Synthesis of isotopically labeled puromycin derivatives for kinetic isotope effect analysis of ribosome catalyzed peptide bond formation

Article abstract of DOI:10.1016/j.tet.2004.10.023

The mechanism by which the ribosome catalyze peptide bond formation remains controversial. Here we describe the synthesis of dinucleotides that can be used in kinetic isotope effect experiments to assess the transition state of ribosome catalyzed peptide

Full text of DOI:10.1016/j.tet.2004.10.023

Tetrahedron 60 (2004) 12101–12112
Synthesis of isotopically labeled puromycin derivatives for
kinetic isotope effect analysis of ribosome catalyzed
peptide bond formation
Kensuke Okuda,a Amy C. Seilab and Scott A. Strobela,c,
*
aDepartment of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA
bDepartment of Genetics, Yale University, New Haven, CT 06520-8114, USA
cDepartment of Chemistry, Yale University, New Haven, CT 06520-8114, USA
Received 22 March 2004; revised 8 October 2004; accepted 8 October 2004
Available online 28 October 2004
Abstract—The mechanism by which the ribosome catalyze peptide bond formation remains controversial. Here we describe the synthesis of
dinucleotides that can be used in kinetic isotope effect experiments to assess the transition state of ribosome catalyzed peptide bond
formation. These substrates are the isotopically labeled dinucleotide cytidylyl-(30-50)-30-amino-30-deoxy-30-L-phenylalanyl-N6,N6-
dimethyladenosine (Cm6ANPhe-NH2) and cytidylyl-(30-50)-30-amino-30-deoxy-30-(L-2-hydroxy-3-phenylpropionyl)-N6,N6-dimethyl-
adenosine (Cm6ANPhe-OH). These substrates are active in peptide bond formation and can be used to measure kinetic isotope effects in
ribosome catalyzed protein synthesis.
q 2004 Elsevier Ltd. All rights reserved.
1. Introduction
as a general base to deprotonate the nucleophile, or
deprotonation may cause a conformation change in the
ribosome that enhances the reaction rate. Several chemical
pathways have been proposed for the reaction, all of which
invoke a tetrahedral transition state.6–8 They differ with
regard to the point along the reaction coordinate at which
the nucleophile is deprotonated and the leaving group is
protonated.
The ribosome is the ribonucleoprotein-complex responsible
for protein synthesis in all cells. The structure of the 50 S
ribosomal subunit was determined recently by X-ray
crystallography.1,2 The structure revealed that the peptidyl
transferase center of the ribosome resides solely within the
23 S ribosomal RNA (rRNA), that is, the ribosome is an
RNA enzyme or ribozyme.1 The ribosome catalyses
peptide bond formation between two substrates, the
aminoacyl tRNA bound in the ribosomal A site, and the
peptidyl tRNA bound in the ribosomal P site. The reaction
involves nucleophilic attack of the A-site tRNA a-amino
group on the carbonyl-ester linking the nascent peptide to
the P-site tRNA. The products of this reaction are a
deacylated P-site tRNA and an A-site tRNA linked to the
nascent peptide, which has been extended by one amino
acid.
Transition state stabilization is a fundamental strategy
employed by enzymes to promote chemical reactions.
Characterization of the transition state would provide
information essential to understanding how the ribosome
enhances the peptidyl transferase reaction; but the transient
nature of the free energy maxima between products and
reactants makes such investigations extremely challenging.
One approach to transition state analysis is the measurement
of kinetic isotope effects.9–12 Reactive functional groups in
the substrates are isotopically labeled and the relative
reaction rates of the heavy and light substrates determined
by enzyme kinetics. Kinetic isotope effects arise from
changes in vibrational states between the ground state and
the transition state in a chemical reaction.13 The magnitude
and direction of these effects provides the information
needed to successfully predict the reaction transition state,
including reactions catalyzed by enzymes such as the
ribosome.13–18
The ribosome enhances the rate of peptide bond formation
by more than 100,000 fold.3 The pH dependence of the
reaction suggests that an ionizable functional group in the
ribosome is catalytically important.3–5 This group could act
Keywords: Ribosome; Puromycin; Kinetic isotope effect; Solid-phase
synthesis.
* Corresponding author. Tel.: C1 203 432 9772; fax: C1 203 432 5767;
e-mail: strobel@csb.yale.edu
0040–4020/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2004.10.023

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K. Okuda et al. / Tetrahedron 60 (2004) 12101–12112
Scheme 1. Schematic of the modified fragment assay.
In order to measure a kinetic isotope effect, the isotope
sensitive step must be rate-limiting. Ribosomal protein
synthesis involves a multi-step process aided by GTP
dependent protein factors. It is known from kinetic studies
that accommodation of the amino-acyl tRNA is rate-
limiting during the elongation step of protein synthesis.19
Therefore, an assay that does not include accommodation is
necessary for kinetic isotope effect measurements. The 50 S
ribosomal subunit alone can catalyze peptide bond for-
mation using two small synthetic substrates that mimic the
A-site and P-site tRNAs (Scheme 1).20 In this modified
fragment assay, cytidylyl-(30-50)-puromycin (CPmn) func-
tions in place of the A-site tRNA, while cytidylyl-(30-50)-
cytidylyl-(30-50)-30-(biotinyl-3-aminocaproyl-L-phenyl-
alanyl)adenosine (CCApcb) serves in place of the P-site
tRNA. Like the standard peptidyl transferase reaction, the
a-amino group of the A-site substrate attacks the ester bond
in the P-site substrate, to produce a new peptide bond.21
This simplified reaction is ideal for measuring kinetic
isotope effects for two reasons: (i) the small substrates can
be chemically synthesized to include specific isotopic
substitutions, and (ii) the reaction is mechanistically
simplified to the reversible binding of two small substrates,
a chemical reaction, and product release.
In order to measure kinetic isotope effects on the ribosome,
it was necessary to synthesize substrates with heavy atom
substitutions on the reactive functional groups. Here we
describe the synthesis of CPmn derivatives with 15N
substitution at the a-amino group and remote positions (1,
1* and 1***), and derivatives in which the a-amino is
substituted with an 18O a-hydroxyl group (2 and 2*)
(Fig. 1). We also demonstrate that these molecules serve as
ribosome substrates.
2. Results and discussion
2.1. Synthesis of Cm6ANPhe-NH2
The CPmn analogs Cm6ANPhe-NH2 (1 and 1*),22 where N
is either 14N or 15N, were prepared by solid phase synthesis.
Figure 1. Synthetic targets Cm6ANPhe-NH2 and Cm6ANPhe-OH and their isotopic derivatives.

K. Okuda et al. / Tetrahedron 60 (2004) 12101–12112
12103
For this purpose, the appropriately protected puromycin
analogs were attached to solid support and coupled to
cytidine as shown in Scheme 2.
additional remote substitutions in the 15N labeled substrate.
This is reminiscent of the remote labeling method that used
radioactive isotopes as markers for heavy atom substitutions
in kinetic isotope effect studies.27 Because the cytidine
moiety does not participate directly in the chemical
reaction, heavy isotope substitution should not affect the
reaction, nor should it show an isotope effect. Any affects
can be controlled for by characterization of a dinucleotide
containing only the remote labels. We selected
[3-15N,4-15NH2]cytidine28 (9**) for this purpose. The
preparation of the [3-15N,4-15NH2]cytidine phosphorami-
dite (8**) was accomplished as follows (Scheme 3).29
The 30-amino group of puromycin aminonucleoside was
selectively derivatized with commercially available N-(9-
fluorenylmethoxycarbonyl)-L-phenylalanine (3 and 3*)
using 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide
hydrochloride (EDCI). Further derivatization of the 50 and
20 hydroxyl groups was performed using p,p0-dimethoxy-
trityl chloride (DMTrCl) and succinic anhydride, respect-
ively. This transformation was accomplished using methods
analogous to those reported in the literature23,24 to yield
suitably protected compounds 6 and 6*. These were
attached to LCAA-polystyrene support and the loading
quantified spectroscopically by the DMTr cation method.25
The solid support (7 and 7*) was coupled to the protected
cytidine phosphoramidite, 4-acetyl-50-O-[benzhydroxybis-
(trimethylsiloxy)silyl]-20-O-[bis(2-acetoxyethoxy)methyl]-
cytidine-30-(methyl-N,N-diisopropyl)phosphoramidite (8).
The dinucleotide was cleaved from the solid support and
deprotected as described previously.26 Subsequent purifi-
cation by reversed-phase HPLC yielded 1 and 1*,
respectively.
Compound 9** was prepared from uridine in five steps.28
The 15N at position N3 was introduced by the rearrangement
reaction caused by the attack of 15NH3 at the C4 of 20,30,50-
tri-O-acetyl-3-nitrouridine. The 15NH2 substitution at the
N4 position was introduced from the 4-(tetrazol-1-yl)
intermediate by the 15NH3 replacement reaction. Compound
9** was treated with 1,3-dichloro-1,1,3,3-tetraisopropyl-
disiloxane (TIPDSCl2) in pyridine to simultaneously protect
the 30 and 50 hydroxyl groups to produce 10**. The N4
amino group was acetyl protected (11**), and the 20
hydroxyl group protected with tris(2-acetoxyethoxy)ortho-
formate in the presence of 4-tert-butyldimethylsiloxy-3-
penten-2-one in CH2Cl2 under reflux to give 12**. The 50-30
silyl protecting group was removed by fluoride (13**), and
the 50 hydroxyl group was again protected by benzhydroxy-
bis(trimethylsiloxy)silyl chloride (BzhCl) and diisopropyl-
amine (14**). Finally the 30 hydroxyl group was derivatized
with methyl tetraisopropyl phosphorodiamidite and 1H-
tetrazole to yield target phosphoramidite (8**). Coupling of
8** to 7* and deprotection was performed by standard
2.2. Synthesis of [3-15N,4-15NH2]Cm6ANPhe-15NH2
The mass difference between Cm6ANPhe-14NH2 (1) and
Cm6ANPhe-15NH2 (1*) is only 1 Da. Whole molecule mass
spectroscopic analysis of the two isotopes would be
complicated by the significant size of the MC1 peaks that
arises from natural 13C abundance. To increase the mass
difference between the two isotopes, we incorporated two
Scheme 2. Synthesis of Cm6ANPhe-NH2 (1, 1*). Reagents and conditions: (a) puromycin aminonucleoside, EDCI, N-hydroxysuccinimide, DMF, 0 8C to room
temperature, 74% (4), 77% (4*); (b) DMTrCl, triethylamine, pyridine, room temperature, 86% (5), 89% (5*); (c) succinic anhydride, DMAP, pyridine, room
temperature, 65% (6), 60% (6*); (d) LCAA-polystyrene, EDCI, DMAP, triethylamine, pyridine, room temperature, 109 mmol/g (7), 110 mmol/g (7*); (e) as
described in Ref. 26.

