A ribozyme with michaelase activity: synthesis of the substrate precursors.

Article abstract of DOI:10.1016/S0968-0896(02)00311-5

The ability to generate RNA molecules that can catalyze complex organic transformations not only facilitates the reconstruction and plausibility of possible prebiotic reaction pathways but is also crucial for elucidating the potential of the application of RNA catalysts in organic syntheses. Iterative RNA selection previously identified a ribozyme that catalyzes the Michael addition of a cysteine thiol to an alpha,beta-unsaturated amide. This reaction is chemically similar to the rate limiting step of the thymidylate synthase reaction, which is the corresponding reaction of a cysteine thiol to the double-bond of the uracil nucleobase. Here we provide a detailed description of the synthesis of the ribozyme substrates and the substrate oligonucleotides used for its characterization and the investigation of the background reaction. We also describe the further characterization of the ribozyme with respect to substrate specificity. We show that the thiol group of the cysteine nucleophile is essential for the reaction to proceed. When substituted for a thiomethyl group, no reaction takes place.

Full text of DOI:10.1016/S0968-0896(02)00311-5

Bioorganic & Medicinal Chemistry 11 (2003) 235–249
A Ribozyme with Michaelase Activity:
Synthesis of the Substrate Precursors
Alexander Eisenfuhr,a,y Paramjit S. Arora,b,y Gerhard Sengle,a
Leo R. Takaoka,b James S. Nowickb and Michael Famuloka,*
a
´
Kekule-Institut fur Organische Chemie und Biochemie, Rheinische Friedrich-Wilhelms-Universitat Bonn,
¨
¨
Gerhard-Domagk-Straße 1, 53121 Bonn, Germany
bDepartment of Chemistry, University of California, Irvine, Irvine, CA 92697-2025, USA
Received 24 May 2002; accepted 18 July 2002
Abstract—The ability to generate RNA molecules that can catalyze complex organic transformations not only facilitates the
reconstruction and plausibility of possible prebiotic reaction pathways but is also crucial for elucidating the potential of the appli-
cation of RNA catalysts in organic syntheses. Iterative RNA selection previously identified a ribozyme that catalyzes the Michael
addition of a cysteine thiol to an a,b-unsaturated amide. This reaction is chemically similar to the rate limiting step of the thymi-
dylate synthase reaction, which is the corresponding reaction of a cysteine thiol to the double-bond of the uracil nucleobase. Here
we provide a detailed description of the synthesis of the ribozyme substrates and the substrate oligonucleotides used for its char-
acterization and the investigation of the background reaction. We also describe the further characterization of the ribozyme with
respect to substrate specificity. We show that the thiol group of the cysteine nucleophile is essential for the reaction to proceed.
When substituted for a thiomethyl group, no reaction takes place.
# 2002 Elsevier Science Ltd. All rights reserved.
Introduction
synthase is the nucleophilic attack of an internal
cysteine residue (C198) to the Michael acceptor system
of 20-deoxyuridine-50-monophosphate (dUMP).18À20
This step activates dUMP for the subsequent methyla-
tion by tetrahydrofolate to yield thymidine monopho-
sphate (TMP). The ribozyme isolated previously by in
vitro selection catalyzes a Michael reaction that is che-
mically analogous to the transformation performed by
thymidylate synthase. The selection scheme used for its
isolation involved covalent attachment of the a,b-unsa-
turated Michael acceptor group to the 50-phosphate of a
guanosine-monophosphate residue that served as an
initiator nucleotide in an in vitro transcription with T7-
RNA polymerase. Thus, every member of the RNA
library was selectively derivatized with the Michael
acceptor at the 50-terminus. Upon reaction with a bioti-
nylated cysteine thiol, active catalytic RNAs became
labeled with a biotin tag that allowed their immobiliza-
tion on streptavidin agarose.
In vitro selection of complex mixtures of oligo-
ribonucleotides has led to the isolation of an impressive
number of new RNA catalysts for a variety of chemical
transformations. Examples include the catalysis of acyl
transfer reactions,1,2 self-alkylation reactions,3,4 RNA
ligation,5 peptide bond formation,6 cleavage of amide
bonds,7 cleavage of DNA,8,9 pericyclic reactions10,11
and isomerization reactions.12
We have recently described the isolation of RNA cata-
lysts for a Michael addition of a thiol Michael donor
substrate to an a,b-unsaturated carbonyl system, cova-
lently attached to an oligonucleotide substrate.13 The
Michael reaction not only is among the most important
in organic synthesis14 but also plays a role in various
cellular processes.15À17 For example, the rate-limiting
step of the reaction sequence catalyzed by thymidylate
Here we provide further details on the design of the in
vitro selection scheme, the synthesis and characteriza-
tion of the required substrates, and the preparation of
appropiately modified RNA libraries. We also report
*Corresponding author. Tel.: +49-228-735661; fax: +49-228-735388;
e-mail: m.famulok@uni-bonn.de
yThese authors contributed equally to this work.
0968-0896/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00311-5

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¨
the background rate of the reaction and the substrate
specificity of the ribozyme with respect to the active
nucleophilic site of the cysteine in greater detail.
fumaramide reaction partner to each member of the
RNA library. The attachment was achieved by linking it
to a guanosine-monophosphate (GMP) (1, Fig. 1A)
moiety for convenient incorporation into RNA mole-
cules by means of the T7 RNA polymerase (T7 RNAP).
It is well established that initiation of enzymatic RNA
synthesis can be achieved by chemically modified gua-
nosine monophosphate21À23 and several examples where
substrate-modified GMP-residues are utilized as initia-
tor nucleotides for the preparation of substrate-mod-
ified RNA libraries for the selection of catalytic RNAs
have been described.1,10,11 The designed initiator
nucleotide was tailored for its application in the current
selection process. Thus, the fumaramide group (D) is
connected to the GMP (A) via a flexible aminoalkyl-
spacer (C) and a photo-cleavable linker (B).24 The flex-
ible aminoalkyl spacer should give potential catalysts an
optimal orientation towards the substrates during the
reaction.
Results and Discussion
Rational design of the selection strategy
The selection scheme was designed so as to allow for the
direct selection of catalytically active RNA molecules
(Scheme 1). A DNA library of partially randomized
sequences with a diversity of approximately 2ꢀ1015 dif-
ferent molecules consisting of 142 randomized nucleo-
tides served as the template for RNA synthesis by T7
transcription. The RNA library was in vitro transcribed
in the presence of fumaramide-derivatized guanosine 1
which served as the initiator nucleotide. Hence, the
transcription reaction yielded RNA molecules that con-
tained the fumaramide group covalently attached to
their 50-end. The biotinylated cysteine 5 served as the
Michael donor substrate in the selection experiments.
Upon formation of the Michael product those RNA
sequences that catalyzed the nucleophilic attack of the
thiol group (red) of 5 to the double bond of the fumar-
amide system (blue) became tagged with a biotin
anchor. Consequently, active ribozymes became immo-
bilized on streptavidin agarose and could be separated
from the non-biotinylated, non-functional RNA species.
To achieve the selective removal of the desired RNAs,
we had incorporated a photolabile group in the initiator
nucleotides. Eluted active sequences were subsequently
amplified by reverse transcription, PCR, and in vitro
transcription.
The photo-labile unit can be cleaved by irradiation with
UV light at 365 nm which allows separation of the RNA
from the product after completion of the Michael addi-
tion. This feature avoids unintentional enrichment of
RNA sequences that become bound to biotin at other
potential Michael acceptor sites contained in RNA
during the course of selection. O-nitrobenzyl groups
have been extensively studied as photo-labile protecting
groups and linker units in solid phase peptide25,26 and
oligonucleotide synthesis.27À29 Prior to our study,13 their
suitability and application for in vitro selections of new
ribozymes had only been tested in principle in a mock
selection.30 To improve the water solubility of the
initiator nucleotide and introduce RNA binding groups
into the Michael acceptor, an aminoalkyl unit (E) was
also incorporated (Fig. 1).
Design of substrates
Figure 1 shows the design of the Michael substrates
synthesized for the preparation of the RNA selection
library. In analogy to the natural Michael donor/accep-
tor system of the thymidylate synthase, we chose an
amide-linked cysteine as the S-nucleophile serving as the
Michael donor (5 in Fig. 1B). This cysteine is covalently
bound to biotin via a short diamino alkyl chain and is
therefore termed ‘biotin cysteine’ throughout the text.
The biotin serves as an affinity anchor that tags reacted
RNA molecules, allowing their selective immobilization
out of a large pool of non-reactive RNA species
(Scheme 1).
For functional verification of the selected ribozyme
activities, it was necessary to synthesize a modified
initiator nucleotide 4 which differs from the one used in
the selection (1) only in having the unsaturated fumar-
amide unit exchanged by the saturated succinamide
unit. The ribozyme molecules modified with 4 cannot be
biotinylated by a Michael reaction. This modification
was studied to rule out the possibility that the catalytic
RNAs were getting attached to the biotin by an
unwanted reaction, such as transaminations. In order to
further characterize the isolated ribozymes, two more
initiator nucleotide derivatives 2 and 3 were synthesized.
Both differ from 1 in the substitution of one of the two
olefinic hydrogen atoms of the fumaramide unit by a
methyl group. Kinetic characterization of ribozymes
derivatized with these initiator nucleotides was expected
to provide further insights into the reaction mechanism
and preferences of the nucleophilic attack on the
Michael acceptor system.
As the Michael acceptor we chose a fumaramide moiety
mimicking the Michael acceptor system of the uracile
nucleobase in the open chain form. A set of reactions of
thiols with different Michael acceptor systems led to the
choice of fumaramide as the acceptor substrate, because
this moiety exhibited a moderate reactivity in the non-
catalyzed background reaction (data not shown). This
appeared to be most promising for a successful selection,
because highly reactive substrates would apply insuffi-
cient selection pressure due to high background reaction
rates, whereas unreactive substrates would decrease the
chance of finding catalytic species at all. The selection
strategy necessitated the covalent attachment of the
As the Michael donor, the free thiol group of biotin
cysteine 5 was used in the ribozyme selection. To test
whether the thiol group or the free amino of cysteine is
the attacking nucleophile, we synthesized compound 6,
where the nucleophilicity of the thiol group is blocked
by a methyl group at the sulfur.

A. Eisenfuhr et al. / Bioorg. Med. Chem. 11 (2003) 235–249
¨
237
Synthesis of the fumaramide-derivatized initiator
nucleotides 1–4
isobutyryl amide34 and the 20- and 30-hydroxy groups
were protected as an acetal group.
The standard procedure for the formation of the phos-
phate group connecting the guanosine and the photo-
clevable linker employs the phosphoramidite coupling
method.31À33 This reaction necessitates appropriate
protection of the N2-amino group of the purine ring and
the ribose 20 and 30- hydroxy groups (Scheme 2A). In
the synthesis of the fumaramide-modified guanosine,
the N2-amine of the purine ring was protected as the
N2-Isobutyryl-20,30-isopropylidene-guanosine 8 was pre-
pared from guanosine as illustrated in Scheme 2A. The
N2-amine of guanosine was protected as the isobutyryl
amide by the ‘transient protection’ method developed
by Jones and coworkers.35,36 The resulting N2-iso-
butyrylguanosine 7 was converted into the protected
guanosine 8 by reaction with 2,2-dimethoxypropane
under acidic catalysis. We proceeded to prepare the pho-
Scheme 1. In vitro selection of catalytic RNA for the Michael reaction: (a) in vitro transcription of randomized DNA template with fumaramide-
derivatized guanosine generates fumaramide-functionalized RNA libraries; (b) Michael reaction of biotinylated cysteine thiol with the RNA library
leads to biotinylation of active RNAs; (c) inactive RNAs are removed by affinity chromatography on streptavidin agarose; (d) active RNAs are
isolated after photocleavage; (e) reverse transcription of the active RNAs followed by PCR amplification of the cDNA library affords DNA pool for
the next cycle.

