Synthesis of isotopically labeled P-site substrates for the ribosomal peptidyl transferase reaction

Article abstract of DOI:10.1021/jo702070m

(Chemical Equation Presented) Isotopomers of the ribosomal P-site substrate, the trinucleotide peptide conjugate CCA-pcb (Zhong, M.; Strobel, S. A. Org. Lett. 2006, 8, 55-58), have been designed and synthesized in 26-35 steps. These include individual iso

Full text of DOI:10.1021/jo702070m

Synthesis of Isotopically Labeled P-Site Substrates for the
Ribosomal Peptidyl Transferase Reaction
Minghong Zhong† and Scott A. Strobel*,†,‡
Department of Molecular Biophysics and Biochemistry and Department of Chemistry, Yale UniVersity,
New HaVen, Connecticut 06520-8114
scott.strobel@yale.edu
ReceiVed September 20, 2007
Isotopomers of the ribosomal P-site substrate, the trinucleotide peptide conjugate CCA-pcb (Zhong, M.;
Strobel, S. A. Org. Lett. 2006, 8, 55-58), have been designed and synthesized in 26-35 steps. These
include individual isotopic substitution at the R-hydrogen, carbonyl carbon, and carbonyl oxygen of the
amino acid, the O2′ and O3′ of the adenosine, and a remote label in the N3 and N4 of both cytidines.
These isotopomers were synthesized by coupling cytidylyl-(3′,5′)-cytidine phosphoramidite isotopomers
as the common synthetic intermediates, with isotopically substituted A-Phe-cap-biotin (A-pcb). The isotopic
enrichment is higher than 99% for 1-13C (Phe), 2-2H (Phe), and 3,4-15N2 (cytidine), 93% for 2′/3′-18O
(adenosine), and 64% for 1-18O (Phe). A new synthesis of highly enriched [1-18O2]phenylalanine has
been developed. The synthesis of [3′-18O]adenosine was improved by Lewis acid aided regioselective
ring opening of the epoxide and by an economical SN2-SN2 method with high isotopic enrichment (93%).
Such substrates are valuable for studies of the ribosomal peptidyl transferase reaction by complete kinetic
isotope effect analysis and of other biological processes catalyzed by nucleic acid related enzymes,
including polymerases, reverse transcriptases, ligases, nucleases, and ribozymes.
Introduction
of the terminal nucleoside of the tRNA, A76. The 2′-OH vicinal
to the P-site ester is essential for the reaction. Deletion or
alteration of this functional group results in a complete loss of
peptidyl transferase activity (>106-fold loss of activity).4 This
contribution is significantly greater than that made by any of
the rRNA nucleotides within the active site, though the 2′-OH
of A2451 also makes an important contribution.5 The ribosome
aligns the two substrates such that reaction proceeds through a
chiral transition state with a S stereochemistry.6 This configu-
ration places the critical A76 2′-OH between the R-amino
The catalytic mechanism of peptide bond formation by the
ribosome is an area of ongoing research. Several aspects of the
reaction mechanism are well-established.2 The reaction uses two
substrate tRNAs, an aminoacyl-tRNA that binds to the A site
and a peptidyl tRNA that binds to the P site. The reaction
involves aminolysis of the P-site ester by the A-site R-amino
group. Relative to the rate in solution, the peptidyl transferase
center of the ribosome provides a rate enhancement of ap-
proximately 107-fold, a contribution that has been largely
attributed to entropy.3 The nascent peptide in the P site and the
amino acid in the A site are both linked via an ester to the 3′-O
(3) (a) Sievers, A.; Beringer, M.; Rodina, M. V.; Wolfenden, R. Proc.
Natl. Acad. Sci. U.S.A. 2004, 101, 7897-7901. (b) Schroeder, G. K.;
Wolfenden, R. Biochemistry 2007, 46, 4037-4044.
(4) (a) Dorner, S.; Panuschka, C.; Schmid, W.; Barta, A. Nucleic Acids
Res. 2003, 31, 6536-6541. (b) Weinger, J. S.; Parnell, K. M.; Dorner, S.;
Green, R.; Strobel, S. A. Nat. Struct. Mol. Biol. 2004, 11, 1101-1106.
(5) Erlacher, M. D.; Lang, K.; Wotzel, B.; Rieder, R.; Micura, R.;
Polacek, N. J. Am. Chem. Soc. 2006, 128, 4453-4459.
* To whom correspondence should be addressed.
† Department of Molecular Biophysics and Biochemistry.
‡ Department of Chemistry.
(1) Zhong, M.; Strobel, S. A. Org. Lett. 2006, 8, 55-58.
(2) Green, R.; Noller, H.F. Annu. ReV. Biochem. 1997, 66, 679-716.
10.1021/jo702070m CCC: $40.75 © 2008 American Chemical Society
Published on Web 12/15/2007
J. Org. Chem. 2008, 73, 603-611
603

Zhong and Strobel
nucleophile and the 3′-O leaving group. Possible roles for this
hydroxyl in proton transfer from the R-amine to the 3′-O have
been proposed.7c,i,k
Although the essential contributors to the reaction have been
largely defined, both biochemically and structurally, several
substantially different mechanisms have been drawn to explain
these observations.7,8 One model assumes a mechanism equiva-
lent to that observed in solution in which the reaction proceeds
through a zwitterionic T( intermediate followed by deprotona-
tion to a T- intermediate that is then resolved into products.7a,b
It is suggested that the A76 2′-OH could accept the proton from
the R-amino group. A second alternative involves resolution of
the T( intermediate directly to products by the simultaneous
transfer of a proton from the amine, through the A76 2′-OH to
the 3′-O leaving group as the C-O bond is broken.7c,i A third
suggestion, which has appeared in several reviews, involves the
simultaneous N-C bond formation with C-O bond cleavage
coupled to proton transfer through the 2′-OH.7d-g This mech-
anism does not involve a tetrahedral intermediate. Other
mechanistic proposals have also been made that invoke ad-
ditional intermediates and transition states.7h,j
In order to understand the role the ribosome plays in
promoting the peptidyl transferase reaction, it is essential
to characterize the reaction mechanism. The most powerful
approach for transition-state characterization is kinetic iso-
tope effect (KIE) analysis in which heavy isotopes are introduced
at atoms expected to have altered bonding in the transition
state. By measuring and analyzing the small differences
in reaction rate that result from isotopic substitution, it is
possible to characterize the nature of the chemical transition
state.
FIGURE 1. Isotopomers of the P-site substrate for kinetic isotope
effect analysis of ribosomal peptide bond formation.
