzoyl p-CH), 7.83 (4 H, d,
J 7.4, benzoyl o-CH), 8.23 and 8.32 [1 H, 2 × s, CH(8) rotamers]
and 8.61 and 8.63 [1 H, 2 × s, CH(2) rotamers]; δC(50.28 MHz;
CDCl3) 27.8 and 28.0 (Boc CH3 rotamers), 42.7 and 42.9
(CH2CO2 rotamers), 48.3 [CH2CH2N(Boc)], 49.5 and 50.6
(ACH2CH2), 52.2 (ester CH3), 81.1 and 81.2 (Boc C rotamers),
127.5 [C(5)], 128.3–134.4 (benzoyl CH), 146.2 [CH(8)], 151.8
[C(4)], 153.7 [C(2)], 155.2 (Boc CO), 169.0 [C(6)], 170.7 and
171.0 (benzoyl CO rotamers) and 172.6 (ester CO); m/z
[CI(NH3)] 581 (M + Na+, 6%), 559 (M + H+, 100), 503
([M Ϫ C4H8 + H]+, 48), 459 ([M Ϫ Boc + 2 H]+, 7), 455
([M Ϫ Bz + 2 H]+, 17), 437 (46), 399 ([M Ϫ C4H8 Ϫ Bz + 2 H]+,
13), 355 ([M Ϫ Bz Ϫ Boc + 3 H]+, 30) and 240 (BzA + H+, 11).
The dibenzoyladenine methyl ester (0.211 g, 0.380 mmol) was
dissolved in acetone (2 ml), aq. NaOH (2.0 ; 2 ml) was added
and the reaction mixture was stirred for 3 h at room tem-
perature. The solvents were evaporated off, the residue was
taken up in water (10 ml) and the pH was adjusted to 2 with aq.
KHSO4. The product was obtained as a solid (0.100 g, 60%)
after extraction into ethyl acetate and trituration with diethyl
ether. Recrystallisation from ethanol–water gave compound 10
as a crystalline solid, mp 185–188 ЊC (decomp.) (Found: C,
56.3; H, 5.3; N, 19.0. C21H24N6O5ؒ0.5H2O requires C, 56.1; H,
5.6; N, 18.7%); δH (200 MHz; NaOD/D2O) 0.62 and 0.90 (9 H,
2 × br s, Boc rotamers), 3.46 [2 H, br m, CH2CH2N(Boc)], 3.42
and 3.60 (2 H, 2 × s, CH2CO2H rotamers), 4.16 (2 H, br m,
BzACH2CH2), 7.23–7.26 (3 H, br m, benzoyl m- and p-CH),
7.69–7.72 (2 H, br m, benzoyl o-CH), 7.91 and 7.94 [CH(8)
rotamers] and 8.30 [CH(2)]; m/z (negative ion ES-MS) 439
([M Ϫ H]Ϫ, 100%); νmax(KBr)/cmϪ1 3327 (O–H) and 1693br
and 57 (12); νmax(KBr)/cmϪ1 3218 (O–H) and 1693s (C᎐O).
᎐
N-tert-Butoxycarbonyl-N-(2-hydroxyethyl)glycine methyl ester 7
N-Boc-N-(2-hydroxyethyl)glycine (1.98 g, 9.04 mmol ) was sus-
pended in diethyl ether (~15 ml). Diazomethane (diluted with
nitrogen)33 was bubbled through the ice-cold solution until the
yellow colour persisted. Excess of diazomethane was destroyed
by addition of a few drops of glacial acetic acid. The resulting
clear solution was evaporated under reduced pressure to give
the methyl ester 7 as a clear oil (2.15 g, quant.). The crude
product was used for the next step without further purification:
δH (200 MHz; CDCl3) 1.42 and 1.47 (9 H, 2 × s, Boc rotamers),
3.47 [2 H, br m, CH2CH2N(Boc)], 3.73 (2 H, br m, HOCH2-
CH2), 3.78 (3 H, s, Me ester) and 3.95 and 3.98 (2 H, 2 × s,
CH2CO2H); δC(50.28 MHz; CDCl3) 28.0 and 28.1 (Boc CH3
rotamers), 50.3, 50.8, 51.9, 52.2 and 52.3 [CH2CO2, CH2CH2-
N(Boc) and ester CH3 rotamers], 60.9 and 61.1 (HOCH2CH2
rotamers), 80.7 (Boc C rotamers), 155.7 and 155.9 (Boc CO
rotamers), 172.5 and 172.9 (benzoyl CO).