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K. Okuda et al. / Tetrahedron 60 (2004) 12101–12112
Scheme 3. Synthesis of [3-15N,4-15NH2]Cm6ANPhe-15NH2 (1***). Reagents and conditions: (a) TIPDSCl2, pyridine, 0 8C to room temperature, 78%;
(b) acetic anhydride, DMF, room temperature, 100%; (c) tris(2-acetoxyethoxy)orthoformate, pyridnium p-toluenesulfonate, 4-tert-butyldimethylsiloxy-3-
penten-2-one, CH2Cl2, reflux, 85%; (d) N,N,N0,N0-tetramethylethylenediamine, 48% HF aq, acetonitrile, room temperature, 99%; (e) BzhCl, diisopropylamine,
CH2Cl2, 0 8C, 96%; (f) methyl tetraisopropyl phosphorodiamidite, 1H-tetrazole, CH2Cl2, 0 8C, 89%; (g) as described in Ref. 26.
methods of solid phase oligoribonucleotide synthesis.26
substrate via a new ester linkage. Pmn-OH has been used to
deconvolute the contribution of the subtrate pKa from that of
the ribosome.3
Purification
by
reverse-phase
HPLC
yielded
[3-15N,4-15NH2]-Cm6ANPhe-15NH2 (1***), which has
three 15N labels.
In order to explore kinetic isotope effects on this
transesterification reaction we set out to prepare the
CPmn-OH derivatives (2 and 2*) with 18O substitution at
the nucleophilic hydroxyl. Preparation of Cm6ANPhe-OH
followed a synthetic scheme analogous to that described
above (Scheme 4). The 2 hydroxyl group of D-methyl 2-
hydroxy-3-phenylpropionate30 (15) was converted to the
triflate by trifluoromethanesulfonic anhydride in the
2.3. Synthesis of Cm6ANPhe-OH
The A-site substrate hydroxy-puromycin (Pmn-OH) has
proven useful for investigating the peptidyl transferase
reaction, because it is a substrate that does not have a neutral
pKa.5 Pmn-OH participates in a transesterification reaction
in which the peptide in the P-site is transferred to the A-site
Scheme 4. Synthesis of Cm6ANPhe-OH (2 and 2*); Reagents and conditions: (a) trifluoromethanesulfonic anhydride, pyridine, CH2Cl2, 0 8C to room
temperature, then acetic 18O2-acid, K2CO3, acetonitrile, room temperature, 83%; (b) 5 N KOH aq, MeOH, room temperature, 54%; (c) acetic anhydride,
pyridine, room temperature, 100% (19), 95% (19*); (d) puromycin aminonucleoside, EDCI, N-hydroxysuccinimide, DMF, 0 8C to room temperature, 73%
(20), 71% (20*); (e) DMTrCl, triethylamine, pyridine, room temperature, 89% (21), 89% (21*); (f) succinic anhydride, DMAP, pyridine, room temperature,
57% (22), 62% (22*); (g) LCAA-polystyrene, EDCI, DMAP, triethylamine, pyridine, room temperature, 70 mmol/g (23), 103 mmol/g (23*); (h) as described in
Ref. 26.

K. Okuda et al. / Tetrahedron 60 (2004) 12101–12112
12105
presence of pyridine and CH2Cl2.31 The crude intermediate
(16) was directly transformed to the 18O-labeled acetoxy
compound (17**) with inverted stereochemistry using
acetic 18O2-acid and K2CO3 in acetonitrile. The L-[2-
18OH]-3-phenyllactic acid (18*) was obtained by alkaline
hydrolysis. L-3-Phenyllactic acid (18 and 18*) was con-
verted to acetate by acetic anhydride in pyridine to give 19
and 19*, respectively. As above, the 30-amino group of
puromycin aminonucleoside was0 selectively derivatized
with 19 or 19* using EDCI, the 5 and 20 hydroxyl groups
protected with DMTrCl and succinic anhydride, respect-
ively, and the resulting compounds attached to solid
support. Solid-phase coupling of the cytidine phosphor-
amidite (8), followed by deprotection and HPLC purifi-
cation yielded substrates, 2 and 2*. Unlike the 15N
containing compound 1*, the difference of two mass units
between 16O and 18O is sufficient for kinetic isotope effect
measurements by whole molecule mass spectrometry.
radiolabeled with the enzyme polynucleotide kinase.
Production of the P-site product CCA was monitored by
gel electrophoresis (Fig. 2a). A new band of increased
mobility increased as a function of time. The aminolysis
and alcoholysis reactions proceed at a rate O100-fold and
O10-fold above the background rate of hydrolysis,
respectively. The A-site product was visualized by a peak
of increased retention on the HPLC and the assignment
confirmed by mass spectroscopic analysis (Fig. 2b, data
shown for 1). Similar results were obtained for compounds
1*, 1***. These data indicate that 1, 1*, 1***, 2 and 2*
serve as acceptors in the peptidyl transferase reaction. These
substrates will make it possible to perform kinetic isotope
experiments on the ribosome.
3. Experimental
3.1. General
2.4. Ribosome 50 S subunit reaction assay
All reactions were monitored by thin-layer chromatography
(TLC) using E. Merck silica gel 60 F254 precoated plates
(0.25 mm). Chromatography was performed with the
indicated solvent system using Silicycle 0.040–0.060 mm
silica gel. NMR spectra were measured on Brucker Avance
We tested if 1 and 2 serve as acceptors in the ribosomal
peptidyl transferase reaction. Each substrate was incubated
with 50 S ribosomal subunits in the presence of the P-site
substrate CCApcb (Dharmacon Inc.)21 that had been 50-32P
Figure 2. a. Demonstration of the peptidyl transferase reaction of 1 (A) and 2 (B) with 32pCCApcb catalyzed by the 50 S ribosome subunit. The top bands are
32pCCApcb and the bottom bands are the deacylated product 32pCCA. b. HPLC trace of the reaction of 1 with CCApcb at an intermediate time point. Peaks for
each of the reactants and nucleotide containing products are visible. Their observed molecule weights are indicated.

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K. Okuda et al. / Tetrahedron 60 (2004) 12101–12112
DPX-400 and Brucker Avance DPX-500 spectrometers. 1H
and 13C NMR chemical shifts were recorded relative to the
standard of tetramethylsilane, 15N NMR chemical shifts
were recorded relative to nitromethane as an external
standard, and 31P NMR chemical shifts were recorded
relative to 85% phosphoric acid as an external standard.
Mass spectra were collected on Waters Micromass LCT and
Waters Micromass ZQ mass spectrometers. Optical rotation
was performed on Perkin–Elmer Polarimeter 341.
OH), 5.98 (d, 1H, JZ0.8 Hz, H010), 50.18 (t, 1H, JZ4.2 Hz,
50-OH), 4.50–4.46 (m, 2H, H2 , H3 ), 4.42–4.37 (m, 1H,
Phe-CH), 4.18–4.08 (m, 3H, Fmoc-CH, Fmoc-CH2), 3.96
(m, 1H, H40), 3.67 (m, 1H, H50), 3.44 (br, 7H, H50, NCH3),
3.04–2.97 (m, 1H, Phe-CH2), 2.84–2.75 (m, 1H, Phe-CH2);
13C NMR (125.8 MHz, DMSO-d6/CDCl3Z3:1): 171.8,
155.6 (d, JCNZ27.5 Hz), 154.3, 151.6, 149.6, 143.7,
140.6, 140.6, 137.9, 137.7, 129.3, 127.9, 127.5, 126.9,
126.9, 126.1, 125.3, 125.1, 119.9, 119.8, 89.4, 83.4, 73.2,
65.7, 60.9, 56.0 (d, JCNZ11.3 Hz), 50.3, 46.6, 37.9 (N6,N6-
dimethyl carbon was overlapped by DMSO-d6, confirmed
by DEPT.); 15N NMR (50.7 MHz, DMSO-d6): K292.4 (d,
JHNZ94.7 Hz); ESI-MS (ESC): m/z calcd for
C36H37N615NO6 664.3, found 665.3 (MHC), 687.3 (MC
NaC); HRMS m/z calcd for C36H37N615NO6 665.2854
(MHC), found 665.2865.
Amino-derivatized polystyrene beads (Primer Support 30
HL, Amino-derivatized with a loading level of 161 mmol/g)
was purchased from Amersham Biosciences. Pyridine was
dried using Molecular Sieves. All other chemicals were
used as received from commercial suppliers. [3-15N,4-
15NH2]cytidine28 (9**) and D-methyl 3-phenyllactate30 (15)
were synthesized according to the literature procedure.