The P-site substrate contains the last three nucleotides at the
3′-end of the tRNA (CCA) linked to a peptidyl mimic (pcb) by
an ester linkage. Kinetic isotope effects of P-site atoms that are
directly or closely involved in the reaction are expected to
provide specific information about the reaction mechanism
(Figure 1, X1 ) N).10 For example, the carbonyl carbon is
directly involved in peptide bond formation, and the 13C isotope
effect at this position is always normal (>1.00) in solution
aminolysis reactions with a magnitude as large as 1.04 (a 4%
KIE). This substitution provides information about the extent
of N-C bond formation in the transition state. Deuterium
substitution at the adjacent R-H is expected to result in an inverse
KIE (kD > kH), and its magnitude (reported to be between 0
and 11%) will define the orbital hybridization at the reaction
center carbon, with large KIEs correlating to sp3 hybridization
of the tetrahedral intermediate and small KIEs to sp2 hybridiza-
tion of a concerted mechanism. The primary isotope effect of
the carbonyl oxygen should be normal, and its magnitude should
also correlate with its bonding characteristics and environment,
such as its bond order with the reaction center carbon in the
transition state (TS). The kinetic isotope effect of the leaving
group (18klg) for the ester linkage can further define the chemical
mechanism of this catalyzed reaction. The18klg for the alkaline
hydrolysis of methyl formate, where formation of tetrahedral
intermediate is rate limiting, is 0.9%, while the hydrazinolysis
reaction has a KIE of 6.2%. This isotope effect provides valuable
information about the degree of C-O3′ bond cleavage in the
transition state. The value of the isotope effect at the O2′ should
correlate with its catalytic role and define the bonding details
of this atom as a possible proton shuttle.
Toward this goal, we prepared an A-site substrate in which
the R-amino group was 15N substituted and used this substrate
to measure a KIE of 1.009 for this substitution.9 This analysis
provided evidence that chemistry is at least partially rate limiting
in the modified fragment reaction between CCA-pcb and
CCPmn catalyzed by 50S ribosomes. The magnitude of the 15
N
KIE suggests either that the transition state is early, with little
N-C bond order, or that there is significant deprotonation of
the amine in the transition state. It is impossible to distinguish
these possibilities or to speculate on the nature of other changes
in the reaction coordinate based upon this single measurement.
Our goal is to expand the KIE measurements to include
substitutions at other atoms involved in the aminolysis reaction,
all of which are located within the P-site substrate. This
necessitates that various isotopic substitutions be introduced into
the substrate CCA-pcb.
(6) Huang, K. S.; Weinger, J. S.; Butler, E. B.; Strobel, S. A. J. Am.
Chem. Soc. 2006, 128, 3108-3109.
(7) (a) Satterthwait, A. C.; Jencks, W. P. J. Am. Chem. Soc. 1974, 96,
7018-703. (b) Jencks, W. P.; Gilchrist, M. J. Am. Chem. Soc. 1968, 90,
2622-2637. (c) Das, G. K.; Bhattacharyya, D.; Burma, D. P. J. Theor.
Bio. 1999, 200, 193-205. (d) Dorner, S., Polacek, N., Schulmeister U.,
Panuschk, C., Barta, A. Biochem. Soc. Trans. 2002, 30, 1131-1136. (e)
Trobro, S.; Aqvist, J. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 12395-
12400. (f) Changalov, M. M. et al. ChemBioChem 2005, 6, 992-996. (g)
Rodnina, M. V.; Beringer, M.; Wintermeyer, W. Trends Biochem. Sci. 2007,
32, 20-26. (h) Rangelov, M. A.; Vayssilov, G. N.; Yomtova, V. M.; Petkov,
D. D. Org. Biomol. Chem. 2005, 3, 737-744. (i) Schmeing, T. M.; Huang,
K. S.; Kitchen, D. E.; Strobel, S. A.; Steitz, T. A. Mol. Cell 2005, 20, 437-
448. (j) Rangelov, M. A.; Vayssilov, G. N.; Yomtova, V. M.; Petkov, D.
D. J. Am. Chem. Soc. 2006, 128, 4964-4965. (k) Rodnina, M. V.; Beringer,
M.; Wintermeyer, W. Q. ReV. Biophys. 2006, 39, 203-225.
In the KIE study of the A-site R-amine, we used a competition
method in which the heavy and the light isotopomer were mixed
together and the change in substrate ratio was monitored using
mass spectroscopy with a remote label. This approach neces-
sitates that isotopic substitutions are also made at a site distant
from the reactive groups. For this purpose, we have selected
the N3 and N4 nitrogens of the two cytidines for 15N substitution
(8) Weinger, J. S.; Strobel, S. A. Biochemistry 2006, 45, 5939-5948.
(9) Seila, A. C.; Okuda, K.; Strobel, S. A. Biochemistry 2005, 44, 4018-
4027.
(10) (a) Hengge, A. C.; Hess, R. J. Am. Chem. Soc. 1994, 116, 1256-
63. (b) Marlier, J. F. Acc. Chem. Res. 2001, 34, 283-290.
604 J. Org. Chem., Vol. 73, No. 2, 2008

Ribosomal Peptidyl Transferase Reaction
(Figure 1). This provides a convenient mass change of 4 Da
that is expected to fully separate the light and heavy substrate
peaks on the mass spectrometer.
selected to protect 2′-OH to exclude possible hydrolysis of AcE
during the cleavage of Fmoc. Both DMTr and ortho ester were
removed under weak acidic conditions to ensure the full
retention of the 3′-ester linkage and chirality within the peptide
chain mimic. After exploring several nucleotide coupling
conditions, we found that the phenylimidazolium salt of the
phosphoramidite provided high coupling yields. Using this
synthetic approach, the isotopomers of the P-site substrate were
synthesized by coupling two fragments of similar sizes: cy-
tidinyl-(3′5′)-cytidine phosphoramidite (or its N3 and N4
substituted isotopomer) and isotopomers of A-pcb with substitu-
tions at each of the relevant positions.
Synthesis of C(3′5′)C Phosphoramidite and Its Isoto-
pomer. There are three general methods to prepare [3-15N,4-
15NH2]cytidine. The compound was synthesized by SNAr
displacement of the 4-oxo of uridine followed by a Dimroth
rearrangement of cytidine.15 An alternative method by a
Dimroth-like rearrangement of N3-activated uridine followed
by SNAr reaction was also reported.16 The compound can also
be prepared by construction of the pyrimidine ring followed by
glycosylation. We used the well-studied second method, and
[3-15N,4-15NH2]cytidine (1b) was prepared according to a
reported procedure by a Dimroth-like rearrangement of 2′,3′,5′-
O-acetyl-N3-nitrouridine and SNAr reaction of the intermediate
2′,3′,5′-O-acetyl[15N3]uridine with in situ produced (15N)H3 as
a nucleophile, respectively, followed by deacetylation.16 Cytidine
(1a) and its isotopomer (1b) were then treated with 1,3-dichloro-
1,1,3,3-tetraisopropyldisiloxane (TIPDSCl2) in pyridine, respec-
tively, to afford 3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)-
cytidine (92%) and its isotopomer (90%).12 Compounds 2a,b
were selectively tritylated at the N4 to produce 3a,b (91%, 88%),
which was further protected at the 2′-position by reflux with a
mixture of trisacetoxyethoxy orthoformate, 4-tert-butyldimeth-
ylsiloxy-3-penten-2-one, and PPTS in DCM. The syrup that
resulted after chromatography was treated with HF-TMEDA
in CH3CN to provide compounds 5a,b (71%, 86%), which were
further protected at the 5′-position by DOD (89%, 89%). The
cytidylyl phosphoramidites 7a,b were obtained by treatment of
compound 6a,b with methyl tetraisopropylphosphorodiamidite
(POMe) in the presence of tetrazole in DCM overnight (81%,
quant).
