Fmoc/Acyl protecting groups in the synthesis of polyamide (peptide) nucleic acid monomers

Article abstract of DOI:10.1039/a907832k

The chemical synthesis of polyamide (peptide) nucleic acid (PNA) monomers 22-25 has been accomplished using Fmoc [N-(2-aminoethyl)glycine backbone], anisoyl (adenine), 4-tert-butylbenzoyl (cytosine) and isobutyryl/ diphenylcarbamoyl (guanine) protecting-group combinations, thus allowing oligomer synthesis on both peptide and oligonucleotide synthesizers. An alternative method for the preparation of (N⁶-anisoyladenin-9-yl)acetic acid 7 is described using partial hydrolysis of a dianisoylated derivative. Different methods were studied for guanine alkylation including (a) Mitsunobu reaction; (b) low-temperature, sodium hydride- and (c) N, N-diisopropylethylaminemediated alkylation reactions to give preferentially N⁹-substituted derivatives. Empirical rules are proposed for differentiating N⁹/N⁷-substituted guanines based on their ¹³C NMR chemical-shift differences. The Royal Society of Chemistry 2000.

Full text of DOI:10.1039/a907832k

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Fmoc/Acyl protecting groups in the synthesis of polyamide
(peptide) nucleic acid monomers
Zoltán Timár,a Lajos Kovács,*a Györgyi Kovácsa and Zoltán Schmélb
1
a Department of Medicinal Chemistry, Dóm tér 8 and Department of Pharmaceutical
b
Analysis, Somogyi B. u. 4, Albert Szent-Györgyi Medical University, H-6720 Szeged,
Hungary. Fax: ϩ36-62-42 52 62; E-mail: kovacs@ovrisc.mdche.u-szeged.hu
Received (in Cambridge, UK) 29th September 1999, Accepted 10th November 1999
The chemical synthesis of polyamide (peptide) nucleic acid (PNA) monomers 22–25 has been accomplished
using Fmoc [N-(2-aminoethyl)glycine backbone], anisoyl (adenine), 4-tert-butylbenzoyl (cytosine) and isobutyryl/
diphenylcarbamoyl (guanine) protecting-group combinations, thus allowing oligomer synthesis on both peptide
and oligonucleotide synthesizers. An alternative method for the preparation of (N6-anisoyladenin-9-yl)acetic acid 7 is
described using partial hydrolysis of a dianisoylated derivative. Different methods were studied for guanine alkylation
including (a) Mitsunobu reaction; (b) low-temperature, sodium hydride- and (c) N,N-diisopropylethylamine-
mediated alkylation reactions to give preferentially N9-substituted derivatives. Empirical rules are proposed for
differentiating N9/N7-substituted guanines based on their 13C NMR chemical-shift differences.
Introduction
Results and discussion
Polyamide (or as originally referred: peptide) nucleic acids
(PNA) are one of the most powerful analogues of oligo-
nucleotides in terms of chemical and enzymic stability,
double- and triple-helix formation, with potential applications
in antisense diagnosis and therapeutics.1,2 In these compounds
the entire sugar–phosphate backbone is replaced with an N-(2-
aminoethyl)glycine moiety and the nucleobases are attached
through an N-acetyl linkage.
The choice of nucleobase-protecting groups was motivated by
different considerations since uniform protection, though
attractive, is not possible. Thymine does not require protection
and our synthesis of the thymine monomer, starting from
acid 1, was based on the procedure of Thomson et al.9 The
anisoylated cytosine derivative 2 was alkylated to give acid 3
(Scheme 1) but the solubilities of these substances were so low
The chemical synthesis of PNA mostly relies on the assembly
of the protected N-(2-aminoethyl)glycine backbone and pro-
tected nucleobase-substituted acetic acid structural units fol-
lowed by standard oligomerization protocols.3,4 The pioneering
efforts of a Danish group resulted in the application of Boc
(backbone) and Z (cytosine, adenine) or O-benzyl (guanine)
protection.5,6 Later the Uhlmann group used a monomethoxy-
trityl (MMTr)/acyl (anisoyl, 4-tert-butylbenzoyl, isobutyryl/
acetyl) strategy.7,8 All these methods require the use of (strong)
acidic conditions (e.g., TFA, HF) in the oligomer construction
and final cleavage from the support. The need for milder
methods led to the employment of the Fmoc group for back-
bone protection and Z9 or MMTr groups10 for the nucleobases.
The Fmoc group is a convenient alternative to acid-sensitive
backbone-protecting groups (Boc, MMTr) and allows easy
monitoring of the coupling process.11 The combination Fmoc/
acyl should also be feasible since the former group can be
cleaved without affecting the more stable base-protecting acyl
groups.12–14 Herein we report on our results concerning the use
of Fmoc (backbone)/acyl (4-tert-butylbenzoyl for cytosine;
anisoyl for adenine; isobutyryl/N,N-diphenylcarbamoyl for
guanine) protecting groups in the synthesis of PNA monomers.
The prior protecting-group combinations (with the exception
of Fmoc/MMTr) were used either for peptide or oligonucleo-
tide synthesis protocols. With biologically important PNA–
DNA and PNA–peptide conjugates in mind our approach
offers a substantial advantage over the existing ones since both
oligomerization methodologies are possible with the same
monomers. Beside this, use of the frequently applied urethane
protecting groups (e.g., Z) for nucleobases is not practical as
in our experience the yields are often very low. This paper
complements and details our preliminary account.15
Scheme 1 Reagents and conditions: a, (1) BrCH2COOMe, K2CO3,
DMF; (2) NaOH, then HCl; b, AnCl, py, 80 ЊC; c, 4-ButC6H4COCl,
Et3N, DMF; d, NaH, DMF, BrCH2COOEt; e, NaOH, aq. 1,4-dioxane,
then HCl. An = 4-MeOC6H4CO.
in common solvents that we had to abandon this group. The
4-tert-butylbenzoyl group proved to be more rewarding; acid 58
was easily obtained via intermediate 4 and used later.
(N6-Anisoyladenin-9-yl)acetic acid 77 was prepared in 28%
overall yield from adenine via the alkylation of compound 6
(Scheme 2). An improved overall yield (40%) was obtained
when tert-butyl bromoacetate was used for the alkylation
J. Chem. Soc., Perkin Trans. 1, 2000, 19–26
This journal is © The Royal Society of Chemistry 2000
19

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Scheme 2 Reagents and conditions: a, 1.5 equiv. AnCl, py, 100 ЊC; b,
NaH, DMF, BrCH2COOMe; c, NaOH, then KHSO4; d, NaH, DMF,
BrCH2COOBut; e, 50% (v/v) TFA–CH2Cl2, ( )-1,4-dithiothreitol; f,
NaH, DMF, BrCH2COOEt; g, 2.5 equiv. AnCl, py, 80 ЊC; h, NaOH,
aq. MeOH, then HCl.