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¨
The derivatized fumaric/succinic acids 22a–d were pre-
pared from amine 20 and monoethyl esters 17a–d
(Scheme 3). Amine 20 was prepared from N-Boc-ethyl-
enediamine in three steps. The Michael reaction of
N-Boc-ethylenediamine 1840 with acrylonitrile, followed
by protection of the resulting secondary amine with a
Boc group yielded nitrile 19.41 Catalytic hydrogenation
of this nitrile with Raney nickel gave the partially pro-
tected aminoalkyl linker 20.
While fumaric acid monoethyl ester 17a is commercially
available and succinic acid monoethyl ester 17d could be
obtained in one step from succinic acid anhydride,42 the
methyl substituted fumaric acid monoethyl esters 17b–c
were synthesized by Wittig or Horner–Emmons reac-
tions. Ester 17c was prepared from hydroxypropane.
After protection of the hydroxyl group,43 ketone 14 was
subjected to a Horner–Emmons reaction to give ester
15.44 Removal of the silyl protecting group and sub-
sequent oxidation of the hydroxyl group using Jones
reagent45 gave the 3-substituted fumaric acid monoester
17c. The 2-methyl derivative 17b was synthesized in a
similar manner. The corresponding carbonyl compound
glycolaldehyde is available in dimerized form, which
serves as an intrinsic protection of the hydroxyl group.
Thus, glycolaldehyde dimer could be employed without
further protection in a Wittig reaction yielding alcohol
13.43,46 The latter was oxidized as above and furnished
2-methylfumaric acid monoethyl ester 17b. Coupling of
monoethyl esters 17a-d to amine 20 with DCC followed
by ester cleavage with aqueous NaOH furnished fuma-
ric/succinic acids 22a–d.
Figure 1. Design of substrates chosen for the isolation and further
characterization of catalytic RNAs for the Michael reaction: (A)
Fumaramide-derivatized guanosines 1–4; (B) biotinylated cysteine
thiols 5 and 6.
tocleavable unit 10 (Scheme 2B). 4-(Bromomethyl)-3-
nitrobenzoic acid26 was hydrolyzed to give alcohol 937
which was in turn subjected to EDC mediated coupling
with N-Boc-diaminobutane38,39 to give alcohol 10. 10 was
subsequently incubated with 2-cyanoethyl N,N-diisopropyl
chlorophosphoramidite to generate the corresponding
phosphoramidite 11. Coupling of the phosphoramidite 11
with guanosine 8, followed by iodine oxidation, generated
the functionalized guanosine 12 (Scheme 2C).
Derivatized guanosine 12 was treated with TFA to
remove the Boc group, and the resulting amine was
Scheme 2. (A) Synthesis of protected guanosine 8; (B) synthesis of the photocleavable unit 10; (C) coupling of guanosine and photocleavable unit to
give derivatized guanosine 12.

A. Eisenfuhr et al. / Bioorg. Med. Chem. 11 (2003) 235–249
¨
239
Scheme 3. Synthesis of the fumaric acid and succinic acid derivatives 22a–d.
coupled to NHS-activated fumaric and succinic acids
22a–d to generate the protected products 23a–d.
Removal of the protecting groups with concentrated
NH4OH followed by aqueous TFA yielded the desired
fumaramide derivatized guanosines 1–4 (Scheme 4).
Synthesis of the Michael donor substrates 5and 6
The biotinylated thiols 547 and 6 were prepared as
shown in Scheme 5. d-Biotin was coupled to N-Boc-
ethylenediamine with EDC to obtain carbamate 24, which
was deprotected with TFA and coupled to NHS-activated
N-Boc-S-tritylcysteine or N-Boc-S-methylcysteine to
afford thioethers 25 and 26, respectively. Triethylsilane
and TFA-mediated deprotection of these provided the
biotinylated cysteine thiol 5 and its S-methylated ana-
logue 6.48
Fumaramide-derivatized RNA libraries
The in vitro selection strategy outlined in Scheme 1
allowed the selection of RNA catalysts for the Michael
reaction between a fumaramide group and a thiol
group.13 This selection strategy requires that each
member of the RNA library contains a fumaramide
Scheme 4. Synthesis of fumaramide-derivatized guanosines 1–4.

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A. Eisenfuhr et al. / Bioorg. Med. Chem. 11 (2003) 235–249
¨
in the presence of varying amounts of GTP and mod-
ified guanosine 1. The T7 RNA polymerase generates a
mixture of the desired RNA (92-mer) and RNA with one
additional nucleotide at the 30-terminus (93-mer).49 RNA
transcripts of 92 (band a) and 93 nucleotides (band b)
were generated from the standard transcription reaction
(no modified guanosine 1). As the concentration of 1 was
increased, two new bands corresponding to fumaramide-
modified 92-mer RNA (band c) and fumaramide-mod-
ified 93-mer RNA (band d) were also obtained.
As shown in Figure 2C, maximum amounts (66%) of
modified RNA transcripts (compared to total amounts
of RNA generated) were obtained when the ratio of
GTP/1 was 1/4. However, at this GTP/1 ratio the total
amount of RNA obtained from the transcription reac-
tion was 30% lower than expected from a standard
transcription reaction (data not shown). At ratios of
GTP/1 of 2/3, 3/2, or 4/1, the presence of 1 did not have
any detectable inhibitory effect on T7 polymerase activ-
ity. Therefore, for further transcription reactions GTP/
1–4 ratios of 2/3 were used.
Scheme 5. Synthesis of biotin cysteine thiol 5 and S-methylated ana-
logon 6.
group at its 50-end. As mentioned earlier, we sought to
prepare the fumaramide-modified RNA library by an in
vitro transcription reaction in the presence of fumar-
amide-derivatized initiator nucleotide 1. To investigate
whether the modified guanosines 1–4 are accepted by T7
RNA polymerase, these compounds were added to the
standard in vitro transcription reactions. The amounts of
GTP and 1–4 were varied to determine the optimum con-
ditions for the generation of fumaramide-modified RNAs.
For these transcription reactions, a 114-mer synthetic
DNA template was used which affords 92-mer RNAs.
The fumaramide-modified guanosines 1–4 contain a
photocleavable unit which allows cleavage of the
fumaramide group from modified-RNA molecules by
irradiation with 365 nm light. We had designed this
feature to allow facile removal of active RNAs from the
streptavidin matrix during in vitro selection steps. Fig-
ure 2B shows that UV irradiation (365 nm, 3 h) allows
successful cleavage of fumaramide from the fumaramide-
modified RNAs. The cleavage efficiency of the photo-
labile unit of the initiator nucleotides was determined to
The RNA molecules produced from the transcrip-
tion reaction were separated on a 10% denaturing
polyacrylamide gel and visualized on a phosphorimager.
Figure 2A shows the results of the transcription reaction
Figure 2. Enzymatic incorporation of initiator nucleotides 1–4. (A) Representative autoradiogram of a transcription reaction of a synthetic 114 bp
DNA-template in the presence of initiator nucleotide 1 run by T7-RNA-polymerase. Bands a, b, c, d represent the desired transcripts. Band a:
unmodified RNA 92-mer (n-mer) transcript, band b: unmodified RNA 93-mer transcript (n+1-mer, the additional nucleotide could be A, C, G or
U), band c: fumaramide–RNA conjugate (n-mer), band d: fumaramide–RNA conjugate (n+1-mer). (B) Gelelectrophoretic separation and auto-
radiography of the same transcripts as in A after exposure to UV light at 365 nm for 3 h. (C) Rate of incorporation for initiator nucleotides 1–4.

A. Eisenfuhr et al. / Bioorg. Med. Chem. 11 (2003) 235–249
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241
for complex organic reactions with relevance for cellular
processes.13 Here, we describe the design and synthesis
of the molecules required for the ribozyme selection
experiment and for the characterization of the isolated
ribozymes. We also report the incorporation of these
molecules into RNA libraries and their role in the
underlying selection strategy. Furthermore, we provide
details about the investigation of the background reac-
tion and report the results of the further characteriza-
tion of the ribozyme substrate specificity with respect to
the Michael donor group. We have now shown that our
ribozyme specifically catalyzes the Michael addition of a
cysteine thiol group to an a,b-unsaturated amide. The
designed selection scheme utilized a photo-induced
ribozyme isolation procedure; we expect this method to
be useful in the isolation of other ribozymes as it allows
for the separation of active RNA molecules from the
mixture in a mild and specific manner.
The in vitro selection methodology, together with the
molecules and methods described in this paper, pro-
mises to be a generally applicable procedure for the
generation of RNA molecules that can catalyze complex
organic reactions.
Figure 3. Time course of the background reaction of the Michael
reaction between fumaramide derivatized RNA-library (pool-RNA
derivatized with 1–3) and biotinylated cysteine 5. The reactions were
carried out under selection conditions [50 mM K-MOPS (pH 7.4), 200
mM NaCl, 5 mM MgCl2, room temperature] at concentrations of
[modified RNA]=1 mM and [biotinylated cysteine]=30–50 mM. As
negative controls, the same experiment was performed using succina-
mide 4 derivatized pool-RNA or using S-methylated cysteine 6.
Experimental
be 80–90% in solution, which can be considered to be
quantitative. Moreover, as expected, the RNA molecules
do not degrade under the cleavage conditions.
Materials and methods
Commercial grade reagents and solvents were used
without further purification, except as indicated below.
Dichloromethane was distilled from calcium hydride.
Tetrahydrofuran was distilled from sodium and benzo-
phenone under nitrogen. Reverse-phase HPLC experi-
ments were conducted with 4.6ꢀ250 mm (analytical scale)
or 21.4ꢀ250 mm (preparative scale) Rainin C18 reverse
phase columns. All reactions were stirred magnetically in
flame- or oven-dried glassware under a positive pressure
of Ar or N2.
Substrate specificity with respect to the nucleophilic
Michael donor site
We have reported before that the Michael reaction
between fumaramides 1–3 and biotin cysteine 5 in the
absence of catalyst takes place with a low but measur-
able rate whereas the succinamide 4 or non-derivatized
RNA show no reactivity.13 Thus, the Michael acceptor
system of the fumaramide could be identified as the
preferred reaction site for the nucleophilic attack of 5.
In order to also show that the biotinylated cysteine
attacks the fumaramide via the thiol group of cysteine,
we incubated the fumaramide derivatized RNA library
with 30 mM of the S-methylated cysteine 6. The amount
of biotinylation detected was negligible (Fig. 3). We also
compared the reactions of the fumaramide derivatized
ribozyme sequence with cysteines 5 and 6 under the
same conditions as in the background reactions.
Whereas complete biotinylation of the ribozyme mole-
cules was accomplished by cysteine 5 within 4 h, the rate
of biotinylation with the methylated analogue 6 during
the same period of time was below 1%. Together, these
experiments indicate that the thiol group of the cysteine
is indeed the acting nucleophile in the Michael reaction
catalyzed by the selected ribozyme.
N2-Isobutyrylguanosine (7). Guanosine hydrate (10 g,
35.3 mmol) was dried by co-evaporation of its suspen-
sion in dry pyridine (3ꢀ100 mL) in vacuo. The residue
was suspended in dry pyridine (250 mL) under a nitro-
gen atmosphere, and chlorotrimethylsilane (28.8 g, 265
mmol) was added. The reaction mixture was stirred at
ambient temperature for 2 h, cooled to 0 ꢁC, and iso-
butyryl chloride (11.3 g, 106 mmol) was added dropwise
over 20 min. The mixture was allowed to warm to room
temperature and stirred for 3 h. The reaction mixture
was cooled to 0 ꢁC, and the reaction was quenched by
addition of H2O (30 mL). After stirring for 5 min at
0 ꢁC and then 5 min at room temperature, concentrated
aqueous NH4OH (65 mL) was added. After stirring for
an additional 15 min at room temperature, the mixture
was diluted with H2O (500 mL) and washed with
CH2Cl2 (200 mL). The aqueous layer was concentrated
by evaporation in vacuo. The residue was recrystallized
from hot H2O to obtain N2-isobutyrylguanosine 7 (9.90
Conclusion
1
g, 79%) as a white solid. H NMR (500 MHz, D2O): d
8.10 (s, 1H), 5.90 (d, J=4.7 Hz, 1H), 4.68 (t, J=5.0 Hz,
1H), 4.45 (app t, J=5.1 Hz, 1H), 3.95 (dd, J=12.7, 3.0
We have previously reported the selection of a ribozyme
for the Michael reaction as an example for RNA catalysts