3.1.3. 30-Amino-30-deoxy-30-[N-(9-fluorenylmethoxycar-
bonyl)-L-phenylalanyl]-50-O-(p,p0-dimethoxytrityl)-
N6,N6-dimethyladenosine (5). 4 (175.0 mg, 0.264 mmol)
was dried by repeated co-evaporation with pyridine, then
dissolved in pyridine (8.0 ml). Triethylamine (0.12 ml,
0.861 mmol) and p,p0-dimethoxytrityl chloride (DMTrCl)
(283.6 mg, 0.795 mmol) were added to the solution, then
stirred at room temperature for 3 h. After addition of MeOH
(2 ml) to quench the reaction, the mixture was evaporated.
Further co-evaporation with toluene twice was followed by
column chromatography (gradient from 1% MeOH in
CH2Cl2 to 2% MeOH in CH2Cl2) to give the pure product
3.1.1. 30-Amino-30-deoxy-30-[N-(9-fluorenylmethoxycar-
bonyl)-L-phenylalanyl]-N6,N6-dimethyladenosine (4). To
the solution of puromycin aminonucleoside (Sigma)
(200.3 mg, 0.681 mmol), N-(9-fluorenylmethoxycarbonyl)-
L-phenylalanine (3, 290.4 mg, 0.750 mmol), and N-
hydroxysuccinimide (290.4 mg, 0.748 mmol) in DMF
(9.0 ml), 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide
hydrochloride (EDCI) (145.0 mg, 0.756 mmol) was added
at 0 8C. The reaction mixture was stirred at 0 8C for 1 h, then
stirred at room temperature for 24 h. After evaporation, the
oily residue was crystallized with ethyl acetate (15 ml). This
crude product was washed with ethyl acetate (30 ml), water
(15 ml), ethyl acetate (10 ml), successively to give the pure
product 4 as a colorless powder (333.9 mg, 74%). 1H NMR
(400 MHz, DMSO-d6): 8.44 (s, 1H, H8), 8.23 (s, 1H, H2),
8.17 (d, 1H, JZ7.2 Hz, 30-NH), 7.87 (d, 2H, JZ8.0 Hz,
Fmoc-aromatic), 7.64 (m, 3H, Fmoc-aromatic, Phe-NH),
7.42–7.18 (m, 9H, Fmoc-aromatic, Phe-0aromatic), 6.10 (d,
1H, JZ40.4 Hz, 20-OH), 6.00 (s, 1H, H1 ), 05.19 (t, 1H, JZ
4.0 Hz, 5 -OH), 4.52–4.47 (m, 2H, H20, H3 ), 4.42–4.37 (m,
1H, Phe-CH), 4.17–4.08 (m, 3H, Fmoc-CH, Fmoc-CH2),
3.94 (m, 1H, H40), 3.72–3.65 (m, 1H, H50), 3.49 (br, 7H,
H50, NCH3), 3.04–2.97 (m, 1H, Phe-CH2), 2.85–2.72 (m,
1H, Phe-CH2); 13C NMR (125.8 MHz, DMSO-d6/CDCl3Z
3:1): 171.8, 155.7, 154.3, 151.6, 149.6, 143.7, 140.7, 140.6,
137.9, 137.7, 129.3, 127.9, 127.5, 126.9, 126.9, 126.1,
125.2, 125.1, 119.8, 119.8, 89.4, 83.5, 73.2, 65.7, 60.9, 56.1,
50.4, 46.6, 37.9 (N6,N6-dimethyl carbon was overlapped by
DMSO-d6, confirmed by DEPT.); ESI-MS (ESC): m/z calcd
for C36H37N7O6 663.3, found 664.5 (MHC); HRMS m/z
calcd for C36H37N7O6 664.2883 (MHC), found 664.2871.
1
(5) as a white foam (219.7 mg, 86%). H NMR (400 MHz,
CDCl3): 8.22 (s, 1H, H8), 7.99 (s, 1H, H2), 7.75 (d, 2H, JZ
7.2 Hz, Fmoc-aromatic), 7.54 (t, 2H, JZ7.0 Hz, Fmoc-
aromatic), 7.40–7.11 (m, 18H, Fmoc-aromatic, DMTr-
aromatic, Phe-aromatic),0 6.76 (d, 4H, JZ8.4 Hz, DMTr-
aromatic), 6.40 (br, 1H, 3 -NH), 5.58 (br, 1H, H10), 5.45 (br,
1H, JZ6.4 Hz, Phe-NH), 4.67 (br, 1H, JZ4.4 Hz, H20),
4.41(br, 1H, H30), 4.36 (br.d, 3H, JZ6.4 Hz, Phe-CH,
Fmoc-CH2), 4.19 (m, 2H, H40, Fmoc-CH), 3.76 (s, 6H,
DMTr-OCH3), 3.54 (br, 6H, NCH3), 3.44 (d, 1H, JZ8.8 Hz,
H50), 3.32 (dd, 1H, JZ10.6, 3.4 Hz, H50), 3.10 (br, 1H, Phe-
CH2), 2.91 (br, 1H, Phe-CH2); 13C NMR (125.8 MHz,
CDCl3): 171.2, 158.5, 155.9, 154.9, 151.7, 149.1, 144.4,
143.8, 143.6, 141.3, 141.3, 136.4, 136.0, 135.7, 135.6,
130.1, 130.0, 129.2, 128.8, 128.2, 127.8, 127.8, 127.7,
127.1, 127.0, 126.8, 125.1, 120.6, 120.0, 119.9, 113.1, 91.3,
86.5, 84.1, 74.5, 67.1, 63.6, 56.4, 55.2, 52.6, 47.1, 39.2, 38.7
(br); ESI-MS (ESC): m/z calcd for C57H55N7O8 965.4,
found 988.6 (MCNaC); HRMS m/z calcd for C57H55N7O8
966.4190 (MHC), found 966.4185.
3.1.2. 30-Amino-30-deoxy-30-[[2-15NH]-N-(9-fluorenyl-
methoxycarbonyl)-L-phenylalanyl]-N6,N6-dimethyl-
adenosine (4*). The product (4*) was obtained from the
coupling of puromycin aminonucleoside (250.7 mg,
0.852 mmol) and [2-15NH]-N-(9-fluorenylmethoxycar-
bonyl)-L-phenylalanine (3*, 398.9 mg, 0.938 mmol) as
described in Section 3.1.1. 4* was obtained as a colorless
powder (435.0 mg, 77%). 1H NMR (400 MHz, DMSO-d6):
8.44 (s, 1H, H8), 8.23 (s, 1H, H2), 8.20 (d, 1H, JZ7.6 Hz,
30-NH), 7.87 (d, 2H, JZ7.6 Hz, Fmoc-aromatic), 7.64 (t,
3.1.4. 30-Amino-30-deoxy-30-[[2-15NH]-N-(9-fluorenyl-
methoxycarbonyl)-L-phenylalanyl]-50-O-(p,p0-dimethoxy-
trityl)-N6,N6-dimethyladenosine (5*). The product (5*)
was obtained as a white powder from 4* (199.4 mg,
0.300 mmol) using the method described in Section 3.1.3
(257.1 mg, 89%). 1H NMR (400 MHz, CDCl3): 8.22 (s, 1H,
H8), 7.98 (s, 1H, H2), 7.74 (d, 2H, JZ7.6 Hz, Fmoc-
aromatic), 7.53 (t, 2H, JZ7.6 Hz, Fmoc-aromatic), 7.40–
7.10 (m, 18H, Fmoc-aromatic, DMTr-aromatic, Phe-
aromatic),0 6.76 (d, 4H, JZ8.4 Hz, DMTr-aromatic), 6.42
(br, 1H, 3 -NH), 5.59–5.35 (m, 2H, H10, Phe-NH), 4.66 (t,
1H, JZ5.0 Hz, H20), 4.41 (m., 1H, H30), 4.35 (br.d, 3H, JZ
6.0 Hz, Phe-CH, Fmoc-CH2), 4.18 (m, 2H, H40, Fmoc-CH),
2H, JZ7.8 Hz, Fmoc-aromatic), 7.63 (dd, 1H, JHN
Z
92.4 Hz, JHHZ8.8 Hz, Phe-NH), 7.42–7.16 (m, 9H,
Fmoc-aromatic, Phe-aromatic), 6.10 (d, 1H, JZ4.0 Hz, 20-

K. Okuda et al. / Tetrahedron 60 (2004) 12101–12112
12107
3.76 (s, 6H, DMTr-OCH3), 3.54 (br, 6H, NCH3), 3.43 (dd,
1H, JZ10.8, 2.0 Hz, H50), 3.29 (dd, 1H, JZ11.0, 3.4 Hz,
H50), 3.08 (br, 1H, Phe-CH2), 2.89 (br, 1H, Phe-CH2); 13C
NMR (125.8 MHz, CDCl3): 171.2, 158.5, 154.9, 151.7,
149.2, 144.4, 143.7 (d, JCNZ13.1 Hz), 141.3, 136.4, 136.0,
135.7, 135.6, 130.1, 129.2, 128.8, 128.2, 127.8, 127.7,
127.1, 126.8, 125.1, 120.6, 120.0, 120.0, 113.1, 91.3, 86.5,
84.2, 74.5, 67.2, 63.7, 56.4 (d, JCNZ12.7 Hz), 55.2, 52.7,
47.1, 39.2, 38.7 (br); 15N NMR (50.7 MHz, CDCl3):
K294.1 (d, JHNZ91.6 Hz); ESI-MS (ESC): m/z calcd for
C57H55N615NO8 966.4, found 989.5 (MCNaC); HRMS m/z
calcd for C57H55N615NO8 967.4160 (MHC), found
967.4136.