Isotopically labeled nucleosides, nucleotides, and their poly-
mers (RNA and DNA) have potential applications in exploring
the mechanistic details of fundamental biological processes such
as protein synthesis, DNA and RNA synthesis, mRNA post-
transcriptional modifications and processing, and formation of
small RNAs, as shown in recent reviews on biological kinetic
isotope effects.11 However, there are few reports of this kind
of synthetic efforts, and the probes that are used are usually
prepared by enzymatic methods on small scales. We have
reported the solid-phase synthesis of CCPmn with 15N substitu-
tions at the R-amine of puromycin and the N3 and N4 of
cytidine.12 However, this synthetic strategy, as well as other
routine synthetic methods for oligonucleotides, cannot be applied
to the synthesis of CCA-pcb isotopomers for the following
reasons. First, the 3′-ester linkage is highly labile and will not
survive the coupling reactions and final basic deprotection steps;
second, the chirality of the amino acids is sensitive to the basic
conditions used in oligonucleotide synthesis. Therefore, we have
developed a compatible solution-phase method to synthesize
these isotopically labeled P-site substrates.1
We have reported the synthesis of CCA-pcb in two different
schemes by phosphoramidite chemistry, using acid-labile 2′-
O-bis(acetoxyethoxy)methyl (AcE),13 2′-O-bis(methoxyethoxy)-
methyl (MeE), and 4/6-N-(4,4′-dimethoxytrityl) (DMTr) as
protecting groups and imidazolium salt as the promoter.1 Here,
we report the synthesis of a complete collection of isotopically
labeled and remotely tagged P-site substrates. Improved methods
for synthesis of 18O-adenosine with the highest reported isotope
enrichment and a general method for incorporation of 18O into
amino acids are also described.
Results and Discussion
The synthesis of CCA-pcb was first reported by coupling the
trinucleotide CCA with cyanomethyl pcb.14 Although this
synthesis provided synthetic access to the molecule, there were
multiple byproducts that were difficult to separate from the
authentic compound resulting in low overall yields. Furthermore,
this approach could not be readily adapted to the synthesis of
the target molecule with [2′-18O]- or [3′-18O]adenosine or with
the remote isotope tags. We recently reported an alternative
synthetic scheme for this compound that produced high yields
and few side products.1 We used bulky bis(trimethylsiloxy)-
cyclododecyloxylsilyl (DOD) at the O5′ of cytidine 3′-O-
phosphoramidite (C74 in tRNA) and AcE at the O2′ of the
second cytidine (C75 in tRNA). This ensured the coupling
between the 3′-O-phosphoramidite of the first nucleoside and
the 5′-OH in the second nucleoside with its free 3′-OH site
reserved for the coupling to aminoacyladenosine, which is the
last coupling step. We used the methyl phosphoramidite, which
can be cleaved under mild conditions, and DMTr and ortho ester
(AcE, MeE) to protect unwanted nucleophilic sites. MeE was
Compounds 7a,b were directly coupled to compound 5a,b
in the presence of PhImOTf17 and molecular sieves in CH3CN/
DCM (1:1), followed by oxidation with t-BuOOH in toluene.
The reaction selectively produced 8a,b in good yield (81% and
quant) after chromatography. Due to the large size of DOD at
the O5′ of compound 7a,b, the relatively large 2′-O-AcE of
compound 5a,b, and large leaving group of phenylimidazole,
only the 3′-5′ coupling products were detected, which were
fully deprotected to give cytidylyl(3′,5′)cytidine. The diribo-
nucleotide was transformed into its phosphoramidite 9a,b as
before (73% and 51%) (Scheme 1). Synthesis of the dinucleotide
with tetrazole and 5-(ethylthio)-1H-tetrazole gave only moderate
yields, which might be caused by the bulky protecting groups
as reasoned above.
Synthesis of [3′/2′-18O]Adenosine Derivative. Methods for
synthesis of 18O nucleosides are limited, and this is especially
(11) Schramm, V.L. Methods Enzymol. 1999, 308, 301-355 and
references therein.
(12) Okuda, K.; Seila, A. C.; Strobel, S. A. Tetrahedron 2004, 60,
12101-12112.
(15) Sako, M.; Kawada, H. J. Org. Chem. 2004, 69, 8148-8150.
(16) Ariza, X.; Vilarrasa. J. J. Org. Chem. 2000, 65, 2827-2829.
(17) (a) Kawai, R.; Sugimoto, J.; Kataoka, M.; Hayakawa, Y. Nucleosides
Nucleotides Nucleic Acids 2001, 20, 1047-1048. (b) Hayakawa, Y.; Kawai,
R.; Hirata, A.; Sugimoto, J.; Kataoka, M.; Sakakura, A.; Hirose, M.; Noyori,
R. J. Am. Chem. Soc. 2001, 123, 8165-8176.
(13) Scaringe, S. A.; Wincott, F. E.; Caruthers, M. H. J. Am. Chem.
Soc. 1998, 120, 11820-11821.
(14) Bobkova, E. V.; Yan, Y. P.; Jordan, D. B.; Kurilla, M. G.;
Pompliano, D. L. J. Biol. Chem. 2003, 278, 9802-9807 and references
therein.
