Preparation of stilbene-tethered nonnatural nucleosides for use with blue-fluorescent antibodies

Article abstract of DOI:10.1021/jo001676f

The synthesis of the first examples of stilbene-tethered hydrophobic C-nucleosides is described. Compounds of this type are targeted for use with our recently reported "blue-fluorescent antibodies with the aim of probing native and nonnatural DNA. The nucleophilic addition of aryl Grignard reagents to either a protected 2′-deoxy-1′-chloro-ribofuranose or a protected 2′-deoxy-ribonolactone was the key synthetic step and afforded C-nucleosides in good yields. Both routes resulted in a final product that was ≥ 90% of the β-anomer. Amide- and ether-based linkers for attachment of trans-stilbene to the nucleobase were assessed for utility during synthesis and in binding of the ligands to a blue-fluorescent monoclonal antibody. X-ray structures of each complex were obtained and serve as a guideline for second-generation stilbene-tethered C-nucleosides. The development of these hydrophobic nucleosides will be useful in current native and nonnatural DNA studies and invaluable for investigations regarding novel, nonnatural genomes in the future.

Full text of DOI:10.1021/jo001676f

J . Org. Chem. 2001, 66, 1725-1732
1725
P r ep a r a tion of Stilben e-Teth er ed Non n a tu r a l Nu cleosid es for Use
w ith Blu e-F lu or escen t An tibod ies
Da-Wei Chen,† Albert E. Beuscher IV,‡,§ Raymond C. Stevens,§ Peter Wirsching,†
Richard A. Lerner,† and Kim D. J anda*,†
Department of Chemistry, The Scripps Research Institute and the Skaggs Institute for Chemical Biology,
10550 N. Torrey Pines Road, La J olla, California 92037, Department of Chemistry, University of
CaliforniasBerkeley, Berkeley, California 94720-1460, and Department of Molecular Biology,
The Scripps Research Institute, 10555 N. Torrey Pines Road, La J olla, California 92037
kdjanda@scripps.edu
Received November 29, 2000
The synthesis of the first examples of stilbene-tethered hydrophobic C-nucleosides is described.
Compounds of this type are targeted for use with our recently reported “blue-fluorescent antibodies”
with the aim of probing native and nonnatural DNA. The nucleophilic addition of aryl Grignard
reagents to either a protected 2′-deoxy-1′-chloro-ribofuranose or a protected 2′-deoxy-ribonolactone
was the key synthetic step and afforded C-nucleosides in good yields. Both routes resulted in a
final product that was g90% of the â-anomer. Amide- and ether-based linkers for attachment of
trans-stilbene to the nucleobase were assessed for utility during synthesis and in binding of the
ligands to a blue-fluorescent monoclonal antibody. X-ray structures of each complex were obtained
and serve as a guideline for second-generation stilbene-tethered C-nucleosides. The development
of these hydrophobic nucleosides will be useful in current native and nonnatural DNA studies and
invaluable for investigations regarding novel, nonnatural genomes in the future.
The advent of the new millenium holds great promise
for revolutions in genomic engineering. In the past few
years, the sequences of the entire genomes of both
prokaryotic and eukaryotic species have been pub-
lished.1,2 The pinnacle, the complete human genome, is
almost in hand. However, full annotation of the human
genome, even with continued advances in bioinformatics,
presents a formidable task and will likely continue well
into this century. While the genetic codes of known
organisms are being solved, Schultz and Romesberg have
also proposed an expansion of the genetic alphabet with
ramifications for the coding of unknown life forms.3 In
light of the onset of these bold ventures, the analysis of
both natural and nonnatural genomes will benefit from
new sequencing, fingerprinting, and mapping technolo-
gies.