(m, 1H, Phe-CH), 4.34–4.20 (m, 2H, Fmoc-CH2), 4.11 (t,
1H, JZ7.2 Hz, Fmoc-CH), 3.83 (br, 1H, H40), 3.74 (s, 3H,
DMTr-OCH3), 3.73 (s, 3H, DMTr-OCH3), 3.51 (br, 6H,
NCH3), 3.40–3.29 (m, 2H, H50), 2.85–2.58 (m, 6H, Phe-
CH2, succinic ester-CH2); 13C NMR (125.8 MHz, CDCl3):
175.2, 171.3, 170.9, 158.5, 156.5 (d, JCNZ27.8 Hz), 154.9,
152.6, 149.8, 144.4, 143.5, 141.2, 141.2, 136.3, 136.1,
135.6, 135.6, 130.2, 129.3, 128.5, 128.4, 127.8, 127.8,
127.2, 126.9, 126.9, 125.1, 125.0, 120.4, 120.0, 113.1, 87.5,
86.5, 82.3, 75.8, 67.5, 62.6, 55.1 (2C), 49.4, 46.9, 40.2, 38.6
(br), 29.8, 29.7; 15N NMR (50.7 MHz, CDCl3): K291.5 (d,
JHNZ91.4 Hz); ESI-MS (ESC): m/z calcd for
C61H59N615NO11 1,066.4, found 1,067.6 (MHC); HRMS
m/z calcd for C61H59N615NO11 1067.4320 (MHC), found
1067.4310.
3.1.5. 30-Amino-30-deoxy-30-[N-(9-fluorenylmethoxy-
carbonyl)-L-phenylalanyl]-50-(p,p0-dimethoxytrityl)-
N6,N6-dimethyladenosine 20-O-succinate (6). 5
(167.7 mg, 0.173 mmol) was dried by repeated co-evapor-
ation with pyridine and dissolved in pyridine (1.4 ml).
Succinic anhydride (52.1 mg, 0.521 mmol) and 4-
(dimethylamino)pyridine (DMAP) (11.0 mg, 0.090 mmol)
was added to the solution and stirred at room temperature
for 24 h. The mixture was evaporated, followed by addition
of 0.1 M NaHCO3 aq (25 ml) and extraction with CH2Cl2
(25 ml!7). The combined organic phase was evaporated.
Further co-evaporation with toluene twice was followed by
column chromatography (gradient from 2% MeOH in
CH2Cl2 to 10% MeOH in CH2Cl2) to give the pure product
3.1.7. 30-Amino-30-deoxy-30-[N-(9-fluorenylmethoxy-
carbonyl)-L-phenylalanyl]-50-O-(p,p0-dimethoxytrityl)-
N6,N6-dimethyladenosine 20-O-(LCAA-polystyrene)suc-
cinate (7). Amino-derivatized polystyrene support
(850.0 mg) was suspended in pyridine (8.5 ml) with 6
(180.0 mg, 0.169 mmol), DMAP (10.7 mg, 0.0876 mmol),
EDCI (327.2 mg, 1.71 mmol), and triethylamine (70 ml,
0.498 mmol). The mixture was rocked gently for 26 h. The
support was filtered and washed successively with pyridine
(8.5 ml), MeOH (17 ml), and CH2Cl2 (25.5 ml). The support
was capped to acetylate unreacted amino residues by
suspension in 0.5 M acetic anhydride, 0.5 M pyridine, 1 M
N-methyl imidazole solution in THF (8.5 ml) and rocked for
2.5 h. The support was filtered and successively washed
with MeOH (17 ml) and CH2Cl2 (25.5 ml) to yield the
product (7). The nucleoside loading was 109 mmol/g.
1
(6) as a white foam (120.7 mg, 65%). H NMR (400 MHz,
CDCl3): 8.30 (s, 1H, H8), 7.94 (s, 1H, H2), 7.75 (d, 2H, JZ
7.6 Hz, Fmoc-aromatic), 7.50 (t, 2H, JZ7.8 Hz, Fmoc-
aromatic), 7.45–7.00 (m, 18H, Fmoc-aromatic, DMTr-
aromatic, Phe-aromatic), 6.80 (d, 4H, JZ7.6 Hz, DMTr-
aromatic), 6.46 (d, 1H, JZ9.2 Hz, 30-NH), 6.10 (d, 1H, JZ
1.2 Hz, H10), 5.79 (dd, 1H, JZ5.4, 1.8 Hz, H20), 5.72 (d,
1H, JZ9.6 Hz, Phe-NH), 5.19 (m, 1H, H30), 4.90 (m, 1H,
Phe-CH), 4.33–4.20 (m, 2H, Fmoc-CH2), 4.11 (t, 1H, JZ
7.0 Hz, Fmoc-CH), 3.83 (br, 1H, H40), 3.74 (s, 3H, DMTr-
OCH3), 3.73 (s, 3H, DMTr-OCH3), 3.51 (br, 6H, NCH3),
3.40–3.28 (m, 2H, H50), 2.85–2.58 (m, 6H, Phe-CH2,
succinic ester-CH2); 13C NMR (125.8 MHz, CDCl3): 175.4
(br), 171.2, 171.0, 158.5, 156.5, 154.9, 152.5, 149.8, 144.4,
143.5, 141.2, 141.2, 136.2, 136.1, 135.6, 130.2, 129.3,
128.5, 128.4, 127.8, 127.8, 127.1, 126.9, 126.9, 125.1,
125.0, 120.4, 120.0, 113.1, 87.6, 86.4, 82.3, 75.7, 67.6, 62.6,
55.2, 49.5, 46.9, 40.2, 38.6 (br), 29.8; ESI-MS (ESC): m/z
calcd for C61H59N7O11 1,065.4, found 1,066.6 (MHC);
HRMS m/z calcd for C61H59N7O11 1066.4350 (MHC),
found 1066.4330.
3.1.8. 30-Amino-30-deoxy-30-[[2-15NH]-N-(9-fluorenyl-
methoxycarbonyl)-L-phenylalanyl]-50-O-(p,p0-dimethoxy-
trityl)-N6,N6-dimethyladenosine 20-O-(LCAA-poly-
styrene)succinate (7*). The product (7*) was obtained
from 6* (180.0 mg, 0.169 mmol) using the method
described in Section 3.1.7. The nucleoside loading was
110 mmol/g.
3.1.9. Cytidylyl-(30-50)-30-amino-30-deoxy-30-L-phenyl-
alanyl-N6,N6-dimethyladenosine (1). The coupling of
4-acetyl-50-O-[benzhydroxybis(trimethylsiloxy)silyl]-20-O-
[bis(2-acetoxyethoxy)methyl]cyti0dine-30-(methyl-N,N-di-
isopropyl)phosphoramidite (8) to 5 hydroxyl group of 7 and
successive deprotection was performed as described pre-
viously.26 After lyophilization, the deprotected product was
purified over a C-18 column with 0.1 M triethylammonium
acetate buffer (pH 6.5) and acetonitrile (from 10:0 to 6:4).
3.1.6. 30-Amino-30-deoxy-30-[[2-15NH]-N-(9-fluorenyl-
methoxycarbonyl)-L-phenylalanyl]-50-O-(p,p0-dimethoxy-
trityl)-N6,N6-dimethyladenosine 20-O-succinate (6*).
The product (6*) was obtained as a white foam from 5*
(242.2 mg, 0.250 mmol) using the method described in
Section 3.1.5 (161.2 mg, 60%). 1H NMR (400 MHz,
CDCl3): 8.30 (s, 1H, H8), 7.94 (s, 1H, H2), 7.75 (d, 2H,
JZ7.6 Hz, Fmoc-aromatic), 7.50 (t, 2H, JZ7.6 Hz, Fmoc-
aromatic), 7.44–7.01 (m, 18H, Fmoc-aromatic, DMTr-
aromatic, Phe-aromatic), 6.80 (d, 4H, JZ8.4 Hz, DMTr-
aro0matic), 6.47 (d, 1H, JZ7.2 Hz, 30-NH), 6.09 (s, 1H,
Lyophilization resulted in the purified product 1. H NMR
1
(500 MHz, D2O): 8.19 (s, 1H, H8-puromycin), 8.04 (s, 1H,
H2-puromycin), 7.54 (d, 1H, JZ7.5 Hz, H6-cytosine),
7.35–7.15 (m, 5H, Phe-aromatic), 5.93 (br, 1H, H10-
puromycin)0, 5.60 (d, 1H, JZ7.5 Hz, H5-cytosine), 5.42
(br, 1H, H1 -cytosine), 4.32 (m, 1H, Phe-CH), 4.22 (dd, 1H,
JZ13.0, 8.1 Hz, H50-cytosine), 4.09 (m, 1H), 4.03 (m, 2H),
3.85 (m, 1H), 3.71 (dd, 1H, JZ12.8, 2.1 Hz, H50-
puromycin), 3.63 (dd, 1H, JZ13.2, 4.2 Hz, H50-puro-
mycin), 3.61 (m, 1H), 3.25 (br, 6H, NCH3), 3.12–2.93 (m,
4H); ESI-MS (ESC): m/z calcd for C30H39N10O11P 746.3,
found 747.0 (MHC); HRMS m/z calcd for C30H39N10O11P
769.2437 (MCNaC), found 769.2433.