242
A. Eisenfuhr et al. / Bioorg. Med. Chem. 11 (2003) 235–249
¨
Hz, 1H), 3.86 (dd, J=12.7, 4.4 Hz, 1H), 2.77 (app sept,
J=6.9 Hz, 1H), 1.25 (d, J=6.8 Hz, 1H), 1.25 (d, J=6.8
Hz, 3H), 1.24 (d, J=6.8 Hz, 3H). 13C NMR (125 MHz,
D2O): d 182.3, 156.8, 149.8, 148.0, 139.6, 119.9, 88.5,
85.1, 74.6, 70.4, 61.5, 36.2, 18.6. IR (CHCl3): 3490–
3100, 3165, 3145, 1677, 1604 cmÀ1. HRMS (FAB): m/e
for C14H20N5O6 [M+H]+, calcd 354.1413, found
354.1421.
by saturated aqueous NaHCO3 (50 mL). The organic
layer was dried with anhydrous MgSO4, filtered, and
concentrated in vacuo to afford 3.5 g of a white solid.
Recrystallization from EtOAc/hexanes provided
4-(4-BOC-aminobutylcarbamoyl)-2-nitrobenzylic alco-
hol 10 (3.0 g, 59%) as a white solid. 1H N MR
(500 MHz, CDCl3): d 8.41 (s, 1H), 8.02 (d, J=7.5 Hz,
1H), 7.80 (d, J=7.9 Hz, 1H), 7.59 (br s, 1H); 4.98 (s,
2H), 4.88 (br s, 1H), 3.93 (br s, 1H), 3.47 (app q, J=5.8
Hz, 2H), 3.12 (app q, J=6.5 Hz, 2H), 1.65 (app quint,
J=6.5 Hz, 2H), 1.57 (app quint, J=6.2 Hz, 2H), 1.41 (s,
9H). 13C NMR (125 MHz, CDCl3): d 165.5, 156.5,
147.8, 140.5, 134.6, 132.1, 129.1, 123.4, 79.5, 61.6, 40.1,
39.9, 28.3, 27.9, 25.9. IR (CHCl3): 3477–3150, 3355,
3327, 1683, 1643 cmÀ1. HRMS (FAB): m/e for
C17H25N3O6 [M+H]+, calcd 368.1821, found 368.1819.
N2-Isobutyryl-20,30-isopropylidene-guanosine (8). A solu-
tion of N2-isobutyrylguanosine 7 (10 g, 28.3 mmol),
2,20-dimethoxypropane (200 mL; 1.63 mol), and
p-toluenesulfonic acid monohydrate (0.6 g, 3.15 mmol)
in DMF (200 mL) was stirred under a nitrogen atmo-
sphere. After 10 h, the reaction mixture was con-
centrated to dryness and the residue partitioned
between equal volumes (100 mL) of CH2Cl2 and satu-
rated aqueous NaHCO3. The layers were separated, and
the aqueous layer was extracted with CH2Cl2 (2ꢀ100
mL). The combined organic layers were dried over
MgSO4, filtered and concentrated in vacuo. The residue
was purified by column chromatography on silica gel
(MeOH/CH2Cl2, 5:95) to afford protected guanosine 8
(9.95 g, 87%) as a white solid. 1H NMR (500 MHz,
CDCl3): d 12.12 (s, 1H), 8.94 (s, 1H), 7.90 (s, 1H), 5.86
(d, J=3.9 Hz, 1H), 5.12 (dd, J=6.2, 3.9 Hz, 1H), 5.10–
5.08 (m, 1H), 5.00 (dd, J=6.2, 2.3 Hz, 1H), 4.41 (app q,
J=2.3 Hz, 1H), 3.92 (d, J=12.2 Hz, 1H), 3.80–3.76 (m,
1H), 2.69 (app sept, J=6.9 Hz, 1H), 1.59 (s, 3H), 1.35
(s, 3H), 1.27 (d, J=7.0 Hz, 3H), 1.25 (d, J=7.0 Hz,
3H). 13C NMR (125 MHz, D2O): d 180.1, 155.6, 148.1,
138.7, 129.7, 113.8, 90.5, 86.8, 84.0, 81.1, 62.1, 35.7,
26.9, 24.8, 18.7. IR (CHCl3): 3250–3075, 1695, 1685,
1610 cmÀ1. HRMS (FAB): m/e for C17H24N5O6
[M+H]+, calcd 394.1726, found 394.1724.
[4-(4-BOC-Aminobutylcarbamoyl)-2-nitrobenzyl]-2-cya-
noethyl diisopropyl phosphoramidite (11). To a solution
of nitrobenzylic alcohol 10 (100 mg, 0.272 mmol) and
N,N0-diisopropylethylamine (105 mg, 0.816 mmol) in
CH2Cl2 (2 mL) was added at 0 ꢁC 2-cyanoethyl diiso-
propylchorophosphoramidite (97 mg, 0.408 mmol).
After 10 min at 0 ꢁC and a further 20 min at room tem-
perature, the mixture was diluted with EtOAc (75 mL)
and washed with saturated aqueous NaHCO3 (25 mL).
The organic layer was dried over MgSO4, filtered, and
concentrated in vacuo. The residue was purified by flash
chromatography on silica gel (tBME/cyclohexane, 3:1)
to afford phosphoramidite 11 (125 mg, 81%) as a col-
orless oil. 1H NMR (400 MHz, CDCl3): d 8.49 (d,
J=1.2 Hz, 1H), 8.10 (dd, J=8.1 Hz, J=1.2 Hz, 1H),
7.84 (d, J=8.1 Hz, 1H), 7.45 (m, 1H), 5.08 (dd, J=16.2,
6.9 Hz, 1H), 5.01 (dd, J=16.1, 7.75 Hz, 1H), 4.80 (t,
J=5.2 Hz, 1H), 3.84 (m, 1H), 3.77 (m, 1H), 3.62 (ddt,
J=13.5, 6.7, 6.8 Hz, 1H), 3.58 (ddt, J=13.4, 6.6, 6.7
Hz, 1H), 3.42 (app q, J=6.1 Hz, 2H), 3.08 (app q,
J=6.2 Hz, 2H), 2.59 (t, J=6.3 Hz, 2H), 1.60 (app quint,
J=6.8 Hz, 2H), 1.51 (app quint, J=6.8 Hz, 2H), 1.35 (s,
9H), 1,14 (dd, J=6.6, 5.4 Hz, 12H). 13C N MR
(100 MHz, CDCl3): d 165.1, 156.5, 146.7, 138.6 (d,
J=7.6 Hz), 134.7, 132.3, 128.9, 123.5, 117.7, 79.4, 62.4
(d, J=20 Hz), 58.5 (d, J=20 Hz), 43.3 (d, J=12.2 Hz),
40.1, 39.9, 28.4, 28.0, 25.9, 24.6 (d, J=8 Hz), 20.4 (d,
J=7 Hz). 31P NMR (121 MHz, CDCl3): d 149.1. IR
(film): 3335, 2970, 2255, 1690, 1650, 1530, 1365, 1175,
1030, 980 cmÀ1. MS (FAB): m/e 568 ([M+H]+).
4-(Hydroxymethyl)-3-nitrobenzoic acid (9). A solution of
4-(bromomethyl)-3-nitrobenzoic acid (4.40 g, 16.92
mmol) and Na2CO3 (8.96 g, 84.6 mmol) in water/ace-
tone (1:1 v/v, 140 mL) was heated at reflux temperature.
After 5 h, the acetone was evaporated. The resulting
aqueous solution was washed with ether (80 mL), acid-
ified with half-concentrated HCl and extracted with
EtOAc (3ꢀ100 mL). The combined organic layers were
washed with water (50 mL), dried over MgSO4 and
concentrated in vacuo to yield 3.30 g (99%) of
4-(hydroxymethyl)-3-nitrobenzoic acid 9 as a yellow
1
solid. H NMR (400 MHz, CD3OD): d 8.57 (d, J=1.7
Hz, 1H), 8.27 (dd, J=8.1, 1.5 Hz, 1H), 7.97 (d, J=8.1
Hz, 1H), 4.99 (s, 2H). 13C NMR (100 MHz, CD3OD): d
167.5 (C-7), 148.4 (C-3), 144.1 (C-4), 135.2 (C-6), 132.1
(C-1), 129.8 (C-5), 126.7 (C-2), 62.0 (C-8). IR (neat):
3595, 1695, 1525, 1315, 1050 cmÀ1. HRMS (EI): m/e for
C8H5NO4 [MÀH2O]+, calcd 179,0219, found 179,0221.
N2-Isobutyryl-20,30-isopropyliden-guanosine-50-monophos-
phate-P-(2-cyanethyl ester)-P-[4-(4-BOC-aminobutylcar-
bamoyl)-2-nitro-benzylic ester] (12) (as a mixture of
diasteromers). To an ice-cooled solution of protected
guanosine 8 (65 mg, 0.164 mmol) and tetrazole (46 mg,
0.656 mmol) in CH3CN(1 mL) was added phosphor-
amidite 11 (125 m, 0.220 mmol) in CH3CN(1.5 mL).
The reaction mixture was stirred at 0 ꢁC for 5 min, and
then at room temperature for an additional 20 min. The
reaction mixture was again cooled to 0 ꢁC, and iodine
(1.64 mL, 0.164 mmol, 0.1 M solution in 2:20:80, H2O/
pyridine/THF) was added dropwise over 30 min. The
reaction was stirred at 0 ꢁC for 10 min and at room tem-
perature for an additional 15 min. The reaction mixture
was concentrated in vacuo, redissolved in 10 mL EtOAc/
4-(4-BOC-Aminobutylcarbamoyl)-2-nitrobenzylic alcohol
(10). A solution of nitrobenzoic acid 9 (2.72 g, 13.8
mmol), N-0BOC-diaminobutane (2.60 g, 13.8 mmol) and
.
N-ethyl-N -(3-dimethylaminopropyl)-carbodiimide HCl
(EDC, 3.16 g, 16.5 mmol) in ethanol (100 mL) was stir-
red for 2 h and then concentrated in vacuo. The residue
was dissolved in EtOAc (200 mL), and the solution was
washed with 5% aqueous acetic acid (100 mL) followed

