Dipeptides bearing nucleobases for the synthesis of novel peptide nucleic acids

Article abstract of DOI:10.1039/a603699f

Three stereoisomers (cis-L, trans-D and cis-D) of N-Fmoc-glycyl-4-(thymin-1-y)proline and their pentafluorophenyl esters have been prepared for use in the synthesis of novel peptide nucleic acids. In addition, N-Fmoc-glycyl-4-(N⁶-benzoyladenin-9-yl)proline, N-Fmoc-glycyl-4-(N⁴-benzoylcytosin-1-yl)proline and N-Fmoc-glycyl-4-(N²-isobutyrylguanin-9-yl)proline and their pentafluorophenyl esters of the cis-D series have been synthesized.

Full text of DOI:10.1039/a603699f

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Dipeptides 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
Three stereoisomers (cis-L, trans-D and cis-D ) of N-F moc-glycyl-4-(thymin-1-yl)proline and their
pentafluorophenyl esters have been prepared for use in the synthesis of novel peptide nucleic acids. In
addition, N-F moc-glycyl-4-(N 6-benzoyladenin-9-yl)proline, N-F moc-glycyl-4-(N 4-benzoylcytosin-1-
yl)proline and N-F moc-glycyl-4-(N 2-isobutyrylguanin-9-yl)proline and their pentafluorophenyl esters of
the cis-D series have been synthesized.
Fmoc/OBut strategy. For these reasons, it was decided to use the
Fmoc instead of Boc as the N-protecting group.
Introduction
The design of two peptide nucleic acid systems, one with the
conformationally restricted glycylproline backbone and the
other with the conformationally more flexible glycyl-N-ethyl-
glycine backbone, have been reported together with the prepar-
ation of the protected amino acids bearing nucleobases 1–3 for
use in their synthesis.1
There were two possible synthetic pathways to the target
dipeptide 4, the two amino acids may be coupled first and the
nucleobase attached later by the Mitsunobu reaction or the
nucleobase may be incorporated before the peptide coupling.
The first approach has the advantage of being a more con-
vergent approach. However, a preliminary investigation sug-
gested that it is not satisfactory because of the extensive cleav-
age of the Fmoc group during the Mitsunobu reaction.4 It also
seemed likely that displacement of a tosyloxy group by a nucleo-
base would give similar premature cleavage of the Fmoc group
since the reactions require basic conditions.
A temporary N-protecting group for the hydroxyproline was
required, therefore, which is stable to the basic conditions of
the Mitsunobu reaction but which can be removed, without
disturbing the carboxy-protecting group, in order to allow
coupling with Fmoc-glycine (or other amino acid) to give the
Fmoc-dipeptide 4. As the carboxy-protecting group must be
selectively removed in the presence of the Fmoc group at the
end of the synthesis, an acid-labile protecting group seemed
appropriate. The combination of the acid labile Boc group and
diphenylmethyl (Dpm)5 ester is ideal because introduction
and cleavage of both groups are simple and give high yields.
The Dpm ester is fully compatible with the N-Fmoc group
and a selective cleavage of a Boc group in the presence of a
diphenylmethyl ester is possible.6
A preliminary investigation of the solid-phase synthesis of
the flexible peptide nucleic acid [Gly-Eg(T)]n from the thymine
monomer (3, B = thymine, R = OH) revealed that the coupling
was inefficient, especially that of the secondary amino group
with Boc-glycine. This prompted us to explore the possibility of
coupling the amino acids bearing nucleobases 1–3 with the
spacer amino acid (e.g. glycine) to form dipeptides with the
intention of performing the solid-phase synthesis using these
dipeptide building blocks.
Although both possible dipeptides 4 and 5 were considered,
Results and discussion
Initial studies were undertaken with the cheap, commercially
available trans-4-hydroxy--proline, which was protected as its
N-Boc/Dpm ester derivative according to the method described
by Tozuka and Takaya.6b The crystalline derivative 6a was
obtained in greater than 80% yield in two steps.
the dipeptide 4 appeared to be a better building block since the
more difficult coupling (i.e. at the secondary amino group)
could be achieved in solution and the product purified before
the solid-phase synthesis was undertaken. Furthermore, if an
optically active spacer amino acid was used instead of glycine,
racemisation of the spacer amino acid during fragment coup-
ling of the dipeptide of type 5 would be a serious problem,
whereas such racemisation is minimised when the C-terminus
of the activated fragment is proline2 because N-acylproline
could not be racemised by the oxazolone mechanism.
Owing to the mild conditions used for the deprotection of the
N-Fmoc group, the Fmoc/OBut strategy in solid-phase peptide
synthesis is favoured over the classical Boc/OBzl strategy.3 Fur-
thermore, most machine synthesizers capable of handling
small-scale synthesis (50 µmol or less) can accommodate only the
The Mitsunobu reaction on compound 6a with N 3-benzoyl-
thymine (BzT)7 gave the thymine derivative 7a, together with a
less polar product, possibly the O2-isomer or the elimination
product. Fortunately, the thymine derivative 7a is crystalline
and after column chromatography and one recrystallisation, the
pure material was obtained in 51% yield.
Deprotection of the N-Boc group of the protected thymine
derivative 7a was accomplished with methanolic HCl according
to the literature.6 The resulting amine salt was treated with
Fmoc-glycine pentafluorophenyl ester in the presence of di-
isopropylethylamine (DIEA) to give the protected dipeptide 8a
in excellent yield. Treatment of compound 8a with trifluoro-
acetic acid (TFA), either as a neat liquid or in the presence of
phenol or anisole as a scavenger,8 at room temperature for a few
hours led to the formation of roughly equal amounts of two
J. Chem. Soc., Perkin Trans. 1, 1997
547

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products as shown by TLC and high-performance liquid
chromatography (HPLC), which could not be separated by
crystallisation. The unexpected product, which was more polar
than the desired product, was identified as the debenzoylated
thymine derivative 9a. Since protection of thymine at N3 was
only required for selective alkylation at thymine-N1, the
debenzoylated thymine derivative 9a was still suitable for the
oligomer synthesis. However, attempts to remove the benzoyl
group completely by prolonged treatment with TFA resulted in
a complex mixture. HBr in acetic acid, which is commonly used
for the removal of the benzyloxycarbonyl (Z) group,9 appeared
to give better results. Brief treatment of the mixture of products
from TFA-cleavage with 10% HBr in acetic acid resulted in
complete cleavage of the benzoyl group as shown by HPLC.
The cleavage conditions have also been applied to the fully pro-
tected dipeptide 8a without pre-treatment with TFA, with equal
success. The synthesis of the Fmoc-dipeptide bearing thymine
at the 4-position in the cis- proline series is summarised in
Scheme 1.
Fig. 1 Analytical reversed-phase chromatogram of trans--thymine
Fmoc-dipeptide 9b; Inset shows the HPLC chromatogram of a 1:1
mixture of trans- (9b) and cis- (9c) Fmoc-dipeptide under identical
conditions (arbitrary scale). The peak marked by an asterisk is the
diastereomeric impurity. HPLC conditions: C-18 Hypersil semipre-
parative reversed-phase HPLC column (3 µ particle size); linear gradient
water–acetonitrile containing 0.1% TFA (75:25) for 5 min, then 10:90
over a period of 35 min; flow rate 1.0 ml minϪ1
.
diastereomers 6b and 6c with N 3-benzoylthymine on a 20
mmol scale gave the products 7b and 7c in 33 and 36% yield,
respectively. The Boc group in compounds 7b and 7c was
removed with methanolic HCl and the products were treated
with Fmoc-glycine pentafluorophenyl ester to give the pro-
tected dipeptides 8b and 8c. After treatment with 10% HBr in
acetic acid the Fmoc-dipeptide acids 9b and 9c were obtained
with concomitant cleavage of the N 3-benzoyl group. The inter-
mediate protected dipeptide 8b and the final product 9b were
not crystallised as readily as their diastereomers 8c and 9c.
However, the purity of the crude Fmoc-dipeptides 9b and 9c
was found to be satisfactory by HPLC (Fig. 1).
Scheme 1 Reagents and conditions: i, N3-BzT, Ph3P, DEAD in THF,
room temp., overnight (51%); ii, (a) THF–satd. HCl in MeOH (1:1),
room temp., 3 h; (b) FmocGlyOPfp, DIEA in 1,4-dioxane, room temp.,
overnight (99%); iii, 10% HBr in HOAc, room temp., 1 h (50%)
The protected cis- and trans-hydroxy--proline compounds
6b and 6c were required for the preparation of the trans- and
cis--proline dipeptides bearing nucleobases. The reaction of
N-Boc-cis-4-hydroxy--proline10 with diphenyldiazomethane11
gave the Dpm ester 6b in 90% yield. Inversion of the 4-OH
group in 6b to give 6c was effected by the Mitsunobu reaction
in the presence of formic acid followed by treatment with
methanolic ammonia as with the methyl ester.1 By this route,
trans-compound 6c was prepared in multigram quantities from
cis-stereoisomer 6b in excellent yield (90%, 2 steps) (Scheme 2).
The Fmoc-dipeptides 9a–c were prepared in gram-
quantities for solid-phase synthesis. Pentafluorophenyl esters of
the diastereomeric thymine dipeptides (compounds 10a–c)
were all prepared by reactions of the free acids with penta-
fluorophenol in the presence of dicyclohexylcarbodiimide
(DCCI) in dichloromethane.12 These active esters were crystal-
line solids which were stable enough to permit purification by
silica gel column chromatography and could be stored for sev-
eral months at Ϫ20 ЊC without apparent decomposition accord-
1
ing to H NMR analysis. These were used for peptide synthesis
and the results compared with direct coupling of the free acids.