N-tert-Butoxycarbonyl-N-[2-(adenin-9-yl)ethyl]glycine methyl
ester 8
DEAD (0.18 ml, 1.2 mmol) was added dropwise to a stirred
cooled (ice-bath) THF suspension (15 ml) of adenine (0.171 g,
1.27 mmol), methyl ester 7 (0.235 g, 10.0 mmol) and triphenyl-
phosphine (0.290 g, 1.20 mmol). The reaction mixture was
warmed to room temperature and stirred overnight. The cloudy
suspension was evaporated to dryness and the residue was
chromatographed on silica gel with acetone–methanol (10:1) as
eluent. The product was obtained as a solid (0.093 g, 27%)
which was recrystallised from ethanol to give N-tert-butoxy-
carbonyl-N-[2-(adenin-9-yl)ethyl]glycine methyl ester 8 as a crys-
talline solid, mp 173–175 ЊC (Found: C, 51.5; H, 5.9; N, 23.9.
C15H22N6O4 requires C, 51.4; H, 6.3; N, 24.0%); δH (200 MHz;
CDCl3) 1.10 and 1.30 (9 H, 2 × s, Boc rotamers), 3.55–3.90 [7
H, br m, CH2CH2N(Boc), Me ester and CH2CO2H], 4.33 (2 H,
m, ACH2CH2), 6.40 (2 H, 2 × s, NH2), 7.82 and 8.00 [1 H, 2 × s,
CH(8)] and 8.30 [1 H, s, CH(2)]; δC(50.28 MHz; CDCl3) 27.7
and 27.9 (Boc CH3 rotamers), 42.3 and 42.7 (CH2CO2 rotam-
ers), 48.6 and 48.7 [CH2CH2N(Boc) rotamers], 49.8 and 50.6
(ACH2CH2), 52.1 and 52.2 (ester CH3 rotamers), 81.1 (Boc C),
119.7 [C(5)], 141.3 and 141.4 [CH(8)], 150.3 [C(4)], 153.1 [C(2)],
(C᎐O).
᎐
N-tert-Butoxycarbonyl-N-[2-(N 3-benzoylthymin-1-yl)ethyl]-
glycine methyl ester 9
DEAD (1.80 ml, 11.0 mmol ) was added dropwise to a stirred
cooled (ice-bath) suspension of N 3-benzoylthymine (1.85 g, 8.0
mmol), the alcohol 7 (2.1 g, 9.0 mmol) and triphenylphosphine
(2.90 g, 11.0 mmol) in THF (25 ml). The reaction mixture was
warmed to room temperature and was stirred overnight. The
light yellow solution was evaporated to dryness and the residue
was purified by column chromatography (SiO2; dichloro-
methane–acetone 15:1). The product 9 (RF 0.33) was obtained
as a solid (4.67 g) which may be used for the next step without
further purification. Recrystallisation from ethanol gave N-tert-
butoxycarbonyl-N-[2-(N3-benzoylthymin-1-yl) ethyl]glycine
methyl ester 9 as crystals (2.26 g, 56%), mp 177–179 ЊC (Found:
C, 59.2; H, 6.0; N, 9.3. C22H27N3O7 requires C, 59.3; H, 6.1; N,
9.4%); δH (200 MHz; CDCl3) 1.43 and 1.48 (9 H, 2 × s, Boc
rotamers), 1.93 and 1.98 (3 H, 2 × s, thymine CH3 rotamers),
J. Chem. Soc., Perkin Trans. 1, 1997
545

View Article Online
O. Buchardt, L. Christensen, C. Behrens, S. M. Freier, D. A. Driver,
R. H. Berg, S. K. Kim, B. Norden and P. E. Nielsen, Nature
(London), 1993, 365, 566.