J. Org. Chem, Vol. 73, No. 2, 2008 605

Zhong and Strobel
during triflation for the synthesis of [3′-18O]adenosine, which
was finally prepared via an epoxide intermediate.20 For pyri-
midine nucleosides, the inversion can be realized by formation
of 2,2′- or 2,3′-anhydronucleosides.19,21
SCHEME 1. Preparation of Cytidinyl-(3′5′)-cytidine
Phosphoramidite and Its Isotopomer
We initially attempted to prepare the [3′-18O]adenosine
according to a reported procedure via an epoxide intermediate.20
The epoxide 17 was prepared as reported. Cesium [18O2]-
propionate was prepared by acidic hydrolysis of propionitrile
in HCl-H218O in a pressure tube, which gave 94% of 18O
enrichment. This was in contrast to less than 50% enrichment
reported for hydrolysis of propionyl chloride. Ring opening of
the epoxide by cesium [18O2]propionate (94% 18O) gave multiple
spots on TLC, in contrast to the reported regioselective ring
opening. After deprotection, ion-exchange chromatography
revealed a 1.6:1 mixture of two major products, due to the two
possible attacking sites. We reasoned that the formed alkoxide
in the ring-opening reaction may cause the disruption of the
glycosidic bond and hydrolysis of the 5′ ester. To avoid these
side reactions, we employed a Lewis acid to neutralize the
resulting alkoxide. In the presence of phenylimidazolium triflate,
TLC showed a clean reaction with two products. A moderate
Lewis acid induced regioselectivity (3.6:1) for the production
of compound 18. This may result from the increased steric
hindrance in the possible hydrogen-bonding complex between
imidazolium and N3. Deprotection of 5′-O and 3′-O gave
compound 18, which was selectively crystallized in the reaction
mixture (63%). The final incorporation of 18O at the 3′ position
was 93%. Silylation of compound 18 by TIDPSCl2 at the 3′
and 5′ positions (73%), followed by similar inversion of
configuration of the 2′-OH, gave compound 21f (42% for three
steps) (Scheme 2).
Although we improved the epoxide method, we still expected
that the steroselective inversion (SN2-SN2) strategy should
provide an economical synthesis of the target compound 16f.
We reasoned that the inaccessible triflation might be due to the
bulky spherical TBS, which could be substituted with more
suitable protecting groups. DMTr is propeller shaped just like
diphenylimidazole, which in a reported alkylation study inef-
ficiently excluded N7 alkylation of purine.22 Selective protection
of 5′-O by DMTr is straightforward and may leave the 3′-OH
accessible to triflation. Based upon this observation, compound
10 was prepared according to a reported procedure.23 The 3′-
OH was triflate substituted in good yield (84%). Nucleophilic
displacement by cesium propionate (70%) and hydrolysis (77%)
gave the xylofuranosyl nucleoside. Reactivation of 3′-OH by
triflation and nucleophilic substitution incorporated 18O at 3′
position to give compound 15 in good yield (82%). Full
deprotection by sequential treatments with NaOH in dioxane-
H2O and HCOOH in MeOH at 50 °C gave compound 16f (61%
with 93% 18O incorporated at 3′). Because 18O was introduced
at a later stage in a stereoselective way, this more economical
SN2-SN2 method was used in subsequent syntheses (Scheme
2).
true for substitutions within the ribosyl sugar.18-21 Hogencamp
et al. synthesized [3′/2′-18O]adenosine by condensation of
adenine with ribosyl chlorides to produce an isotope enrichment
of 27-33%.18 Reversible hydration (HCl/H218O) of a 3′-
ketouridine derivative followed by reduction (NaBH4) and
separation from the xylo epimer (major) gave [3′-18O]uridine
in low yield.21 Alternatively, the configurations of the 2′-C or
3′-C were inverted, the resulting 2′-OH or 3′-OH were trans-
formed into good leaving groups, and these were replaced to
incorporate 18O. Using this strategy, Jiang et al. reported the
synthesis of [2′-18O]adenosine; however, they had difficulty
(18) Follman, H.; Hogencamp, H. P. C. J. Am. Chem. Soc. 1970, 92,
671-677.
Compound [2′-18O]adenosine (16e) was synthesized according
to the reported procedure from arabinofuranosyladenine,20 with
high enrichment of 18O (>90%), using the highly 18O-enriched
cesium propionate. Compounds 21a,e,f were selectively trity-
lated at the N6 to produce 22a,e,f (91%, 84%, and 93%), which
were further protected at the 2′-position by reflux with a mixture
(19) Wnuk, S. F.; Chowdhury, S. M.; Garcia, P. I., Jr.; Robins, M. J. J.
Org. Chem. 2002, 67, 1816-1819.
(20) Jiang, C.; Suhadolnik, R. J.; Baker, D. C. Nucleosides Nucleotides
1988, 7, 271-94.
(21) (a) Pang, H.; Schram, K. H.; Smith, D. L.; Gupta, S. P.; Townsend,
L. B.; McCloskey, J. A. J. Org. Chem. 1982, 47, 3923-3932. (b) Solsten,
R. T.; McCloskey, J. A.; Schram, K. H. Nucleosides Nucleotides 1982, 1,
57-64. (c) Schubert, E. M.; Schram, K. H. J. Labelled Compd. Radiopharm.
1982, 19, 929-935. (d) Schwartz, H. M.; MacCoss, M.; Danyluk, S. S. J.
Am. Chem. Soc. 1983, 105, 5901-5911. (e) Schwartz, H. M.; MacCoss,
M.; Danyluk, S. S. Magn. Reson. Chem. 1985, 23, 885-894.
(22) Zhong, M.; Robins, M. J. J. Org. Chem. 2006, 71, 8901-8906.
(23) Serebryany, V.; Beigelman, L. Nucleosides Nucleotides Nucleic
Acids 2003, 22, 1007-1009.
606 J. Org. Chem., Vol. 73, No. 2, 2008

Ribosomal Peptidyl Transferase Reaction
SCHEME 2. Preparation of [3′-18O]Adenosine
SCHEME 3. Synthesis of Adenosine and [2′-18O/
3′-18O]Adenosine Intermediates
of trismethoxyethoxy orthoformate, 4-tert-butyldimethylsiloxy-
3-penten-2-one, and PPTS in DCM. The syrups that resulted
after chromatography were treated with HF-TMEDA in CH3-
CN to provide compounds 23a,e,f (71%, 74%, and 70%), which
were further protected by DOD to give 24a,e,f (89%, 57%, and
55%) (Scheme 3).
Synthesis of Isotopically Substituted N-Fmoc-L-phenyla-
niline. To synthesize isotopically substituted CCA-pcb, isoto-
pically substituted N-Fmoc-L-phenylaniline was first prepared.
Stereoselective preparation of L-[R-D]phenylaniline is challeng-
ing because of the sensitivity of its chirality to even weak bases.
The reported syntheses include the introduction of deuterium
by an enzymatic reductive amination24 and full isotope exchange
or chemical preparation of a mixture followed by enzymatic
resolution of two enantiomers.25,26
Because of the readily available deuterium source and acylase,
we synthesized L-[R-D]phenylaniline by a reported method using
enzymatic resolution.26 N-Acetyl-DL-phenylalanine was treated
with NaOH-D2O at room temperature, and the resulting highly
(24) Wong, C. H.; Whitesides, G. W. J. Am. Chem. Soc. 1983, 105,
5012-5014.
deuterium enriched (>98% by NMR) N-acetyl-[R-D]-DL-phe-
nylalanine was resolved by porcine kidney acylase at pH 7 to
give L-[R-D]phenylaniline in high purity, which was protected
(25) Yamamoto, D. M.; Upson, D. A.; Linn, D. K.; Hruby, V. J. J. Am.