Binding studies between the 10-mers derived from coupling
of compounds 10a–c and poly(dA) showed that the oligomer
derived from isomer 10c binds most strongly. The cis--proline
series was selected therefore for further investigation. The pro-
tected cis-hydroxy--proline 6b was converted into the crystal-
line trans--toluene-p-sulfonyl ester 11 in 68% yield by a
Mitsunobu reaction with methyl toluene-p-sulfonate in the
presence of triphenylphosphine and diethyl azodicarboxylate
(DEAD).10 Reaction of compound 11 and N 6-benzoyladenine
in the presence of K2CO3 and a catalytic amount of 18-crown-6
in dimethylformamide (DMF) afforded the N9-isomer of
Boc--Pro(cis-4-BzA)-ODpm 12 in 42% yield. However, on
scaling up, a small amount of another isomer (~5%) was also
isolated. This was probably the N7-isomer according to the
Scheme 2 Reagents and conditions: i, Ph2CN2, EtOAc, room temp.,
overnight (90%); ii, HCO2H, Ph3P, DEAD in THF, room temp.,
overnight (quant.); iii, aq. NH3, MeOH, 1 h (90%)
The specific rotation of the product 6c {[α]2D5 +53.0 (c 1.0,
EtOH)} when compared with that of the trans- isomer {[α]2D5
Ϫ54.3 (c 1.0, EtOH)} indicated that inversion was essentially
complete.
Mitsunobu reaction on the protected hydroxyproline
548
J. Chem. Soc., Perkin Trans. 1, 1997

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-proline 6c was carried out in an analogous way to the methyl
ester derivative.1 The product, however, could not be isolated
free from diethyl hydrazinedicarboxylate. Treatment with 1,8-
diazabicyclo[5.4.0]undec-7-ene (DBU) in pyridine to remove
the O 6-nitrophenylethyl protecting group followed by column
chromatography gave the pure N 9-substituted isobutyryl-
guanine derivative 18 as a solid in 43% overall yield. Removal
of the Boc group and reaction of the product with Fmoc-
glycine pentafluorophenyl ester as before gave the protected
guanine dipeptide 19 in 52% yield. Removal of the carboxy-
protecting group and reaction of the product with pentafluoro-
phenol and DCCI gave the isobutyrylguanine dipeptide and its
pentafluorophenyl ester 20.
Solid-phase synthesis of peptide nucleic acids from the
dipeptide acids 9 and pentafluorophenyl esters 10, 14, 17 and 20
will be described elsewhere.
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 units of 10Ϫ1
deg cm2 gϪ1. IR spectra were recorded on a Perkin-Elmer 1750
Fourier Transform Infrared spectrometer. Elemental analyses
were performed on a Carlo Erba CHN analyser model 1106.
Routine 1H and 13C NMR spectra were recorded 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 dis-
tortionless enhancement by polarisation transfer (DEPT)
experiment performed on the Varian Gemini 200 spectrometer.
upfield 13C chemical shift of adenine C5 (δC 115.0 and 114.6,
rotamers) relative to the major product (δC 123.4).13
Deprotection of the Boc group in compound 12 was first
attempted by methanolic HCl as described previously for the
thymine derivatives; however, the selectivity was somewhat less
than had been hoped. On the other hand, toluene-p-sulfonic
acid (PTSA) in acetonitrile, which has been successfully applied
to deprotect the N-Boc group during the synthesis of cephalo-
sporin derivatives,14 cleanly removed the Boc group without
cleaving the Dpm ester. The product was treated with Fmoc-
glycine pentafluorophenyl ester to give the Fmoc-dipeptide
diphenylmethyl ester 13 in 85% yield. Deprotection of the Dpm
ester with TFA in the presence of anisole gave the free acid,
which was directly converted into the pentafluorophenyl ester
14 by treatment with pentafluorophenol in the presence of
DCCI.12 The N6-benzoyl group on adenine remained intact
throughout the reaction sequence. Attempted purification of
the highly polar pentafluorophenyl ester 14 by column chrom-
atography found only limited success. However, the crude prod-
uct obtained after trituration and washing with hexane was
shown by 1H NMR spectroscopy to contain approximately 10%
of dicyclohexylurea (DCU) as the only contaminant, and was
used successfully for the solid-phase synthesis.
1
Highfield H NMR spectra were recorded on a Bruker AMX
500 spectrometer (500 MHz). 19F NMR spectra were recorded
1
on a Bruker AM 250 instrument at 235.35 MHz. H and 13C
chemical shifts are quoted in ppm relative to tetramethylsilane
and were internally referenced to the residual protonated sol-
vent signal. 19F chemical shifts were externally referenced to
CFCl3 in CHCl3.
Chemical ionisation and fast-atom bombardment mass
spectra were recorded on a VG 20-250 masslab and a VG Micro-
mass ZAB-1F mass spectrometer. Electrospray mass spectra
were recorded on a VG Biotech BioQ or VG Biotech Platform.
Masses are quoted as m/z-values unless otherwise stated, only
the molecular ions and major fragments being quoted.
Reaction of the trans--toluene-p-sulfonyl ester 11 with N 4-
benzoylcytosine in the presence of K2CO3/18-crown-6 in DMF
gave the desired N1-isomer, Boc--Pro(cis-4-N 1-BzC)-ODpm,
15 in 25% yield along with the less polar O2-isomer in 41%
yield, which could be readily separated by chromatography on
silica gel. The identity of the two isomers was further con-
firmed by the characteristic downfield shift of the 13C resonance
of C4Ј of the O2-isomer compared to the N1-isomer as discussed
earlier for the methyl ester derivatives.1 Since N 4-benzoyl-
cytosine was shown to be partially hydrolysed in hot 85% acetic
acid to give uracil,15 the stability of this group towards acids
was tested before we attempted deprotection of the Boc group
or the diphenylmethyl ester. The Boc-protected amino acid 15
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,17 if
necessary. N-Boc-trans-4-hydroxy--proline and Fmoc-glycine
pentafluorophenyl ester were obtained from Calbiochem-
Novabiochem Ltd. Toluene-p-sulfonyl chloride was purified by
recrystallisation from light petroleum (distillation range 60–
80 ЊC). DMF was peptide synthesis grade obtained from Rath-
burn Chemical Ltd. and was used without further purification
except when strictly anhydrous conditions were required, where
it was re-distilled from calcium hydride under reduced pressure.
Acetonitrile was HPLC grade obtained from Rathburn and
used without further purification. Tetrahydrofuran (THF) and
1,4-dioxane were distilled from sodium wire/benzophenone
under argon and stored over 4 Å molecular sieves. Pyridine was
distilled from calcium hydride and stored over 4 Å molecular
sieves. Moisture-sensitive reactions were performed under
argon in flame-dried glassware.
1
was treated with TFA in the presence of anisole for 2 h. H
NMR analysis of the product showed that the Boc and Dpm
groups were completely removed whereas the benzoyl group
was stable, thus demonstrating that the deprotection conditions
were satisfactory.
Removal of the Boc group of compound 15 and reaction
of the product with Fmoc-glycine pentafluorophenyl ester as
described for the adenine analogue gave the protected cytosine
dipeptide 16 in 70% overall yield. The benzoylcytosine di-
peptide and its pentafluorophenyl ester 17 were synthesized in
essentially the same way as the thymine and adenine analogues.
The Mitsunobu reaction between N 2-isobutyryl-O 6-(4Ј-
nitrophenylethyl)guanine16 and the protected trans-4-hydroxy-
N-tert-Butoxycarbonyl-trans-4-hydroxy-L-proline diphenyl-
methyl ester 6a and N-tert-butoxycarbonyl-cis-4-hydroxy-D-
proline diphenylmethyl ester 6b
To a solution of freshly prepared diphenyldiazomethane11 (3.50
g, 18.0 mmol) in ethyl acetate (20 ml) was added a solution of
N-Boc-trans-4-hydroxy--proline (3.25 g, 14.0 mmol) in ethyl
J. Chem. Soc., Perkin Trans. 1, 1997
549

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acetate (30 ml). Nitrogen gas was slowly evolved from the solu-
tion and the intense purple colour of diphenyldiazomethane
was gradually discharged. The solution was stirred at room
temperature overnight using a CaCl2 guard tube. Evaporation
off of the solvent followed by precipitation of the product from
ethyl acetate–light petroleum (distillation range 40–60 ЊC) gave
a solid, N-tert-butoxycarbonyl-trans-4-hydroxy--proline di-
phenylmethyl ester 6a (5.10 g, 91%), mp 93–95 ЊC (lit.,6b 103–
104 ЊC); δH (200 MHz; CDCl3) 1.22 and 1.47 (9 H, 2 × s, Boc
rotamers), 1.95–2.50 [3 H, br m, CH2(3) and OH], 3.45–3.74
[2 H, br m, CH2(5)], 4.40–4.65 [2 H, br m, CH(2) and CH(4)],
6.95 (1 H, br s, CHPh2), 7.25–7.55 (10 H, br m, phenyl CH);
rotamers], 54.7 [CH2(5)], 57.8 and 58.1 [CH(2) rotamers], 69.2
and 70.0 [CH(4) rotamers], 77.2 and 77.6 (CHPh2 rotamers),
80.6 (Boc C), 127.1–128.8 (phenyl CH rotamers), 140.0 and
140.2 (phenyl C rotamers), 154.4 (Boc CO) and 172.0 (ester
CO); νmax(KBr)/cmϪ1 3491br (O–H), 1728s (C᎐O ester) and
᎐
1693s (C᎐O urethane); [α]25 +53.0 (c 1.0, EtOH).