H1 ), 5.78 (d, 1H, JZ5.6 Hz, H20), 5.73 (dd, 1H, JHN
Z
91.6 Hz, JHHZ8.4 Hz, Phe-NH), 5.18 (m, 1H, H30), 4.90

12108
K. Okuda et al. / Tetrahedron 60 (2004) 12101–12112
3.1.10. Cytidylyl-(30-50)-30-amino-30-deoxy-30-([2-15NH2]-
L-phenylalanyl)-N6,N6-dimethyladenosine (1*). The
product (1*) was obtained from 7* using the method
described in Section 3.1.9; ESI-MS (ESC): m/z calcd for
C30H39N915NO11P 747.3, found 748.0 (MHC); HRMS m/z
calcd for C30H39N915NO11P 748.2586 (MHC), found
748.2585.
disiloxanediyl)cytidine (12**). To a stirred solution of
11** (2.65 g, 5.00 mmol) in CH2Cl2 were added tris(2-
acetoxyethoxy)orthoformate tris (ACE)orthoformate
(4.51 g, 14.0 mmol), pyridinium p-toluenesulfonate
(251 mg, 1.00 mmol) and 4-tert-butyldimethylsiloxy)-3-
penten-2-one (2.13 ml, 9.01 mmol). The reaction mixture
was refluxed under Ar atmosphere for 10 h, cooled to the
room temperature, and quenched by the addition of
N,N,N 0,N 0-tetramethylethylenediamine (0.38 ml,
2.50 mmol). The mixture was subjected directly to a
chromatography column (gradient from 50% ethyl acetate
in n-hexane to 100% ethyl acetate) to afford the pure
3.1.11. [3-15N,4-15NH2]-30,50-O-(1,1,3,3-Tetraisopropyl-
1,3-disiloxanediyl)cytidine (10**). [3-15N,4-15NH2]-
cytidine28 (9**, 1.63 g, 6.65 mmol) was dried by co-
evaporation twice with pyridine (25 ml) and then dissolved
in pyridine (55 ml). 1,3-Dichloro-1,1,3,3-tetraisopropyl-
disiloxane (2.34 ml, 7.31 mmol) was added to the mixture
dropwise at 0 8C over 1 min. The reaction mixture was
stirred for 1 h at 0 8C, then stirred at room temperature for
13 h. After evaporation, addition of water (100 ml) was
followed by extraction using CH2Cl2 (100 ml!3). The
organic phases were combined, dried over MgSO4, then
evaporated followed by co-evaporation twice with toluene
(40 ml). The pure product 10** was obtained by column
chromatography (gradient from 4% MeOH in CH2Cl2 1to
10% MeOH in CH2Cl2) as a white solid (2.53 g, 78%). H
NMR (400 MHz, CDCl3/CD3ODZ3:1): 7.96 (d, 1H, JZ
7.2 Hz, H6), 5.81 (d, 1H, JZ7.2 0Hz, H5), 5.68 (s, 1H, H10),
4.29–4.17 (m, 3H, H30, H40, H5 ), 4.09 (d, 1H, JZ3.6 Hz,
H20), 4.02 (dd, 1H, JZ13.2, 2.0 Hz, H50), 1.13–0.96 (m,
28H, 4!CH(CH3)2); 13C NMR (125.8 MHz, CDCl3/
CD3ODZ3:1): 166.3 (dd, JCNZ21.4, 4.2 Hz), 156.5 (d,
JCNZ5.0 Hz), 141.0, 94.7, 91.8, 81.8, 75.3, 68.4, 60.2, 17.6,
17.6, 17.4, 17.4, 17.1, 17.1, 17.0, 16.9, 13.7, 13.3, 13.2,
12.7; 15N NMR (50.7 MHz, CDCl3/CD3ODZ3:1): K176.9
(d, JHNZ4.5 Hz), K292.5 (br); ESI-MS (ESC): m/z calcd
for C21H39N15N2O6Si2 487.2, found 488.5 (MHC); HRMS
m/z calcd for C21H39N15N2O6Si2 488.2396 (MHC), found
488.2414.
1
product 12** as a white foamy powder (3.19 g, 85%). H
NMR (400 MHz, CDCl3): 9.94 (d, 1H, JHNZ89.6 Hz, NH),
8.30 (d, 1H, JZ7.2 Hz, H6), 7.44 (d, 1H, JZ7.2 Hz, H5),
5.85 (s, 1H, H10), 5.84 (s, 1H, ACE-CH), 4.32–4.17 (m, 8H,
2!ACE-CH2, H20, H30, H40, H50), 4.02–3.84 (m, 5H, 2!
ACE-CH2, H50), 2.28 (d, 3H, JHNZ1.2 Hz, N4-COCH3),
2.07 (s, 3H, ACE-COCH3), 2.06 (s, 3H, ACE-COCH3),
1.11–0.91 (m, 28H, 4!CH(CH3)2); 13C NMR (125.8 MHz,
CDCl3): 170.9, 170.9, 170.8 (d, JCNZ12.3 Hz), 163.1 (dd,
JCNZ18.3, 7.1 Hz), 154.7 (dd, JCNZ6.5, 5.2 Hz), 144.2,
111.9, 96.4, 89.9, 82.0, 77.3, 67.5, 63.5, 63.3, 61.7, 61.2,
59.3, 24.9 (d, JCNZ9.1 Hz), 20.9, 20.8, 17.5, 17.4, 17.3,
17.3, 17.1, 16.9, 16.9, 16.8, 13.4, 13.1, 12.9, 12.6; 15N NMR
(50.7 MHz, CDCl3): K151.0 (d, JHNZ5.9 Hz), K233.3
(dd, JHNZ89.7, 6.1 Hz); ESI-MS (ESC): m/z calcd for
C32H55N15N2O13Si2 747.3, found 770.5 (MCNaC); HRMS
m/z calcd for C32H55N15N2O13Si2 770.3112 (MCNaC),
found 770.3100.
3.1.14. [3-15N,4-15NH2]-4-Acetyl-20-O-[bis(2-acetoxy-
ethoxy)methyl]cytidine (13**). To the mixture of aceto-
nitrile (42 ml) and N,N,N0,N0-tetramethylethylenediamine
(3.13 ml, 4.14 mmol) 48% HF aq (0.53 ml, 14.6 mmol) was
added dropwise over 1 min at 0 8C. 12** (3.10 g,
4.14 mmol) was added to the solution, then stirred at room
temperature for 4 h. The reaction mixture was evaporated.
Pure product 13** was obtained by column chromatography
(gradient from 5% MeOH in CH2Cl2 to 10% MeOH in
CH2Cl2) as a white foamy powder (2.08 g, 99%). 1H NMR
(400 MHz, CDCl3): 9.67 (d, 1H, JHNZ89.6 Hz, NH), 8.30
(d, 1H, JZ7.6 Hz, H6), 7.43 (d, 1H, JZ7.2 Hz, H5), 5.80
(d, 1H, JZ2.4 Hz, H10),0 5.62 (s, 1H, ACE-CH), 4.63 (d0d,
1H, JZ5.4, 3.0 Hz, H2 ), 4.39 (q, 1H, JZ5.7 Hz, H3 ),
4.26–4.02 (m, 7H, 2!ACE-CH2, H40, H50, 50-OH), 3.89–
3.78 (m, 5H, 2!ACE-CH2, H50), 3.54 (d, 1H, JZ6.0 Hz,
30-OH), 2.26 (d, 3H, JHNZ0.8 Hz, N4-COCH3), 2.07 (s, 3H,
ACE-COCH3), 2.06 (s, 3H, ACE-COCH3); 13C NMR
3.1.12. [3-15N,4-15NH2]-4-Acetyl-30,50-O-(1,1,3,3-tetra-
isopropyl-1,3-disiloxanediyl)cytidine (11**). To a mixture
of 10** (2.47 g, 5.06 mmol) and DMF (50 ml) was added
acetic anhydride (2.39 ml, 25.3 mmol). The reaction
mixture became clear soon after acetic anhydride addition.
The solution was stirred for additional 6 h, evaporated, and
co-evaporated twice with MeOH (30 ml). The pure product
11** was obtained by column chromatography (3% MeOH
1
in CH2Cl2) as a white foamy powder (2.72 g, 100%). H
NMR (400 MHz, CDCl3): 10.11 (d, 1H, JHNZ90.4 Hz,
NH), 8.21 (d, 1H, JZ7.2 Hz, H6), 7.44 (d, 1H,0 JZ7.2 Hz,
H5), 5.82 (s, 1H, H10), 4.29–4.20 (m, 4H, H2 , H30, H40,
H50), 4.01 (dd, 1H, JZ13.6, 2.8 Hz, H50), 2.30 (d, 3H,
JHNZ1.6 Hz, N4-COCH3), 1.11–0.90 (m, 28H, 4!
CH(CH3)2); 13C NMR (125.8 MHz, CDCl3): 171.2 (d,
JCNZ11.2 Hz), 163.2 (dd, JCNZ18.1, 6.9 Hz), 155.0 (dd,
JCNZ6.9, 4.8 Hz), 144.4, 96.6, 91.5, 82.0, 75.2, 68.6, 60.0,
24.9 (d, JCNZ9.1 Hz), 17.5, 17.4, 17.3, 17.3, 17.0, 17.0,
16.9, 16.8, 13.4, 13.0, 12.9, 12.5; 15N NMR (50.7 MHz,
CDCl3): K152.0, K233.1 (d, JHNZ86.0 Hz); ESI-MS
(ESC): m/z calcd for C23H41N15N2O7Si2 529.2, found
530.5 (MHC); HRMS m/z calcd for C23H41N15N2O7Si2
530.2502 (MHC), found 530.2511.
(100.6 MHz, CDCl3): 171.1 (2C), 171.0 (d, JCN
13.3 Hz), 162.9 (dd, JCNZ18.4, 6.9 Hz), 155.5 (dd, JCN
Z
Z
5.1, 4.5 Hz), 146.6, 112.7, 96.9, 92.1, 85.1, 76.8, 68.4, 63.1,
63.0, 62.9, 62.8, 60.6, 24.9 (d, JCNZ9.2 Hz), 20.9, 20.9; 15
N
NMR (50.7 MHz, CDCl3): K149.5 (d, JHNZ4.7 Hz),
K233.2 (dd, JHNZ90.2, 6.6 Hz); ESI-MS (ESC): m/z
calcd for C20H29N15N2O12 505.2, found 506.4 (MHC);
HRMS m/z calcd for C20H29N15N2O12 506.1770 (MHC),
found 506.1793.