A. Eisenfuhr et al. / Bioorg. Med. Chem. 11 (2003) 235–249
¨
243
2-butanol (7:3) and the solution was washed with aqu-
eous NaHSO3 (0.5 M, 2.5 mL) followed by water (2.5
mL). The organic layer was dried over MgSO4, filtered
and concentrated in vacuo. The residue was purified by
flash chromatography on silica gel (MeOH/CH2Cl2,
5:95) to isolate 100 mg (70%) of modified GMP 12 as a
white solid. 1H NMR (400 MHz, CDCl3): d 12.21 (s, 1H),
10.61 (s, 1H), 8.42 (m, 1H), 8.04 (m, 1H), 8.00 (s, 1H),
7.90 (m, 1H), 7.51 (br. s, 1H), 5.92 (m, 1H), 5.39 (m, 2H),
5.23 (m, 1H), 5.12 (br. s, 1H), 4.92 (m, 1H), 4.42 (m, 1H),
4.35 (m, 2H), 4.27 (m, 2H), 3.42 (m, 2H), 3.09 (m, 2H),
2.81 (m, 2H), 2.72 (m, 1H), 1.62 (m, 2H), 1.55 (m, 2H),
1.53 (s, 3H), 1.35 (s, 9H), 1.30 (s, 3H), 1.18 (m, 6H). 13C
NMR (100 MHz, CDCl3): d 180.3, 165.1, 156.5, 154.9,
148.7, 146.2, 139.1, 136.6, 133.3 (d, J=9 Hz), 132.6,
128.0, 124.2, 118.5, 116.3, 114.9, 91.7, 84.9 (d, J=7 Hz),
84.0, 81.2, 79.3, 66.5 (d, J=3 Hz), 62.8 (d, J=5 Hz), 61.7
(d, J=3 Hz), 40.1, 36.0, 28.4, 27.7, 27.3, 26.1, 25.5, 19.8
(d, J=7 Hz), 19.2. 31P NMR (121 MHz, CDCl3): d
À2.9,À3.0. IR (neat): 3380, 2930, 1685, 1530, 1365, 1255,
1160, 1030 cmÀ1. HRMS (FAB): m/e for C37H51N9O14P
[M+H]+, calcd 876.3293, found 876.3283.
TBDMS-hydroxypropanone 14 (2.37 g, 12.58 mmol)
was added dropwise with cooling to below 30 ꢁC. After
2 h of stirring at ambient temperature, the mixture was
poured onto water (80 mL) and extracted with ether
(4ꢀ100 mL). The organic combined organic layers were
dried over MgSO4 and concentrated in vacuo. Purifica-
tion of the residue by flash chromatography on silica gel
(cyclohexane/EtOAc, 20:1) yielded 1.79 g (55%) of
g-hydroxy ester 15 as a colorless oil. 1H N MR
(400 MHz, CDCl3): d 5.90 (m, 1H), 4.09 (q, J=7.1 Hz,
2H), 4.02 (m, 2H), 1.96 (m, 3H), 1.20 (t, J=7.1 Hz, 3H),
0.84 (s, 9H), 0.00 (s, 6H). 13C NMR (100 MHz, CDCl3):
d 167.0, 157.1, 113.3, 67.1, 59.5, 25.6, 18.3, 15.4,
14.3,À5.5. IR (film): 2930, 2860, 1720, 1220, 1155, 1110
cmÀ1. HRMS (EI): m/e for C9H17O3Si [MÀC4H9]+,
calcd 201.0947, found 201.0938.
4-Hydroxy-3-methylbut-2-enoic acid ethyl ester (16). To
a solution of 4-(TBDMS-hydroxy)-3-methylbut-2-enoic
ethyl ester 15 (1.7 g, 6.58 mmol) in THF (60 mL) was
added dropwise at 0 ꢁC a 1 M solution of TBAF in THF
(7.3 mL, 7.3 mmol). The mixture was stirred at ambient
temperature for 30 min and then concentrated in vacuo.
The residue was purified by flash chromatography on
silica gel (ether/petrol ether, 3:1) to afford the depro-
tected g-hydroxy ester 16 (540 mg, 57%) as a colorless
4-Hydroxy-2-methylbut-2-enoic acid ethyl ester (13). To
a refluxing solution of glycolaldehyde dimer (790 mg,
6.57 mmol) in CH2Cl2 (75 mL) was added dropwise a
solution of (1-ethoxycarbonylethyliden)-triphenylphos-
phorane (5 g, 13.8 mmol) in CH2Cl2 (50 mL). After 4 h
at refluxing temperature, the solvent was evaporated
and the residue purified by flash chromatography on
silica gel (ether/petrol ether, 3:1) to give 1.45 g (77%) of
g-hydroxy ester 13 as a colorless oil. 1H N MR
(400 MHz, CDCl3): d 6,75 (tq, J=6.0, 1.4 Hz, 1H), 4.27
(dq, J=6.0, 0.9 Hz, 2H), 4.12 (q, J=7,1 Hz, 2H), 2.75
(s, 1H), 1.76 (app q, J=1.1 Hz, 3H), 1.22 (t, J=7.1 Hz,
3H). 13C NMR (100 MHz, CDCl3): d 167.9, 140.3,
128.4, 60.8, 59.6, 14.2, 12.6. IR (film): 3360, 2975, 1690,
1365, 1165 cmÀ1. MS (EI): m/e 126 ([MÀH2O]+).
1
oil. H NMR (400 MHz, CDCl3): d 5.90 (m, 1H), 4.09
(q, 2H), 4.05 (d, J=7,1 Hz, 2H), 2.82 (t, J=5,8 Hz, 1H),
2.00 (m, 3H), 1.21 (t, J=7.1 Hz, 3H). 13C N MR
(100 MHz, CDCl3): d 167.1, 157.6, 113.5, 66.9, 59.8,
15.6, 14.2. IR (film): 3490–3370, 2980, 1715, 1695, 1660,
1225, 1150 cmÀ1. HRMS (EI): m/e for C7H12O3, calcd
144.0786, found 144.0775.
2-Methylfumaric acid monoethyl ester (17b). To a solu-
tion of 4-hydroxy-2-methylbut-2-enoic acid ethyl ester
13 (1.35 g, 9.36 mmol) was added at 0 ꢁC Jones reagent,
consisting of chrome(VI)-oxide (2.15 g, 21.53 mmol),
water (6,8 mL) and concentrated H2SO4 (2 mL). After
stirring at ambient temperature for 30 min, EtOH (3.2
mL) and water (20 mL) were added and the mixture was
extracted with ether (2ꢀ50 mL). The combined organic
layers were dried over MgSO4 and concentrated in
vacuo. 2-Methylfumaric acid monoethyl ester 17b was
obtained as a colorless oil (1.13 g, 76%). 1H N MR
(400 MHz, CDCl3): d 10.83 (br. s, 1H), 6.73 (q, J=1.3
Hz, 1H), 4.20 (q, J=7.1 Hz, 2H), 2.25 (d, J=1.5 Hz,
3H), 1.27 (t, J=7.0 Hz, 3H). 13C NMR (100 MHz,
CDCl3): d 171.4, 166.9, 146.4, 125.7, 61.9, 14.6, 14.1. IR
(film): 3100–2900, 1720, 1690, 1645, 1260 cmÀ1. HRMS
(EI): m/e for C7H8O3 [MÀH2O]+, calcd 140.0473,
found 140.0476.
TBDMS-Hydroxypropanone (14). Hydroxypropanone
(1.3 mL, 18.6 mmol), TBDMSCl (3.65 g, 24.2 mmol)
and imidazole (1.77 g, 26 mmol) were dissolved in DMF
(20 mL) and stirred at ambient temperature for 16 h.
The mixture was poured onto water (100 mL) and
extracted with cyclohexane (3ꢀ100 mL). The combined
organic layers were dried over MgSO4 and concentrated
in vacuo. The residue was purified by flash chromato-
graphy on silica gel (cyclohexane/EtOAc, 8:1) to afford
TBDMS-hydroxypropanone 14 (2.91 g, 83%) as a color-
less oil. 1H NMR (400MHz, CDCl3): d 4.06 (s, 2H), 2.08
(s, 3H), 0.83 (s, 9H), 0.00 (s, 6H). 13C NMR (100MHz,
CDCl3): d 209.1, 69.5, 25.6, 18.3,À5.5. IR (film): 2930,
2860, 1720, 1255, 1120 cmÀ1. HRMS (EI): m/e for
C8H17O2Si [MÀCH3]+, calcd 173.0998, found 173.0998.
3-Methylfumaric acid monoethyl ester (17c). 4-Hydroxy-
2-methylbut-2-enoic acid ethyl ester 16 (500 mg, 3.47
mmol) was oxidized as described above to give
3-methylfumaric acid monoethyl ester 17c as a colorless
4-(TBDMS-Hydroxy)-3-methylbut-2-enoic acid ethyl es-
ter (15). Sodium hydride (60% suspension in natural
oil, 670 mg, 16.8 mmol) was washed with pentane and
then suspended in THF (40 mL). To this suspension was
added dropwise with cooling to 20 ꢁC a solution of
phosphonoacetic acid triethyl ester (3.10 mL, 15.5
mmol) in THF (8 mL). After stirring at ambient tem-
perature for 1 h until the end of hydrogen formation,
1
oil (540 mg, 98%). H NMR (400 MHz, CDCl3): d 9.90
(br. s, 1H), 6.82 (q, J=1.6 Hz, 1H), 4.17 (q, J=7.1 Hz,
2H), 2.21 (d, J=1.5 Hz, 3H), 1.25 (t, J=7.1 Hz, 3H).
13C NMR (100 MHz, CDCl3): d 172.4, 165.7, 142.5,
128.7, 60.9, 14.1, 13.9. IR (film): 3100–2900, 1720, 1690,
1645, 1285, 1220 cmÀ1. MS (EI): m/e 143 ([MÀCH3]+).