Scheme 4 Reagents and conditions: a, DIAD, Ph3P, THF, HOCH2-
COOBut; b, DIAD, 4-Me2NC6H4PPh2, THF, HOCH2COOBut; c,
DIAD, Bu3P, THF, HOCH2COOBut; d, NaH, Ϫ20 ЊC, DMF, BrCH2-
COOBut; e, 8% (v/v) TFA–CH2Cl2, 1,3-(MeO)2C6H4, 0 ЊC, 18 h; f,
BrCH2COOMe, EtNPri2, DMF; g, NaOH, aq. 1,4-dioxane–MeOH,
then HCl.
instead of methyl bromoacetate followed by acidolysis. In
an alternative approach ethyl (adenin-9-yl)acetate 85 was
anisoylated with excess of anisoyl chloride and the resulting
N6,N6-dianisoylated derivative (not isolated) was subjected to
partial hydrolysis to give acid 7 in an improved yield (70%;
overall 45% from adenine).
reaction24,25 provided the product (13, Scheme 4) with good
regioselectivity; however, its purification was very difficult and
it was contaminated with significant amounts of triphenyl-
phosphine oxide. (4-Dimethylaminophenyl)diphenylphos-
phine,26,27 claimed to give a phosphine oxide which can be
removed by acidic extraction,28 proved to be unsatisfactory
since the product was still contaminated with the corresponding
phosphine oxide. Tributylphosphine, the oxide of which is
water-soluble, gave a cleaner product but the yield was low
(36%). In the next experiments sodium hydride-mediated
alkylation with tert-butyl bromoacetate was used to obtain the
desired compound. We noticed that at ambient temperature the
relative proportion of N7-regioisomer was relatively high, while
lowering the temperature favoured the formation of the desired
N9-regioisomer. At Ϫ20 ЊC a clean reaction gave negligible
amounts of the N7-isomer but the yield of N9-isomer was still
low (40%). Acidolysis of ester 13 in dilute TFA–CH2Cl2 (0 ЊC;
18 h) removed the N,N-diphenylcarbamoyl (Dpc) group with-
out affecting the tert-butyl ester functionality (→ 11). Albeit
there is some evidence for the lability of this group in 50% (v/v)
TFA–CH2Cl2 8 or in the presence of Lewis acids29 it was surpris-
ing that the Dpc group was more sensitive towards acid than
was the tert-butyl group. The latter was expected to cleave
under similar conditions.14,30
The substitution of guanine is notorious for giving N9/N7-
regioisomers.16 Although the 2-amino group is not really nucleo-
philic enough to interfere with many transformations, the poor
solubility of unprotected guanine excludes its use in most reac-
tions. The application of N2-acyl (acetyl, propionyl, isobutyryl,
etc.)-protected derivatives increases the solubility but N2-
acylation alone cannot solve the fundamental problem of the
selectivity of alkylation.16 Constraining guanine from its dom-
inant 6-lactam structure to lactim (enolate) form by different
groups has a beneficial effect on the ratio of N9/N7-regio-
isomers. The most successful in this respect is the N,N-diphenyl-
carbamoyl protecting group17 which reportedly gives in some
cases a 100:1 ratio in favour of the N9-regioisomer.18,19 To see
how the introduction of this group alters the selectivity of
alkylation, first 2-N-isobutyrylguanine 920 was alkylated with
tert-butyl bromoacetate in the presence of sodium hydride to
afford a nearly 1:1 ratio of N9/N7-isomers (11 and 12, respect-
ively) in 74% yield (Scheme 3). The selection of the isobutyryl
The application of N,N-diisopropylethylamine as a hindered
base and methyl bromoacetate8 to circumvent premature cleav-
age of the Dpc group in the subsequent hydrolysis gave a 71%
yield of the product 14, of which 58% was available without
chromatography, along with 15% of the N7-isomer 15. Basic
hydrolysis of ester 14 led to acid 16 in a clean transformation.
The surprisingly high yield of the unwanted isomer 15 in the
first reaction underlines the fact that even the sterically hin-
dered Dpc protecting group is not sufficient to steer the reaction
to complete regioselectivity. Thus the claim that the use of the
Dpc group has solved the historic problem of regioselective N9 -
substitution of guanine18,19 seems to be restricted to the realm
of glycosylation reactions, while alkylation transformations
require further experimentation.
Scheme 3 Reagents and conditions: a, (PriCO)2O, DMF, 150 ЊC; b,
Ac2O, DMF, 100 ЊC; c, Ph2NCOCl, EtNPri2, py; d, NaH, DMF, 0 ЊC,
BrCH2COOBut. Ibu = PriCO; Dpc = Ph2NCO.
group was motivated by the fact that although its removal
under basic conditions is more sluggish than that of other
simple acyl groups (acetyl, propionyl)17,21 it confers steric
hindrance on the 2-amino group and thus prevents unwanted
alkylation/glycosylation on it.19
The coupling of nucleobase-substituted acetic acids 1, 5, 7,
16 with the backbone unit 179 under standard peptide-coupling
conditions afforded the PNA esters 18–21 which were sub-
sequently acidolysed (TFA in dichloromethane) to give the
PNA monomers 22–25 (Scheme 5). As expected from our pre-
vious experience (13 → 11, Scheme 4) in the latter reaction
the Dpc protecting group was removed along with the tert-butyl
group. It is clear that in this final deprotection step the protect-
Next the N,N-diphenylcarbamoyl derivative 1019,22 was
chosen for alkylation studies under different conditions. Its
transformation with tert-butyl glycolate23 in the Mitsunobu
20
J. Chem. Soc., Perkin Trans. 1, 2000, 19–26

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Fig. 1 13C NMR chemical-shift parameters of N9/N7-substituted
guanines.
sophisticated assignment techniques (selective INEPT, HMQC,
HMBC, etc.).
Scheme 5 Reagents and conditions: a, HBTU, HOBt, DMF, EtNPri2;
b, 17–43% (v/v) TFA–CH2Cl2, 1,3-(MeO)2C6H4.
2. The parameter b shows similar characteristics {N9: 16.10–
26.91 ppm; N7: 29.70–41.17 ppm; for regioisomers the differ-
ence [∆ = b(N9) = b(N7)] is Ϫ11.68 to Ϫ16.15 ppm} and its utility
is further enhanced by the fact that δC-5 is unmistakable
among the skeletal carbons and δC-8 can simply be located in a
J-modulated spin-echo experiment.
ing groups of ester 21 (Fmoc, Ibu, Dpc, But) are not completely
orthogonal and further research is required. Although the pres-
ence of the Dpc group is not essential in oligomer synthesis, it
may confer better solubility on the guanine subunits.
3. The parameter c can be clustered according to the nature
of attached substituent rather than lactam/lactim tautomerism
and it gives useful values for glycosylated derivatives {N9:
33.38–35.81 ppm; N7: 16.30–23.38 ppm; for regioisomers the
difference [∆c = c(N9) Ϫ c(N7)] is 10.61–12.79 ppm} while for
(cyclo)alkylated compounds it is of less use {N9: 53.40–83.45
ppm; N7: 54.57–71.15 ppm; for regioisomers the difference
[∆c = c(N9) Ϫ c(N7)] is 10.68–13.35 ppm}. The identification
of δC-1Ј, involved in this parameter, often requires more sophisti-
cated techniques.
As a conclusion it can be seen that the values a, b [both for
(cyclo)alkyl and glycosylated derivatives] and c (for glyco-
sylated derivatives) are useful for characterizing the N9/N7-
substitution pattern of guanines. From a practical point of view
the parameter b is the most convenient one for the reasons
explained above. It is noteworthy that δC-5 (118.81 ppm) and the
parameters a (35.38 ppm) and b (26.06 ppm) for compound
1019 are in good agreement with those for N9-substituted
derivatives, suggesting that its dominant tautomer is 9H in
DMSO-d6 solution.
UV spectroscopy is often used to locate substituents on
nucleobases. However, this technique is not reliable in the case
of guanines especially if only one regioisomer is available.31
Moreover, there are no data on the influence of lactam/lactim
tautomerism of guanines on UV properties. Indeed, the UV
spectra of N9/N7-regioisomers 11/12 (lactams) and 14/15
(lactims), respectively, in buffered ethanolic solutions (pH 0,
6 and 13) revealed that there are no significant differences
which would justify the assignment based solely on UV data.
Therefore the site of alkylation in guanine derivatives 13–15,
2632 was established in 2D NMR (HMQC33,34 and HMBC35,36
)
experiments.