3.1.15. [3-15N,4-15NH2]-4-Acetyl-50-O-[benzhydroxy-
bis(trimethylsiloxy)silyl]-20-O-[bis(2-acetoxyethoxy)-
methyl]cytidine (14**). A solution of 13** (2.02 g,
4.00 mmol) and diisopropylamine (0.56 ml, 4.00 mmol)
3.1.13. [3-15N,4-15NH2]-4-Acetyl-20-O-[bis(2-acetoxy-
ethoxy)methyl]-30,50-O-(1,1,3,3-tetraisopropyl-1,3-

K. Okuda et al. / Tetrahedron 60 (2004) 12101–12112
12109
in CH2Cl2 (22 ml) was cooled to 0 8C. Benzhydroxy-
bis(trimethylsiloxy)silyl chloride (BzhCl) (3.47 g,
8.17 mmol) was dissolved in CH2Cl2 (13.5 ml), then
diisopropylamine (0.56 ml, 4.00 mmol) was added drop-
wise to this mixture at 0 8C. This mixture was added
dropwise to the previous solution over 2 h at 0 8C. Upon
completion, the addition of 5% NaHCO3 aq (50 ml)
quenched the reaction. The mixture was separated, then
the aqueous phase was extracted with CH2Cl2 (30 ml!3).
The organic layers were combined, washed with brine, dried
over MgSO4, and evaporated. The pure product 14** was
obtained by column chromatography (gradient from 50%
ethyl acetate in n-hexane to 10% MeOH in CH2Cl2) as a
(161.9 MHz, CDCl3): 151.8, 151.0; ESI-MS (ESC): m/z
calcd for C46H73N215N2O16PSi3 1054.4, found 1055.6
(MHC).
3.1.17. [3-15N, 4-15NH2]Cytidylyl-(30-50)-30-amino-30-
deoxy-30-([2-15NH]-phenylalanyl)-N6,N6-dimethyladeno-
sine (1***). The product (1***) was obtained from 9* and
8** using the method described in Section 3.1.9; ESI-MS
(ESC): m/z calcd for C30H39N715N3O11P 749.2, found 750.3
(MHC); HRMS m/z calcd for C30H39N715N3O11P 750.2526
(MHC), found 750.2537.
3.1.18. D-Methyl 2-trifluoromethanesulfonyl-3-phenyl-
lactate (16). Trifluoromethanesulfonic anhydride (2.4 ml,
14.3 mmol) was added dropwise over 2 min to a solution of
D-methyl 3-phenyllactate30 (15, 500.0 mg, 2.77 mmol) in
CH2Cl2 (25 ml) and pyridine (1.2 ml) at 0 8C. After addition
of CH2Cl2 (20 ml), the mixture was stirred at room
temperature for 2 h. The mixture was further diluted by
addition of CH2Cl2 (60 ml), and successively washed with
0.8 N NaHCO3 aq (120 ml), 1 N HCl aq (120 ml), and brine
(120 ml). The organic layer was dried over MgSO4, and
evaporated to give crude product (16) as an oil (0.70 g). 1H
NMR (400 MHz, CDCl3): 7.37–7.20 (m, 5H, phenyl), 5.25
(dd, 1H, JZ8.8, 4.4 Hz, CH), 3.84 (s, 3H, CH3), 3.35 (dd,
1H, JZ14.8, 4.4 Hz, CH2), 3.21 (dd, 1H, JZ14.8, 8.4 Hz,
CH2).
1
colorless oil (3.43 g, 96%). H NMR (400 MHz, CDCl3):
8.98 (d, 1H, JHNZ89.6 Hz, NH), 8.33 (d, 1H, JZ7.6 Hz,
H6), 7.36–7.25 (m, 9H, H5, phenyl), 7.22–7.17 (m, 2H,
phenyl), 5.94 (s, 1H, Bzh-CH), 5.93 (s, 1H, H10), 5.71 (s,
1H, ACE-CH), 4.28–4.21 (m, 5H, 2!ACE-CH2, H20),
4.11–3.98 (m, 03H, H30, H40, H50), 3.93–3.83(m, 5H, 2!
ACE-CH2, H5 ), 2.85 (d, 1H, JZ8.0 Hz, 30-OH), 2.25 (d,
3H, JHNZ1.6 Hz, N4-COCH3), 2.08 (s, 3H, ACE-COCH3),
2.05 (s, 3H, ACE-COCH3), 0.09 (s, 9H, Si(CH3)3), 0.08 (s,
9H, Si(CH3)3); 13C NMR (125.8 MHz, CDCl3): 170.9,
170.9, 169.9 (d, JCNZ11.2 Hz), 162.5 (dd, JCNZ19.1,
7.5 Hz), 155.0 (dd, JCNZ6.9, 5.3 Hz), 144.7, 143.9 (d,
JCNZ5.4 Hz), 128.3, 127.3, 126.4, 126.3, 113.0, 96.2, 89.6,
83.9, 78.6, 67.1, 63.1, 63.1, 63.0, 60.5, 25.0 (d, JCN
Z
9.4 Hz), 20.9, 20.8, 1.5 (6C); 15N NMR (50.7 MHz, CDCl3):
K149.8 (d, JHNZ6.9 Hz), K233.8 (dd, JHNZ88.6, 7.2 Hz);
ESI-MS (ESC): m/z calcd for C39H57N15N2O15Si3 893.3,
found 894.6 (MHC); HRMS m/z calcd for C39H57N15N2-
O15Si3 916.2936 (MCNaC), found 916.2926.
3.1.19. L-Methyl [2-18O,10-18O]-2-acetoxy-3-phenyl-
lactate (17**). Crude 16 (0.70 g) was dissolved in an acetic
18O2-acid (200 mg, 3.12 mmol) solution in acetonitrile
(15 ml). K2CO3 (402.6 mg, 2.914 mmol) was added and
the mixture was stirred at room temperature for 29 h.
CH2Cl2 (100 ml) was added and the organic phase was
washed successively with 0.8 N NaHCO3 aq (100 ml), 1 N
HCl aq (100 ml), and brine (120 ml). The organic layer was
dried over MgSO4, and evaporated to give product (17**) as
an oil (521.6 mg, 83%). 1H NMR (400 MHz, CDCl3): 7.33–
7.21 (m, 5H, phenyl), 5.22 (dd, 1H, JZ8.8, 4.4 Hz, CH),
3.73 (s, 3H, CO2CH3), 3.17 (dd, 1H, JZ14.2, 4.6 Hz, CH2),
3.09 (dd, 1H, JZ14.0, 8.8 Hz, CH2), 2.08 (s, 3H, acetoxy-
CH3); ESI-MS (ESC): m/z calcd for C12H14O218O2 226.1,
found 249.2 (MCNaC).
3.1.16. [3-15N,4-15NH2]-4-Acetyl-50-O-[benzhydroxy-
bis(trimethylsiloxy)silyl]-20-O-[bis(2-acetoxyethoxy)-
methyl]cytidine-30-(methyl-N,N-diisopropyl)phosphor-
amidite (8**). To a stirred solution of 14** (3.38 g,
3.78 mmol) in CH2Cl2 (25 ml) were added methyl tetra-
isopropyl phosphorodiamidite (3.05 ml, 10.6 mmol) and
1H-tetrazole (291 mg, 4.16 mmol). The resulting solution
was stirred for 19 h. The reaction was quenched by the
addition of 5% NaHCO3 aq (50 ml). The organic phase was
separated, and the aqueous phase was extracted by CH2Cl2
(30 ml!4). The organic fractions were combined, dried
over MgSO4, and evaporated. The pure product 8** was
obtained by column chromatography (gradient from 30%
CH2Cl2: 60% n-hexane: 10% triethylamine to 50% CH2Cl2:
40% n-hexane: 10% triethylamine) as a colorless oil (3.55 g,
89%). 1H NMR (400 MHz, CDCl3): (mixture of dia-
stereomers) 10.05 (d, 1H, JHNZ88.8 Hz, NH), 8.37 (d,
0.4H, JZ7.6 Hz, H6), 8.35 (d, 0.6H, JZ7.6 Hz, 0H6), 7.38–
7.18 (m, 0 11H, H5, phenyl), 6.02 (s, 0.4H, H1 ), 6.00 (s,
0.6H, H1 ), 5.97 (s, 0.4H, Bzh-CH), 5.96 (s, 0.6H, Bzh-CH),
5.81 (s, 0.6H, ACE-CH), 5.75 (s, 0.4H, ACE-CH), 4.31–
4.16 (m, 8H), 4.11–4.03 (m, 1H), 3.95–3.80 (m, 4H), 3.61–
3.53 (m, 2H, CH(CH3)2), 3.35 (d, 1.6H, JZ13.6 Hz, OCH3),
3.32 (d, 1.4H, JZ13.6 Hz, OCH3), 2.28 (s, 3H, N4-
COCH3), 2.05 (s, 1.5H, ACE-COCH3), 2.05 (s, 1.5H,
ACE-COCH3), 2.04 (s, 1.5H, ACE-COCH3), 2.04 (s, 1.5H,
ACE-COCH3), 1.17–1.14 (m, 12H, CH(CH3)2), 0.09 (s,
4.5H, Si(CH3)3), 0.09 (s, 4.5H, Si(CH3)3), 0.07 (s, 4.5H,
Si(CH3)3), 0.06 (s, 4.5H, Si(CH3)3); 15N NMR (50.7 MHz,
CDCl3): K151.8, K233.2 (d, JHNZ88.4 Hz); 31P NMR
3.1.20. L-[2-18OH]-3-Phenyllactic acid (18*). 17**
(496.1 mg, 2.19 mmol) was dissolved in MeOH (10 ml)
and 5 N KOH aq (10 ml), and stirred at room temperature
for 5 h. After addition of conc. HCl (10 ml), the mixture was
evaporated at 60 8C to give a white semi-solid. The crude
product was washed with ethyl acetate (40 ml!4). The
combined organic phase was filtered and evaporated.