244
A. Eisenfuhr et al. / Bioorg. Med. Chem. 11 (2003) 235–249
¨
Succinic acid monoethyl ester (17d). Succinic anhydride
(50 g, 500 mmol) was dissolved in EtOH (35 mL, 600
mmol) and refluxed for 1 h. Excess EtOH was evapo-
rated to give monoethyl ester 17d (71.16 g, 97%) as a
1165 cmÀ1. HRMS (EI): m/e for C15H31N3O4, calcd
317.2315, found 317.2322.
3-{3-[BOC-(2-BOC-Aminoethyl)-amino]-propylcarba-
moyl}-acrylic acid ethyl ester (21a). A solution of fuma-
ric acid ethyl ester 17a (4.09 g, 28.4 mmol), amine 20
(9.00g, 28.4 mmol) and dicyclohexylcarbodiimide
(DCC, 7.03 g, 34.1 mmol) in CH2Cl2 (100 mL) was
stirred at room temperature. After 3 h, the reaction
mixture was filtered to remove DCU and the filtrate
concentrated in vacuo. The residue was dissolved in
EtOAc (200 mL) and the solution washed sequentially
with 5% aqueous acetic acid (100 mL) and saturated
aqueous NaHCO3 (100 mL). The organic layer was
dried over anhydrous MgSO4, filtered and concentrated
in vacuo. The residue was purified by column chroma-
tography on silica gel (MeOH/CH2Cl2, 5:95) to afford
1
colorless oil. H NMR (400 MHz, CDCl3): d 10.66 (s,
1H), 4.08 (q, J=7.14 Hz, 2H), 2.61 (t, J=6.6 Hz, 2H),
2.55 (t, J=6.3 Hz, 2H), 1.18 (t, J=7.14 Hz, 3H). 13C
NMR (100 MHz, CDCl3): d 178.0, 172.2, 60.7, 28.8,
28.8, 13.9. IR (film): 2985, 1735, 1170 cmÀ1. HRMS
(EI): m/e for C6H8O3 [M-H2O]+, calcd 128.0473, found
128.0477.
N-BOC-1,2-Diaminoethane (18). To
a solution of
1,2-diaminoethane (28 mL, 400 mmol) in CHCl3 (400
mL) was added dropwise at 0 ꢁC a solution of di-tert-
butyldicarbonate (8.74 g, 40 mmol) in CHCl3 (200 mL)
over a period of 3 h. After stirring at ambient tempera-
ture for 16 h, the mixture was washed with brine (6ꢀ100
mL) and water (1ꢀ100 mL), dried over MgSO4 and
concentrated in vacuo to afford 6.40 g (quantitative
yield) of N-BOC-1,2-diaminoethane 18 as a colorless
oil. 1H NMR (400 MHz, CDCl3): d 5.01 (br. s, 1H), 3.10
(app q, J=5.7 Hz, 2H), 2.73 (t, J=6.0 Hz, 2H), 1.38 (s,
9H), 1.12 (s, 2H). 13C NMR (100 MHz, CDCl3): d
156.2, 79.1, 43.4, 41.9, 28.4. IR (film): 3500–3100, 2975,
1
12.2 g (97%) of ester 21a as a colorless oil. H N MR
(500 MHz, CDCl3, 330 K): d 7.14–7.02 (br s, 1H), 6.89
(d, J=15.5 Hz, 1H), 6.74 (d, J=15.5 Hz, 1H), 4.88 (br
s, 1H), 4.22 (q, J=7.1 Hz, 2H), 3.30–3.23 (m, 8H), 1.72
(app quint, J=6.3 Hz, 2H), 1.44 (s, 9H), 1.41 (s, 9H),
1.27 (t, J=7.1 Hz, 3H). 13C NMR (125 MHz, CDCl3,
330 K): d 165.5, 163.8, 156.3, 156.0, 136.7, 129.9, 80.3,
79.4, 60.8, 46.9, 45.6, 39.6, 36.5, 28.3, 27.9, 14.0. IR
(CHCl3): 3680–3395, 1641 cmÀ1. HRMS (FAB): m/e for
C21H38N7O7 [M+H]+, calcd 444.2709, found 444.2710.
1690, 1525, 1365, 1250, 1170 cmÀ1
.
3-[BOC-(2-BOC-Aminoethyl)-amino]-propionitrile (19).
A solution of N-BOC-1,2-diaminoethane 18 (10.5 g,
65.54 mmol) and acrylonitrile (6.96 g, 131.08 mmol) in
MeOH (150 mL) was stirred for 12 h and then con-
centrated in vacuo. The residue was redissolved in
CH2Cl2 (20 mL), di-tert-butylcarbonate (16.45 g, 75.37
mmol) was added, and the resulting mixture was stirred
at room temperature. After 12 h, the reaction mixture
was concentrated in vacuo and chromatographed on
silica gel (ether/petrol ether, 3:1) to obtain nitrile 19
3-{3-[BOC-(2-BOC-Aminoethyl)-amino]-propylcarba-
moyl}-2-methyl-acrylic acid ethyl ester (21b). 2-Methyl-
fumaric acid monoethyl ester 17b (1.11 g, 7 mmol) was
coupled to amine 20 as described above to give 2.16 g
(67%) of ester 21b as a colorless oil. 1H NMR (400 MHz,
CDCl3): d 6.99+6.27 (br. s, 1H, NH-rotameres), 6.74 (s,
1H), 5.02+4.76 (br. s, 1H, NH-rotameres), 4.17 (q,
J=7.1 Hz, 2H), 3.20 (m, 8H), 2.19 (d, J=1.2 Hz, 3H),
1.64 (m, 2H), 1.40 (s, 9H), 1.36 (s, 9H), 1.25 (t, J=7,1
Hz, 3H). 13C NMR (100 MHz, CDCl3): d 165.6, 156.4,
156.0, 138.9, 130.2, 80.4, 79.4, 61.3, 46.6, 43.9, 39.5,
35.6, 30.8, 28.4, 14.2, 13.9. IR (film): 3325, 2975, 1750–
1650, 1530, 1415, 1365, 1270, 1170 cmÀ1. MS (FAB): m/
e 458 ([M+H]+).
1
(12.90 g, 63%) as a white solid. H NMR (400 MHz,
CDCl3): d 4.89+4.73 (br. s, 1H, NH-rotameres), 3.43 (t,
J=6.6 Hz, 2H), 3.33 (t, J=6.2 Hz, 2H), 3.20 (m, 2H),
2.60 (t, J=5.3 Hz, 1H), 2.52 (t, J=5.3 Hz, 1H), 1.41 (s,
9H), 1.37 (s, 9H). 13C NMR (100 MHz, CDCl3): d
155.9+155.7, 155.0, 118.1+117.6, 80.7, 79.2,
47.9+46.9, 44.1+43.8, 39.1, 28.1, 17.3+16.7. IR (neat):
3360, 2980, 1680, 1520, 1165 cmÀ1. HRMS (EI): m/e for
C15H27N3O4, calcd 313.2002, found 313.2002.
3-{3-[BOC-(2-BOC-Aminoethyl)-amino]-propylcarba-
moyl}-3-methyl-acrylic acid ethyl ester (21c). 3-Methyl-
fumaric acid monoethyl ester 17c (520 mg, 3.3 mmol) was
coupled to amine 20 as described above to give 320 mg
(21%) of ester 21c as a colorless oil. 1H NMR (400MHz,
CDCl3): d 7.49+6.35 (br. s, 1H, NH-rotameres), 6.46 (s,
1H), 5.02+4.77 (br. s, 1H, NH-rotameres), 4.14 (q,
J=7.1 Hz, 2H), 3.21 (m, 8H), 2.28 (s, 3H), 1.63 (m, 2H),
1.41 (s, 9H), 1.36 (s, 9H), 1.23 (t, J=7.1 Hz, 3H). 13C
NMR (100 MHz, CDCl3): d 166.3, 166.0, 156.6, 156.0,
143.5, 127.3, 80.7, 79.6, 60.4, 46.5, 43.7, 39.4, 35.9, 30.8,
28.4, 14.2. IR (film): 3335, 2930, 1700, 1670, 1530, 1420,
1370, 1250, 1170. MS (FAB): m/e 458 ([M+H]+), 480
([M+Na]+).
3-[BOC-(2-BOC-Aminoethyl)-amino]-propylamine (20).
A solution of nitrile 19 (1.57 g, 5 mmol), Raney Ni41
(0.4 g) and KOH (0.32 g, 5.75 mmol) in EtOH (40 mL)
under H2 (3.5 bar) was shaken in a parr apparatus.
After 5 h, the reaction mixture was filtered over Celite,
and the filtrate was concentrated in vacuo. The residue
was dissolved in CH2Cl2 (40 mL) and the solution was
washed with saturated aqueous NaHCO3 (20 mL). The
organic layer was dried over MgSO4 and concentrated
in vacuo to afford 1.58 g (quantitative yield) of amine 20
as a colorless oil. 1H NMR (400 MHz, CDCl3): d
5.11+4.93 (br. s, 1H, NH-rotameres), 3.20 (m, 6H),
2.64 (t, J=6.5 Hz, 2H), 1.94 (m, 2H), 1.61 (br. s, 2H),
1.40 (s, 9H), 1.36 (s, 9H). 13C NMR (100 MHz, CDCl3):
d 155.9+155.8, 80.0, 79.1, 46.5+46.2, 45.2+44.4, 39.5,
38.9, 32.5+31.3, 28.4. IR (film): 3360, 2975, 1695, 1365,
3-{3-[BOC-(2-BOC-Aminoethyl)-amino]-propylcarba-
moyl}-propionic acid ethyl ester (21d). Succinic acid
monoethyl ester 17d (1.56 g, 5 mmol) was coupled to
amine 20 as described above to give 1.74 g (80%) of
ester 21d as a colorless oil. 1H NMR (400 MHz,

A. Eisenfuhr et al. / Bioorg. Med. Chem. 11 (2003) 235–249
¨
245
CDCl3): d 6.81+6.11 (br. s, 1H, NH-rotameres),
5.02+4.77 (br. s, 1H, NH-rotameres), 4.07 (q, J=7.1
Hz, 2H), 3.19 (m, 8H), 2.60 (t, J=6.5 Hz, 2H), 2.44 (m,
2H), 1.61 (m, 2H), 1.40 (s, 9H), 1.37 (s, 9H), 1.18 (t,
J=7,1 Hz, 3H). 13C NMR (100 MHz, CDCl3): d 173.0,
171.7, 156.3, 155.9, 80.3, 79.4, 60.6, 46.6, 43.9, 39.5,
35.9, 31.0, 29.6, 28.4, 27.7, 14.2. IR (film): 3330, 2975,
1750–1650, 1540, 1415, 1365, 1250, 1170 cmÀ1. MS
(FAB): m/e 446 ([M+H]+), 468 ([M+Na]+).
(m, 2H), 1.39 (s, 9H), 1.36 (s, 9H). 13C NMR (100 MHz,
CDCl3): d 175.3, 172.8, 156.3, 156.0, 80.5, 79.8, 46.7,
44.2, 39.3, 36.4, 31.0, 30.0, 28.4, 27.6. IR (neat): 3360,
3140, 2975, 2935, 1720, 1660, 1530, 1420, 1275, 1245,
1180 cmÀ1. MS (FAB): m/e 418 ([M+H]+), 440
([M+Na]+).
N2-Isobutyryl-20,30-isopropyliden-guanosine-50-monophos-
phate-P-(2-cyanethylester)-P-{4-[4-(3-{3-[BOC-(2-BOC-
aminoethyl) - amino] - propylcarbamoyl} - acryloylamino)-
butylcarbamoyl]-2-nitro-benzylic ester} (23a) (as a mix-
ture of diasteromers). Fumaric acid 22a (715 mg, 1.72
mmol) was activated by stirring with N-hydroxy-
succinimide (217 mg, 1.89 mmol) and DCC (389 mg,
1.89 mmol) in CH2Cl2 (2 mL) at room temperature.
After 30 min, the mixture was filtered and the filtrate
was concentrated in vacuo to give the NHS-activated
acid as a white solid. At the same time, a solution of
modified GMP 12 (1.15 g, 1.32 mmol) and tri-
fluoroacetic acid (TFA, 1.5 mL) in CH2Cl2 (15 mL) was
stirred under argon. After 30 min, the reaction mixture
was concentrated in vacuo and the residue was redis-
solved in saturated aqueous NaHCO3 (25 mL). The pH
of the mixture was adjusted to pH 8 with 1 M NaOH,
and a solution of the NHS-activated acid in CH3CN(25
mL) was added. The reaction mixture was stirred for 1 h
and then extracted with CH2Cl2 (3ꢀ50 mL). The com-
bined organic layers were dried over MgSO4, filtered
and concentrated in vacuo. The residue was purified by
column chromatography (MeOH/CH2Cl2, 7:93) to
obtain 1.01g (65%) of modified GMP 23a as a colorless
3-{3-[BOC-(2-BOC-Aminoethyl)-amino]-propylcarba-
moyl}-acrylic acid (22a). A solution of ethyl ester 21a
(4.93 g, 11.1 mmol) in THF (75 mL) was stirred for 1 h
with 1 M NaOH (22.3 mL, 22.3 mmol). The reaction
mixture was cooled with an ice bath, acidified to pH 2
with concentrated HCl and extracted with CHCl3
(3ꢀ100 mL). The combined organic layers were dried
over MgSO4, filtered, and concentrated in vacuo to
afford 3.80 g (82%) of fumaric acid 22a as a white solid.
1H NMR (500 MHz, CDCl3, 330 K): d 9.0 (br s, 1H),
7.4 (br s, 1H), 7.18 (d, J=15.4 Hz, 1H), 6.84 (d, J=15.5
Hz, 1H), 5.1 (br s, 1H), 3.36–3.28 (m, 8H), 1.79 (app
quint, J=6.3 Hz, 2H), 1.48 (s, 9H), 1.44 (s, 9H). 13C
NMR (125 MHz, CDCl3, 330 K): d 168.0, 164.2, 156.4,
138.0, 129.7, 80.9, 79.8, 47.0, 45.2, 39.7, 36.9, 28.4, 27.6.
IR (CHCl3): 3400–3250, 3320, 1717, 1698, 1670, 1653
cmÀ1. HRMS (FAB): m/e for C19H34N3O7 [M+H]+,
calcd 416.2396, found 416.2396.
3-{3-[BOC-(2-BOC-Aminoethyl)-amino]-propylcarba-
moyl}-2-methyl-acrylic acid (22b). Ethyl ester 21b (2.12
g, 4.63 mmol) was hydrolyzed as described above to
give 1.91 g, (96%) of fumaric acid 22b as a white solid.
1H NMR (400 MHz, CDCl3): d 8.16 (br. s, 1H),
7.34+6.87 (br. s, 1H, NH-rotameres), 6.96 (s, 1H),
5.20+4.86 (br. s, 1H, NH-rotameres), 3.21 (m, 8H),
2.21 (s, 3H), 1.67 (m, 2H), 1.40 (s, 9H), 1.36 (s, 9H). 13C
NMR (100 MHz, CDCl3): d 170.5, 166.0, 156.5, 156.0,
138.9, 131.1, 81.0, 79.7, 46.6, 44.4, 39.3, 35.9, 30.8, 28.4,
13.9. IR (neat): 3350, 2975, 1720, 1655, 1515, 1425,
1370, 1250, 1170 cmÀ1. MS (FAB): m/e 430 ([M+H]+).
1
oil. H NMR (500 MHz, CD3OD, 330 K): d 8.53 (d,
J=1.7 Hz, 0.5H), 8.49 (d, J=1.7 Hz, 0.5H), 8.13 (dd,
J=8.0, 1.7 Hz, 0.5H), 8.07 (dd, J=8.1, 1.7 Hz, 0.5H),
8.99 (s, 0.5H), 7.98 (s, 0.5H), 7.78 (d, J=8.1 Hz, 0.5H),
7.71 (d, J=8.1 Hz, 0.5H), 6.86 (s, 2H), 6.12 (d, J=2.0
Hz, 0.5H), 6.10 (d, J=2.2 Hz, 0.5H), 5.51 (d, J=7.0 Hz,
0.5H), 5.46 (d, J=7.1 Hz, 0.5H), 5.31 (d, J=2.1 Hz,
0.5H), 5.29 (d, J=2.1 Hz, 0.5H), 5.21 (dd, J=6.2, 3.7
Hz, 0.5H), 5.19 (dd, J=6.3, 3.1 Hz, 0.5H), 4.52–4.45
(m, 2H), 4.39–4.25 (m, 3H), 3.46 (t, J=5.9 Hz, 2H), 3.35
(d, J=6.4 Hz, 2H), 3.29 (app t, J=5.0 Hz, 6H), 3.19 (t,
J=6.3 Hz, 2H), 2.91 (t, J=6.0 Hz, 1H), 2.87 (t, J=5.8
Hz, 1H), 2.76 (app sept, J=6.9 Hz, 1H), 1.79 (app
quint, J=7.0 Hz, 2H), 1.74–1.68 (m, 4H), 1.58 (s, 1.5H),
1.56 (s, 1.5H), 1.45 (s, 9H), 1.42 (s, 9H), 1.38 (s, 1.5H),
1.36 (s, 1.5H), 1.24–1.21 (m, 6H); 13C NMR (125 MHz,
CD3OD, 330 K): d 181.7, 181.6, 174.5, 166.9, 166.7,
158.2, 157.4, 157.2, 149.6, 149.5, 148.2, 148.1, 140.3,
140.2, 137.23, 137.20, 135.4 (d, J=8.2 Hz), 135.3 (d,
J=8.3 Hz), 133.9, 133.8, 133.43, 133.37, 130.0, 129.9,
124.8, 122.2, 122.1, 118.3 (d, J=4.3 Hz), 115.7 (d,
J=4.2 Hz), 92.1, 91.8, 86.7 (d, J=7.2 Hz), 86.5 (d,
J=7.4 Hz), 85.7, 85.5, 82.4 (d, J=7.7 Hz), 81.2, 80.1,
69.1, 67.4 (d, J=3.7 Hz), 64.5 (d, J=5.4 Hz), 48.0, 46.5,
40.8, 40.3, 40.2, 38.3, 36.9, 29.1, 28.8, 28.7, 27.7, 27.5,
26.3, 25.7, 20.1 (d, J=7.0 Hz), 19.4, 19.3 (d, J=7.7 Hz);
IR (film): 3309, 3178, 1683, 1649 cmÀ1. MS (FAB): m/e
for C51H74N12O18P [M+H]+, calcd 1173.5, found
1173.6.
3-{3-[BOC-(2-BOC-Aminoethyl)-amino]-propylcarba-
moyl}-3-methyl-acrylic acid (22c). Ethyl ester 21c (320
mg, 0.70 mmol) was hydrolyzed as described above to
give (300 mg, 99%) of fumaric acid 22c as a white solid.
1H NMR (400 MHz, CDCl3): d 7.77 (br. s, 1H),
7.51+6.32 (br. s, 1H, NH-rotameres), 6.43 (s, 1H),
5.18+4.86 (br. s, 1H, NH-rotameres), 3.21 (m, 8H),
2.27 (s, 3H), 1.65 (m, 2H), 1.40 (s, 9H), 1.36 (s, 9H). 13C
NMR (100 MHz, CDCl3): d 170.1, 168.4, 156.6, 156.0,
149.5, 127.4, 80.9, 79.6, 46.5, 43.9, 39.3, 36.1, 29.7, 28.4,
14.3. IR (neat): 3335, 2930, 1720–1650, 1535, 1420,
1365, 1250, 1170 cmÀ1. MS (FAB): m/e 430 ([M+H]+),
452 ([M+Na]+).
3-{3-[BOC-(2-BOC-Aminoethyl)-amino]-propylcarba-
moyl}-propionic acid (22d). Ethyl ester 21d (1.72 g, 3.85
mmol) was hydrolyzed as described above to give (1.55
1
g, 96%) of succinic acid 22d as a white solid. H N MR
(400 MHz, CDCl3): d 9.05 (br. s, 1H), 7.10+6.58 (br. s,
1H, NH-rotameres), 5.18+4.86 (br. s, 1H, NH-rota-
meres), 3.19 (m, 8H), 2.63 (m, 2H), 2.47 (m, 2H), 1.63
N2-Isobutyryl-20,30-isopropyliden-guanosine-50-monophos-
phate-P-(2-cyanethylester)-P-{4-[4-(3-{3-[BOC-(2-BOC-