DNA-duplex stability and sequence specificity are
founded on the complementary Watson-Crick hydrogen-
bonding patterns of adenine with thymine and cytosine
with guanine. However, over the past several years, the
concept of hydrophobicity has been evaluated as a driving
force in DNA structure and synthesis that could lead to
an alternative mode of selective information storage and
replication. Efforts to modify the genetic alphabet rely
on the specific thermodynamic and kinetic parameters
of nucleosides containing hydrophobic, nonnatural bases.
The nonnatural bases should form stable pairs in B-form
DNA with high selectivity relative to mispairing with the
native bases. Furthermore, a DNA polymerase must be
capable of efficiently utilizing both the nonnatural nu-
cleosides and the DNA containing them, and must do so
with high fidelity. Kool and co-workers4 introduced the
concept of nonpolar nucleoside isosteres (hydrophobic
“shape analogues”) and demonstrated stability in sub-
stituted DNA, as well as good substrate activity with
DNA polymerases, providing surrogates for the natural
base pairings. Schultz and Romesberg, et al.3 have
focused on the dominant role of hydrophobicity to achieve,
with some nonnatural pairings, rates comparable to those
for the synthesis of native DNA.3b,c The ability to replicate
DNA containing hydrophobic, nonnatural nucleobases
would also allow for the enzymatic incorporation of
additional functional moieties as handles for analytical
procedures.
† Department of Chemistry, The Scripps Research Institute and the
Skaggs Institute for Chemical Biology.
‡ University of CaliforniasBerkeley.
§ Department of Molecular Biology, The Scripps Research Institute.
(1) (a) Borinskaya, S. A.; Yankovsky, N. K. Mol. Biol. 1999, 33, 831-
845. (b) Karlin, S.; Campbell, A. M.; Mrazek, J . Annu. Rev. Genet. 1998,
32, 185-225.
(2) (a) The Arabidopsis Genome Initiative. Nature 2000, 408, 796-
815. (b) Rubin, G. M.; Yandell, M. D.; Wortman, J . R.; Miklos, G. L.
G.; Nelson, C. R., et al. Science 2000, 287, 2204-2215. (c) Adams, M.
D.; Celniker, S. E.; Holt, R. A.; Evans, C. A.; Gocayne, J . D., et al.
Science 2000, 287, 2185-2195. (d) Bassett, D. E., J r.; Basrai, M. A.;
Connelly, C.; Hyland, K. M.; Kitagawa, K.; Mayer, M. L.; Morrow, D.
M.; Page, A. M.; Resto, V. A.; Skibbens, R. V.; Hieter, P. Curr. Opin.
Genet. Dev. 1996, 6, 763-766.
(3) (a) Ogawa, A. K.; Wu, Y.; Berger, M.; Schultz, P. G.; Romesberg,
F. E. J . Am. Chem. Soc. 2000, 122, 8803-8804. (b) Wu, Y.; Ogawa, A.
K.; Berger, M.; McMinn, D. L.; Schultz, P. G.; Romesberg, F. E. J . Am.
Chem. Soc. 2000, 122, 7621-7632. (c) Ogawa, A. K.; Wu, Y.; McMinn,
D. L.; Liu, J .; Schultz, P. G.; Romesberg, F. E. J . Am. Chem. Soc. 2000,
122, 3274-3287. (d) Berger, M.; Ogawa, A. K.; McMinn, D. L.; Wu, Y.;
Schultz, P. G.; Romesberg, F. E. Angew. Chem., Int. Ed. 2000, 39,
2940-2942. (e) McMinn, D. L.; Ogawa, A. K.; Wu, Y.; Liu, J .; Schultz,
P. G.; Romesberg, F. E. J . Am. Chem. Soc. 1999, 121, 11585-11586.
We recently reported antibody-stilbene complexes,
coined “blue-fluorescent antibodies,” that emitted a bright-
blue fluorescence of very high quantum yield.5 Aside from
important fundamental principles concerning protein-
ligand dynamics engendered by these antibodies, a
(4) Kool, E. T.; Morales, J . C.; Guckian, K. M. Angew. Chem., Int.