᎐
D
N-tert-Butoxycarbonyl-trans-4-(p-tolylsulfonyloxy)-D-proline
diphenylmethyl ester 11
A solution of N-Boc-cis-4-hydroxy--proline diphenylmethyl
ester 6b (6.40 g, 16.1 mmol), triphenylphosphine (4.50 g, 16.8
mmol) and methyl toluene-p-sulfonate (3.04 g, 16.4 mmol) in
THF was treated with DEAD (2.80 ml, 4.2 mmol) dropwise at
Ϫ78 ЊC. The reaction was allowed to warm gradually to room
temperature and was stirred overnight. Evaporation followed
by column chromatography (SiO2; diethyl ether; RF 0.61) gave
the crude product as an oil, which was reprecipitated from
diethyl ether–light petroleum (40–60 ЊC) to give the essentially
pure product as a solid (6.01 g, 68%). Recrystallisation from
ethyl acetate–light petroleum (40–60 ЊC) gave N-tert-butoxy-
carbonyl-trans-4-(p-tolylsulfonyloxy) --proline diphenylmethyl
ester 11 as crystals, mp 147–149 ЊC (Found: C, 65.2; H, 5.8; N,
2.4. C30H33NO7S requires C, 65.3; H, 6.0; N, 2.5%); δH (200
MHz; CDCl3) 1.22 and 1.44 (9 H, 2 × s, Boc rotamers), 1.89–
2.18 and 2.32–2.66 [2 H, m, CH2(3)], 2.46 (3 H, s, tosyl CH3),
3.54–3.74 [2 H, m, CH2(5)], 4.50 [1 H, m, CH(2)], 4.98 [1 H, br
m, CH(4)], 6.87 and 6.92 (1 H, 2 × s, CHPh2 rotamers), 7.25–
7.38 (12 H, m, arom CH) and 7.77 (2 H, d, J 8.2, tosyl CH);
δC(50.28 MHz; CDCl3) 21.6 (tosyl CH3), 27.9 and 28.2 (Boc
CH3 rotamers), 35.6 and 37.0 [CH2(3) rotamers], 51.8 and
52.1 [CH2(5) rotamers], 57.4 and 57.6 [CH(2) rotamers], 78.3
and 78.9 [CH(4) rotamers], 78.2 and 78.7 (CHPh2 rotamers),
80.7 and 80.9 (Boc C rotamers), 127.0–130.3 (aromatic CH),
133.5, 139.9 and 145.3 (aromatic C), 154.6 and 154.8 (Boc CO
rotamers) and 171.3 (ester CO rotamers); νmax(KBr)/cmϪ1 1742s
(C᎐O ester), 1704 (C᎐O urethane), 1402s (᎐SO O᎐) and 1174s
νmax(KBr)/cmϪ1 3491br (O–H), 1728s (C᎐O ester) and 1693s
᎐
(C᎐O urethane); m/z (ES-MS) 436 (M + K+, 55%), 420 (M +
᎐
Na+, 100), 415 (M + NH4+, 15) and 398 (M + H+, 72); [α]2D5
Ϫ54.3 (c 1.0, EtOH).
The cis--diastereoisomer 6b was prepared similarly starting
from N-Boc-cis-hydroxy--proline10 (11.6 g, 50.0 mmol) and
diphenyldiazomethane (11.3 g, 58.0 mmol) in ethyl acetate (150
ml). N-tert-Butoxycarbonyl-cis-4-hydroxy--proline diphenyl-
methyl ester 6b was obtained as a solid after precipitation from
ethyl acetate–light petroleum (40–60 ЊC) (17.9 g, 90%), mp 102–
105 ЊC (Found: C, 69.5; H, 6.8; N, 3.3. C23H27NO5 requires C,
69.5; H, 6.8; N, 3.5%); δH (200 MHz; CDCl3) 1.26 and 1.30 (9 H,
2 × s, Boc rotamers), 2.08 and 2.35 [2 H, m, CH2(3)], 2.88 and
3.09 (1 H, 2 × d, J 9.6, OH rotamers), 3.57–3.66 [2 H, br m,
CH2(5)], 4.32 [1 H, m, CH(4)], 4.42–4.58 [1 H, m, CH(2)], 6.91
and 6.99 (1 H, 2 × s, CHPh2 rotamers) and 7.25–7.48 (10 H,
br m, Ph); δC(50.28 MHz; CDCl3) 28.0 and 28.3 (Boc CH3
rotamers), 37.6 and 38.6 [CH2(3) rotamers], 55.4 and 55.9
[CH2(5) rotamers], 58.0 and 58.1 [CH(2) rotamers], 70.1 and
71.2 [CH(4) rotamers], 78.1 and 78.6 (CHPh2 rotamers), 80.4
and 80.6 (Boc C), 127.2–128.8 (phenyl CH rotamers), 139.6 and
139.8 (phenyl C rotamers), 154.1 (Boc CO) and 174.0 (ester CO
rotamers); νmax(KBr)/cmϪ1 3466br (O–H), 1749s (C᎐O ester)
᎐
and 1687s (C᎐O urethane); [α]25 +41.2 (c 1.0, EtOH).
᎐
᎐
᎐
D
2
(᎐SO2O᎐).
N-tert-Butoxycarbonyl-trans-4-hydroxy-D-proline
diphenylmethyl ester 6c
N-tert-Butoxycarbonyl-4-(N 3-benzoylthymin-1-yl)proline
N-Boc-cis-hydroxy--proline diphenylmethyl ester 6b (0.20 g,
0.50 mmol), triphenylphosphine (0.160 g, 0.60 mmol) and for-
mic acid (25 µl, 0.65 mmol) were dissolved in dry THF (10 ml)
and the solution was cooled in an ice-bath. DEAD (100 µl, 0.60
mmol) was added dropwise. The reaction mixture was stirred
under nitrogen at room temperature overnight. The solvent was
evaporated off and the residue was chromatographed on silica
gel with dichloromethane–acetone (20:1) as eluent to give the
4-formate ester (RF 0.50) as an oil (0.248 g, quant.); δH (200
MHz; CDCl3) 1.25 and 1.47 (9 H, 2 × s, Boc rotamers), 2.10–
2.55 [2 H, br m, CH2(3)], 3.57–3.80 [2 H, br m, CH2(5)], 4.48–
4.64 [1 H, m, CH(2)], 5.35–5.43 [1 H, br m, CH(4)], 6.91 and
6.95 (1 H, 2 × s, CHPh2 rotamers), 7.25–7.42 (10 H, br m,
phenyl CH) and 8.03 [1 H, s, HC(O)].
The oil was taken up in methanol (10 ml), and conc. aq.
ammonia (d 0.880; 0.5 ml) was added. TLC analysis indicated
complete reaction after stirring at room temperature for 1 h.
The solvent was removed under reduced pressure and the resi-
due was chromatographed on a silica gel column with diethyl
ether as eluent to give the product (RF 0.30) as a foam (0.179 g,
90% from 6b), which was further purified by reprecipitation
from ethyl acetate–light petroleum (40–60 ЊC) to give N-
tert-butoxycarbonyl-trans-4-hydroxy--proline diphenylmethyl
ester 6c as a solid, mp 105–108 ЊC (Found: C, 69.8; H, 6.9; N,
3.3. C23H27NO5 requires C, 69.5; H, 6.8; N, 3.5%); δH (200 MHz;
CDCl3) 1.22 and 1.47 (9 H, 2 × s, Boc rotamers), 2.02 and 2.30
[2 H, 2 × br m, CH2(3)], 2.70 and 2.82 (1 H, 2 × br d, J 3.1 and
3.4, OH rotamers), 3.45–3.70 [2 H, br m, CH2(5)], 4.42–4.65 [2
H, br m, CH(2) and CH(4)], 6.90 and 6.95 (1 H, 2 × s, CHPh2)
and 7.25–7.45 (10 H, br m, phenyl CH); δC(50.28 MHz; CDCl3)
27.9 and 28.3 (Boc CH3 rotamers), 38.1 and 38.9 [CH2(3)
diphenylmethyl esters 7a–c
N-Boc-trans-4-hydroxy--proline diphenylmethyl ester 6a
(0.425 g, 1.07 mmol), triphenylphosphine (0.290 g, 1.10 mmol)
and N 3-benzoylthymine (0.250 g, 1.09 mmol) were dissolved in
dry THF (10 ml) and the solution was cooled to Ϫ15 ЊC.
DEAD (180 µl, 1.10 mmol) was then added dropwise to the
stirred mixture. The reaction mixture was stirred under argon
at room temperature overnight. The solvent was evaporated
off and the residue was chromatographed on silica gel with
dichloromethane–acetone (20:1) as eluent to give N-tert-
butoxycarbonyl-cis-4-(N3-benzoylthymin-1-yl)--proline diphen-
ylmethyl ester 7a (RF 0.56), which was recrystallised from
ethanol to give a fluffy solid (0.310 g, 51%), mp 183–185 ЊC
(Found: C, 68.9; H, 5.5; N, 6.7. C35H35N3O7 requires C, 69.0; H,
5.8; N, 6.9%); δH (200 MHz; CDCl3) 1.30 and 1.49 (9 H, 2 × br s,
Boc rotamers), 1.82 (3 H, br s, thymine CH3), 2.05 and 2.85 [2
H, br m, CH2(3Ј)], 3.65 (1 H, br m) and 4.02 (1 H, dd, J 12.0
and 8.0) [CH2(5Ј)], 4.54 [1 H, br m, CH(2Ј)], 5.26 [1 H, br m,
CH(4Ј)], 6.94 (1 H, s, CHPh2), 7.12 and 7.18 [1 H, 2 × br s,
CH(6) rotamers], 7.30–7.42 (10 H, br m, phenyl CH), 7.50 (2 H,
t, J 7.0, benzoyl m-H), 7.67 (1 H, t, J 7.0, benzoyl p-H) and 7.92
(2 H, d, J 7.0, benzoyl o-H); δC(50.28 MHz; CDCl3) 12.3 (thy-
mine CH3), 27.9 and 28.2 (Boc CH3 rotamers), 34.9 and 35.3
[CH2(3Ј) rotamers], 49.3 and 49.4 [CH2(5Ј) rotamers], 52.1 and
52.5 [CH(4Ј) rotamers], 57.5 [CH(2Ј)], 78.1 and 78.4 (CHPh2
rotamers), 81.3 (Boc C), 111.7 [C(5)], 126.9–130.6 (aromatic
CH), 131.6 (benzoyl C), 135.3 (benzoyl p-CH), 136.0 and 136.2
[CH(6) rotamers], 139.4 (phenyl C), 150.1 [C(2)], 153.6 (Boc
CO), 162.6 [C(4)], 169.1 (benzoyl CO) and 171.6 (ester CO);
m/z (FAB) 632 (M + Na+, 10%), 610 (M + H+, 39), 554
([M Ϫ C4H8 + H]+, 27), 506 ([M Ϫ PhCO + H]+, 18), 338
550
J. Chem. Soc., Perkin Trans. 1, 1997

View Article Online
([M Ϫ PhCO Ϫ Ph2CH]+, 20), 266 (31), 231 (BzT + H+, 52),
167 (Ph2CH+, 100), 105 (PhCO+, 24) and 57 (C4H9+, 28);
νmax(KBr)/cmϪ1 1751, 1694 and 1659 (C᎐O); [α]25 Ϫ17.2 (c 1.0,
C), 123.4 [C(5)], 127.0–129.0 (aromatic CH), 132.9 (aromatic
CH), 133.9 (aromatic C), 139.4 and 139.5 (aromatic C), 141.5
[CH(8)], 149.8 [C(4)], 152.0 [C(6)], 152.7 [CH(2)], 153.6 and
154.0 (Boc CO rotamers), 165.1 (benzoyl CO) and 170.9 (ester
CO); m/z (ES-MS) 619 (M + H+, 100%); νmax(KBr)/cmϪ1 1748
(C᎐O) and 1697s (C᎐O); λmax(CHCl3)/nm 285 (ε/dm3 molϪ1
᎐
D
DMF).