246
A. Eisenfuhr et al. / Bioorg. Med. Chem. 11 (2003) 235–249
¨
aminoethyl)-amino]-propylcarbamoyl}-2-methyl-acryloy-
lamino)-butylcarbamoyl]-2-nitro-benzylic ester} (23b) (as
a mixture of diasteromers). Fumaric acid 22b (73 mg,
0.175 mmol) was activated and coupled to modified
GMP 12 as described above to give 65 mg (56%) of
modified GMP 23b as a colorless oil. 1H N MR
(400 MHz, CDCl3): d 8.52 (m, 1H), 8.04 (s, 1H), 8.02
(m, 1H), 7.71 (m, 1H), 6.46 (m, 1H), 6.12 (m, 1H), 4.99
(m, 2H), 4.88 (m, 1H), 4.60 (m, 1H), 4.48 (m, 1H), 4.34
(m, 2H), 3.44 (m, 2H), 3.31 (m, 4H), 3.26 (m, 4H), 3.17
(m, 2H), 2.91 (m, 1H), 2.82 (m, 2H), 2.18 (m, 3H), 2.03
(m, 2H), 1.74 (m, 2H), 1.68 (m, 2H), 1.45 (s, 9H), 1.41
(s, 9H), 1.28 (s, 6H), 1.24 (s, 6H). 13C NMR (100 MHz,
CDCl3): d 181.8, 168.3, 167.5, 167.1, 158.5, 157.5, 157.3,
147.8, 144.6, 142.6, 140.2, 137.0, 133.7, 132.5, 129.6,
126.3, 125.0, 120.1, 118.5, 115.8, 92.1, 87.1 (d, J=8 Hz),
85.6, 82.4, 81.3, 80.1, 69.4 (d, J=4 Hz), 67.6 (d, J=4
Hz), 64.6 (d, J=5 Hz), 50.9, 48.1, 46.1, 40.9, 40.6, 37.7,
37.0, 28.9, 28.8, 27.9, 27.6, 25.7, 20.2 (d, J=7 Hz), 19.6,
14.7. 31P NMR (121 MHz, CDCl3): d À2.3,À2.4. IR
(film): 3420–3350, 2925, 2855, 1685, 1530, 1465, 1250,
1030 cmÀ1. MS (ESI): m/e 1187 ([M+H]+), 1209
([M+Na]+).
(m, 2H), 2.53 (m, 4H), 1.94 (m, 2H), 1.58 (m, 2H), 1.49
(m, 2H), 1.35 (s, 9H), 1.32 (s, 9H), 1.18 (s, 6H), 1.15 (s,
6H). 13C NMR (100 MHz, CDCl3): d 181.8, 174.7,
167.1, 158.4, 157.5, 157.3, 147.9, 144.4, 140.2, 136.0 (d,
J=6 Hz), 133.6, 130.4, 129.6, 125.0, 120.1, 118.5, 115.8,
92.1, 86.7 (d, J=8 Hz), 85.6, 82.4, 81.3, 80.1, 69.4 (d,
J=6 Hz), 67.5 (d, J=4 Hz), 64.6 (d, J=5 Hz), 50.9,
48.1, 46.1, 40.9, 40.1, 37.9, 37.0, 33.1, 28.8, 28.8, 27.9,
27.6, 25.7, 20.2 (d, J=7 Hz), 19.5. 31P NMR (121 MHz,
CDCl3): d À2.3,À2.4. IR (film): 3420–3300, 2925, 1685,
1535, 1365, 1255, 1160, 1035 cmÀ1. MS (FAB): m/e 1175
([M+H]+).
Guanosine-50-monophosphate-P-[4-(4-{3-[3-(2-aminoethyl-
amino)-propylcarbamoyl]-acryloylamino}-butylcarbamoyl)-
2-nitro-benzylic ester] (1) (as a mixture of diasteromers).
A solution of protected modified GMP 23a (1.00 g,
0.853 mmol) in MeOH (5 mL) and concentrated
NH4OH (30 mL) was heated at 55 ꢁC in a pressure tube.
After 20 h, the reaction mixture was concentrated in
vacuo to obtain a yellow oil. The oil was redissolved in
H2O (21 mL) and trifluoroacetic acid (9 mL). The solu-
tion was stirred under N2 for 12 h and then con-
centrated in vacuo to afford a yellow oil. Purification by
preparative HPLC (MeCN/0.15% aqueous TFA, 13:87,
25 mL/min) afforded 0.723 g (82%) of fumaramide
N2-Isobutyryl-20,30-isopropyliden-guanosine-50-monophos-
phate-P-(2-cyanethylester)-P-{4-[4-(3-{3-[BOC-(2-BOC-
aminoethyl)-amino]-propylcarbamoyl}-3-methyl-acryloy-
lamino)-butylcarbamoyl]-2-nitro-benzylic ester} (23c) (as
a mixture of diasteromers). Fumaric acid 22c (73 mg,
0.175 mmol) was activated and coupled to modified
GMP 12 as described above to give 58 mg (43%) of
modified GMP 23c as a colorless oil. 1H N MR
(400 MHz, CDCl3): d 8.42 (d, J=2 Hz, 1H), 8.05 (dd,
J=8, 2 Hz, 1H), 7.92 (s, 1H), 7.88 (d, J=8 Hz, 1H),
6.38 (m, 1H), 6.02 (d, J=2HZ, 1H), 4.89 (m, 2H), 4.77
(m, 1H), 4.49 (m, 1H), 4.38 (m, 1H), 4.24 (m, 2H), 3.33
(m, 2H), 3.21 (m, 4H), 3.18 (m, 4H), 3.08 (m, 2H), 2.81
(app sept, J=5 Hz, 1H), 2.72 (m, 2H), 2.08 (m, 3H),
1.94 (m, 2H), 1.65 (m, 2H), 1.57 (m, 2H), 1.36 (s, 9H),
1.32 (s, 9H), 1.18 (s, 6H), 1.15 (s, 6H). 13C N MR
(100 MHz, CDCl3): d 181.9, 168.4, 167.6, 167.1, 158.5,
157.6, 157.3, 148.5, 144.4, 142.6, 137.0, 135.7, 133.6,
133.0, 129.6, 126.5, 124.6, 120.1, 118.5, 115.6, 92.2, 87.1
(d, J=7 Hz), 85.6, 82.4, 81.4, 80.2, 69.3 (d, J=2 Hz),
67.7 (d, J=5 Hz), 64.6 (d, J=5 Hz), 50.9, 48.0, 46.0,
40.8, 40.0, 38.5, 37.0, 28.9, 28.8, 27.9, 27.5, 25.7, 20.2 (d,
J=5 Hz), 19.5, 14.6. 31P NMR (121 MHz, CDCl3):
delta; À2.3,À2.4. IR (film): 3420, 2925, 1655, 1540,
1465, 1365, 1250, 1155 cmÀ1. MS (FAB): m/e 1187
([M+H]+).
1
modified GMP 1 as a yellow oil. H NMR (500 MHz,
D2O): d 8.88 (s, 1H), 8.29 (d, J=1.3 Hz, 1H), 7.93 (d,
J=8.1 Hz, 1H), 7.74 (d, J=8.1 Hz, 1H), 6.70 (d,
J=15.3 Hz, 1H), 6.62 (d, J=15.3 Hz, 1H), 5.87 (d,
J=4.1 Hz, 1H), 5.10 (t, J=5.4 Hz, 2H), 4.70 (t, J=4.7
Hz, 1H), 4.39 (t, J=5.1 Hz, 1H), 4.31 (s, 1H), 4.17 (s,
2H), 3.35–3.27 (m, 8H), 3.25 (t, J=5.7 Hz, 2H), 3.09 (t,
J=7.6 Hz, 2H), 1.90 (app quint, J=7.4 Hz, 2H), 1.59
(br s, 4H). 13C NMR (125 MHz, D2O): d 167.4, 167.2,
166.6, 163.0 (q, J=36 Hz), 155.5, 154.9, 149.7, 146.1,
137.3 (d, J=8.9 Hz), 136.4, 134.4, 133.0, 132.8, 132.1,
128.9, 123.9, 116 .5 (q, J=290 Hz), 108.5, 90.4, 84.7 (d,
J=8.1 Hz), 73.9, 70.0, 65.4 (d, J=5.2 Hz), 64.5, 46.0,
44.6, 40.1, 39.7, 36.7, 35.8. 26.0, 25.9, 25.8. IR (film):
3554–2593, 3307, 3079, 2941, 1675, 1639 cmÀ1. MS (ESI):
m/e for C31H45N11O13P [M+H]+, calcd 810, found 810.
Guanosine-50-monophosphate-P-[4-(4-{3-[3-(2-aminoethyl-
amino)-propylcarbamoyl]-2-methyl-acryloylamino}-butyl-
carbamoyl)-2-nitro-benzylic ester] (2) (as a mixture of
diasteromers). Modified GMP 23b (40 mg, 0.034 mmol)
was deprotected as described above and the product
purified by MPLC (MeCN/0.15% aqueous TFA, 8:92)
to give 6 mg (21%) of fumaramide modified GMP 2 as a
1
yellow oil. H NMR (400 MHz, CDCl3): d 8.88 (br. s,
N2-Isobutyryl-20,30-isopropyliden-guanosine-50-monophos-
phate-P-(2-cyanethylester)-P-{4-[4-(3-{3-[BOC-(2-BOC-
aminoethyl)-amino]-propylcarbamoyl}-propionylamino)-
butylcarbamoyl]-2-nitro-benzylic ester} (23d) (as a mix-
ture of diasteromers). Succinic acid 22d (73 mg, 0.175
mmol) was activated and coupled to modified GMP 12
as described above to give 52 mg (39%) of modified
GMP 23d as a colorless oil. 1H NMR (400 MHz,
CDCl3): d 8.42 (m, 1H), 7.98 (s, 1H), 7.94 (m, 1H), 7.60
(m, 1H), 6.02 (m, 1H), 4.98 (m, 2H), 4.77 (m, 1H), 4.50
(m, 1H), 4.38 (m, 1H), 4.24 (m, 2H), 3.35 (m, 2H), 3.21
(m, 4H), 3.14 (m, 4H), 3.04 (m, 2H), 2.82 (m, 1H), 2.72
1H), 8.32 (s, 1H), 7.95 (d, J=8 Hz, 1H), 7.75 (d, J=8
Hz, 1H), 6.28 (s, 1H), 5.88 (d, J=4 Hz, 1H), 5.10 (app t,
J=5 Hz, 2H), 4.71 (app t, J=5 Hz, 1H), 4.38 (app t,
J=5 Hz, 1H), 4.31 (m, 1H), 4.15 (app t, J=3 Hz, 2H),
3.41 (t, J=7 Hz, 2H), 3.33 (m, 4H), 3.27 (m, 4H), 3.08
(t, J=8 Hz, 2H), 2.02 (s, 3H), 1.88 (app quint, J=7 Hz,
2H), 1.59 (m, 4H). 13C NMR (100 MHz, CDCl3): d
173.8, 171.4, 157.7, 148.3, 145.5, 139.7 (d, J=9 Hz),
136.7, 135.1, 131.2, 127.4, 126.2, 120.4, 92.6, 87.0 (d,
J=8 Hz), 76.0, 72.3, 67.7 (d, J=5 Hz), 66.7 (d, J=3
Hz), 53.0, 48.3, 46.8, 42.4, 42.0, 38.6, 38.0, 28.3, 28.2,
16.6. 31P NMR (121 MHz, CDCl3): d À0.8. IR (film):