Chem. Soc. 1977, 99, 1564-1570.
(26) Fujihara, H.; Schowen, R. J. Org. Chem. 1984, 49, 2819-2820.
J. Org. Chem, Vol. 73, No. 2, 2008 607

Zhong and Strobel
SCHEME 5. Preparation of Protected A-pcb and Its
SCHEME 4. Preparation of l-[18O2]Phenylaniline
Isotopomers
with Fmoc by treatment of Fmoc O-succinimide in CH3CN-
H2O (1:1) in the presence of NaHCO3 (quant).
We were unable to identify a synthesis of L-[18O2]phenylaniline
despite an extensive literature search. Sharpless and co-workers
reported a stereoselective synthesis of an L-phenylgylcinonitrile
derivative.27 We thought acidic hydrolysis of a nitrile may not
epimerize the chiral center. According to this procedure, we
synthesized compound 27 by acetylating L-phenylglycinamide
and treating the resulting N-acetyl-L-phenylglycinamide with
cyanuric chloride.28 The overall yield was 85% for two steps.
N-Fmoc-L-[18O2]phenylaniline was then synthesized by hy-
drolysis of compound 27 to give [18O2]phenylaniline with high
18O enrichment (quant, >92% 18O). Because of 18O/16O
exchange between amino acids and H2O in aqueous solution,
compound 28a was treated with Fmoc N-hydroxysuccinimide
ester (Fmoc-OSu) (85%) in pyridine (Scheme 4). Significant
epimerization was observed in spite of the much lower pKa of
pyridine than that of R-H in phenylalanine.
Synthesis of A-pcb and Its Isotopomers. Aminoacylation
of adenosine derivatives (24a,e,f) was challenging because of
the steric hindrance caused by the bulky 5′- and 2′-protecting
groups (DOD and MeE) and the relatively low nucleophilicity
of the 3′-OH. Several coupling strategies such as acylation by
acyl halides and DCC-promoted esterification were attempted,
but we achieved our best coupling using the imidazolium salt
of the amino acid. Compounds 24a,e,f were treated with
N-Fmoc-L-phenylalanines in the presence of mesitylenesulfonyl
tetrazole (MST)29,30 and excess N-methylimidazole in DCM at
ambient temperature to produce the fully protected derivatives
30a,d-h in reasonable to excellent yields (59% to quant). The
reaction involves the imidazolium cation as shown by 1H NMR.
The imidazole was nucleophilically displaced as a neutral
leaving group31 by the 3′-OH of 24a,e,f. All isotopomers are
by treatment with HF-TMEDA in CH3CN gave 34a,d-h in
fair to good yields (Scheme 5). Compound 32 was prepared as
reported1 with an improved high yield tritylation of a methyl
ester of biotin in the presence of both Et3N and DMAP in
pyridine at 70 °C (88%).
Synthesis of Protected CCA-pcb Isotopomers and Their
Deprotection. With compounds 9a,b and 34a,d-h in hand, we
performed the final steps of CCA-pcb isotopomer syntheses
using the previously reported coupling method.1 Diribonucle-
otide phosphoramidites 9a,b were coupled to 1 molar equiv of
compound 34a,d-h using phenylimidazolium triflate (PhIm-
OTf) and molecular sieves as the promotor in CH3CN/DCM
(1:1) to form the fully protected CCA-pcb 35a-h in reasonable
yields (33-72%). The efficient 1:1 coupling reaction involves
imidazolium cation, which was displaced by the 5′-OH to release
a neutral leaving group. Desilylation of compound 35a-h by
treatment with HF-TMEDA in CH3CN and cleavage of methyl
phosphate ester with S2Na2,13 followed by complete removal
of DMTr and orthoformate ester in 0.5 M HCOOH in MeOH-
DCM (1:1) at 55 °C for 5 h, gave compounds 1a (160 mg,
59%), 1b (40 mg, 26%), 1c (53 mg, 28%), 1d (21 mg, 13%),
and 1h (54 mg, 62%) as essentially single isotopomers and
isotopically highly enriched 1e (86 mg, 30% with 93% of 18O
in A76 (2′-O)), 1f (16 mg, 8% with 93% of 18O in A76 (3′-O)),
and 1g (12 mg, 13% with 64% of 18O (CdO)) as a mixture of
1
single distereomers as shown by H NMR, except for 30g.
Compound 30g is a mixture of two distereomers as a result of
the corresponding racemic N-Fmoc-[18O2]phenylalanine. Re-
moval of Fmoc by treatment of 30a,d-h with 20% piperidine
in CH3CN resulted in 31a,d-h. The two diostereomers of 31g
were separated by careful column chromatography, and the
correct component was used for later steps. DCC-mediated
amide formation with compound 32 followed by desilylation
(27) Demko, Z. P.; Sharpless, K. B. Org. Lett. 2002, 4, 2525-2527.
(28) Spero, D. M.; Kapadia, S. R. J. Org. Chem. 1996, 61, 7398-7401.
(29) Stawinski, J.; Hozumi, T.; Narang, S. A.; Bahl, C. P.; Wu, R. Nucleic
Acids Res. 1977, 4, 353-371.
(30) (a) Kumar, G.; Celewicz, L.; Chla`dek, S. J. Org. Chem. 1982, 47,
634-644. (b) Happ, E.; Scalfi-Happ, C.; Chla`dek, S. J. Org. Chem. 1987,
52, 5387-5391. (c) Hagen, M. and Chla`dek, S. J. Org. Chem. 1989, 54,
3189-3195. (d)Hagen, M.; Scalfi-Happ, C.; Happ, E.; Chla`dek, S. J. Org.
Chem. 1988, 53, 5040-5045.
1
two regioisomers (2′/3′-ester as shown by H NMR), respec-
tively (Scheme 6). The final relatively low enrichment (64%)
of 18O in the carbonyl of compound 1g may be caused by 18O/
16O exchange with adventitious H2O and with triflate in MST-
promoted aminoacylation.
(31) Zhong, M.; Nowak, I.; Robins, M. J. J. Org. Chem. 2006, 71, 7773-
7779.
608 J. Org. Chem., Vol. 73, No. 2, 2008

Ribosomal Peptidyl Transferase Reaction
SCHEME 6. Coupling and Deprotection for CCA-pcb and
Its Isotopomers
give a solid residue. DCM (10 mL) and methyl tetraisopropylphos-
phorodiamidite (1.3 mL, 1.2 g, 1.8 mmol) were added, and the
resulting solution was stirred under Ar at room temperature for 11
h. TLC (EtOAc/hexanes, 85:15) showed complete reaction. The
reaction mixture was then diluted by addition of DCM (50 mL),
and the resulting solution was washed with NaCl/H2O (30 × 2 mL).