N-tert-Butoxycarbonyl-trans-4-(N3-benzoylthymin-1-yl)--
proline diphenylmethyl ester 7b was similarly prepared starting
from the cis--alcohol 6b (8.0 g, 20 mmol). The product was
obtained as a solid after column chromatography [SiO2;
dichloromethane–acetone (20:1)] and trituration with diethyl
ether (4.20 g, 33%). Recrystallisation from ethyl acetate–hexane
gave an analytically pure sample as crystals, mp 189–192 ЊC
(Found: C, 68.9; H, 5.5; N, 6.7%); δH (200 MHz; CDCl3) 1.30
and 1.47 (9 H, 2 × s, Boc rotamers), 1.80 (3 H, s, thymine CH3),
2.40 and 2.58 [2 H, m, CH2(3Ј)], 3.55–4.02 [2 H, m, CH2(5Ј)],
4.53–4.64 [1 H, m, CH(2Ј)], 5.12 [1 H, m, CH(4Ј)], 6.90 and 6.94
(1 H, 2 × s, CHPh2 rotamers), 7.00 [1 H, s, CH(6) rotamers],
7.35–7.45 (10 H, br m, phenyl CH), 7.52 (2 H, t, J 7.2, benzoyl
m-H), 7.69 (1 H, t, J 7.2, benzoyl p-H) and 7.94 (2 H, d, J 7.2,
benzoyl o-H); δC(50.28 MHz; CDCl3) 12.6 (thymine CH3), 27.9
and 28.2 (Boc CH3 rotamers), 33.4 and 35.1 [CH2(3Ј) rotamers],
49.1 and 49.5 [CH2(5Ј) rotamers], 53.8 and 54.4 [CH(4Ј) rotam-
ers], 57.7 and 58.0 [CH(2Ј) rotamers], 77.9 and 78.2 (CHPh2
rotamers), 81.2 and 81.3 (Boc C rotamers), 111.9 [C(5)], 127.0–
130.6 (aromatic CH), 131.6 (benzoyl C), 135.3 (benzoyl p-CH),
136.2 [CH(6)], 139.4 and 139.7 (phenyl C rotamers), 149.9
[C(2)], 153.6 (Boc CO), 162.7 [C(4)], 169.1 (benzoyl CO) and
171.0 (ester CO); νmax(KBr)/cmϪ1 1739s (C᎐O), 1703s (C᎐O)
᎐
᎐
cmϪ1 2.1 × 104); [α]2D5 +14.1 (c 0.63, CHCl3).
N-tert-Butoxycarbonyl-cis-4-(N4-benzoylcytosin-1-yl)-D-proline
diphenylmethyl ester 15 and N-tert-butoxycarbonyl-cis-4-(4-
benzoylaminopyrimidin-2-yloxy)-D-proline diphenylmethyl ester
A reaction mixture containing the trans--toluene-p-sulfonyl
ester 11 (1.10 g, 2.00 mmol), N 4-benzoylcytosine (0.475 g, 2.20
mmol), anhydrous K2CO3 (0.300 g, 2.20 mmol) and 18-crown-6
(200 mg) in DMF (10 ml) was stirred at 70–80 ЊC under argon
overnight. The suspension was diluted with dichloromethane
(75 ml), filtered through Celite, and the organic phase was
washed with water. Evaporation gave the crude product as an
oil, which was purified by column chromatography (SiO2; ethyl
acetate). The more polar fractions (RF 0.33) were combined and
evaporated to give the N 1-isomer (0.299 g, 25%) as a foam.
Recrystallisation from ethanol–water gave crystals of N-tert-
butoxycarbonyl-cis-4-(N 4-benzoylcytosin-1-yl)--proline diphen-
ylmethyl ester 15, mp 133–135 ЊC (Found: C, 65.8; H, 6.5; N,
8.8. C34H34N4O6ؒC2H5OHؒH2O requires C, 65.6; H, 6.4; N,
8.5%); δH (200 MHz; CDCl3) 1.30 and 1.49 (9 H, 2 × s, Boc
rotamers), 2.20 and 2.90 [2 H, br m, CH2(3Ј)], 3.50–3.80 and
3.95–4.15 [2 H, 2 × br m, CH2(5Ј)], 4.45–4.70 [1 H, br m,
CH(2Ј)], 5.28 [1 H, br m, CH(4Ј)], 6.87 (1 H, s, CHPh2), 7.15–
7.40 (10 H, m, phenyl CH), 7.40–7.75 [5 H, m, CH(5), CH(6)
and benzoyl m- and p-H], 7.89 (2 H, d, J 7.4, benzoyl o-H) and
8.83 (1 H, br s, NH); δC(50.28 MHz; CDCl3) 27.7 and 28.0 (Boc
CH3 rotamers), 34.4 and 36.0 [CH2(3Ј) rotamers], 49.6 and 50.5
[CH2(5Ј) rotamers], 54.3 and 54.9 [CH(4Ј) rotamers], 57.6
[CH(2Ј)], 78.0 and 78.3 (CHPh2 rotamers), 81.3 (Boc C), 96.8
[CH(5)], 127.0–129.2 (aromatic CH), 133.4 (benzoyl C), 139.5
(aromatic C), 145.2 and 145.7 [CH(6) rotamers], 153.8 (Boc CO
rotamers), 155.6 [C(2)], 162.0 [C(4)], 166.8 (benzoyl CO) and
171.3 (ester CO); m/z (ES-MS) 595 (M + H+, 100%); νmax(KBr)/
cmϪ1 1743 (C᎐O) and 1704s (C᎐O); λmax(CHCl3)/nm 266 (ε/dm3
᎐
᎐
and 1664s (C᎐O); [α]25 +11.3 (c 1.03, DMF).
᎐
D
N-tert-Butoxycarbonyl-cis-4-(N3-benzoylthymin-1-yl)--
proline diphenylmethyl ester 7c was similarly prepared starting
from the trans--alcohol 6c (7.62 g, 19.2 mmol). The product
was obtained as a crystalline solid after column chromato-
graphy [SiO2; dichloromethane–acetone (20:1)] and recrystal-
lisation from ethanol (4.20 g, 36%), mp 183–186 ЊC (Found: C,
69.1; H, 5.8; N, 6.8%); δH (200 MHz; CDCl3) 1.30 and 1.49 (9 H,
2 × br s, Boc rotamers), 1.81 (3 H, br s, thymine CH3), 2.04 and
2.86 [2 H, 2 × br m, CH2(3Ј)], 3.66 (1 H, br m) and 4.02 (1 H,
dd, J 12.0 and 8.0) [CH2(5Ј)], 4.53 [1 H, br m, CH(2Ј)], 5.26 [1
H, br m, CH(4Ј)], 6.95 (1 H, s, CHPh2), 7.12 and 7.18 [1 H,
2 × br s, CH(6) rotamers], 7.30–7.44 (10 H, br m, phenyl CH),
7.50 (2 H, t, J 7.0, benzoyl m-H), 7.67 (1 H, t, J 7.0, benzoyl p-
H), 7.92 (2 H, d, J 7.0, benzoyl o-H); νmax(KBr)/cmϪ1 1751s
(C᎐O), 1699s (C᎐O) and 1661s (C᎐O); [α]25 +16.9 (c 1.03,
᎐
᎐
molϪ1 cmϪ1 8.9 × 104) and 312 (3.4 × 104); [α]2D3 Ϫ13.6 (c 0.50,
CHCl3).
The less polar fractions (RF 0.61) were combined and
rechromatographed [SiO2; dichloromethane–acetone (10:1)] to
give the O 2-isomer as a foam (0.489 g, 41%), which was
recrystallised from ethanol to give needles of N-tert-butoxy-
carbonyl-cis-4-(4-benzoylaminopyrimidin-2-yloxy)--proline di-
phenylmethyl ester, mp 145–147 ЊC (Found: C, 68.6; H, 5.5; N,
9.4. C34H34N4O6 requires C, 68.7; H, 5.8; N, 9.4%); δH (200
MHz; CDCl3) 1.27 and 1.46 (9 H, 2 × s, Boc rotamers), 2.42
and 2.63 [2 H, br m, CH2(3Ј)], 3.60–4.20 [2 H, br m, CH2(5Ј)],
4.50 and 4.70 [1 H, 2 × m, CH(2Ј)], 5.40 [1 H, br m, CH(4Ј)],
6.91 and 6.98 (1 H, s, CHPh2), 7.15–7.32 (10 H, m, phenyl CH),
7.46–7.62 (3 H, m, benzoyl m- and p-H), 7.70–7.94 [3 H, m,
benzoyl o-H and CH(5)], 8.37 [1 H, d, J 5.7, CH(6)] and 8.67 (1
H, br s, NH); δC(50.28 MHz; CDCl3) 27.9 and 28.3 (Boc CH3
rotamers), 35.1 and 36.0 [CH2(3Ј) rotamers], 51.7 and 52.1
[CH2(5Ј) rotamers], 57.6 and 57.9 [CH(2Ј) rotamers], 74.1 and
75.2 [CH(4Ј) rotamers], 77.3 and 77.5 (CHPh2 rotamers), 80.2
and 80.4 (Boc C rotamers), 104.7 [CH(5)], 127.1–129.2 (aro-
matic CH), 133.1 and 133.4 (benzoyl C), 140.0–140.3 (aromatic
C), 154.0 and 154.4 (Boc CO rotamers), 159.6 [C(2)], 160.5
[CH(6) rotamers], 163.8 [C(4)], 166.3 (benzoyl CO) and 170.9
and 171.1 (ester CO); m/z (ES-MS) 595 (M + H+, 100%);
νmax(KBr)/cmϪ1 1738s (C᎐O) and 1687s (C᎐O).