A. Eisenfuhr et al. / Bioorg. Med. Chem. 11 (2003) 235–249
¨
247
3420, 1680, 1640, 1535, 1205 cmÀ1. MS (FAB): m/e 824
CDCl3) d 176.4, 166.2, 158.6, 80.2, 63.4, 61.7, 57.0, 41.1,
40.5, 36.9, 29.8, 29.5, 28.8, 26.8. IR (film): 3300, 2935,
1695, 1650, 1535, 1265, 1175 cmÀ1. HRMS (FAB): m/e
for C17H31N4O4S [M+H]+, calcd 387.2066, found
387.2066.
([M+H]+), 846 ([M+Na]+).
Guanosine-50-monophosphate-P-[4-(4-{3-[3-(2-aminoethyl-
amino)-propylcarbamoyl]-3-methyl-acryloylamino}-butyl-
carbamoyl)-2-nitro-benzylic ester] (3) (as a mixture of
diasteromers). Modifed GMP 23c (58 mg, 0.049 mmol)
was deprotected as described above and the product
purified by MPLC (MeCN/0.15% aqueous TFA, 8:92)
to give 9 mg (22%) of fumaramide modified GMP 3 as a
N-[2-(D-Biotinylamino)-ethyl]-(N-BOC-S-trityl-L-cy-
steine)-amide (25). A solution of N-BOC-S-trityl-l-
cysteine (428 mg, 0.92 mmol), NHS (117 mg,1.02 mmol)
and DCC (210 mg, 1.02 mmol) in CH2Cl2 (2 mL) was
stirred for 30 min at room temperature and then filtered
and concentrated in vacuo. The residue was recrys-
tallized from iPrOH to give the NHS-ester. A solution
of protected amine 24 (238 mg, 0.62 mmol) and tri-
fluoroacetic acid (0.71 mL, 9.24 mmol) in CH2Cl2 (6
mL) was stirred for 1 h at room temperature and then
concentrated in vacuo. The residue was redissolved in
saturated aqueous NaHCO3 (8 mL) and a solution of
the NHS-ester in CH3CN(6 mL) was added. The mix-
ture was stirred for 10 h and then extracted with EtOAc
(3ꢀ30 mL). The organic layers were dried over MgSO4
and concentrated in vacuo. The residue was purified by
column chromatography on silica gel (CH2Cl2/MeOH,
90:10) to give 379 mg (84%) of biotin modified cysteine
25 as a white solid. 1H NMR (500 MHz, CDCl3): d 7.39
(dd, J=7.5, 1.2 Hz, 6H), 7.28 (t, J=7.3 Hz, 6H), 7.21 (t,
J=7.2 Hz, 3H), 7.05 (br s, 2H), 6.93 (br s, 1H), 6.19 (br
s, 1H), 5.23 (br s, 1H), 4.42 (dd, J=7.6, 4.9 Hz, 1H),
4.24 (dd, J=7.5, 4.6 Hz, 1H), 3.90 (app q, J=5.3 Hz,
1H), 3.39–3.32 (m, 2H), 3.27–3.19 (m, 2H), 3.11–3.07
(m, 1H), 2.82 (dd, J=12.8, 4.7 Hz, 1H), 2.68 (d, J=12.7
Hz, 1H), 2.61 (dd, J=12.6, 7.1 Hz, 1H), 2.53 (dd,
J=12.6, 5.6 Hz, 1H), 2.25 (br s, 1H), 2.08 (t, J=7.2 Hz,
2H), 1.75–1.53 (m, 4H), 1.40 (s, 9H), 1.40–1.27 (m, 1H);
13C NMR (125 MHz, CDCl3): d 173.9, 171.4, 164.5,
155.4, 144.7, 144.3, 129.5, 128.0, 127.9, 126.9, 80.1, 67.1,
61.7, 60.3, 55.7, 53.7, 40.4, 39.6, 39.1, 35.7, 34.1, 28.3,
28.1, 28.0, 25.3. IR (CHCl3): 3520–3170, 3305, 1690
cmÀ1. HRMS (FAB): m/e for C39H50N5O5S2 [M+H]+,
calcd 732.3253, found 732.3252.
1
yellow oil. H NMR (400 MHz, CDCl3): d 8.79 (br. s,
1H), 8.47 (s, 1H), 8.08 (d, J=8 Hz, 1H), 7.87 (d, J=7
Hz, 1H), 6.52 (s, 1H), 5.97 (d, J=5 Hz, 1H), 5.23 (app t,
J=6 Hz, 2H), 4.84 (m, 1H), 4.52 (app t, J=5 Hz), 4.43
(m, 1H), 4.28 (m, 2H), 3.47 (m, 6H), 3.38 (m, 4H), 3.20
(t, J=8 Hz, 2H), 2.09 (s, 3H), 2.02 (m, 2H), 1.73 (m,
4H). 13C NMR (100 MHz, CDCl3): d 174.2, 174.0,
171.0, 159.3, 148.4, 148.1, 144.7, 143.4, 140.0 (d, J=9
Hz), 136.6, 135.1, 131.1, 130.0, 128.8, 126.1, 120.5, 91.9,
86.9 (d, J=8 Hz), 75.8, 72.5, 67.9 (d, J=5 Hz), 66.7 (d,
J=2 Hz), 53.0, 48.3, 46.8, 42.4, 41.6, 39.0, 38.0, 28.4,
28.2, 16.4. 31P NMR (121 MHz, CDCl3): d À0.8. IR
(film): 3425, 1685, 1640, 1540, 1205 cmÀ1. MS (FAB):
m/e 824 ([M+H]+), 846 ([M+Na]+).
Guanosine-50-monophosphate-P-[4-(4-{3-[3-(2-aminoethyl-
amino) - propylcarbamoyl] - propionylamino} - butylcarba-
moyl)-2-nitro-benzylic ester] (4) (as
a mixture of
diasteromers). Modifed GMP 23d (52 mg, 0.044 mmol)
was deprotected as described above and the product
purified by MPLC (MeCN/0.15% aqueous TFA, 8:92)
to give 7 mg (20%) of fumaramide modified GMP 4 as a
1
yellow oil. H NMR (400 MHz, CDCl3): d 8.70 (br. s,
1H), 8.38 (s, 1H), 7.98 (d, J=8 Hz, 1H), 7.80 (d, J=8
Hz, 1H), 5.87 (m, 1H), 5.12 (app t, J=6 Hz, 2H), 4.73
(m, 1H), 4.42 (app t, J=5 Hz), 4.32 (m, 1H), 4.18 (m,
2H), 3.38 (m, 2H), 3.34 (m, 4H), 3.17 (m, 4H), 3.05 (t,
J=8 Hz, 2H), 2.45 (m, 4H), 1.82 (app quint, J=8 Hz,
2H), 1.57 (m, 4H). 13C NMR (100 MHz, CDCl3): d
177.9, 177.2, 170.0, 157.7, 148.3, 146.0, 139.8 (d, J=7
Hz), 136.7, 135.1, 132.0, 131.2, 126.2, 120.4, 92.6, 87.0
(d, J=9 Hz), 76.0, 72.3, 67.6 (d, J=5 Hz), 66.7 (d, J=4
Hz), 53.0, 48.2, 46.8, 42.4, 41.6, 38.5, 38.0, 33.7, 33.6,
28.5, 28.3. 31P NMR (121 MHz, CDCl3): d À0.8. IR
(film): 3315, 1675, 1640, 1535, 1385, 1200 cmÀ1. MS
(ESI): m/e 812 ([M+H]+), 834 ([M+Na]+).
N-[2-(D-Biotinylamino)-ethyl]-(N-BOC-S-methyl-L-cys-
teine)-amide (26). N-BOC-S-methyl-l-cysteine (217 mg,
0.92 mmol) was coupled to protected amine 24 as
described above to afford 343 mg (99%) of biotin mod-
ified S-methylcysteine 26 as a white solid. 1H N MR
(400 MHz, CDCl3): d 4.43 (dd, J=7.8, 4.8 Hz, 1H), 4.24
(dd, J=7.8, 4.3 Hz, 1H), 4.08 (dd, J=7.5, 6.0 Hz, 2H),
3.21 (m, 4H), 3.13 (ddd, J=8.4, 5.5, 4.9 Hz, 1H), 2.85
(dd, J=12.8, 4.9 Hz, 1H), 2.78 (dd, J=13.8, 5.7, 1H),
2.61 (d, J=12.8 Hz, 1H), 2.59 (d, J=13.8 Hz, 1H),
2.11 (t, J=7.3 Hz, 2H), 2.03 (s, 3H), 1.69–1.45 (m,
2H), 1.55 (quint, J=6.8 Hz, 2H), 1.36 (s, 9H), 1.19 (s,
2H). 13C NMR (100 MHz, CDCl3): ꢀ 176.5, 174.2,
166.0, 157.9, 81.0, 63.4, 61.8, 57.0, 55.6, 41.1, 40.1,
37.4, 36.9, 29.8, 29.5, 28.8, 26.8, 15.8. IR (film): 3650–
N-BOC- N-[2-(D -Biotinylamino)-ethyl] -amin (24). A
solution of d-biotin (1000 mg, 4.09 mmol), N-BOC-1,2-
diaminoethane 18 (787 mg, 4.91 mmol) and EDC (1020
mg, 5.32 mmol) in MeOH (10 mL) and CH3CN(30 mL)
was stirred for 5 h at room temperature and then con-
centrated in vacuo. The residue was resuspended in
MeOH and filtrated over Celite. The filtrate was con-
centrated in vacuo, and the residue was purified by col-
umn chromatography on silica gel (CH2Cl2/MeOH,
90:10) to give 882 mg (56%) of protected amine 24 as a
3200, 1685 cmÀ1
.
1
white solid. H NMR (400 MHz, CDCl3): d 4.93 (dd,
J=7.9, 4.4 Hz, 1H), 4.21 (dd, J=7.9, 4.4 Hz, 1H), 3.13
(q, J=6.1 Hz, 2H), 3.10 (dd, J=5.4, 4.9 Hz, 1H), 3.04
(t, J=5.8 Hz, 2H), 2.82 (dd, J=12.7, 5 Hz, 1H), 2.60 (d,
J=12.8 Hz, 1H), 2.10 (t, J=7.5 Hz, 2H), 1.68–1.45 (m,
4H), 1.33 (s, 9H), 1.19 (s, 2H). 13C NMR: (100 MHz,
N-[2-(D-Biotinylamino)-ethyl]-L-cysteine-amide (5). A
solution of protected cysteine 25 (260 mg, 0.329 mmol),
triethylsilane (77 mg, 0.658 mmol), and trifluoroacetic
acid (5 mL, 65.8 mmol) in CH2Cl2 (5 mL) was stirred
under a nitrogen atmosphere. After 20 min, the reaction