Scrutinizing the 13C NMR chemical-shift parameters of
compounds 11–16, 21, 25, 26 (Table 1) and a further 45 N9/N7-
substituted guanines19,37–39 (altogether 54 compounds) show
that δC-5 is the most sensitive to the N9/N7-substitution pattern
(Fig. 1, N9: 113.75–123.70 ppm; N7: 104.56–115.09 ppm; for
regioisomers the difference [∆δC-5 = δC-5(N9) Ϫ δC-5(N7)] is 7.86–
9.82 ppm), insensitive to the lactam/lactim tautomerism (data
not shown) and this signal alone can be of diagnostic value,
especially if data for both regioisomers are available. However,
due to the overlapping of chemical-shift ranges for regioisomers
this parameter might not be sufficient for unambiguous
assignment. Kjellberg and Johansson37 suggested that δC-1Ј, δC-5
and δC-8 could be used to differentiate N9/N7-substituted
guanines. This was also corroborated, in part, by our findings;
however, some further tendencies were also observed. The util-
Further studies relating to the application of the above
monomers in the preparation of PNA oligomers and our quest
for novel combinations of protecting groups are in progress and
will be reported in due time.
Experimental
General
The following abbreviations are employed: diisopropyl azodi-
carboxylate (DIAD); N,N-diisopropylethylamine (DIPEA);
2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexa-
fluorophosphate (HBTU); 1-hydroxybenzotriazole (HOBt);
trifluoroacetic acid (TFA). Chemicals were purchased from
Aldrich or Fluka. Sodium hydride refers to a 55% suspension in
mineral oil. Anhydrous solvents were prepared as described.40
Light petroleum refers to the fraction with distillation range
40–60 ЊC. Thymine derivatives 1, 18 and 22 were prepared
using the procedure of Thomson et al.9 N7-Benzylguanine
hydrochloride 2632 was prepared for comparison of its 13C NMR
parameters with those of other guanine derivatives (see Table
1). Organic solutions were dried using magnesium sulfate and
evaporated in Büchi rotary evaporators. TLC: Kieselgel 60 F254
(Merck), visualization: UV light. Column chromatography:
Kieselgel 60 (0.063–0.200 mm, Merck). Mp: Electrothermal
IA 8103 apparatus. Elemental analysis: Perkin-Elmer CHN
ity of differential parameters a = δC-4 Ϫ δC-5, b = δC-8 Ϫ δC-5
,
c = δC-5 Ϫ δC-1Ј, (Fig. 1) has been assessed and the following
conclusions could be drawn:
1. The parameter a distinctly differs for the regioisomers {N9:
28.20–35.41 ppm; N7: 41.35–54.25 ppm; for regioisomers the
difference [∆a = a(N9) Ϫ a(N7)] is Ϫ9.93 to Ϫ20.53 ppm},
shows little variation for lactam/lactim tautomerism (data not
shown) and presents no overlapping ranges. The diagnostic
value of this observation is slightly diminished since δC-4 usually
cannot be simply identified without having recourse to more
J. Chem. Soc., Perkin Trans. 1, 2000, 19–26
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analyzer model 2400. UV: PE Lambda 10 spectrometer, λmax
nm (lg ε), sh: shoulder. IR: Bio-Rad FTS-60A (KBr pellets, νmax
/
/
(6.47 g, 24.0 mmol) suspended in anhydrous DMF (120 mL)
was added sodium hydride (1.05 g, 24.0 mmol) in portions and
the mixture was stirred at room temperature for 30 min. tert-
Butyl bromoacetate (3.9 mL, 26.4 mmol) was added dropwise
and stirring was continued for 2 h. The mixture was evaporated
in vacuo and the residue was suspended in a mixture of
water (200 mL) and dichloromethane (200 mL). The resulting
precipitate was filtered off and dried (5.66 g, 61%). The major-
ity (5.50 g, 14.3 mmol) of this substance was stirred with 50%
(v/v) TFA in dichloromethane (40 mL) and ( )1,4-dithio-
threitol (0.20 g, 1.3 mmol) at room temperature for 4 h.
The solution was evaporated in vacuo and the residue was
coevaporated with EtOAc (5×). The residue was dissolved
in 5% (w/v) aq. NaHCO3 (150 mL), filtered and acidified with
10% (w/v) aq. NaHSO4 (100 mL). The precipitated solid was
filtered off (4.11 g, 88%), to give 7 mp 213 ЊC (darkens), 250 ЊC
(decomp.). The characteristics (1H NMR and mass spectra)
of this substance were in good agreement both with the pub-
lished values7 and with those of the substance prepared in
procedure B.
cmϪ1; s, strong; m, medium; w, weak). NMR: Bruker Avance
DRX 400 and 500 spectrometers (1H: 400.13 MHz and 500.13
MHz; 13C: 100.62 MHz and 125.76 MHz, respectively),
DMSO-d6 solutions, δ (ppm), J (Hz). Spectral patterns: s, sing-
let; d, doublet; dd, double doublet; t, triplet; m, multiplet; br,
broad; deut, deuterable. The superscripts *, # denote inter-
changeable assignments. For the 2D experiments (HMQC,
HMBC) the standard Bruker software packages (INV4GSSW,
INV4GSLRNDSW) were applied. For 13C NMR data of guan-
ine derivatives see Table 1. Mass spectrometry: Finnigan MAT
TSQ 7000, electrospray (ESI) and atmospheric pressure chem-
ical ionization (APCI) techniques. IUPAC names: AutoNomTM
2.1 (as implemented in ChemDraw 5.0). Statistical analysis of
13C NMR chemical-shift parameters of guanine derivatives
(Fig. 1): Corel Quattro Pro 8.0, @ functions and SigmaPlot
4.01. Details are available upon request.
[4-(4-Methoxybenzoylamino)-2-oxo-1,2-dihydropyrimidin-1-
yl]acetic acid 3
B. Controlled hydrolysis of a dianisoylated derivative. Ethyl
(adenin-9-yl)acetate 85 (4.42 g, 20 mmol) was suspended in
anhydrous pyridine (50 mL), heated to 80 ЊC for 30 min, then
cooled to room temperature. 4-Methoxybenzoyl chloride (8.53
g, 50.0 mmol) was added in portions and the mixture was
stirred for 18 h, then evaporated in vacuo and the residue was
coevaporated with toluene (3×). The residue was dissolved in
dichloromethane (70 mL), and the solution was washed with
10% (w/v) citric acid (2 × 30 mL), dried and evaporated in
vacuo. The crude product (14.96 g) was dissolved in warm
ethanol (100 mL), cooled to room temperature, 2 M aq. NaOH
(30 mL) was added, and the solution was left at room temper-
ature and checked from time to time by TLC. After 175 min
more aq. NaOH (5 mL) was added. The reaction was stopped
after 4 h by addition of 1 M HCl (35 mL), pH ≈5, and the
solution was evaporated in vacuo. The crude product was
recrystallized from methanol (1 L) to afford a white powder
(4.58 g, 70%), mp 215 ЊC (darkens), 254 ЊC (decomp.) [lit.,7
222–223 ЊC (decomp.)]. The 1H NMR and mass spectra
of this compound were in good agreement with the published
values.7
Cytosine (1.11 g, 10.0 mmol) suspended in pyridine (50 mL)
was stirred at room temperature while 4-methoxybenzoyl chlor-
ide (2.56 g, 15.0 mmol) was added and the reaction mixture was
stirred in an oil-bath at 80 ЊC. The cytosine rapidly dissolved,
and then the product precipitated from the solution. After 2 h
the mixture was evaporated in vacuo and coevaporated with
methanol (2×). The residue was suspended in methanol and the
mixture was filtered to afford compound 3 (2.28 g, 93%) as a
white powder, mp > 260 ЊC. The product [4-methoxy-N-(2-oxo-
1,2-dihydropyrimidin-4-yl)benzamide, 2] was insoluble in
practically all solvents, therefore it was used without further
purification; Rf 0.58 (CH2Cl2–MeOH 8:2); λmax[0.20% (v/v)
TFA in EtOH]/nm 216 (lg ε 4.00), 276 (4.35); νmax/cmϪ1 3276w,
3150w, 3073w, 2994w, 2846w, 1714s, 1692s, 1621m, 1609m,
1578m, 1497s, 1406w, 1260s, 1189m, 801m.