5 J. D. Buttrey, A. S. Jones and R. T. Walker, Tetrahedron, 1975, 31,
73; E. N. Olsufevas, S. V. Zenin and Y. P. Shvachkin, J. Gen. Chem.
USSR, 1979, 49, 1004; S. B. Huang, J. S. Nelson and D. D. Weller,
J. Org. Chem., 1991, 56, 6007; H. De Koning and U. K. Pandit, Recl.
Trav. Chim. Pays-Bas, 1971, 90, 1069.
6 O. Buchardt, M. Egholm, R. H. Berg and P. E. Nielsen, Trends
Biotechnol., 1993, 11, 384.
7 D. D. Weller, D. T. Daly, W. K. Olson and J. E. Summerton, J. Org.
Chem., 1991, 56, 6000.
8 W. G. Richards, A. E. Elcock and G. Lowe, unpublished results.
9 After this work was near completion, a report on the identical
flexible system was published, although it was synthesized by a
rather different approach (see ref. 2d).
10 F. M. Kaspersen and U. K. Pandit, J. Chem. Soc., Perkin. Trans. 1,
1975, 1617, 1798.
3.60 [2 H, br m, CH2CH2N(Boc)], 3.78 (3 H, s, Me ester), 3.85–
4.05 [4 H, br m, CH2CH2N(Boc) and CH2CO2Me], 7.28 [1 H, s,
CH(6)], 7.35–7.70 (3 H, br m, benzoyl m- and p-CH) and 7.85–
7.95 and 8.05–8.15 (2 H, br m, benzoyl o-CH rotamers);
δC(50.28 MHz; CDCl3) 12.2 (thymine CH3), 28.0 and 28.1 (Boc
CH3 rotamers), 47.1 and 47.3 [CH2CH2N(Boc) rotamers], 47.6
and 48.3 (BzTCH2CH2 rotamers), 49.5 and 50.9 (CH2CO2Me
rotamers), 52.2 (ester CH3), 81.0 and 81.5 (Boc C), 109.7 [C(5)],
129.2 and 129.4 (benzoyl m-CH), 130.6 and 131.0 benzoyl o-
CH), 131.7 and 131.9 (benzoyl C rotamers), 135.1 and 135.3
(benzoyl p-CH rotamers), 141.3 and 141.9 [CH(6)], 150.3
[C(2)], 155.4 (Boc CO), 163.9 [C(4)], 169.9 (benzoyl CO) and
171.0 and 171.2 (ester CO); m/z [CI(NH3)] 446 (M + H+,
25%), 390 ([M Ϫ C4H8 + H]+, 97), 346 ([M Ϫ Boc + 2 H]+, 26),
286 ([M Ϫ C4H8 Ϫ PhCO + 2 H]+, 22), 242 ([M Ϫ Boc Ϫ Ph-
CO + 3 H]+, 68), 115 ([M Ϫ BzT Ϫ Boc]+, 52), 105 (PhCO+,
100) and 102 (54); νmax(KBr)/cmϪ1 1751s (C᎐O), 1696s (C᎐O)
11 G. Shaw and R. N. Warrener, J. Chem. Soc., 1958, 157; M. R. Atkin-
son, M. H. Maguire, R. K. Ralph, G. Shaw and R. N. Warrener,
J. Chem. Soc., 1957, 2363; G. Shaw, R. N. Warrener, M. H. Maguire
and R. K. Ralph, J. Chem. Soc., 1958, 2294.
᎐
᎐
and 1639s (C᎐O).
᎐
12 A. R. Todd, J. Chem. Soc., 1946, 647; R. A. Baxter and F. S. Spring,
J. Chem. Soc., 1948, 523.
13 W. A. Thomas and M. K. Williams, J. Chem. Soc., Chem. Commun.,
1972, 994; M. M. Bowers-Nemia and M. M. Joullie, Heterocycles,
1983, 20, 817.
14 The Chemistry of Amides, ed. J. Zabicky and S. Patai, Wiley
Interscience, New York, 1970, pp. 1–72.
15 M. W. Bullock, J. J. Hand and E. L. R. Stockstad, J. Org. Chem.,
1957, 22, 568.