248
A. Eisenfuhr et al. / Bioorg. Med. Chem. 11 (2003) 235–249
¨
mixture was concentrated in vacuo and redissolved in a
mixture of H2O (5 mL) and CH2Cl2 (5 mL). The layers
were separated and the aqueous layer was washed with
CH2Cl2 (2ꢀ15 mL). The aqueous layer was con-
centrated in vacuo to afford 160 mg (97%) of biotiny-
References and Notes
1. Jenne, A.; Famulok, M. Chem. Biol. 1998, 5, 23.
2. Suga, H.; Lohse, P. A.; Szostak, J. W. J. Am. Chem. Soc.
1998, 120, 1151.
3. Wilson, C.; Szostak, J. W. Nature 1995, 374, 777.
4. Wecker, M.; Smith, D.; Gold, L. RNA 1996, 2, 982.
5. Ekland, E. H.; Szostak, J. W.; Bartel, D. P. Science 1995,
269, 364.
6. Zhang, B. L.; Cech, T. R. Nature 1997, 390, 96.
7. Dai, X. C.; Demesmaeker, A.; Joyce, G. F. Science 1995,
267, 237.
8. Tsang, J.; Joyce, G. F. Biochemistry 1994, 33, 5966.
9. Tsang, J.; Joyce, G. F. J. Mol. Biol. 1996, 262, 31.
10. Seelig, B.; Jaschke, A. Chem. Biol. 1999, 6, 167.
11. Tarasow, T. M.; Tarasow, S. L.; Eaton, B. E. Nature
1997, 389, 54.
1
lated cysteine 5 as a colorless oil. H NMR (500 MHz,
D2O): d 4.42 (app t, J=6.0 Hz, 1H), 4.24 (dd, J=6.5,
4.5 Hz, 1H), 3.97 (app t, J=5.6 Hz, 1H), 3.29 (app t,
J=6.9 Hz, 1H), 3.17–3.12 (m, 4H), 2.90–2.60 (m, 4H),
2.08 (t, J=7.1 Hz, 2H), 1.56–1.35 (m, 4H), 1.21 (q,
J=6.9 Hz, 2H). 13C NMR (125 MHz, D2O): d 177.4,
168.3, 165.5, 62.5, 60.7, 55.7, 54.9, 40.0, 39.3, 38.7, 35.8,
28.3, 28.0, 25.4, 25.2. IR (film): 3410–3200, 3295, 1675
cmÀ1. HRMS (FAB): m/e for C15H28N5O3S2 [M+H]+,
calcd 390.1633, found 390.1630.
12. Prudent, J. R.; Uno, T.; Schultz, P. G. Science 1994, 264,
1924.
13. Sengle, G.; Eisenfuhr, A.; Arora, P. S.; Nowick, J. S.;
Famulok, M. Chem. Biol. 2001, 8, 459.
14. Sibi, M. P.; Manyem, S. Tetrahedron 2000, 56, 8033.
15. Shimizu, S.; Shiozaki, S.; Ohshiro, T.; Yamada, H. Eur.
J. Biochem. 1984, 141, 385.
16. Requena, J. R.; Fu, M. X.; Ahmed, M. U.; Jenkins, A. J.;
Lyons, T. J.; Baynes, J. W.; Thorpe, S. R. Biochem. J. 1997,
322, 317.
17. Sugumaran, M.; Semensi, V.; Dali, H.; Nellaiappan, K.
Arch. Insect Biochem. Physiol. 1991, 16, 31.
18. Carreras, C. W.; Santi, D. V. Annu. Rev. Biochem. 1995,
64, 721.
19. Montfort, W. R.; Perry, K. M.; Fauman, E. B.; Finer-
Moore, J. S.; Maley, G. F.; Hardy, L.; Maley, F.; Stroud,
R. M. Biochemistry 1990, 29, 6964.
N-[2-(D -Biotinylamino)-ethyl]-(S-methyl-L -cysteine)-
amide (6). Methylcysteine 26 (50 mg, 0.1 mmol) was
deprotected as described above to give 40 mg (98%) of
biotinylated S-methylcysteine 6 as a colorless oil. 1H
NMR (400 MHz, D2O): d 4.45 (dd, J=7.9, 4.4 Hz, 1H),
4.27 (dd, J=7.9, 4.4 Hz, 1H), 3.99 (dd, J=7.9, 5.7 Hz,
1H), 3.32 (td, J=8.9, 4.1 Hz, 1H), 3.22–3.15 (m, 4H),
2.90–2.60 (m, 4H), 2.11 (t, J=7.3 Hz, 2H), 2.00 (s, 3H),
1.62–1.37 (m, 2H), 1.51 (q, J=7.3 Hz, 2H), 1.26 (q,
J=7.5 Hz, 2H). 13C NMR (100 MHz, D2O): d 179.8,
171.2, 167.2, 64.7, 62.9, 57.9, 54.4, 42.3, 40.8, 38.1, 37.0,
30.6, 30.3, 27.7, 17.2. IR (film): 3600–3100, 1680 cmÀ1
.
MS (FAB): m/e 404 ([M+H]+), 426 ([M+Na]+).
Kinetic study of background Michael reactions by
affinity chromatography
20. Walsh, C. T. Enzymatic Reaction Mechanisms; W.H.
Freeman: San Francisco, CA, 1979.
The 32P-labeled RNA library derivatized with one of the
compounds 1–4 was dissolved in aqueous buffer (50
mM K-MOPS, 200 mM NaCl, pH 7.4) to a final con-
centration of 1 mM, and heated to 90 ꢁC for 5 min to
denature the RNA. The solution was cooled to room
temperature for 20 min, and MgCl2 followed by 5 or 6
was added to obtain a final concentration of 5 mM
MgCl2 and 30–50 mM of 5/6. The reaction mixture was
incubated at 25 ꢁC. After 0–6 h, the reaction was quen-
ched by precipitating RNA with ethanol. The pellet was
redissolved in water, and the unreacted biotinylated
cysteine was removed by filtering the solution through a
G50 column. The isolated RNA was precipitated with
ethanol dissolved in streptavidin agarose coupling buf-
fer (25 mM Na2HPO4/NaH2PO4, 150 mM NaCl, pH
6.9). The solution was incubated for 45 min with
appropriate amounts of streptavidin-derivatized agar-
ose. The mixture containing agarose and RNA was fil-
tered, and the agarose was washed with 4 column
volumes of denaturating buffer (8 M urea, 5 mM
EDTA, adjusted with K-MOPS to pH 7.4) and water.
The amount of RNA linked to the matrix was measured
by scintillation counting.
21. Pitulle, C.; Kleineidam, R. G.; Sproat, B.; Krupp, G. Gene
1992, 112, 101.
22. Logsdon, N.; Lee, C. G.; Harper, J. W. Anal. Biochem.
1992, 205, 36.
23. Martin, C. T.; Coleman, J. E. Biochemistry 1989, 28, 2760.
24. Hausch, F.; Jaschke, A. Bioconjug. Chem. 1997, 8, 885.
25. Rodebaugh, R.; FraserReid, B.; Geysen, H. M. Tetra-
hedron Lett. 1997, 38, 7653.
26. Barany, G.; Albericio, F. J. Am. Chem. Soc. 1985, 107,
4936.
27. Ordoukhanian, P.; Taylor, J. S. J. Am. Chem. Soc. 1995,
117, 9570.
28. Givens, R. S.; Kueper, L. W. Chem. Rev. 1993, 93, 55.
29. Greenberg, M. M. Tetrahedron Lett. 1993, 34, 251.
30. Hausch, F.; Jaschke, A. Tetrahedron 2001, 57, 1261.
31. Beaucage, S. L.; Iyer, R. P. Tetrahedron 1993, 49, 1925.
32. Caruthers, M. H. Acc. Chem. Res. 1991, 24, 274.
33. Goodchild J. Bioconjug. Chem. 1990, 1, 165.
34. Scaringe, S. A.; Wincott, F. E.; Caruthers, M. H. J. Am.
Chem. Soc. 1998, 120, 11820.
35. Ti, G. S.; Gaffney, B. L.; Jones, R. A. J. Am. Chem. Soc.
1982, 104, 1316.
36. McLaughlin, L. W.; Piel, N.; Hellmann, T. Synthesis 1985,
322.
37. Berteina, S.; De Mesmaeker, A. Synlett 1998, 1227.
38. Pons, J. F.; Fauchere, J. L.; Lamaty, F.; Molla, A.;
Lazaro, R. Eur. J. Org. Chem. 1998, 853.
39. Muller, D.; Zeltser, I.; Bitan, G.; Gilon, C. J. Org. Chem.
1997, 62, 411.
Acknowledgements
40. Kneeland, D. M.; Ariga, K.; Lynch, V. M.; Huang, C. Y.;
Anslyn, E. V. J. Am. Chem. Soc. 1993, 115, 10042.
41. Shih, T. L.; Ruiz-Sanchez, J.; Mrozik, H. Tetrahedron
Lett. 1987, 28, 6015.
This work was supported by a grant to M.F. from the
Deutsche Forschungsgemeinschaft and in part by
NATO grants SRG 921372 and SRG 961195 to J.S.N.

A. Eisenfuhr et al. / Bioorg. Med. Chem. 11 (2003) 235–249
¨
249
42. Smith, A. B.; Fukui, M.; Vaccaro, H. A.; Empfield, J. R.
J. Am. Chem. Soc. 1991, 113, 2071.
43. Oppolzer, W.; Kingma, A. J. Helv. Chim. Acta 1989, 72,
1337.
46. Ainsworth, P. J.; Craig, D.; Reader, J. C.; Slawin, A. M. Z.;
White, A. J. P.; Williams, D. J. Tetrahedron 1995, 51, 11601.
47. Tolbert, T. J.; Wong, C. H. J. Am. Chem. Soc. 2000, 122,
5421.
44. Jew, S.; Kim, H.; Song, S.; Jang, J.; Park, H. Heterocycles
1998, 47, 153.
48. Pearson, D. A.; Blanchette, M.; Baker, M. L.; Guindon,
C. A. Tetrahedron Lett. 1989, 30, 2739.
45. Baker, R.; Rao, V. B.; Ravenscroft, P. D.; Swain, C. J.
Synthesis 1983, 572.
49. Milligan, J. F.; Groebe, D. R.; Witherell, G. W.; Uhlen-
beck, O. C. Nucleic Acids Res. 1987, 15, 8783.