᎐
᎐
᎐
D
DMF).
N-tert-Butoxycarbonyl-cis-4-(N 6-benzoyladenin-9-yl)-D-proline
diphenylmethyl ester 12
A mixture of the trans--toluene-p-sulfonyl ester 11 (0.552 g,
1.00 mmol), N 6-benzoyladenine (0.595 g, 2.50 mmol), anhy-
drous K2CO3 (0.700 g, 5.00 mmol) and 18-crown-6 (0.100 g) in
DMF (5 ml) was stirred under argon at 80 ЊC overnight. The
reaction mixture was diluted with dichloromethane (20 ml) and
washed with water, dried (MgSO4) and evaporated to give the
crude pruduct, which was purified by column chromatography
(SiO2; 2.5% methanol in dichloromethane; RF 0.27) to give the
product as a foam (0.260 g, 42%) which was spectroscopically
pure. Further recrystallisation from ethanol–water gave analy-
tically pure N-tert-butoxycarbonyl-cis-4-(N6-benzoyladenin-9-
yl)--proline diphenylmethyl ester 12 as needles, mp 115–119 ЊC
(Found: C, 65.9; H, 5.5; N, 13.1. C35H34N6O5ؒH2O requires C,
66.0; H, 5.7; N, 13.2%); δH (200 MHz; CDCl3) 1.31 and 1.49 (9
H, 2 × s, Boc rotamers), 2.52 and 2.90 [2 H, 2 × br m, CH2(3Ј)],
3.90–4.20 [2 H, br m, CH2(5Ј)], 4.52 and 4.63 [1 H, 2 × br m,
CH(2Ј) rotamers], 5.14 [1 H, br m, CH(4Ј)], 6.83 (1 H, s,
CHPh2), 7.15–7.28 (10 H, m, phenyl CH), 7.35–7.60 (3 H, m,
benzoyl m- and p-H), 7.95–8.05 [3 H, m, CH(8) and benzoyl
o-H], 8.68 [1 H, s, CH(2)] and 9.39 (1 H, s, NH); δC(50.28 MHz;
CDCl3) 28.0 and 28.2 (Boc CH3 rotamers), 34.5 and 35.7
[CH2(3Ј) rotamers], 49.9 and 50.5 [CH2(5Ј) rotamers], 52.3 and
52.8 [CH(4Ј) rotamers], 57.6 [CH(2Ј)], 77.8 (CHPh2), 81.3 (Boc
᎐
᎐
N-tert-Butoxycarbonyl-cis-4-(N 2-isobutyrylguanin-9-yl)-D-
proline diphenylmethyl ester 18
To a stirred suspension of the trans--alcohol 6c (0.400 g, 1.00
mmol), N 2-isobutyryl-O 6-(nitrophenylethyl)guanine (0.360 g,
J. Chem. Soc., Perkin Trans. 1, 1997
551

View Article Online
1.00 mmol) and triphenylphosphine (0.294 g, 1.10 mmol) in
anhydrous 1,4-dioxane (10 ml) at room temperature was slowly
added DEAD (182 µl, 1.10 mmol) under argon. Another two
aliquots of DEAD (91 µl, 0.55 mmol each) were added during
a period of 36 h. The resulting clear yellow solution was evap-
orated and the residue was chromatographed (SiO2; ethyl acet-
ate; RF 0.46) to give the O 6-nitrophenylethyl derivative as a
foam (0.634 g, contaminated with diethyl hydrazinedicarboxy-
late). This was dissolved in dry pyridine (5 ml) containing DBU
(300 µl, 2.00 mmol) and the solution was stirred at room tem-
perature overnight under argon. The reaction mixture was
diluted with dichloromethane, washed successively with 5%
HCl and water, and then evaporated to dryness. The residue
was purified by column chromatography [SiO2; ethyl acetate–
methanol (20:1)] to give the product as a foam (0.258 g, 43%
from 6c). Recrystallisation from ethyl acetate–light petroleum
(40–60 ЊC) gave N-tert-butoxycarbonyl-cis-4-(N2-isobutyryl-
guanin-9-yl)--proline diphenylmethyl ester 18 as a crystalline
solid, mp 140–145 ЊC (Found: C, 64.0; H, 5.7; N, 13.6.
C32H36N6O6 requires C, 64.0; H, 6.0; N, 14.0%); δH (200 MHz;
CDCl3) 1.18–1.41 [15 H, m, Boc and (CH3)2CH], 2.30 (1 H, br
m) and 2.75–2.95 (2 H, br m) [CH2(3Ј) and (CH3)2CH], 3.78 and
4.05 [2 H, br m, CH2(5Ј)], 4.45–4.63 [1 H, 2 × m, CH(2Ј)], 4.90
[1 H, br m, CH(4Ј)], 6.77 and 6.80 (1 H, s, CHPh2), 7.15–7.30
(10 H, m, phenyl CH) and 7.60 and 7.66 [1 H, 2 × s, CH(8)];
δC(50.28 MHz; CDCl3) 18.9 [(CH3)2CH], 27.9 and 28.2
(Boc CH3 rotamers), 35.8 [CH2(3Ј)], 50.2 and 50.7 [CH2(5Ј)
rotamers], 51.9 and 52.3 [CH(4Ј) rotamers], 57.6 [CH(2Ј)], 60.4
[(CH3)2CH], 77.7 and 77.9 (CHPh2 rotamers), 81.1 (Boc C),
121.1 [C(5)], 127.0–128.7 (aromatic CH), 137.2 [CH(8)], 139.5
(aromatic C), 148.2 and 149.1 [C(2)/C(6)], 153.6 and 154.2 (Boc
CO rotamers), 156.1 [C(4)], 171.0 (ester CO) and 180.5 (amide
CO); m/z (ES-MS) 601 (M + H+, 100%); λmax(CHCl3)/nm 255sh
(ε/dm3 molϪ1 cmϪ1 1.5 × 104) and 282 (1.2 × 104); [α]2D3 +37.8 (c
0.545, CHCl3).
CH), 135.8 [CH(6)], 139.4 and 139.6 (phenyl C rotamers), 141.7
and 144.0 (Fmoc aromatic C), 150.0 [C(2)], 156.2 (Fmoc CO),
162.4 [C(4)], 167.8 (benzoyl CO), 168.9 (peptide CO) and 170.8
(ester CO); m/z (FAB) 811 (M + Na+, 21%), 789 (M + H+, 5),
179 {[(C H ) C᎐CH + H]+, 23}, 167 (Ph CH+, 100) and 105
᎐
6
4
2
2
2
(PhCO+, 22); νmax(KBr)/cmϪ1 1751s, 1737s, 1697 and 1657s
᎐
(C᎐O); λmax(CHCl3)/nm 260 (ε/dm3 molϪ1 cmϪ1 3.1 × 104); [α]2D2
Ϫ41.2 (c 0.50, CHCl3).
N-[N-(Fluoren-9-ylmethoxycarbonyl)glycyl]-trans-4-(N3-
benzoylthymin-1-yl)--proline diphenylmethyl ester 8b was
obtained as a solid (85%, starting from 5.3 mmol of substrate
7b) after column chromatography. Recrystallisation from
ethanol–water gave a solid, mp 125–128 ЊC (Found: C, 71.4; H,
5.0; N, 6.6%); δH (200 MHz; CDCl3) 1.88 and 1.94 (3 H, 2 × s,
thymine CH3 rotamers), 2.30 and 2.60 [2 H, br m, CH2(3Ј)
rotamers], 3.70–4.10 [4 H, br m, CH2(5Ј) and Gly CH2], 4.22 (1
H, t, J 7.1, Fmoc aliphatic CH), 4.39 (2 H, d, J 7.0, Fmoc CH2),
4.88 [1 H, br m, CH(2Ј)], 5.13 [1 H, br m, CH(4Ј)], 5.90 and 5.98
(1 H, 2 × br m, Gly NH), 6.91 and 6.94 (1 H, 2 × s, CHPh2
rotamers), 7.16 [1 H, s, CH(6)], 7.29–8.00 (m, phenyl, benzoyl
and Fmoc aromatic CH); δC(50.28 MHz; CDCl3) 12.4 (thymine
CH3), 32.0 [CH2(3Ј) rotamers], 43.2 and 43.4 (Gly CH2 rotam-
ers), 47.0 (Fmoc aliphatic CH), 48.3 [CH2(5Ј)], 55.2 and 55.3
[CH(4Ј) rotamers], 57.9 [CH(2Ј)], 67.3 (Fmoc CH2), 78.6 and
79.3 (CHPh2 rotamers), 112.0 [C(5)], 120.2 (Fmoc aromatic
CH), 125.4–131.6 (aromatic CH), 135.6 [CH(6)], 137.2 and
139.2 (phenyl C rotamers), 141.5 and 144.1 (Fmoc aromatic C),
150.0 [C(2)], 156.9 (Fmoc CO), 162.9 [C(4)], 168.4 (benzoyl
CO), 169.4 (peptide CO) and 170.1 and 170.5 (ester CO); m/z
(ES-MS) 806 (M + NH4+, 28%) and 789 (M + H+, 100);
νmax(KBr)/cmϪ1 1749, 1702 and 1660 (C᎐O).