Recrystallization from CHCl3/n-hexane gave the product
1
(18*) as needles (200.6 mg, 54%). H NMR (400 MHz,
CDCl3): 7.35–7.26 (m, 5H, phenyl), 4.53 (br.s, 1H, CH),
3.23 (d, 1H, JZ11.6 Hz, CH2), 3.01 (dd, 1H, JZ13.4,
7.0 Hz, CH2); ESI-MS (ESC): m/z calcd for C9H10O218O
168.1, found 190.9 (MCNaC); [a]D22: K27.8 (c0.18,
MeOH).
3.1.21. L-2-Acetoxy-3-phenyllactic acid.32 (19) Acetic
anhydride (0.3 ml, 3.15 mmol) was added to a L-3-
phenyllactic acid (18) (250.4 mg, 1.48 mmol) solution in
pyridine (3.0 ml), and the mixture was stirred at room

12110
K. Okuda et al. / Tetrahedron 60 (2004) 12101–12112
temperature for 1 day. The reaction was quenched by
addition of MeOH (5 ml), and the mixture was evaporated.
The resulting oil was dissolved in 0.1 N HCl aq (20 ml), and
extracted with CH2Cl2 (20 ml!3). The combined organic
phase was washed with brine (30 ml), dried over MgSO4,
and evaporated to give product (19) as a colorless oil
(313.2 mg, 100%). 1H NMR (400 MHz, CDCl3): 7.34–7.24
(m, 5H, phenyl), 5.25 (dd, 1H, JZ9.0, 4.2 Hz, CH), 3.23
(dd, 1H, JZ14.2, 3.8 Hz, CH2), 3.12 (dd, 1H, JZ14.4,
8.8 Hz, CH2), 2.08 (s, 3H, CH3); ESI-MS (ESC): m/z calcd
for C11H12O4 208.1, found 231.1 (MCNaC).
HRMS m/z calcd for C23H28N6O518O 487.2191 (MHC),
found 487.2209.
3.1.25. 30-Amino-30-deoxy-30-(L-2-acetoxy-3-phenylpro-
pionyl)-50-(p,p0-dimethoxytrityl)-N6,N6-dimethyladeno-
sine (21). The product (21) was obtained as a white foam
from 20 (242.0 mg, 0.499 mmol) using the method
described in Section 3.1.3 (350.9 mg, 89%). 1H NMR
(400 MHz, CDCl3): 8.26 (s, 1H, H8), 7.97 (s, 1H, H2), 7.35–
7.14 (m, 14H, DMTr-aromatic, Phe-aromatic), 6.77 (d, 4H,
JZ9.2 Hz, DMTr-aromatic), 6.65 (d, 1H, JZ5.2 Hz, 30-
NH), 6.01 (s, 1H, 20-OH), 5.77 (d, 1H, JZ4.0 Hz, H10), 5.32
(dd, 1H, JZ7.2, 5.6 Hz, Phe-CH), 4.65 (t, 1H, JZ5.4 Hz,
H200), 4.44 (dd, 1H, JZ11.6, 5.6 Hz, H30), 4.33 (m, 1H,
H4 ), 3.77 (s, 6H, DMTr-OCH3), 3.54 (br, 6H, NCH3), 3.47
(dd, 1H, JZ10.4, 2.4 Hz, H50), 3.37 (dd, 1H, JZ10.8,
3.6 Hz, H50), 3.17 (dd, 1H, JZ14.2, 5.4 Hz, Phe-CH2), 3.09
(dd, 1H, JZ14.2, 7.4 Hz, Phe-CH2), 2.07 (s, 3H, acetyl-
CH3); 13C NMR (125.8 MHz, CDCl3): 169.7, 169.6, 158.5,
155.0, 151.7, 149.2, 144.4, 136.1, 135.9, 135.7, 135.6,
130.1, 129.5, 128.5, 128.2, 127.8, 127.0, 126.8, 120.7,
113.1, 91.5, 86.5, 84.4, 74.6, 74.4, 63.7, 55.2, 52.6, 38.6
(br), 37.7, 20.8; ESI-MS (ESC): m/z calcd for C44H46N6O8
786.4, found 787.5 (MHC), 809.5 (MCNaC); HRMS m/z
calcd for C44H46N6O8 787.3435 (MHC), found 787.3447.
3.1.22. L-[2-18O]-Acetoxy-3-phenyllactic acid (19*). The
product (19*) was obtained as a colorless oil from 18*
(154.6 mg, 0.919 mmol) using method described in Section
3.1.21 (184.0 mg, 95%). 1H NMR (400 MHz, CDCl3):
7.34–7.24 (m, 5H, phenyl), 4.53 (dd, 1H, JZ9.0, 4.2 Hz,
CH), 3.24 (dd, 1H, JZ14.4, 4.0 Hz, CH2), 3.12 (dd, 1H, JZ
14.2, 8.6 Hz, CH2), 2.08 (s, 3H, CH3); ESI-MS (ESC): m/z
calcd for C11H12O318O 210.1, found 232.9 (MCNaC).
3.1.23. 30-Amino-30-deoxy-30-(L-2-acetoxy-3-phenylpro-
pionyl)-N6,N6-dimethyladenosine (20). EDCI (176.0 mg,
0.900 mmol) was added at 0 8C to a solution of puromycin
aminonucleoside (240.4 mg, 0.817 mmol), 19 (172.5 mg,
0.828 mmol), and N-hydroxysuccinimide (107.1 mg,
0.903 mmol) in DMF (10 ml). The reaction mixture was
stirred at 0 8C for 1 h, then stirred at room temperature for
24 h. After evaporation, the oily residue was subjected to
column chromatography (gradient from 2% MeOH in
CH2Cl2 to 4% MeOH in CH2Cl2) to give the crude product
as a white powder. This material was washed with ethyl
acetate (10 ml) to give the pure product 20 (288.9 mg, 73%)
3.1.26. 30-Amino-30-deoxy-30-(L-[2-18O]-2-acetoxy-3-
phenylpropionyl)-50-(p,p0-dimethoxytrityl)-N6,N6-di-
methyladenosine (21*). The product (21*) was obtained as
a white foam from 20* (249.4 mg, 0.513 mmol) using the
1
method described in Section 3.1.3 (359.9 mg, 89%). H
NMR (400 MHz, CDCl3): 8.27 (s, 1H, H8), 7.96 (s, 1H,
H2), 7.32–7.16 (m, 14H, DMTr-aromatic, Phe-aromatic),
6.75 (d, 4H, JZ9.2 Hz, DMTr-aromatic), 6.69 (d, 1H, JZ
4.80Hz, 30-NH), 6.02 (s, 1H, 20-OH), 5.73 (d, 1H, JZ4.4 Hz,
H1 ), 5.34 (dd, 1H, JZ7.0, 5.4 Hz, Phe-CH), 4.71 0(t, 1H,
JZ5.6 Hz, H20), 4.41 (dd, 1H, JZ11.4, 5.0 Hz, H3 ), 4.37
(m, 1H, H40), 3.78 (s, 3H, DMTr-OCH3), 3.77 (s, 3H,
DMTr-OCH3), 3.54 (br, 6H, NCH3), 3.46 (dd, 1H, JZ10.8,
2.8 Hz, H50), 3.37 (dd, 1H, JZ10.6, 3.4 Hz, H50), 3.18 (dd,
1H, JZ14.0, 5.2 Hz, Phe-CH2), 3.10 (dd, 1H, JZ14.2,
7.4 Hz, Phe-CH2), 2.08 (s, 3H, acetyl-CH3); ESI-MS (ESC):
m/z calcd for C44H46N6O718O 788.3, found 811.4 (MC
NaC); HRMS m/z calcd for C44H46N6O718O 789.3487
(MHC), found 789.3477.
1
as a colorless fine powder. H NMR (400 MHz, CDCl3/
CD3ODZ1:1): 8.29 (s, 1H, H8), 8.24 (s, 1H, H2), 7.32–7.21
(m, 5H, phenyl), 5.85 (d, 1H, JZ2.8 Hz, H10), 50.30 (dd, 1H,
JZ7.4, 5.8 Hz, Phe-CH), 4.049–4.41 (m, 2H, H2 , H30), 4.05
(dt, 1H, JZ7.2, 2.0 Hz, H4 ), 3.95 (dd, 1H,0JZ12.8, 2.0 Hz,
H50), 3.69 (dd, 1H, JZ12.8, 2.4 Hz, H5 ), 3.53 (br, 6H,
NCH3), 3.36 (m, 1H, 30-NH), 3.18 (dd, 1H, JZ14.2, 5.4 Hz,
Phe-CH2), 3.11 (dd, 1H, JZ13.8, 7.4 Hz, Phe-CH2), 2.12 (s,
3H, acetyl-CH3); 13C NMR (125.8 MHz, CDCl3/CD3ODZ
1:1): 171.4, 171.1, 155.5, 152.2, 149.4, 138.2, 136.2, 129.9,
129.0, 127.5, 121.3, 91.5, 84.6, 75.1, 74.3, 61.8, 50.8, 39.0
(br), 38.3, 20.7; ESI-MS (ESC): m/z calcd for C23H28N6O6
484.2, found 485.2 (MHC), 507.2 (MCNaC); HRMS m/z
calcd for C23H28N6O6 485.2148 (MHC), found 485.2154.