The organic layer was separated and dried (MgSO4), and volatiles
were evaporated under vacuum. The derived residue was chro-
matographed (DCM/hexanes, 3:7 f 2:3 with 10% Et3N) to give
1
compound 9b (1.3 g, 51%) (four distereomers) with H and 31P
NMR spectra identical to those of compound 9a; ESI-MS (ES+)
m/z 2156.88 ([M + H]+ [C104H147N315N4O32P2Si3] ) 2156.88).
6-N-Benzoyl-2′-O-tert-butyldimethylsilyl-5′-O-(4,4′-dimethox-
ytrityl)-3′-O-trifluoromethanesulfonyladenosine (11). 2′-O-tert-
Butyldimethylsilyl-5′-O-(4,4′-dimethoxytrityl)-6-N-benzoyladenos-
ine (0.98 g, 1.2 mmol) and DMAP (230 mg, 1.9 mmol) were dried
by coevaporation with DCM (10 mL). The mixture was then
dissolved in DCM (15 mL) and cooled in ice-water bath under
Ar. To this solution was added trifluoromethanesulfonyl chloride
(0.32 g, 1.9 mmol), and the reaction mixture was stirred at 0 °C
under Ar for about 1 h (TLC showed complete reaction). The
reaction mixture was poured into ice-cold saturated NaHCO3-H2O
(50 mL) and extracted with DCM (50 × 2 mL). The organic layer
was separated, washed twice with saturated NaHCO3-H2O (50 ×
2 mL), and dried (MgSO4). Volatiles were evaporated under
vacuum, and the resulting mixture was chromatographed (EtOAc/
hexanes, 3:7) to give compound 11 (0.96 g, 84%): 1H NMR (500
MHz, DMSO-d6) δ 11.36 (s, 1H), 8.81 (s, 1H), 8.67 (s, 1H), 8.12
(d, J ) 7.3 Hz, 2H), 7.74-6.94 (m, 16H), 6.22 (d, J ) 7.5 Hz,
1H), 5.64-5.66 (m, 1H), 5.57-5.58 (m, 1H), 4.61 (t, J ) 5.4 Hz,
1H), 3.82 (s, 6H), 3.65 (dd, J ) 5.5, 8.3 Hz, 1H), 3.50 (dd, J )
5.5, 8.3 Hz, 1H), 0.77 (s, 9H), 0.00 (s, 3H), -0.41 (s, 3H).
6-N-Benzoyl-[2′-O-tert-butyldimethylsilyl-5′-O-(4,4′-dimethox-
ytrityl)-3′-O-propionyl-9-xylofuranosyl]adenine (12). A mixture
of compound 11 (0.96 g, 1 mmol) and cesium propionate (0.46 g,
2.1 mmol) was dissolved in anhydrous DMF (10 mL), and the
solution was stirred under Ar at room temperature overnight.
Volatiles were evaporated under vacuum, and the residue was
chromatographed (EtOAc/hexanes, 3:7) to give compound 12 (0.61
g, 70%): 1H NMR (500 MHz, DMSO-d6) δ 11.17 (s, 1H), 8.67 (s,
1H), 8.34 (s, 1H), 8.00 (d, J ) 7.4 Hz, 2H), 7.06-6.81 (m, 16H),
6.07 (d, J ) 3.4 Hz, 1H), 5.21-5.19 (m, 1H), 4.97-4.96 (m, 1H),
4.57-4.54 (m, 1H), 3.69 (s, 6H), 3.36 (dd, J ) 6.5, 8.4 Hz, 1H),
3.15 (dd, J ) 4.7, 7.5 Hz, 1H), 2.03-1.92 (m, 2H), 0.78 (s, 9H),
0.75 (t, J ) 7.5 Hz, 3H), 0.00 (s, 3H), -0.10 (s, 3H).
Summary and Conclusions
(2′-O-tert-Butyldimethylsilyl-5′-O-(4,4′-dimethoxytrityl)-9-xy-
lofuranosyl)adenine (13). To a solution of compound 12 (0.61 g,
0.72 mmol) in MeOH (20 mL) was added K2CO3 (0.5 g, 3.6 mmol),
and the mixture was stirred at room temperature overnight (TLC
showed complete reaction). Volatiles were evaporated under
vacuum, and the residue was chromatographed (EtOAc/hexanes,
1:1 f 7:3 f EtOAc) to give compound 13 (0.38 g, 77%): 1H
NMR (500 MHz, DMSO-d6) δ 8.07 (s, 1H), 8.01 (s, 1H), 7.37
-6.85 (m, 15H), 5.86 (br s, J ) 1.5 Hz, 1H), 5.68 (d, J ) 4.8 Hz,
1H), 4.38 (br s, 1H), 4.28-4.25 (m, 1H), 3.92-3.90 (m, 1H), 3.68
(s, 6H), 3.45 (dd, J ) 9.9, 10.2 Hz, 1H), 3.50 (dd, J ) 1.4, 5.2 Hz,
1H), 0.80 (s, 9H), 0.00 (s, 6H).
[2′-O-tert-Butyldimethylsilyl-5′-O-(4,4′-dimethoxytrityl)-3′-O-
trifluoromethanesulfonyl-9-xylofuranosyl]adenine (14). Com-
pound 13 (0.38 g, 0.6 mmol) and DMAP (115 mg, 0.9 mmol) were
dried by coevaporation with DCM (10 mL). The mixture was then
dissolved in DCM (15 mL) and cooled in an ice-water bath under
Ar. To this solution was added trifluoromethanesulfonyl chloride
(0.16 g, 0.98 mmol), and the reaction mixture was stirred at 0 °C
under Ar for about 1 h (TLC showed complete reaction). The
reaction mixture was poured into ice-cold saturated NaHCO3-H2O
(50 mL) and extracted with DCM (50 × 2 mL). The organic layer
was separated, washed twice with saturated NaHCO3-H2O (50 ×
2 mL), and dried (MgSO4). Volatiles were evaporated under
In conclusion, we have accomplished the syntheses of a
complete set of CCA-pcb isotopomers that will be used for a
kinetic isotope effect analysis of the ribosomal peptidyl transfer
reaction. These syntheses were made possible by improved
methods for high-yield synthesis of CCA-pcb.1 With its compat-
ible coupling methods for nucleotide synthesis, amino acylation,
and mild deprotection conditions, 3′,5′-linkages were constructed
exclusively, and the chirality of the amino acid and the labile
2′/3′-ester linkage was conserved. The improved syntheses of
3′-[18O]adenosine with high heavy isotope enrichment have been
developed, which provide an important small molecule probe
for studies of nucleic acid related enzymes.