The majority (2.20 g, 8.97 mmol) of this substance was sus-
pended in anhydrous DMF (25 mL), anhydrous K2CO3 (1.24 g,
8.97 mmol) and methyl bromoacetate (0.86 mL, 8.97 mmol)
were added and the mixture was stirred for 24 h. The reaction
mixture was filtered, washed with DMF and the filtrate was
evaporated. Water (9 mL) and 4 M HCl (0.4 mL) were added to
the residue and stirred for 15 min. The ester was filtered off and
then added to a mixture of aq. 2 M NaOH (6.5 mL, 13.0 mmol)
and water (12 mL) and the reaction mixture was sonicated. The
substance dissolved and after 30 min no starting material was
present (TLC). The reaction mixture was acidified with 4 M
HCl (3.6 mL, 14.4 mmol) and the precipitated substance was
filtered off. Purification of the crude product (2.94 g) was
attempted by recrystallization from methanol. 1 L of solvent
was not enough to dissolve the above amount, and so 0.45 g was
filtered off to give a white powder, TLC: single spot, mp 220 ЊC
(darkens), 239 ЊC (decomp.). From the methanolic solution a
second crop (0.86 g) was obtained as a white powder, TLC:
single spot, mp 166 ЊC (darkens), 227 ЊC (decomp.). Overall
yield: 1.31 g (48%); Rf 0.58 (MeCN–MeOH–AcOH 4:1:1)
(Found: C, 55.5; H, 4.2; N, 13.7. Calc. for C14H13N3O5: C, 55.45;
H, 4.3; N, 13.9%); λmax(EtOH)/nm 208 (lg ε 4.58), 252 (4.55), 287
(4.27); νmax/cmϪ1 3147w, 3076w, 1711s, 1631m, 1615w, 1503m,
1419w, 1251m, 1183m, 755w; δH (400 MHz) 3.84 (3 H, s,
OCH3), 4.56 (2 H, s, CH2), 7.04 and 8.03 (2 × 2 H, 2 × d, J 8.9,
ArH), 7.30 (1 H, d, J 7.2, H-5*), 8.08 (1 H, d, J 7.2, H-6*), 11.03
(1 H, br s, deut, OH#), 11.97 (1 H, br s, deut, NH#); m/z (ESI)
304 (44%, [M ϩ H]ϩ). This compound was mentioned by
Breipohl et al.8 but not described in detail.
(2-Isobutyrylamino-6-oxo-1,6-dihydropurin-9-yl)acetic acid tert-
butyl ester 11 and (2-isobutyrylamino-6-oxo-1,6-dihydropurin-7-
yl)acetic acid tert-butyl ester 12
N-(6-Oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide 919,22 (1.11
g, 5.0 mmol) was suspended in anhydrous DMF and the mix-
ture was chilled to 0 ЊC. Sodium hydride (0.36 g, 8.25 mmol)
was added and the mixture was stirred at 0 ЊC for 30 min. tert-
Butyl bromoacetate (0.81 mL, 5.5 mmol) was added and the
reaction was stopped after 2 h by addition of a small amount of
solid CO2 and methanol (2 mL). The reaction mixture was
evaporated in vacuo and the residue was chromatographed
using 0–5% (v/v) methanol in dichloromethane. Eluted first was
the less polar N7-isomer 12, (0.43 g, 26%), second a mixture
1
(in ≈1:1 ratio as judged by TLC and H NMR) of N7- and
N9-isomer (0.24 g, 14%), and third the pure N9-isomer 11 (0.56
g, 34%).
Ester 11: white powder, mp 204 ЊC (decomp., from EtOH); Rf
0.13 (CH2Cl2–MeOH 95:5) (Found: C, 53.9; H, 6.4; N, 21.1.
Calc. for C15H21N5O4: C, 53.7; H, 6.3; N, 20.9%); λmax[50% (v/v)
1 M HCl in EtOH, pH 0]/nm 206 (lg ε 4.26), 265 (4.23); λmax[50%
(v/v) phosphate buffer in EtOH, pH 6]/nm 260 (lg ε 4.17), 282sh
(4.02); λmax[50% (v/v) 0.1 M NaOH in EtOH, pH 13]/nm 216 (lg ε
4.39), 263 (4.06); νmax/cmϪ1 3151w, 2980w, 2932w, 1753s, 1698m,
1673s, 1614m, 1562m, 1549m, 1483w, 1411m, 1233m, 1154m,
1143m, 795w; δH (500 MHz) 1.11 [6 H, d, J 6.8, (CH3)2CH],
1.40 (9 H, s, But), 2.78 [1 H, pseudoquintet, (CH3)2CH], 4.88
[6-(4-Methoxybenzoylamino)purin-9-yl]acetic acid 7
A. Alkylation of [6-(4-methoxybenzoylamino)purine] and sub-
sequent acidolysis. To [6-(4-methoxybenzoylamino)purine] 67
22
J. Chem. Soc., Perkin Trans. 1, 2000, 19–26

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Table 1 13C NMR chemical shifts of guanine derivatives (δ, ppm)a
Compd.
Subst.
C-2
C-4
C-5
C-6
C-8
Other carbons
irrelevant
10b,c
11d
—
152.34
148.49
154.19
155.17
118.81
119.98
157.41
149.30
144.87
140.72
N9
19.22 [(CH3)2CH], 28.03 [(CH3)3C], 35.01 [(CH3)2CH], 45.17 (CH2COO),
82.69 [(CH3)3C], 167.03 (COOBut), 180.56 (PriCO)
12
N7
N9
147.55
153.24
157.20
155.88
112.08
120.47
152.95
155.95
144.50
147.01
19.23 [(CH3)2CH], 28.00 [(CH3)3C], 35.06 [(CH3)2CH], 48.26 (CH2COO),
82.36 [(CH3)3C], 167.20 (COOBut), 180.32 (PriCO)
13e,f
20.08 [(CH3)2CH], 28.52 [(CH3)3C], 35.18 [(CH3)2CH], 45.82 (CH2COO),
83.40 [(CH3)3C], 127.76, 128.15, 130.26 (arom. Cs), 142.51 (arom. quater-
nary C), 151.03 (OCON), 167.33 (COOBut), 175.97 (PriCO)
20.08 [(CH3)2CH], 35.25 [(CH3)2CH], 45.14 (CH2COO), 53.48 (CH3O),
127.79, 128.14, 130.26 (arom. Cs), 142.51 (arom, quaternary C), 151.03
(OCON), 168.85 (COOMe), 175.90 (PriCO)
19.20 [(CH3)2CH], 34.26 [(CH3)2CH], 47.50 (CH2COO), 52.50 (CH3O),
128.91, 129.04, 129.36 (arom. Cs), 141.28 (arom. quaternary C), 152.02
(OCON), 167.77 (COOMe), 174.94 (PriCO)
19.08 [(CH3)2CH], 34.22 [(CH3)2CH], 66.22 (CH2COO), 126.90, 129.17,
129.37, 129.90 (arom. Cs), 141.49 (arom. quaternary C), 150.07 (OCON),
174.93 (PriCO, COOH)
18.98 [(CH3)2CH], 27.47/27.50 [(CH3)3C], 34.17 [(CH3)2CH], 46.56
(OCH2CH), 43.73/43.99, 47.09, 48.76, 49.96 (4 × CH2), 65.28 (OCH2CH),
80.88/81.98 [(CH3)3C], 119.88, 124.79/124.88, 126.81/126.85, 126.95,
127.40, 128.71, 129.20 (arom. Cs), 140.54, 141.45, 143.61/143.65 (arom.