Ed. 2000, 39, 990-1009.
10.1021/jo001676f CCC: $20.00 © 2001 American Chemical Society
Published on Web 02/13/2001

1726 J . Org. Chem., Vol. 66, No. 5, 2001
Chen et al.
number of practical applications should also be possible
and include uses in immunochemistry, in vitro and in
vivo histological assays, and genomic studies. It is in
regard to the last of these applications, that we have
embarked on a program to create a novel set of aryl
C-nucleosides possessing a tethered stilbene molecule for
probing native and nonnatural DNA. The eventual goal
is the assembly of nonnatural oligonucleotides by chemi-
cal means for use as primers and for hybridization
experiments, and ultimately the construction of full
length genomic transcripts by enzymatic runoff. In this
way, blue-fluorescent antibody binding to gene sequences
could be used to create markers that would be valuable
for a variety of structural and functional genomic studies.
Herein, we describe our initial efforts by synthesis of the
simplest archetype of a stilbene-tethered hydrophobic
C-nucleoside.
A second C-nucleoside incorporating an alternative
linker was similarly founded on the most fundamental
structure involving a benzene nucleobase. In this case,
the para-substituted aromatic ring was introduced using
recent methodology developed by Woski and co-workers
that utilized the ribonolactone 19 (Scheme 2).8
Unlike 10, the lactone is a very shelf-stable reagent
suitable for long-term storage. Furthermore, organome-
tallic additions to 19 result in an anomeric mixture with
a high percentage of the â-configuration. The drawbacks
of the approach are that the initial addition gives a
hemiketal which requires a silane reduction operation
and that the overall yield for the C-nucleoside is generally
lower. After coupling to obtain 15, selective removal of
the methoxymethyl ether with TMSBr afforded the
deprotected alcohol.9 For this synthesis, we used a
benzylic alcohol as a nucleobase so that a glycol linker
could be attached via conversion of 16 to the trifluo-
romethane-sulfonate followed by reaction with 23. Al-
though the yield was poor, no other methodology was
successful for ether formation in these compounds.10
Fluoride cleavage of the tetraisopropyldisiloxane of 17
afforded the C-nucleoside 18 that was 90% â-isomer. At
no point in the synthesis could the anomers be distin-
guished by silica gel chromatography, and so the final
product ratio was fixed by the addition to 19 and hydride
reduction of the hemiketal. Whether the inability to
separate isomers will be a general occurrence using this
route, remains to be determined. However, several cases
were reported8 where the â-isomer was obtained as >90%
of the mixture which bodes well for other C-nucleosides
in our plans.
Resu lts a n d Discu ssion
Two substituted benzene C-nucleosides were prepared
that differed in the composition of the linker between
stilbene and nucleobase. Each linker type could have
unique advantages during polymer-supported oligonucle-
otide synthesis or in enzymatic reactions. In addition, two
synthetic approaches were explored to assess their utility
for future work. In the preparation of the first compound
9, the key step depended on formation of the Grignard
reagent derived from 2 and its coupling with 1,2-dideoxy-
3,5-di-O-p-toluoyl-R-1-chloro-D-ribofuranose 10 (Scheme
1).6, 7
These substitution reactions do not proceed with inver-
sion, but yield a mixture of isomers (anomers) in which
the R-isomer is predominant.7 Here, the nucleobase was
installed to give 3 in 60% yield comprised of 78% of the
R-anomeric configuration. Notably, the yield was better
than generally observed for such couplings. Assignment
Each C-nucleoside was tested for the ability to bind to
the blue-fluorescent monoclonal antibody (mAb) 19G2.
Indeed, the bright, powder-blue fluorescence character-
istic of the mAb 19G2-stilbene interaction was observed,
and the quantum yield (φf) for each of the two complexes
were comparable to that measured previously for 19G2-
12 with a value of φf ∼ 0.80.5 Also, soaking a crystal of
mAb 19G2 with 9 or 18 resulted in the blue emission.