Experimental Section
2′-O-((Bisacetoxyethoxy)methyl)-5′-O-bis(trimethylsiloxy)cy-
clododecyloxysilyl-4-N-(4,4′-dimethoxytrityl)[3-15N,4-15NH2]-
cytidylyl-(3′,5′)-2′-O-((bisacetoxyethoxy)methyl)-4-N-(4,4′-
dimethoxytrityl)[3-15N,4-15NH2]cytidine 3′-(N,N-diisopropyl-
methoxy)phosphoramidite (9b). To compound 8b (3.2 g, 1.6
mmol) in a dry flask was added tetrazole in CH3CN (3 wt %, 5.5
mL, 1.9 mmol) and volatiles were evaporated under vacuum to
J. Org. Chem, Vol. 73, No. 2, 2008 609

Zhong and Strobel
vacuum, and the resulting mixture was chromatographed (MeOH/
DCM, 1:30) to give the compound (0.46 g, quant): 1H NMR (500
MHz, DMSO-d6) δ 8.19 (s, 1H), 8.12 (s, 1H), 7.42-6.83 (m, 13H),
6.03 (d, J ) 4.8 Hz, 1H), 5.71-5.69 (m, 1H), 5.42-5.40 (m, 1H),
4.67-4.64 (m, 1H), 3.72 (s, 6H), 3.49-3.48 (m, 1H), 3.25-3.20
(m, 1H), 0.77 (s, 9H), 0.00 (s, 3H), -0.29 (s, 3H).
2′-O-tert-Butyldimethylsilyl-5′-O-(4,4′-dimethoxytrityl)-[3′-
18O]-3′-O-propionyladenosine (15). [18O2]Propionate cesium
was prepared by acidic hydrolysis of propionitrile in HCl-H218O.
H218O (10 g) was saturated with HCl at 0 °C, and to this cold
solution was added propionitrile (3 mL, 2.32 g, 42 mmol).
The solution was stirred in a pressure tube at 120 °C for 48 h.
The yellow solution was cooled in an ice-water bath and the solid
precipitated out. The acidic H218O was collected for recycling, and
the solid was dissolved in DCM (50 mL). The solution was
extracted with ice-cold NaHCO3-H2O, and the aqueous solution
was washed with DCM. The aqueous solution was acidified by
addition of HCl-H2O to about pH 3 and extracted with DCM.
The organic layer was separated and dried (MgSO4). Vacuum
fractional distillation gave [18O2]propionoic acid as a clear colorless
liquid.
which was treated with TIPDSCl2 to give silylated byproduct (0.20
g).
L-[18O2]Phenylaniline (28a). H218O (10 g) was saturated with
HCl at 0 °C, and to this cold solution added compound 27 (1.5 g,
7.8 mmol). The suspension was stirred at 120 °C to give a clear
brown solution after 10 min, and the solution was further stirred
for 48 h. The yellow solution was cooled in ice-water bath and
solid precipitated out. The acidic H218O was collected for future
use, and the solid was dissolved in MeOH (50 mL). Volatiles were
evaporated in vacuo, and the residue was thoroughly washed with
DCM to give crude compound 28a (including NH4Cl): ESI-MS
(ES+) m/z 168 (M + H+ [C9H1218OO] ) 168, 9.5%), 170 (M +
H+ [C9H1218O2] ) 170, 90.5%).
Cytidylyl-(3′,5′)-cytidylyl-(3′,5′)-3′(2′)-O-(N-(6-D-(+)-biotinoy-
laminohexanoyl)-L-phenylalanyl)adenosine Isotopomers (CCA-
pcb, 1a-h). A freshly prepared HF-TMEDA solution in DMF (0.4
M, 12.5 equiv) was added to a flask containing compound 35a/b/
c/d/e/f/g/h (0.95 g, 0.27 mmol/0.21 g, 0.06 mmol/0.50 g, 0.14
mmol/0.41 g, 0.12 mmol/0.77 g, 0.21 mmol/0.53 g, 0.15 mmol/
0.23 g, 0.06 mmol/0.28 g, 0.08 mmol), respectively, and the
resulting solution was stirred for 0.5 h. TLC showed complete
reaction. Volatiles were evaporated under vacuum and coevaporated
with toluene, and the residue was separated by preparative TLC
(MeOH/DCM, 1:15 f 1:10) to give a solid.
S2Na2‚3H2O was dissolved in H2O/DMF (0.1 M, 2% H2O). To
the clear lightly yellow solution was added the solid, and the
resulting clear solution was stirred at room temperature for 1 h.
TLC showed complete reaction. S2Na2 was then partially precipi-
tated by addition of DCM (25 mL) and removed by centrifuge.
The solid was extracted with DCM (5 mL), and the clear solutions
were combined. Volatiles were evaporated under vacuum, and the
residue was suspended in limited amount of MeOH/DCM (1:3).
The resulting solution was separated by preparative TLC (MeOH/
DCM, 1:6 f 1:3.6) to give a solid.
The liquid was added to MeOH (40 mL) in a flask, and to this
solution was added Cs2CO3 (1.0 g, 3.1 mmol). The mixture was
stirred at room temperature for 3 h, and volatiles were evaporated
under vacuum to give a solid residue, which was thoroughly washed
with Et2O to give the cesium salt (1. 4 g, 95%): ESI-MS (ES-)
m/z 75 ([M - H]- [C3H518OO] ) 75, 12%), 77 ([M - H]-
[C3H518O2] ) 77, 88%).
Compound 14 (0.46 g, 0.56 mmol) and cesium [18O2]propionate
(0.23 g, 1.1 mmol) were dissolved in anhydrous DMF (10 mL),
and the resulting solution was stirred at room temperature under
Ar overnight. Volatiles were removed under vacuum at 37 °C, and
the solid residue was chromatographed (EtOAc/hexanes, 1:1 f 7:3)
to give compound 15 (0.34 g, 81%): 1H NMR (500 MHz, DMSO-
d6) δ 8.44 (s, 1H), 8.16 (s, 1H), 7.55-6.97 (m, 13H), 6.03 (d, J )
7.1 Hz, 1H), 5.51-5.51 (m, 1H), 5.40-5.39 (m, 1H), 4.36 (br s,
1H), 3.86 (s, 6H), 3.48-3.44 (m, 2H), 2.58-2.49 (m, 2H), 1.19 (t,
J ) 7.6 Hz, 3H), 0.78 (s, 9H), 0.77 (t, J ) 7.5 Hz, 3H), 0.00 (s,
3H), -0.23 (s, 3H).