3.1.27. 30-Amino-30-deoxy-30-(L-2-acetoxy-3-phenyl-
propionyl)-50-O-(p,p0-dimethoxytrityl)-N6,N6-dimethyl-
adenosine 20-O-succinate (22). The product (22) was
obtained as a white foam from 21 (299.7 mg, 0.380 mmol)
using the method described in Section 3.1.5 (192.7 mg,
3.1.24. 30-Amino-30-deoxy-30-(L-[2-18O]-2-acetoxy-3-
phenylpropionyl)-N6,N6-dimethyladenosine (20*). The
product (20*) was obtained as a colorless powder from
19* (173.1 mg, 0.823 mmol) using the method described in
Section 3.1.23 (306.1 mg, 76%). 1H NMR (400 MHz,
CDCl3/CD3ODZ3:1): 8.28 (s, 1H, H8), 8.24 (s, 1H, H2),
7.32–7.21 (m, 5H, phenyl), 5.85 (d, 1H, JZ2.8 Hz, H10),
5.30 (dd, 1H, JZ7.2, 5.6 Hz, Phe-CH), 4.49–4.42 (m, 2H,
H20, H30), 4.06 (dt, 1H, JZ6.9, 2.1 Hz, H40), 3.95 (dd, 1H,
JZ13.0, 2.2 Hz, H50), 3.70 (dd, 1H, JZ12.8, 2.4 Hz, H50),
3.54 (br, 6H, NCH3), 3.36 (m, 1H, 30-NH), 3.18 (dd, 1H,
JZ13.8, 5.8 Hz, Phe-CH2), 3.11 (dd, 1H, JZ14.2, 7.4 Hz,
Phe-CH2), 2.12 (s, 3H, acetyl-CH3); ESI-MS (ESC): m/z
calcd for C23H28N6O518O 486.2, found 487.2 (MHC);
1
57%). H NMR (400 MHz, CDCl3): 8.29 (s, 1H, H8), 7.94
(s, 1H, H2), 7.32–7.16 (m, 14H, DMTr-aromatic, Phe-
aromatic), 6.77 (d, 4H, JZ8.8 Hz, DMTr-aromatic), 6.43
(br, 1H, 30-NH), 6.10 (s, 1H, H10), 5.61 (d, 1H, JZ3.2 Hz,
H20), 5.41 (t, 1H, JZ5.2 Hz, Phe-CH), 5.18 (dd, 1H, JZ
15.0, 8.2 Hz, H30), 3.91 (br, 1H, H40), 3.73 (s, 3H, DMTr-
OCH3), 3.72 (s, 3H, DMTr-OCH3), 3.50 (br, 6H, NCH3),
3.38–3.31 (m, 2H, H50), 3.03 (dd, 1H, JZ14.0, 7.6 Hz, Phe-
CH2), 2.92 (dd, 1H, JZ14.2, 5.4 Hz, Phe-CH2), 2.65 (br,
2H, succinic ester-CH2), 2.60 (br, 2H, succinic ester-CH2),
2.08 (s, 3H, acetyl-CH3); 13C NMR (100.6 MHz, CDCl3):
170.7, 170.2, 169.1, 158.4, 154.9, 152.3, 149.4, 144.3,

K. Okuda et al. / Tetrahedron 60 (2004) 12101–12112
12111
139.4, 136.3, 135.7, 135.6, 135.5, 130.1, 129.7, 129.5,
129.1, 128.5, 128.2, 127.9, 127.8, 127.1, 127.0, 126.8,
120.3, 113.1, 113.0, 87.2, 86.6, 77.2, 75.9, 74.1, 62.5, 55.3,
55.2, 49.6, 38.6 (br), 37.8, 29.9, 29.7 (br), 20.9; ESI-MS
(ESC): m/z calcd for C48H50N6O11 886.4, found 887.6
(MHC); HRMS m/z calcd for C48H50N6O11 887.3538
(MHC), found 887.3550.
coliMRE600 cells by a procedure modified from the
literature.33 CCApcb was 50-32P end labeled by phosphoryl-
ation with T4 polynucleotide kinase and [g-32P]ATP. The
reaction of 1.0 mM 1 or 2 with 32pCCApcb and 9 mM 50 S
ribosome was performed in 7 mM Mg2C, 7 mM KC,
166 mM NH4C, 0.1 mM EDTA, 0.2 mM DTT, 25 mM
MES, 25 mM MOPS, 50 mM Tris-HCl buffer (pH 7.0) at
25 8C. The ribosomes were incubated for 2 min at 37 8C
before beginning the reaction. The samples were analyzed
by polyacrylamide gel electrophoresis (7 M urea/50 mM
Tris-sodium phosphate (pH 6.5)/12% polyacrylamide gel
with 50 mM Tris-sodium phosphate buffer (pH 6.5) at
30 W).
3.1.28. 30-Amino-30-deoxy-30-(L-[2-18O]-2-acetoxy-3-
phenylpropionyl)-50-O-(p,p0-dimethoxytrityl)-N6,N6-di-
methyladenosine 20-O-succinate (22*). The product (22*)
was obtained as a white foam from 21* (312.5 mg,
0.396 mmol) using the method described in Section 3.1.5
(217.0 mg, 62%). 1H NMR (400 MHz, CDCl3): 8.29 (s, 1H,
H8), 7.94 (s, 1H, H2), 7.32–7.16 (m, 14H, DMTr-aromatic,
Phe-aromatic),0 6.77 (d, 4H, JZ8.8 Hz, DMTr-0aromatic),
6.43 (br, 1H, 3 -NH), 6.10 (d, 1H, JZ2.0 Hz, H1 ), 5.62 (d,
1H, JZ4.4 Hz, H20), 5.41 (t, 1H, JZ6.4 Hz, Phe-CH), 5.18
(dd, 1H, JZ14.2, 8.6 Hz, H30), 3.91 (br, 1H, H40), 3.74 (s,
3H, DMTr-OCH3), 3.73 (s, 3H, DMTr-OCH3), 3.50 (br, 6H,
NCH3), 3.39–3.32 (m, 2H, H50), 3.03 (dd, 1H, JZ14.4,
7.2 Hz, Phe-CH2), 2.93 (dd, 1H, JZ13.6, 5.2 Hz, Phe-CH2),
2.65 (br, 2H, succinic ester-CH2), 2.61 (br, 2H, succinic
ester-CH2), 2.08 (s, 3H, acetyl-CH3); ESI-MS (ESC): m/z
calcd for C48H50N6O1018O 888.4, found 889. 6 (MHC),
911.7 (MCNaC); HRMS m/z calcd for C48H50N6O1018O
889.3658 (MHC), found 889.3656.
For HPLC analysis, 20 nmol of A-site substrate was added
to 30 nmol of CCApcb (Fig. 2b). The buffer conditions were
the same as above. 4.5 mM 50 S ribosomal subunits were
added to begin the reaction. Once the reaction had
proceeded to greater than 50% reacted, it was quenched
by addition of w50 mM EDTA. The reaction was purified
on an Agilent Technologies XBD-C18 reverse phase HPLC
column using 10 mM triethylamine acetate (TEAA), pH 6.5
as the mobile phase. Substrates and products were separated
by HPLC using a gradient of 0–30% acetonitrile over
30 min followed by an isocratic run for 10 min at 30%
acetonitrile. Mass spectrometry was used to determine the
identity of each HPLC peak. All HPLC fractions were
frozen, lyophilized to dryness, and desalted by multiple
rounds of lyophilization. The samples were analyzed on an
Applied Biosystems PE SciEX API 3000 triple quadrupole
mass spectrometer with an electrospray ion source (ESI-
MS). For ESI-MS analysis, samples were resuspended in 1:1
10 mM TEAA:acetonitrile and injected by direct infusion at
a rate of 10 ml/min. The same procedure was used for
reactions with 1, 1*, and 1*** each serving as the A-site
substrate. As expected, the 15N substitutions did not effect
the HPLC retention time. The exact mass for each of the
A-site substrates 1, 1*, and 1*** and their products
Cm6ANPheN-pcb, Cm6ANPhe15Npcb, and [3-15N,4-15NH2]-
Cm6ANPhe15Npcb are 746.25, 747.25, 749.25, 1262.49,
1263.49, and 1265.49, respectively.
3.1.29. 30-Amino-30-deoxy-30-(L-2-acetoxy-3-phenyl-
propionyl)-50-O-(p,p0-dimethoxytrityl)-N6,N6-dimethyl-
adenosine 20-O-(LCAA-polystyrene)succinate (23). The
product (23) was obtained from 22 (180.0 mg, 0.169 mmol)
using the solid support derivatization method described in
Section 3.1.7. The nucleoside loading was 70 mmol/g.
3.1.30. 30-Amino-30-deoxy-30-(L-[2-18O]-2-acetoxy-3-
phenylpropionyl)-50-O-(p,p0-dimethoxytrityl)-N6,N6-
dimethyladenosine 20-O-(LCAA-polystyrene)succinate
(23*). The product (23*) was obtained from 22*
(180.0 mg, 0.169 mmol) using the solid support derivatiza-
tion method described in Section 3.1.7. The nucleoside
loading was 103 mmol/g.
3.1.31.
Cytidylyl-(30-50)-30-amino-30-deoxy-30-(L-2-
hydroxy-3-phenylpropionyl)-N6,N6-dimethyladenosine
(2). The product (2) was obtained by coupling the cytidine
phosphoramidite (8) to 23 using the method described in
Section 3.1.9. ESI-MS (ESC): m/z calcd for C30H38N9O12P
747.2, found 748.3 (MHC); HRMS m/z calcd for
C30H38N9O12P 770.2275 (MCNaC), found 770.2284.
Acknowledgements
We thank D. Kitchen for assistance with solid-phase
synthesis, E. Pfund for assistance with product character-
ization, J. C. Cochrane for comments on the manuscript.
This research was supported by American Cancer Society
Beginning Investigator Grant RSG-02-052-GMC to S. A. S.
3.1.32.
Cytidylyl-(30-50)-30-amino-30-deoxy-30-(L-[2-
18OH]-2-hydroxy-3-phenylpropionyl)-N6,N6-dimethyl-
adenosine (2*). The product (2*) was obtained by coupling
the cytidine phosphoramidite (8) to 23* using the method
described in Section 3.1.9. ESI-MS (ESC): m/z calcd for
C30H38N9O1118OP 749.2, found 750.4 (MHC); HRMS m/z
calcd for C30H38N9O1118OP 750.2498 (MHC), found
750.2509.
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