᎐
N-[N-(Fluoren-9-ylmethoxycarbonyl)glycyl]-cis-4-(N3-
benzoylthymin-1-yl)--proline diphenylmethyl ester 8c was
obtained as a solid (98%, starting from 5.7 mmol of substrate
7c) after column chromatography (RF 0.30). Recrystallisation
from ethanol–water gave needles, mp 201–203 ЊC (Found: C,
72.4; H, 4.9; N, 7.2%); δH (200 MHz; CDCl3) 1.71 and 1.83 (3 H,
2 × s, thymine CH3 rotamers), 2.06, 2.42, 2.78 and 2.92 [2 H, m,
CH2(3Ј) rotamers], 3.60–3.82 and 3.90–4.10 [4 H, br m, CH2(5Ј)
and Gly CH2], 4.23 (1 H, t, J 7.0, Fmoc aliphatic CH), 4.39 (2
H, d, J 7.1, Fmoc CH2), 4.73 [1 H, br m, CH(2Ј) rotamers], 5.14
and 5.42 [1 H, 2 × m, CH(4Ј) rotamers], 5.60 and 5.68 [1 H,
2 × br m, Gly NH rotamers], 6.87 and 6.91 (1 H, 2 × s, CHPh2
rotamers), 6.96 and 7.09 [1 H, 2 × s, CH(6) rotamers] and 7.21–
7.92 (m, phenyl, Fmoc and benzoyl aromatic CH); m/z (ES-MS)
806 (M + NH4+, 98%) and 789 (M + H+, 100); νmax(KBr)/cmϪ1
Procedure for selective deprotection of the N-Boc group in
diphenylmethyl esters 7a, 7b and 7c and synthesis of N-Fmoc
dipeptide diphenylmethyl esters 8a, 8b and 8c
The Boc-protected monomer (7a–c) was dissolved in THF (~10
ml mmolϪ1), saturated methanolic HCl (~10 ml mmolϪ1) was
added, and the solution was stirred at room temperature for 3–
12 h. The solvents were removed under reduced pressure. The
residue was taken up in dry 1,4-dioxane and DIEA (~2 mol
equiv. excess) was added until the solution was slightly basic
(pH 8) when applied to a piece of moist pH paper. Fmoc-
glycine pentafluorophenyl ester (1 mol equiv. excess) was then
added and the solution was stirred at room temperature over-
night. The reaction mixture was evaporated to dryness and the
residue was purified by column chromatography [SiO2; dichloro-
methane–acetone (10:1)].
1751, 1737, 1697 and 1657 (C᎐O); λmax(CHCl3)/nm 260 (ε/dm3
᎐
molϪ1 cmϪ1 3.4 × 104); [α]2D2 +41.5 (c 0.50, CHCl3).
Procedure for selective deprotection of the N-Boc group in
diphenylmethyl esters 12, 15 and 18 and synthesis of N-Fmoc
dipeptide diphenylmethyl esters 13, 16 and 19
N-[N-(Fluoren-9-ylmethoxycarbonyl)glycyl]-cis-4-(N3-
benzoylthymin-1-yl)--proline diphenylmethyl ester 8a was
obtained as a solid (99%, starting from 5.8 mmol of substrate
7a) after column chromatography. Recrystallisation from
ethanol gave fine needles, mp 201–204 ЊC (Found: C, 71.5; H,
5.0; N, 7.0. C47H40N4O8 requires C, 71.6; H, 5.1; N, 7.0%);
δH (200 MHz; CDCl3) 1.72 and 1.85 (3 H, 2 × s, thymine CH3
rotamers), 2.10, 2.45, 2.72 and 2.95 [2 H, 4 × m, CH2(3Ј)
rotamers], 3.60–3.82 and 3.95–4.09 [4 H, br m, CH2(5Ј) and Gly
CH2], 4.25 (1 H, t, J 7.1, Fmoc aliphatic CH), 4.40 (2 H, d, J
7.1, Fmoc CH2), 4.77 [1 H, br m, CH(2Ј) rotamers], 5.15 and
5.35 [1 H, 2 × m, CH(4Ј) rotamers], 5.60 and 5.78 (1 H, 2 × br
m, glycine NH rotamers), 6.86 and 6.93 (1 H, 2 × s, CHPh2
rotamers), 6.96 and 7.11 [1 H, 2 × s, CH(6) rotamers] and 7.25–
7.95 (m, phenyl, Fmoc and benzoyl aromatic CH); δC(50.28
MHz; CDCl3) 12.1 (thymine CH3), 32.5 [CH2(3Ј) rotamers],
43.5 (Gly CH2), 47.0 (Fmoc aliphatic CH), 48.5 [CH2(5Ј)], 53.2
[CH(4Ј)], 57.8 [CH(2Ј)], 67.3 (Fmoc CH2), 78.7 (CHPh2),
112.3 [C(5)], 120.1 (Fmoc aromatic CH), 125.5–131.8 (aromatic
The Boc-protected monomer (12, 15, 18) and PTSA monohy-
drate (5 mol equiv.) was dissolved in acetonitrile (~5 ml mmolϪ1
)
and the resulting solution was stirred at room temperature
overnight. The solvent was removed under reduced pressure
and the residue was dissolved in DMF (~5–10 ml mmolϪ1).
DIEA (5 mol equiv. excess) was added until the solution was
slightly basic (pH ~8) when applied to a piece of moist pH
paper, followed by HOBtؒH2O (1.2 mol equiv.) and Fmoc-
glycine pentafluorophenyl ester (1.2 mol equiv.) and the reac-
tion mixture was stirred at room temperature overnight. The
reaction mixture was diluted with dichloromethane and washed
successively with saturated aq. NaHCO3 and water. Evapor-
ation gave the crude product, which was purified by column
chromatography.
N-[N-(Fluoren-9-ylmethoxycarbonyl)glycyl]-cis-4-(N6-
benzoyladenin-9-yl)--proline diphenylmethyl ester 13 was
obtained as a foam (85%, starting from 1.28 mmol of substrate
12) after column chromatography (SiO2; 10% methanol in ethyl
552
J. Chem. Soc., Perkin Trans. 1, 1997

View Article Online
acetate), mp 130–133 ЊC (Found: C, 71.0; H, 4.8; N, 12.2.
C47H39N7O6 requires C, 70.8; H, 4.9; N, 12.3%); δH (200
MHz; CDCl3) 2.58 and 2.83 [2 H, 2 × m, CH2(3Ј)], 3.78–4.40
[m, unresolved CH2(5Ј), Gly CH2, Fmoc aliphatic CH and
CH2], 4.78 [1 H, m, CH(2Ј)], 5.05 and 5.28 [1 H, 2 × m, CH(4Ј)
rotamers], 6.13 (1 H, br t, Gly NH), 6.76 (1 H, 2 × s, CHPh2
rotamers), 7.20–7.75 (m, benzoyl m- and p-H, phenyl and Fmoc
aromatic CH), 7.96–8.00 (2 H, d, J 7.1, benzoyl o-H), 8.23 [1 H,
s, CH(8)], 8.70 [1 H, s, CH(2)] and 9.48 (1 H, br s, benzamide
NH); δC(50.28 MHz; CDCl3) 33.5 [CH2(3Ј)], 43.3 (Gly CH2),
47.0 (Fmoc aliphatic CH), 49.4 [CH2(5Ј)], 53.2 [CH(4Ј)], 57.8
[CH(2Ј)], 67.1 (Fmoc CH2), 78.6 (CHPh2), 120.1 (Fmoc aro-
matic CH), 123.5 [C(5)], 125.2–128.9 and 133.0 (aromatic CH),
133.8, 139.4 and 141.4 (aromatic C), 142.0 [CH(8)], 144.1
(aromatic C), 150.0 [C(4)], 152.1 [C(6)], 152.7 [CH(2)], 156.9
(Fmoc CO), 165.4 (benzoyl CO), 168.6 (Gly CO) and 170.4
(ester CO); m/z (ES-MS) 798 (M + H+, 100%); νmax(KBr)/cmϪ1
1718 (C᎐O) and 1668 (C᎐O); [α]22 +18.6 (c 0.21, CHCl3).
1 h. The volatiles were evaporated under reduced pressure, the
residue was triturated with diethyl ether and then washed with
methanol–diethyl ether.
N-[N-(Fluoren-9-ylmethoxycarbonyl)glycyl]-cis-4-(thymin-1-
yl)--proline 9a was obtained as a solid (50%, starting from 2.5
mmol of substrate 8a). Recrystallisation from ethanol–water
gave a solid, mp >200 ЊC (Found: C, 62.5; H, 5.1; N, 10.6.
C27H26N4O7 requires C, 62.5; H, 5.1; N, 10.8%); δH [200 MHz;
(CD3)2SO] 1.75 (3 H, br s, thymine CH3), 2.00–2.35 [2 H, br m,
CH2(3Ј) rotamers], 3.30–4.00 [br m, CH2(5Ј) and Gly CH2
obscured by the water signal], 4.15–4.30 (3 H, br m, Fmoc ali-
phatic CH and CH2), 4.50–4.65 [1 H, br m, CH(2Ј) rotamers],
4.80–4.85 and 4.95–5.05 [1 H, br m, CH(4Ј) rotamers], 7.25–
7.45 (4 H, m, Fmoc aromatic CH), 7.52 [1 H, br s, CH(6)
rotamers], 7.70 and 7.85 (4 H, 2 × d, J 7.1, Fmoc aromatic CH);
m/z (FAB) 541 (M + Na+, 9%), 179 {[(C H ) C᎐CH + H]+,
᎐
6
4
2
2
81}, 165 (32), 119 (30), 103 (44), 85 (83), 77 (32), 59 (85) and
47 (100); νmax(KBr)/cmϪ1 1731 (C᎐O), 1703s (C᎐O) and 1678
᎐
᎐
᎐
᎐
D
N-[N-(Fluoren-9-ylmethoxycarbonyl)glycyl]-cis-4-(N4-
(C᎐O); [α]23 Ϫ4.13 (c 0.63, DMF).