To the obtained solid in a dry flask was added freshly prepared
0.5 M HCOOH in DCM/CH3OH (1:1). The clear solution was
stirred at 55 °C for 5 h with precipitated product observed. Volatiles
were evaporated and coevaporated with toluene twice. The residue
was dried under vacuum overnight and then washed with MeOH/
DCM (1:2) at 37 °C to give compound 1a-h (two regioisomers).
Compound 1a (160 mg, 59%): ESI-MS (ES-) m/z 1362.51 ([M
- H]- [C53H70N15O22P2S] )1362.40), 1384.54 ([M - 2H + Na]-
[C53H69N15O22P2S Na] ) 1384.40).
[3′-18O]Adenosine (16f). To a solution of compound 15 (0.34
g, 0.46 mmol) in dioxane (5 mL) was added 1 M NaOH-H2O
(5 mL), and the turbid solution was stirred at room temperature
overnight. The solution was neutralized by bubbling through
CO2. Volatiles were evaporated under vacuum, and the residue was
chromatographed (MeOH/DCM. 1:15) to give a white solid.
This white solid was then dissolved in MeOH/DCM (1:1, 10
mL), and to the clear solution was added 88% formic acid (0.22
mL, 0.26 g, 5 mmol). The solution was stirred at 50 °C for 11 h
(TLC showed complete reaction). The volatiles were evaporated
under vacuum, and the residue was thoroughly washed (DCM) to
Compound 1b (40 mg, 26%): ESI-MS (ES-) m/z 682.90 ([M
- 2H]2- [C53H6915N4N11O22P2S] ) 682.70), 1366.81 ([M - H]-
[C53H7015N4N11O22P2S] )1366.40), 1388.77 ([M - 2H + Na]-
[C53H6915N4N11O22P2S Na] ) 1386.40).
Compound 1c (53 mg, 28%): ESI-MS (ES-) m/z 683.49 ([M -
2H]2- [C5213CH6915N4N11O22P2S] ) 683.20), 1367.95 ([M - H]-
[C5213CH7015N4N11O22P2S] ) 1367.40), 1389.91 ([M - 2H + Na]-
[C5213CH6915N4N11O22P2S Na] ) 1389.40) with an extremely
abundant peak at 170.7 ppm in 13C NMR.
1
give a white solid (77 mg, 63%) with H and 13C NMR spectra
identical to the authentic sample: ESI-MS (ES+) m/z 268 ([M +
H]+ [C10H13N5O4] ) 268, 8.4%), 270 ([M + H]+ [C10H13N5O318O]
) 270, 91.6%).
Compound 1d (21 mg, 13%): ESI-MS (ES-) m/z 683.41 ([M
- 2H]2- [C532HH6815N4N11O22P2S] ) 683.20), 1367.82 ([M - H]-
[C532HH6915N4N11O22P2S] ) 1367.40).
(9-â-D-[3′-18O]-Arabinofuranosyl)adenine (18). A mixture of
compound 17 (0.86 g, 3.0 mmol), phenylimidazolium triflate (0.97
g, 3.3 mmol), and [18O2]propionate cesium (0.93 g, 4.4 mmol) in
DMF was stirred at 120 °C in the presence of dried molecular sieves
for 6 h. Volatiles were evaporated under vacuum. The derived
residue was dissolved in MeOH (40 mL) in a sealed flask, and to
this solution was added NH3‚H2O (4 mL). The mixture was stirred
at ambient temperature overnight. The precipitated solid was
collected by filtration to give compound 18 (0.5 g, 63%) with with
1H and 13C NMR spectra identical to the authentic unlabeled
sample: ESI-MS (ES+) m/z 268 ([M + H]+ [C10H13N5O4] ) 268,
7.4%), 270 ([M + H]+ [C10H13N5O318O] ) 270, 92.6%). The
mother liquor was dried by evaporation in vacuo, and the solid
residue was washed thoroughly with MeOH to give a crude mixture
of the needed product and (9-â-D-2′-18O-xylofuranosyl)adenine,
Compound 1e (86 mg, 30%): ESI-MS (ES-) m/z 681.65 ([M -
2H]2- [C53H69N15O2118OP2S] ) 681.70), 1364.40 ([M - H]-
[C53H70N15O2118O P2S] ) 1364.40), 1375.90 ([2M + Na - 2H]2-
[C106H140N30O4218O2P4S2Na] ) 1375.90), 1386.37 ([M - 2H +
Na]- [C53H69N15O2118O P2SNa] ) 1386.40).
Compound 1f (16 mg, 8%): ESI-MS (ES-) m/z 681.69 ([M -
2H]2- [C53H69N15O2118OP2S] ) 681.70), 1364.42 ([M - H]-
[C53H70N15O2118O P2S] )1364.40), 1386.39 ([M - 2H + Na]-
[C53H69N15O2118O P2SNa] ) 1386.40).
Compound 1g (12 mg, 13%): ESI-MS (ES-) m/z 680.72 ([M -
2H]2- [C53H69N15O22 P2S] ) 680.70, 35.7%), 681.72 ([M - 2H]2-
[C53H69N15O2118OP2S] ) 681.70, 64.3%), 1362.38 ([M - H]-
[C53H70N15O22 P2S] ) 1362.40, 33.5%), 1364.38 ([M - H]-
[C53H70N15O2118O P2S] ) 1364.40, 66.5%).
610 J. Org. Chem., Vol. 73, No. 2, 2008

Ribosomal Peptidyl Transferase Reaction
Compound 1h (54 mg, 62%): ESI-MS (ES-) m/z 681.45 ([M
- 2H]2- [C5213C H69N15O22P2S] ) 681.20), 1363.74 ([M - H]-
[C5213CH70N15O22P2S] ) 1363.40), 1375.24 ([2M + Na - 2H]2-
[C10413C2H140N30O44P4S2Na] ) 1375.20), 1385.73 ([M - 2H +
Na]- [C5213C H69N15O22 P2SNa] ) 1386.40) with an extremely
abundant peak at 170.7 ppm in 13C NMR.
Supporting Information Available: General experimental
procedures, experimental details, and synthetic procedures for
compounds 2b, 3b, 5b-8b, 22-24e,f, 27, 29a,b, 30-31a,
d-h, 34a,d-h, 35a-h; 1H NMR for new compounds (11-15, 27,
1
29b, and 31g) and H-15N HMBC for compounds 6b and 9b;
and mass spectra of 1a-h, 9b, 16f, 28a, 34e, and 34f. This ma-
terial is available free of charge via the Internet at http://pubs.acs.
org.
Acknowledgment. We thank D. Hiller and E. Pfund for
helpful discussions. This research was supported by NIH Grant
No. GM54839 to S.A.S.
JO702070M
J. Org. Chem, Vol. 73, No. 2, 2008 611