quaternary C), 150.00 (OCON), 155.13, 155.95/156.21, 166.24/166.74,
167.69/168.32 (4 × CO), 175.00 (PriCO)
14e,f
15f
N9
N7
N9
N9
153.29
149.53
152.19
152.01
155.91
164.63
154.81
154.72
120.51
112.19
119.62
119.40
155.91
141.28
155.03
155.05
146.90
150.44
146.50
146.31
16e
21e,g
25e,g
N9
N7
147.79, 149.16, 119.49
147.82
154.79
153.92
140.45
141.06
18.76 [(CH3)2CH], 34.58 [(CH3)2CH], 46.66 (OCH2CH), 43.86/44.00,
46.85/46.97, 47.76, 49.11 (4 × CH2), 65.43 (OCH2CH), 120.01/120.04,
124.94/125.01, 126.95, 127.53 (arom. Cs), 140.64/140.68, 143.76/143.78
(arom. quaternary C), 156.07/156.30, 166.37/166.91, 170.28/170.72
(3 × CO), 180.05 (PriCO, COOH)
51.09 (CH2), 128.70, 129.15, 129.62 (arom. Cs), 136.38 (arom. quaternary
C)
149.24
26d,f,h
154.95
151.46
108.07
a In DMSO-d6; 125.76 MHz; J-modulated spin-echo experiments; for guanine numbering see ester 11, Scheme 3. b Ref. 19. c The C-4, C-5, C-6, C-8
signals were observed only after adding trifluoroacetic acid. d The assignment of signals corresponding to C-2 and C-6 carbons is tentative. e The
assignment of signals corresponding to C-4 and C-6 carbons is tentative. f Assignment based on HMQC and HMBC experiments. g Some signals
were doubled due to the presence of rotamers. h N7-Benzylguanine hydrochloride, prepared according to Bridson et al.32
(2 H, s, CH2COO), 7.95 (1 H, s, H-8), 11.65 (1 H, br s, deut,
NH), 12.10 (1 H, br s, deut, NH); m/z (ESI) 693 (20%,
[2M ϩ Na]ϩ), 671 (55, [2M ϩ H]ϩ), 336 (100, [M ϩ H]ϩ).
Ester 12: white powder, mp 202.5 ЊC (decomp., from EtOH);
Rf 0.19 (CH2Cl2–MeOH 95:5) (Found: C, 53.65; H, 6.15; N,
21.1%); λmax[50% (v/v) 1 M HCl in EtOH, pH 0]/nm 206 (lg ε
4.24), 263 (4.20); λmax[50% (v/v) phosphate buffer in EtOH, pH
6]/nm 221 (lg ε 4.24), 265 (4.11), 282sh (3.98); λmax[50% (v/v) 0.1
contaminated with triphenylphosphine oxide. Further fractions
were also obtained containing varying proportions of the
product and triphenylphosphine oxide. The different, partly
crystalline fractions were triturated with methanol upon which
the product crystallized. This was filtered off and washed with
light petroleum. The cleanest product (0.40 g, 31%), a white
powder, melted at 183.2–185.5 ЊC. A further crystalline crop
(0.39 g) containing the product and triphenylphosphine oxide
(TLC) was also obtained; Rf 0.50 (CH2Cl2–MeOH 95:5)
(Found: C, 63.5; H, 5.5; N, 15.7. Calc. for C28H30N6O5: C, 63.4;
H, 5.7; N, 15.8%); λmax(EtOH)/nm 205 (lg ε 4.60), 229 (4.53),
258sh (4.15), 279 (4.08); λmax/cmϪ1 3462w, 3346w, 2979w, 2934w,
1738s, 1715m, 1624m, 1587m, 1524m, 1449m, 1411m, 1305m,
1240m, 1187s, 1164s, 1056m, 758w, 700m; δH (500 MHz) 1.09
[6 H, d, J 6.8, (CH3)2CH], 1.43 (9 H, s, But), 2.87 [1 H, pseudo-
quintet, J 6.8, (CH3)2CH], 5.03 (2 H, s, CH2), 7.29–7.53 (10 H,
m, ArH), 8.45 (1 H, s, H-8), 10.69 (1 H, br s, deut, NH); m/z
(ESI) 557 (8%, [2Ph3PO ϩ H]ϩ), 531 (100, [M ϩ H]ϩ).
M NaOH in EtOH, pH 13]/nm 224 (lg ε 4.31), 269 (4.01); νmax
/
cmϪ1 3240w, 2981w, 2937w, 1741m, 1695s, 1677s, 1604s, 1535w,
1421w, 1390m, 1370m, 1238m, 1160m, 747w; δH (500 MHz) 1.11
[6 H, d, J 6.8, (CH3)2CH], 1.39 (9 H, s, But), 2.73 [1 H, pseudo-
quintet, (CH3)2CH], 5.07 (2 H, s, CH2COO), 8.11 (1 H, s, H-8),
11.55 (1 H, br s, deut, NH), 12.14 (1 H, br s, deut, NH); m/z
(ESI) 693 (40%, [2M ϩ Na]ϩ), 671 (25, [2M ϩ H]ϩ), 358 (27,
[M ϩ Na]ϩ), 336 (100, [M ϩ H]ϩ).
[6-Diphenylcarbamoyloxy-2-(isobutyrylamino)purin-9-yl]acetic
acid tert-butyl ester 13
A2. With 4-(dimethylamino)phenyl(diphenyl)phosphine.26,27
—
Reaction time: 2.5 h at 0 ЊC. Work-up: the crude product was
dissolved in dichloromethane (50 mL) and extracted succes-
sively with 4 M HCl (3 × 25 mL) and with 5% (w/v) aq.
NaHCO3 (50 mL). TLC revealed that most of the 4-(di-
methylamino)phenyl(diphenyl)phosphine oxide remained in the
organic phase. The organic phase was dried, and purified
by column chromatography using 0–1% (v/v) methanol in
dichloromethane. Methanolic trituration and filtration (light
petroleum) afforded the product (0.42 g, 33%), mp 182.5–
185.0 ЊC. The IR, 1H NMR and mass spectra of this compound
were in good agreement with those of the substance obtained
in procedure A1.
A. Mitsunobu reaction, general procedure. Compound 1019,22
(1.00 g, 2.40 mmol) was suspended in anhydrous THF (50 mL)
and the mixture was refluxed for 20 min to achieve partial dis-
solution of the starting material.20 The suspension was cooled
to room temperature, tert-butyl glycolate23 (0.40 g, 3.0 mmol),
the appropriate phosphine (3.19 mmol) and DIAD (0.62 mL,
3.19 mmol) were added dropwise, and the mixture was stirred at
room temperature. The reaction mixture completely dissolved
and became yellow coloured. After completion of the reaction
(TLC) the solution was evaporated in vacuo and the residue was
subjected to chromatographic purification.
A1. With triphenylphosphine.—Reaction time: 4 h at room
temperature. Chromatography: 50–70% (v/v) ethyl acetate in
light petroleum. Eluted first was the product 13 (0.31 g), slightly
A3. With tributylphosphine.—Reaction time: 1.5 h at 0 ЊC.
Work-up: the crude product was dissolved in dichloromethane
(50 mL) and extracted with water (3 × 25 mL) to remove
J. Chem. Soc., Perkin Trans. 1, 2000, 19–26
23

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the tributylphosphine oxide. Chromatography: 0–1.5% (v/v)
methanol in dichloromethane. Methanolic trituration and
filtration (light petroleum) afforded the product (0.46 g, 36%),
mp 182.2–184.8 ЊC. The IR, 1H NMR and mass spectra of this
compound were in good agreement with those of the substance
obtained in procedure A1.
276 (4.03); νmax/cmϪ1 3295w, 2996w, 2956w, 1756m, 1726s,
1681s, 1631m, 1599m, 1491m, 1384m, 1340s, 1233s, 1223s,
1192m, 1063m, 789w, 758w, 704w; δH (500 MHz) 1.08 [6 H, d,
J 6.8, (CH3)2CH], 2.83 [1 H, pseudoquintet, J 6.8, (CH3)2CH],
3.73 (3 H, s, CH3), 5.15 (2 H, s, CH2), 7.31–7.49 (10 H, m,
ArH), 8.43 (1 H, s, H-8), 10.64 (1 H, br s, deut, NH); m/z (ESI)
511 (25%, [M ϩ Na]ϩ), 489 (100, [M ϩ H]ϩ).