᎐
D
benzoylcytosin-1-yl)--proline diphenylmethyl ester 16 was
obtained as a foam (70%, starting from 1.12 mmol of substrate
15) after column chromatography [SiO2; ethyl acetate–methanol
(20:1)]. Recrystallisation from ethanol gave a solid, mp 131–
133 ЊC (Found: C, 71.2; H, 4.9; N, 9.0. C46H39N5O7 requires C,
71.4; H, 5.1; N, 9.0%); δH (200 MHz; CDCl3) 2.20 and 2.85 [2 H,
2 × m, CH2(3Ј)], 3.61–4.40 [m, unresolved CH2(5Ј), Gly CH2,
Fmoc aliphatic CH and CH2], 4.76–4.81 [1 H, m, CH(2Ј) rotam-
ers], 5.22 and 5.41 [1 H, 2 × m, CH(4Ј) rotamers], 6.03 and 6.12
(1 H, 2 × br t, Gly NH rotamers), 6.78 (1 H, br s, CHPh2),
7.05–8.00 [m, CH(5), CH(6), benzoyl, phenyl and Fmoc aro-
matic CH] and 9.32 (1 H, br s, benzamide NH); δC(50.28 MHz;
CDCl3) 33.2 [CH2(3Ј)], 43.5 (Gly CH2), 47.0 (Fmoc aliphatic
CH), 49.2 [CH2(5Ј)], 55.5 [CH(4Ј)], 57.8 [CH(2Ј)], 67.1 (Fmoc
CH2), 78.6 (CHPh2), 97.3 [CH(5)], 120.1 (Fmoc aromatic CH),
125.4–129.1 and 133.1 (aromatic CH), 133.3, 139.4 and 141.1
(aromatic C), 144.1 (aromatic C), 145.6 [CH(6)], 155.8 [C(2)],
156.7 (Fmoc CO), 162.6 and 163.0 [C(4) rotamers], 167.1 (ben-
zoyl CO), 168.6 (Gly CO) and 170.6 (ester CO); m/z (ES-MS)
N-[N-(Fluoren-9-ylmethoxycarbonyl)glycyl]-trans-4-(thymin-
1-yl)--proline 9b was obtained as a solid (57%, starting
from 5.3 mmol of substrate 8b), mp >200 ЊC; δH [200 MHz;
(CD3)2SO] 1.70 (3 H, br s, thymine CH3), 2.05–2.15 and 2.40–
2.60 [br m, CH2(3Ј) rotamers, obscured by the Me2SO (DMSO)
signal], 3.50–4.00 [br m, CH2(5Ј) and Gly CH2], 4.15–4.30 (3 H,
br m, Fmoc aliphatic CH and CH2), 4.35–4.45 and 4.75–4.85 [1
H, br m, CH(2Ј) rotamers], 4.90–5.00 and 5.05–5.10 [1 H, m,
CH(4Ј) rotamers], 7.25–7.45 (4 H, m, Fmoc aromatic CH), 7.55
[1 H, br s, CH(6)], 7.68 (4 H, 2 × d, J 7.1, Fmoc aromatic CH);
m/z (FAB) 519 (M + H+, 6%), 179 {[(C H ) C᎐CH + H]+, 34},
᎐
6
4
2
2
85 (100), 59 (23) and 47 (32).
N-[N-(Fluoren-9-ylmethoxycarbonyl)glycyl]-cis-4-(thymin-
1-yl)--proline 9c was obtained as a solid (42%, starting from
6.8 mmol of substrate 8c), mp >200 ЊC; δH [200 MHz;
(CD3)2SO] 1.75 (3 H, br s, thymine CH3), 2.11 and 2.52 [2 H,
2 × br m, CH2(3Ј) rotamers], 3.50–4.00 [br m, CH2(5Ј) and Gly
CH2], 4.18–4.30 [4 H, br m, CH(2Ј) and Fmoc aliphatic CH and
CH2], 4.73 and 4.98 [1 H, 2 × br m, CH(4Ј) rotamers], 7.28–7.41
(4 H, m, Fmoc aromatic CH), 7.51 (1 H, m, Gly NH), 7.54 [1 H,
br s, CH(6) rotamers] and 7.72 and 7.88 (4 H, 2 × d, J 7.1,
Fmoc aromatic CH); m/z (FAB) 541 (M + Na+, 2%), 519
(M + H+, 5), 179 (33), 103 (17), 85 (100), 77 (18), 59 (43) and
47 (45); [α]2D3 +4.26 (c 0.61, DMF).
774 (M + H+, 100%); νmax(KBr)/cmϪ1 1750–1665br (C᎐O);
᎐
[α]2D2 +20.9 (c 0.21, CHCl3).
N-[N-(Fluoren-9-ylmethoxycarbonyl)glycyl]-cis-4-(N2-
isobutyrylguanin-9-yl)--proline diphenylmethyl ester 19 was
obtained as a solid (52%, starting from 0.73 mmol of substrate
18) after column chromatography (SiO2; 10% methanol in ethyl
acetate). Recrystallisation from ethyl acetate–light petroleum
(40–60 ЊC) gave a crystalline solid, mp 145–150 ЊC (Found: C,
68.0; H, 5.2; N, 12.6. C44H41N7O7 requires C, 67.8; H, 5.3; N,
12.6%); δH (200 MHz; CDCl3) 1.18 and 1.21 [6 H, d, J 6.7,
(CH3)2CH], 2.31 (1 H, br m) and 2.61–2.79 (2 H, br m) [CH2(3Ј)
and (CH3)2CH], 3.89–4.20 [m, unresolved CH2(5Ј), Gly CH2
and Fmoc aliphatic CH], 4.37 (2 H, d, J 6.7, Fmoc CH2), 4.63 [1
H, m, CH(2Ј)], 4.82 [1 H, m, CH(4Ј)], 6.10 (1 H, br m, Gly NH),
6.77 (1 H, s, CHPh2), 7.12–7.36 (m, phenyl and Fmoc aromatic
CH), 7.49–7.57 [3 H, m, Fmoc aromatic CH and CH(8)], 7.69–
7.73 (2 H, d, J 7.4, Fmoc aromatic CH) and 9.83 (1 H, br s,
isobutyramide NH); δC(50.28 MHz; CDCl3) 18.9 [(CH3)2CH],
35.7 [CH2(3Ј)], 42.9 (Gly CH2), 46.5 (Fmoc CH), 49.0 [CH2(5Ј)],
52.9 [CH(4Ј)], 57.7 [CH(2Ј)], 66.9 (Fmoc CH2), 78.3 (CHPh2),
120.1 (Fmoc aromatic CH), 120.9 [C(5)], 125.2–128.8 (Fmoc
aromatic CH), 137.5 [CH(8)], 139.4 and 139.6 (aromatic C),
144.0 (aromatic C), 148.3 and 148.8 [C(2)/C(6)], 155.8 [C(4)],
157.2 (Fmoc CO), 169.0 (Gly CO), 170.2 (ester CO) and 180.6
(isobutyramide CO); m/z (ES-MS) 780 (M + H+, 100%);
λmax(CHCl3)/nm 270sh (ε/dm3 molϪ1 cmϪ1 11.9 × 104); [α]2D3
+36.7 (c 0.645, CHCl3).
N-[N-(Fluoren-9-ylmethoxycarbonyl)glycyl]-4-(thymin-1-
yl)proline pentafluorophenyl esters 10a–c
A suspension of the Fmoc-dipeptide (9a, 9b or 9c) (1.0 mmol),
pentafluorophenol (1.1 mmol) and DCCI (1.1 mmol) in
dichloromethane (5 ml) was stirred at room temperature for 2–
3 h. The precipitated DCU was filtered off and washed with
dichloromethane. Evaporation of the filtrate followed by
column chromatography (SiO2; ethyl acetate) gave the product
as a foam which in most cases could be made crystalline by tri-
turation with diethyl ether–light petroleum, then being filtered
and air dried.
N-[N-(Fluoren-9-ylmethoxycarbonyl)glycyl]-cis-4-(thymin-
1-yl)--proline pentafluorophenyl ester 10a was obtained from
substrate 9a as a solid (0.574 g, 84%), mp 124–126 ЊC; δH (200
MHz; CDCl3) 1.94 (3 H, s, thymine CH3), 2.26–2.42 and 2.85–
3.00 [2 H, m, CH2(3Ј)], 3.66–3.75 and 3.95–4.26 [m, unresolved
CH2(5Ј), Gly CH2, Fmoc aliphatic CH], 4.38–4.42 (2 H, d, J
7.1, Fmoc CH2), 4.85–4.93 [1 H, m, CH(2Ј)], 5.34–5.42 [1 H, m,
CH(4Ј)], 5.80–5.82 (1 H, br t, Gly NH), 7.10 [1 H, s, CH(6)],
7.31–7.44, 7.60–7.63 and 7.75–7.79 (8 H, m, Fmoc aromatic
CH) and 9.50 (1 H, s, thymine NH); δF (235.35 MHz; CDCl3)
Ϫ162.0 (dd, J 18.1 and 21.4) and Ϫ161.2 (t, J 19.6) (m-F major
and minor rotamers), Ϫ157.0 (t, J 21.8) and Ϫ156.2 (t, J 21.7)
(p-F major and minor rotamers) and Ϫ153.1 (d, J 18.5) and
Ϫ152.8 (d, J 17.7) (o-F minor and major rotamers). The ratio
of major :minor rotamers was ~15:1; m/z (ES-MS) 685.1
N-[N-(Fluoren-9-ylmethoxycarbonyl)glycyl]-4-(thymin-1-yl)-
prolines 9a–c
The protected dipeptide (8a, 8b or 8c) was treated with 10%
HBr in acetic acid (5–10 ml mmolϪ1) at room temperature for
J. Chem. Soc., Perkin Trans. 1, 1997
553

View Article Online
(M + H+, 100%); νmax(KBr)/cmϪ1 1801 (C᎐O) and 1675br
obtained as a solid (81%, starting from 0.17 mmol of substrate
᎐
(C᎐O); [α]23 Ϫ15.9 (c 0.630, CHCl3).
16), mp 133–137 ЊC; δH (200 MHz; CDCl3) 2.46–2.60 and 2.90–
3.12 [2 H, m, CH2(3Ј)], 3.80–4.43 [7 H, m, CH2(5Ј), Gly CH2
and Fmoc aliphatic CH and CH2], 4.93–5.01 [1 H, t, J 7.7,
CH(2Ј)], 5.41–5.49 [1 H, m, CH(4Ј)], 5.72–5.78 (1 H, br t, J 4.5,
Gly NH), 7.27–7.79 [m, CH(6), CH(5), Fmoc aromatic CH and
benzoyl CH] and 7.90 (1 H, br s, benzamide NH); δF (235.35
MHz; CDCl3) Ϫ162.0 (t, J 21.1) and Ϫ161.3 (t, J 19.1) (m-F
major and minor rotamers), Ϫ157.2 (t, J 19.4) and Ϫ156.4 (t, J
19.4) (p-F major and minor rotamers) and Ϫ152.6 (d, J 20.0)
(o-F); m/z (ES-MS) 774 (M + H+, 100%); νmax(KBr)/cmϪ1 1797
᎐
D
N-[N-(Fluoren-9-ylmethoxycarbonyl)glycyl]-trans-4-(thymin-
1-yl)--proline pentafluorophenyl ester 10b was obtained from
substrate 9b as a solid (0.400 g, 58%), mp 115–124 ЊC; δH (200
MHz; CDCl3) 1.94 (3 H, s, thymine CH3), 2.49–2.61 and
2.79–2.95 [2 H, m, CH2(3Ј)], 3.79–4.13 [4 H, m, CH2(5Ј)
and Gly CH2], 4.22 (1 H, t, J 6.7, Fmoc aliphatic CH), 4.37–
4.41 (2 H, d, J 7.1, Fmoc CH2), 5.04–5.18 [2 H, m, CH(2Ј)
and CH(4Ј)], 5.78–5.82 (1 H, br t, Gly NH), 7.00 and 7.02 [1 H,
2 × s, CH(6) rotamers], 7.27–7.44, 7.58–7.61 and 7.74–7.78 (8
H, m, Fmoc aromatic CH) and 9.17 and 9.21 (1 H, 2 × s, thy-
mine NH rotamers); δF (235.35 MHz; CDCl3) Ϫ162.1 (t, J 19.3)
and Ϫ161.4 (t, J 21.8) (m-F major and minor rotamers), Ϫ157.2
(t, J 22.5) and Ϫ156.3 (t, J 19.4) (p-F major and minor
rotamers) and Ϫ153.3 (d, J 19.5) and Ϫ153.0 (d, J 20.0) (o-F
(C᎐O) and 1669 (C᎐O).