16 M.-T. Chenon, R. J. Pugmire, D. M. Grant, R. P. Panzica and
L. B. Townsend, J. Am. Chem. Soc., 1975, 97, 4627.
17 D. M. Brown, A. Todd and S. Varadajan, J. Chem. Soc., 1956, 2384.
18 M.-J. Perez-Perez, J. Rozenski, R. Busson and P. Herdewijn, J. Org.
Chem., 1995, 60, 1531.
19 K. A. Cruickshank, J. Jiricny and C. B. Reese, Tetrahedron Lett.,
1984, 25, 681.
20 T. F. Jenny, K. C. Schneider and S. A. Benner, Nucleosides, Nucleot-
ides, 1992, 11, 1257.
N-tert-Butoxycarbonyl-N-[2-(thymin-1-yl)ethyl]glycine 11
To a solution of the methyl ester 9 (50 mg, 0.11 mmol) in acet-
one (2 ml) was added aq. NaOH (0.2 ; 2 ml). The solution
was left overnight in a refrigerator, then was evaporated to dry-
ness. The residue was taken up in water (10 ml) and acidified to
pH 2 with 10% aq. KHSO4. The acid was extracted with ethyl
acetate. Evaporation to almost dryness followed by addition of
diethyl ether gave a solid, which was collected by suction fil-
tration and washed several times with diethyl ether. Recrystal-
lisation from ethanol–water gave N-tert-butoxycarbonyl-N-[2-
(thymin-1-yl)ethyl]glycine 11 as a solid (30 mg, 83%), mp 202–
205 ЊC (decomp.) (Found: C, 51.1; H, 6.4; N, 12.8. C14H21N3O6
requires C, 51.4; H, 6.5; N, 12.8%); δH (200 MHz; D2O/NaOD)
1.05 and 1.10 (9 H, 2 × s, Boc rotamers), 1.55 (3 H, s, thymine
CH3), 3.32 [2 H, br m, CH2CH2N(Boc)], 3.40–3.45 and 3.50–
3.60 [4 H, br m, CH2CH2N(Boc) and CH2CO2Me] and 6.95 [1
H, s, CH(6)]; m/z (negative ion ES-MS) 326 ([M Ϫ H]Ϫ, 100%);
21 M. L. Peterson and R. Vince, J. Med. Chem., 1991, 34, 2787.
22 T. F. Jenny, N. Previsani and S. A. Benner, Tetrahedron Lett., 1991,
32, 7029; T. F. Jenny, J. Horlacher, N. Previsani and S. A. Benner,
Helv. Chim. Acta, 1992, 75, 1944.
νmax(KBr)/cmϪ1 1753s, 1732s and 1675s (C᎐O).
᎐
23 O. Mitsunobu, Synthesis, 1981, 1.
24 (a) J. P. Greenstein and M. Winitz, Chemistry of the Amino Acids,
Wiley, New York, 1961, p. 2037; (b) G. L. Baker, S. J. Fritschel,
J. R. Stille and J. K. Stille, J. Org. Chem., 1981, 46, 2954.
25 D. L. Hughes, Org. React., 1992, 42, 335 and references therein.
26 T. W. Greene, Protective Groups in Organic Synthesis, Wiley, New
York, 1981, p. 52.
Acknowledgements
We acknowledge the Thai government’s DPST scholarship (to
T. V.). We also thank Mrs E. McGuinness and Dr T. Claridge
for high field 1H NMR and specialised NMR experiments, Miss
T. Jackson for 19F NMR spectra and Dr R. T. Aplin for electro-
spray mass spectra.
27 Amino Acids and Peptides, ed. J. S. Davies, Chapman and Hall,
London, 1985, p. 195.
28 E. R. H. Jones and W. Wilson, J. Chem. Soc., 1949, 547; F. H. C.
Stewart, J. Org. Chem., 1962, 27, 687; G. E. Struve, C. Gazzola and
G. L. Kenyon, J. Org. Chem., 1977, 42, 4035.
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Paper 6/03696A
Received 28th May 1996
Accepted 3rd October 1996
546
J. Chem. Soc., Perkin Trans. 1, 1997