Ester 15: amorphous foam; Rf 0.35 (CH2Cl2–MeOH 9:1)
(Found: C, 61.3; H, 5.1; N, 17.0); λmax[50% (v/v) 1 M HCl in
EtOH, pH 0]/nm 207 (lg ε 4.59), 227 (4.56), 252sh (4.26), 283
(4.09); λmax[50% (v/v) phosphate buffer in EtOH, pH 6]/nm 230
(lg ε 4.61), 256sh (4.17), 282 (4.02); λmax[50% (v/v) 0.1 M NaOH
in EtOH, pH 13]/nm 229 (lg ε 4.53), 287sh (3.90); νmax/cmϪ1
3438w, 2972w, 1753s, 1707m, 1639m, 1592w, 1493s, 1445m,
1301s, 1187s, 761m, 701m; δH (500 MHz) 1.11 [6 H, d, J 6.8,
(CH3)2CH], 2.85 [1 H, pseudoquintet, J 6.8, (CH3)2CH], 3.56
(3 H, s, CH3), 5.20 (2 H, s, CH2), 7.28–7.48 (10 H, m, ArH), 8.53
(1 H, s, H-8), 10.57 (1 H, br s, deut, NH); m/z (ESI) 511 (40%,
[M ϩ Na]ϩ), 489 (100, [M ϩ H]ϩ).
B. Low-temperature, sodium hydride-mediated alkylation. To
compound 1019,22 (1.40 g, 3.36 mmol) suspended in anhydrous
DMF (20 mL) was added sodium hydride (0.16 g, 3.70 mmol) at
room temperature. After 30 min the reaction mixture was
chilled to Ϫ20 ЊC and maintained at this temperature. tert-
Butyl bromoacetate (0.60 mL, 4.0 mmol) was added dropwise.
After 2 h the reaction was stopped by addition of a small
amount of solid CO2 and methanol, and the mixture was
evaporated in vacuo and coevaporated with toluene (2×). The
residue was dissolved in a mixture of water and dichloro-
methane (20 mL each). The aqueous phase was extracted with
dichloromethane (3 × 15 mL), and the combined organic
phases were dried, and evaporated in vacuo. Chromatography:
0–1% (v/v) methanol in dichloromethane. Methanolic tritur-
ation and subsequent crystallization from methanol (15 mL)
afforded the product (0.70 g, 40%) as a white powder, mp
[6-Diphenylcarbamoyloxy-2-(isobutyrylamino)purin-9-yl]acetic
acid 16 (cf. ref. 8)
Ester 14 (2.24 g, 4.59 mmol) was suspended under sonication in
a mixture of methanol (6 mL), 1,4-dioxane (24 mL) and water
(12 mL). 1 M aq. NaOH (5 mL) was added and the mixture was
stirred for 30 min. The pH of the mixture was brought to ≈6 by
addition of 1 M HCl and the organics were evaporated off in
vacuo. The solution was diluted with water (120 mL) and acid-
ified to pH ≈3 by addition of 1 M HCl. The precipitate was
filtered off, and washed with ice–water to give acid 16 (1.99 g,
91%), white powder, mp 156 ЊC (decomp.). Attempted recrystal-
lization from EtOAc resulted in gel formation; Rf 0.40 (MeCN–
MeOH–AcOH 8:1:1) (Found: C, 60.5; H, 4.4; N, 17.4. Calc.
for C24H22N6O5: C, 60.75; H, 4.7; N, 17.7%); λmax(EtOH)/nm
229 (lg ε 4.51), 260sh (4.11), 279 (4.06); νmax/cmϪ1 3379w, 1723s,
1627m, 1591m, 1522m, 1493m, 1441m, 1411m, 1307m, 1200s,
1187s, 760w, 701m; δH (500 MHz) 1.09 [6 H, s, (CH3)2CH], 2.83
[1 H, m, (CH3)2CH], 5.05 (2 H, s, CH2), 7.29–7.50 (10 H, m,
ArH), 8.46 (1 H, s, H-8), 10.65 (1 H, br s, deut, NH); m/z [ESI
(CHCl3 ϩ MeOH)], 971 (8%, [2M ϩ Na]ϩ), 514 (8, [M ϩ K]ϩ),
497 (20, [M ϩ Na]ϩ), 475 (100, [M ϩ H]ϩ).
1
183.1–184.5 ЊC. The IR, H NMR and mass spectra of this
compound were in good agreement with those of the substance
obtained in procedure A1.
Hydrolysis of [6-diphenylcarbamoyloxy-2-(isobutyrylamino)-
purin-9-yl]acetic acid tert-butyl ester (13 → 11)
To ester 13 (0.210 g, 0.38 mmol) dissolved in anhydrous
dichloromethane (6 mL) were added 1,3-dimethoxybenzene
(0.070 mL, 0.53 mmol) and TFA (0.50 mL, 6.5 mmol) at 0 ЊC
and the mixture was stirred for 18 h. The reaction mixture was
diluted with dichloromethane (20 mL), and extracted with satd.
aq. NaHCO3 solution (3 × 10 mL) to remove the excess of acid.
Chromatography: 0–10% (v/v) methanol in dichloromethane to
give the lactam 11 (0.069 g, 55%) as an amorphous foam. The
1
IR, H NMR and mass spectra of this product were in good
agreement with those of the substance obtained in a previous
experiment (vide supra).
[6-Diphenylcarbamoyloxy-2-(isobutyrylamino)purin-9-yl]acetic
acid methyl ester 14 and [6-diphenylcarbamoyloxy-2-(isobutyryl-
amino)purin-7-yl]acetic acid methyl ester 15 (cf. ref. 8)
Esters 19–21
General procedure. To acid 5,7 7 or 16 (2.0 mmol) dissolved in
anhydrous DMF (20 mL) were added HOBt hydrate (0.61 g, 4.0
mmol) and HBTU (1.52 g, 4.0 mmol). Meanwhile ester 179
(1.95 g, 3.0 mmol for acids 5, 7 and 1.30 g, 2.0 mmol for acid 16)
was suspended in dichloromethane (20/30 mL), extracted with
satd. aq. NaHCO3 (10 mL) and dried. The above dichloro-
methane solution of free 17 base and DIPEA (0.70 mL, 4.0
mmol) were added after 5 min and the reaction mixture was
stirred at room temperature. Work-up: after evaporation of the
solution in vacuo, the residue was dissolved in dichloromethane
(30 mL), extracted with 1 M HCl (19: 3 × 10 mL) or with satd.
aq. NaHSO4 (20: 5 × 10 mL), and washed successively with
satd. aq. NaHCO3 (25 mL) and brine (25 mL). In the case of
21, after evaporation of the reaction mixture the residue was
triturated with EtOAc (5 mL) and filtered, followed by crystal-
lization. The resulting crude products were purified chromato-
graphically (19, 20) or by crystallization (21).