᎐
᎐
N-[N-(Fluoren-9-ylmethoxycarbonyl)glycyl]-cis-4-(N 2-iso-
butyrylguanin-9-yl)--proline pentafluorophenyl ester 20 was
obtained as a solid (63%, starting from 0.16 mmol of substrate
19), mp 146–150 ЊC; δH (200 MHz; CDCl3) 1.22–1.27 [6 H,
2 × d, J 6.9, (CH3)2CH], 2.60–2.82 and 2.97–3.12 [3 H, m,
CH2(3Ј) and (CH3)2CH ], 4.08–4.30 [5 H, m, CH2(5Ј), Gly CH2
and Fmoc aliphatic CH], 4.37–4.41 (2 H, d, J 7.2, Fmoc CH2),
4.85–4.94 [1 H, t, J 8.3, CH(2Ј)], 4.99–5.06 [1 H, m, CH(4Ј)],
5.79–5.84 (1 H, br t, J 4.5, Gly NH), 7.27–7.44 (4 H, m, Fmoc
aromatic CH), 7.56–7.60 (2 H, d, J 7.4, Fmoc aromatic CH),
7.67 [1 H, s, CH(8)], 7.74–7.78 (2 H, d, J 7.4, Fmoc aromatic
CH) and 8.95 (1 H, s, isobutyramide NH); m/z (ES-MS) 780
minor and major rotamers); νmax(KBr)/cmϪ1 1797 (C᎐O) and
᎐
1679br (C᎐O); [α]23 +30.0 (c 0.73, CHCl3).
᎐
D
N-[N-(Fluoren-9-ylmethoxycarbonyl)glycyl]-cis-4-(thymin-1-
yl)--proline pentafluorophenyl ester 10c was obtained from
substrate 9c as a solid (0.520 g, 76%), mp 122–126 ЊC; δH (200
MHz; CDCl3) 1.94 (3 H, s, thymine CH3), 2.26–2.42 and 2.85–
3.00 [2 H, m, CH2(3Ј)], 3.66–3.75 and 3.95–4.26 [5 H, m,
CH2(5Ј), Gly CH2 and Fmoc aliphatic CH], 4.38–4.42 (2 H, d, J
7.1, Fmoc CH2), 4.85–4.93 [1 H, m, CH(2Ј)], 5.34–5.42 [1 H, m,
CH(4Ј)], 5.80–5.82 (1 H, br t, Gly NH), 7.10 [1 H, s, CH(6)],
7.31–7.44, 7.60–7.63 and 7.75–7.79 (8 H, m, Fmoc aromatic
CH) and 9.50 (1 H, s, thymine NH); δF (235.35 MHz; CDCl3)
Ϫ162.0 (dd, J 18.1 and 21.4) and Ϫ161.2 (t, J 19.6) (m-F major
and minor rotamers), Ϫ157.0 (t, J 21.8) and Ϫ156.2 (t, J 21.7)
(p-F major and minor rotamers) and Ϫ153.1 (d, J 18.5) and
Ϫ152.8 (d, J 17.7) (o-F minor and major rotamers). The ratio
of major :minor rotamers was ~15:1; νmax(KBr)/cmϪ1 1800
(C᎐O) and 1683br (C᎐O); [α]23 +16.3 (c 0.645, CHCl3).
(M + H+, 100%); νmax(KBr)/cmϪ1 1798 (C᎐O) and 1680br
᎐
(C᎐O).
᎐
Acknowledgements
We gratefully acknowledge the Thai government’s DPST
scholarship (to T. V.). We also would like to thank Dr R. T.
Aplin for electrospray mass spectra, Mrs E. McGuinness and
1
Miss T. Jackson for highfield H NMR and 19F NMR spectra,
and Mrs V. Lamburn for microanalyses.
᎐
᎐
D
Procedure for deprotection of diphenylmethyl esters and
synthesis of Fmoc-dipeptide pentafluorophenyl esters 14, 17 and
20
References
1 G. Lowe and T. Vilaivan, J. Chem. Soc., Perkin Trans. 1, 1997, 539,
preceding article.
The Fmoc dipeptide diphenylmethyl ester 13, 16 or 19 was
treated with TFA acid (~ 5–10 ml mmolϪ1) containing anisole (50
µl mlϪ1 TFA) for 2–3 h. The volatiles were evaporated off under
reduced pressure and the residue was triturated and washed
with diethyl ether. The free acid was obtained as a solid in
nearly quantitative yield after drying over NaOH pellets in
vacuo. This was dissolved in 1:1 DMF–dichloromethane (5
ml mmolϪ1) and pentafluorophenol (1.5 mol equiv.) and DCCI
(1.5 mol equiv.) were added with stirring of the mixture at room
temperature. The reaction mixture was stirred at room tem-
perature for 1–3 h (monitored by TLC). The DCU precipitate
was filtered off, and washed with dichloromethane. The com-
bined organic phase was washed with water, dried (MgSO4) and
evaporated under reduced pressure. The residue was triturated
with diethyl ether–light petroleum (40–60 ЊC) or reprecipitated
from suitable solvents to give the product which contained a trace
2 J. Kovacs, in The Peptides, ed. E. Gross and J. Meienhofer, Academic
Press, New York, 1980, vol. 2, pp. 486–536.
3 G. B. Fields and R. L. Noble, Int. J. Pept. Protein Res., 1990, 35,
161.
4 S. Clark, Part II Thesis, Oxford University, 1994.
5 R. Hiskey and J. B. Adams, Jr., J. Am. Chem. Soc., 1965, 87, 3969.
6 (a) T. W. Greene and P. G. M. Wuts, Protective Groups in Organic
Synthesis, Wiley, New York, 2nd edn., 1991, p. 328; (b) Z. Tozuka
and T. Takaya, J. Antibiot., 1983, 36, 142.
7 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.
8 G. C. Stelakatos, A. Paganon and L. Zervas, J. Chem. Soc. C, 1966,
1191.
9 D. Ben-Ishai and A. Berger, J. Org. Chem., 1952, 17, 1564.
10 M. L. Peterson and R. Vince, J. Med. Chem., 1991, 34, 2787.
11 J. B. Miller, J. Org. Chem., 1959, 24, 560.
12 L. Kisfaludy and I. Schon, Synthesis, 1983, 325; J. Kovacs, R. E.
Cover, R. H. Johnson, T. J. Kalas, G. L. Mayers and J. E. Roberts,
J. Org. Chem., 1973, 38, 2518; J. Kovacs, L. K. Kisfaludy and
M. Q. Ceprini, J. Am. Chem. Soc., 1967, 89, 183.
13 M. T. Chenon, R. J. Pugmire, D. M. Grant, R. P. Panzica and
L. B. Townsend, J. Am. Chem. Soc., 1975, 97, 4627.
14 R. R. Chauvette, R. A. Pennington, C. W. Ryan, R. D. E. Cooper,
L. Jose, I. G. Wright, E. M. Heyningen and G. W. Huffmann, J. Org.
Chem., 1971, 36, 1259.
1
of DCU (~10%) as the only impurity according to H NMR
analysis, but which was pure enough for solid phase synthesis.
N-[N-(Fluoren-9-ylmethoxycarbonyl)glycyl]-cis-4-(N 6-
benzoyladenin-9-yl)--proline pentafluorophenyl ester 14 was
obtained as a solid (83%, starting from 0.5 mmol of substrate
13), mp 125–130 ЊC; δH (200 MHz; CDCl3) 2.86–3.02 and 3.10–
3.24 [2 H, m, CH2(3Ј)], 3.98–4.42 [7 H, m, CH2(5Ј), Gly CH2
and Fmoc aliphatic CH and CH2], 4.97–5.06 [1 H, t, J 8.5,
CH(2Ј)], 5.31–5.42 [1 H, m, CH(4Ј)], 5.70–5.74 (1 H, br t, J 3.8,
Gly NH), 7.27–7.79 (11 H, m, Fmoc aromatic CH and benzoyl
m- and p-H), 8.02–8.06 (2 H, d, J 6.7, benzoyl o-H), 8.13 [1 H, s,
CH(8)], 8.80 [1 H, s, CH(2)] and 9.00 (1 H, br s, benzamide
NH); m/z (ES-MS) 798 (M + H+, 100%); νmax(KBr)/cmϪ1 1798
15 D. M. Brown, A. Todd and S. Varadarajan, J. Chem. Soc., 1956,
2384.
16 T. F. Jenny, K. C. Schneider and S. A. Benner, Nucleosides,
Nucleotides, 1992, 11, 1257.
17 D. D. Perrin and W. L. F. Amarego, Purification of Laboratory
Chemicals, Pergamon Press, Oxford, 3rd edn., 1988.
(C᎐O) and 1671br (C᎐O).
Paper 6/03699F
Received 28th May 1996
Accepted 3rd October 1996
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N-[N-Fluoren-9-ylmethoxycarbonyl)glycyl]-cis-4-(N 4-
benzoylcytosin-1-yl)--proline pentafluorophenyl ester 17 was
554
J. Chem. Soc., Perkin Trans. 1, 1997