({2-[4-(4-tert-Butylbenzoylamino)-2-oxo-1,2-dihydropyrim-
idin-1-yl]acetyl}-[2-(9H-fluoren-9-ylmethoxycarbonylamino)-
ethyl]amino)acetic acid tert-butyl ester 19.—Reaction time: 20
h at room temperature. Chromatography: 1–3% (v/v) methanol
in dichloromethane, yield 0.99 g (70%), colourless oil. In a
repeated experiment the extractive work-up was omitted and
the crude product was triturated with methanol (2 mL) to yield
a cleaner product (0.82 g, 58%), amorphous foam; Rf 0.49
(CH2Cl2–MeOH 95:5) (Found: C, 67.6; H, 6.4; N, 9.7. Calc. for
To compound 1019,22 (3.53 g, 8.47 mmol) suspended in
anhydrous DMF (40 mL) was added DIPEA (2.87 mL, 16.74
mmol) and the mixture was briefly heated to 80 ЊC until a clear
solution was obtained (10 min). The mixture was cooled to
room temperature, methyl bromoacetate (0.87 mL, 9.32 mmol)
was added, and the mixture was stirred for 20 h. The reaction
mixture was evaporated in vacuo and the residue was coevapo-
rated with methanol (3×). The partly crystalline material was
suspended in methanol (40 mL) and added dropwise to water
(120 mL) with vigorous stirring. The precipitate was filtered off
(3.99 g, 96%) and recrystallized from EtOAc (190 mL) to afford
the title products (2.01 g, 49%), mp 167.0–168.4 ЊC; from the
mother liquor was obtained a further crop (0.37 g, 9%), mp
168.0–170.4 ЊC. The mother liquor was evaporated and chrom-
atographed by using 1–4% (v/v) methanol in dichloromethane.
Eluted first was ester 14 (0.54 g, 13%) then its regioisomer 15
(0.60 g, 15%). Overall yield of 14: 2.92 g, 71%.
Ester 14: white powder, mp 170.0–171.4 ЊC (from EtOAc); Rf
0.37 (CH2Cl2–MeOH 95:5) (Found: C, 61.35; H, 5.1; N, 17.4.
Calc. for C25H24N6O5: C, 61.5; H, 4.95; N, 17.2%); λmax[50%
(v/v) 1 M HCl in EtOH, pH 0]/nm 207 (lg ε 4.50), 226 (4.53),
253sh (4.17), 279 (4.07); λmax[50% (v/v) phosphate buffer in
EtOH, pH 6]/nm 227 (lg ε 4.54), 259sh (4.14), 277 (4.08);
λmax[50% (v/v) 0.1 M NaOH in EtOH, pH 13]/nm 230 (lg ε 4.45),
24
J. Chem. Soc., Perkin Trans. 1, 2000, 19–26

View Article Online
C40H46N5O7: C, 67.8; H, 6.5; N, 9.9%); λmax(EtOH)/nm 205 (lg ε
4.19), 265 (3.94), 288 (3.52), 300 (3.52); νmax/cmϪ1 3450m,
3150w, 3070w, 2966w, 2936w, 1710s, 1670s, 1629m, 1563m,
1492s, 1408w, 1367s, 1254s, 1156s, 1113s, 851w, 759w, 740w;
δH (500 MHz, rotamers) 1.29 (9 H, s, But), 1.40 (9 H, s, But),
1.46 (2 H, s, CH2), 3.23 (2 H, m, CH2), 3.44 (2 H, m, CH2), 3.97
(3 H, s, CH3O), 4.26 (1 H, m, CH), 4.68/4.90 (2 H, 2 s, CH2),
6.27 (1 H, s, H-5*), 7.34 (2 H, dd, J 7.4 and 7.2, fluorenyl CH),
7.41 (2 H, dd, J 7.4 and 7.2, fluorenyl CH), 7.52 (2 H, d, J 8.3,
4-ButC6H4CO), 7.83 (2 H, d, J 7.5, fluorenyl CH), 7.87 (2 H, d,
J 7.5, fluorenyl CH), 7.98 (2 H, d, J 8.3, 4-ButC6H4CO), 8.00
(1 H, s, H-6*); m/z (ESI) 730 (78%, [M ϩ Na]ϩ), 708 (100,
[M ϩ H]ϩ).
([2-(9H-Fluoren-9-ylmethoxycarbonylamino)ethyl]-{2-[6-(4-
methoxybenzoylamino)purin-9-yl]acetyl}amino)acetic acid tert-
butyl ester 20.—Reaction time: 1.5 h at room temperature.
Chromatography: 0–20% (v/v) EtOAc in methanol, yield 1.20 g
(85%), amorphous foam; Rf 0.42 (CH2Cl2–MeOH 95:5)
(Found: C, 64.8; H, 5.4; N, 13.7. Calc. for C38H39N7O7: C, 64.7;
H, 5.6; N, 13.9%); λmax(EtOH)/nm 206 (lg ε 4.78), 266 (4.41), 278
(4.41), 289 (4.42), 300sh (4.32); νmax/cmϪ1 3065w, 2980w, 2943w,
1705m, 1670m, 1609m, 1586m, 1513w, 1458m, 1411w, 1252s,
1157m, 845s, 762m, 743m; δH (500 MHz, rotamers) 1.32 (9 H, s,
But), 2.81 (2 H, s, CH2), 3.25 (2 H, m, CH2), 3.65 (2 H, m, CH2),
3.94 (3 H, s, CH3O), 4.50 (3 H, m, CH, CH2), 5.25/5.44 (2 H, 2 s,
CH2), 7.16 (2 H, d, J 8.7, anisoyl CH), 7.39 (2 H, dd, J 7.4 and
7.2, fluorenyl CH), 7.49 (2 H, dd, J 7.4 and 7.2, fluorenyl CH),
7.76 (2 H, d, J 7.4, fluorenyl CH), 7.96 (2 H, d, J 7.4, fluorenyl
CH), 8.13 (2 H, d, J 8.6, anisoyl CH), 8.40 (1 H, s, H-8*), 8.70
(1 H, s, H-2*), 11.10 (1 H, br s, NH); m/z (ESI) 706 (100%,
[M ϩ H]ϩ).
({2-[6-Diphenylcarbamoyloxy-2-(isobutyrylamino)purin-9-
yl]acetyl}-[2-(9H-fluoren-9-ylmethoxycarbonylamino)ethyl]-
amino)acetic acid tert-butyl ester 21.—Reaction time: 1.5 h at
room temperature. The crude product was obtained as
described above and was recrystallized from ethanol (750
mL) to give a white powder (1.22 g, 40%), mp 209.0–209.5 ЊC
(decomp.); Rf 0.36 (CH2Cl2–MeOH 95:5) (Found: C, 66.3; H,
5.5; N, 12.9. Calc. for C47H48N8O8: C, 66.2; H, 5.7; N, 13.1%);
λmax(EtOH)/nm 221 (lg ε 4.57), 228 (4.53), 256 (4.39), 266sh
(4.38), 279sh (4.27), 289sh (4.07), 300 (3.81); νmax/cmϪ1 3302m,
2979w, 1750m, 1732s, 1704s, 1656s, 1624w, 1591w, 1545m,
1493w, 1450m, 1192s, 1156m, 1055m, 762m, 697w; δH (500
MHz, rotamers) 1.06 [6 H, d, J 6.2, (CH3)2CH], 1.35/1.47 (9 H,
2 s, But), 2.83 [1.3 H, m, (CH3)2CH, NH], 3.52 (4 H, m, partly
shielded by the water signal, 2 × CH2), 3.95 (1 H, s, CH), 4.19–
4.29 (2 H, m, CH2), 4.32–4.35 (2 H, m, CH2), 5.07/5.25 (2 H,
2 × s, guanyl CH2), 7.22–7.48 (14 H, m, 2 × Ph, fluorenyl
CH), 7.64 (2 H, d, J 7.3, fluorenyl CH), 7.86 (2 H, dd, J 7.9 and
7.9, fluorenyl CH), 8.30 (1 H, s, H-8), 10.57 (1 H, s, NH); m/z
(ESI) 891 (1%, [M ϩ K]ϩ); 875 (5, [M ϩ Na]ϩ); 853 (100,
[M ϩ H]ϩ).
rotamers) 1.34 (9 H, s, But), 3.14/3.25 (1 H, 2 m, partly shielded
by the water signal, CH2), 3.38/3.47 (1.4 H, 2 m, partly shielded
by the water signal, CH2), 4.03/4.22 (2 H, 2 s, CH2), 4.26 (1 H,
m, CH), 4.31/4.36 (2 H, 2 d, J 6.7, CH2), 4.70/4.89 (2 H, 2 s,
cytosinyl CH2), 7.27/7.41 (1 H, 2 d, J 7.5, H-5*), 7.33 (3 H,
dd, J 7.5 and 6.8, fluorenyl CH, NH), 7.42 (2 H, d, J 6.8, fluor-
enyl CH), 7.53 (2 H, d, J 8.4, 4-ButC6H4CO), 7.69 (2 H, dd,
J 7.5 and 6.8, fluorenyl CH), 7.89 (2 H, d, J 7.5, fluorenyl
CH), 7.94 (1 H, d, J 7.3, H-6*), 7.98 (2 H, dd, J 2.7, 8.4,
4-ButC6H4CO), 11.20 (1 H, br s, NH#), 12.80 (1 H, br s, OH#);
m/z (ESI) 652 (100%, [M ϩ H]ϩ).
([2-(9H-Fluoren-9-ylmethoxycarbonylamino)ethyl]-{2-[6-(4-
methoxybenzoylamino)purin-9-yl]acetyl}amino)acetic acid 24
To ester 20 (0.92 g, 1.30 mmol) dissolved in dichloromethane
(20 mL) were added 1,3-dimethoxybenzene (0.23 mL, 1.82
mmol) and TFA (15.0 mL, 196.0 mmol) and the mixture was
stirred for 6 h at room temperature. The solution was evapor-
ated in vacuo, and the residue was coevaporated with
acetonitrile (5×). The residue was dissolved in methanol (1
mL), diethyl ether (4.5 mL) was added, and the mixture was
stored at 4 ЊC overnight. The resulting gum was triturated with
diethyl ether, filtered and recrystallized from methanol (80 mL)
to afford a white powder (0.59 g, 70%), mp 160.8–163.9 ЊC; Rf
0.76 (CH2Cl2–MeOH 6:4) (Found: C, 62.8; H, 4.65; N, 14.9.
Calc. for C34H31N7O7: C, 62.9; H, 4.8; N, 15.1%); λmax(EtOH)/
nm 206 (lg ε 4.74), 266 (4.43), 278 (4.43), 289 (4.44), 299sh (4.35);
νmax/cmϪ1 3440m, 3222w, 3102w, 3069w, 2978w, 2946w, 1713m,
1693w, 1647m, 1603m, 1582w, 1525m, 1500m, 1411w, 1252s,
1178m, 762m; δH (500 MHz, rotamers) 3.15/3.59 (2 H, 2 m,
CH2), 3.18 (2 H, s, CH2), 3.86 (3 H, s, CH3O), 4.03/4.10 (2 H, 2
m, CH2), 4.22 (1 H, m, CH), 4.30/4.39 (2 H, 2 m, CH2), 5.20/
5.37 (2 H, 2 s, adenyl CH2), 7.08 (2 H, d, J 8.7, anisoyl CH),
7.29/7.42 (1 H, 2 br t, NH), 7.32 (2 H, dd, J 7.4 and 7.3, fluor-
enyl CH), 7.41 (2 H, dd, J 7.4 and 7.3, fluorenyl CH), 7.70 (2 H,
d, J 7.4, fluorenyl CH), 7.88 (2 H, d, J 7.4, fluorenyl CH), 8.06
(2 H, d, J 8.7, anisoyl CH), 8.33 (1 H, s, H-8*), 8.62/8.67 (1 H, 2
s, H-2*); m/z (ESI) 650 (100%, [M ϩ H]ϩ).
{[2-(9H-Fluoren-9-ylmethoxycarbonylamino)ethyl]-[2-(2-iso-
butyrylamino-6-oxo-1,6-dihydropurin-9-yl)acetyl]amino}acetic
acid 25
To ester 21 (0.68 g, 0.79 mmol) suspended in dichloromethane
(20 mL), was added 1,3-dimethoxybenzene (0.125 mL, 0.95
mmol) followed by TFA (7.34 mL, 95.3 mmol) and the mixture
was stirred for 6 h at room temperature. The solution was evap-
orated in vacuo, and the residue was coevaporated with EtOAc
(4×). The solid residue was triturated with EtOAc, and filtered
(0.48 g, quant.), mp 202.0–206.0 ЊC and then recrystallized
from ethanol (40 mL) to furnish a white powder (0.21 g, 45%),
mp 208.8–210.6 ЊC (decomp.); from the mother liquor a further
crop was obtained (0.026 g, 5%), mp 206.0–208.1 ЊC. Overall
yield of the recrystallized product: 0.236 g, 50%. This reaction
was repeated on a 3.0 mmol scale and afforded a quantitative
yield of the crude acid 25 (1.80 g), mp 202.0–206.0 ЊC; Rf 0.16
(MeCN–MeOH–AcOH 8:1:1) (Found: C, 59.7; H, 5.3;
N, 16.1. Calc. for C30H31N7O7: C, 59.9; H, 5.2; N, 16.3%);
λmax(EtOH)/nm 205 (lg ε 4.79), 221sh (4.24), 256sh (4.45), 262
(4.48), 278sh (4.29), 289sh (4.13), 300 (4.03); νmax/cmϪ1 3350w,
3131w, 3067w, 2965w, 2940w, 1693s, 1674m, 1610m, 1570m,
1542m, 1485w, 1411m, 1250m, 1154w, 757w, 743w; δH (500
MHz, rotamers) 1.11 [6 H, d, J 6.6, (CH3)2CH], 2.75 [1 H,
pseudoquintet, J 6.6, (CH3)2CH], 3.13 (1 H, m), 3.35 (1.8 H,
m, partly shielded by the water signal) and 3.49 (1.2 H, m,
2 × CH2), 4.02/4.29 (2 H, 2 s, CH2), 4.25 (1 H, m, CH), 4.32/4.38
(2 H, 2 d, J 5.8, 6.5, CH2), 4.97/5.13 (2 H, 2 s, guanyl CH2), 7.26/
7.46 (1 H, 2 br t, NH*), 7.33 (2 H, m, fluorenyl CH), 7.41 (2 H,
dd, J 7.2 and 7.2, fluorenyl CH), 7.68 (2 H, dd, J 7.5, 7.2,
fluorenyl CH), 7.82 (1 H, s, H-8), 7.88 (2 H, d, J 7.5, fluorenyl
({2-[4-(4-tert-Butylbenzoylamino)-2-oxo-1,2-dihydropyrimidin-
1-yl]acetyl}-[2-(9H-fluoren-9-ylmethoxycarbonylamino)ethyl]-
amino)acetic acid 23
To ester 19 (0.40 g, 0.56 mmol) dissolved in dichloromethane
(20 mL) was added 1,3-dimethoxybenzene (0.19 mL, 1.45
mmol) followed by TFA (4.0 mL, 52.3 mmol) and the mixture
was stirred for 6 h at room temperature. The solution was evap-
orated in vacuo, and the residue was coevaporated with
acetonitrile (5×). The residue was triturated under diethyl ether,
filtered and recrystallized from methanol (20 mL) to give a
white powder (0.27 g, 73%), mp 197.4–199.0 ЊC (decomp.); Rf
0.86 (CH2Cl2–MeOH 6:4) (Found: C, 66.3; H, 5.7; N, 10.6.
Calc. for C36H38N5O7: C, 66.2; H, 5.9; N, 10.7%); λmax(EtOH)/
nm 205 (lg ε 4.88), 265 (4.68), 289 (4.25), 300 (4.27); νmax/cmϪ1
3147w, 3067w, 2964w, 1708s, 1664s, 1610m, 1562m, 1490s,
1409w, 1367m, 1255m, 1113w, 853w, 760w, 741w; δH (500 MHz,
J. Chem. Soc., Perkin Trans. 1, 2000, 19–26
25

View Article Online
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Acknowledgements
This research has been supported by grants MKM 626, OTKA
T 22551 and OTKA W 15521 (purchase of ChemProtect 1.0).
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