Subsequently, we acquired X-ray crystallographic data
on both complexes primarily to obtain information re-
garding positioning of the nucleobase moiety with regard
to the protein framework. The structures of the 19G2-9
and 19G2-18 complexes were determined to a resolution
of 2.45 and 2.20 Å, respectively (Table 1). In both cases,
the antibody structures differ by an RMSD of 0.61 and
0.50 Å from that of the previously determined 19G2-12
complex.5 The stilbene portion of both 9 and 18 is clear
in 2σ density and could be readily modeled into the
density as the trans-isomer (Figure 1, A and B). For both
complexes, binding site amino acid residues within 5 Å
of the stilbene are observed to be in the same conforma-
tion as in 19G2-12.
1
of R- and â-isomers was carried out using H NMR in
conjunction with literature data. For aromatic C-nucleo-
sides, R-isomers are characterized by an apparent triplet
(J ) 6-7 Hz) and â-isomers by a doublet of doublets (J
) 10-11, 5-6 Hz) for proton 1′-H on the deoxyribose
ring.7 Since the R-isomer is undesirable for our DNA
studies, it was necessary to isolate the â-isomer. How-
ever, upon examination of 3, no separation was evident
by thin-layer chromatography. To make the anomer ratio
more favorable, the mixture was subjected to acid-
catalyzed isomerization that resulted in a new mixture
4 now slightly enriched in the â-isomer. Finally, upon
formation of the azido compound 6, one chromatographic
operation could be used to separate the two closely
eluting anomers to yield pure material that was 95%
â-isomer. Reduction of the azido group with triphen-
ylphosphine followed by EDC-mediated amide bond
formation with 12, the original substrate for blue-
fluorescent antibodies,5 afforded the protected compound
8. Hydrolysis of the p-toluate esters resulted in 9, the
first of our stilbene-tethered C-nucleosides.
In 19G2-9, the electron density is clear for ∼7.5 Å from
the stilbene out to the second amide group of the linker
and then diverges in multiple directions. This indicates
that the linker terminus and appended C-nucleoside
assume multiple conformations at the outer rim of the
antibody binding site. The two conformations most obvi-
ous from the electron density were modeled (Figure 1A)
(5) Simeonov, A.; Matsushita, M.; J uban, E. A.; Thompson, E. H.
Z.; Hoffman, T. Z.; Beuscher, A. E., IV.; Taylor, M. J .; Wirsching, P.;
Rettig, W.; McCusker, J . K.; Stevens, R. C.; Millar, D. P.; Schultz, P.
G.; Lerner, R. A.; J anda, K. D. Science 2000, 290, 307-313.
(6) Preparation of 10: Takeshita, M.; Chang, C.-N.; J ohnson, F.;
Will, S.; Grollman, A. J . Biol. Chem. 1987, 262, 10171-10179.
(7) (a) Chaudhuri, N. C.; Ren, R. X.-F.; Kool, E. T. SYNLETT 1997,
341-347. (b) Ren, R. X.-F.; Chaudhuri, N. C.; Paris, P. L.; Rumney,
S., IV.; Kool, E. T. J . Am. Chem. Soc. 1996, 118, 7671-7678.
(8) Wichai, U.; Woski, S. A. Org. Lett. 1999, 1, 1173-1175.
(9) Hanessian, S.; Delorme, D.; Dufresne, Y. Tetrahedron Lett. 1984,
25, 2515-2518.
(10) Berry, J . M.; Hall, L. D. Carbohydrate Res. 1976, 47, 307-310.

Stilbene-Tethered Nonnatural Nucleosides
J . Org. Chem., Vol. 66, No. 5, 2001 1727
Sch em e 1. Syn th esis of a Stilben e-Teth er ed C-Nu cleosid e Usin g a n Am id e Lin k er a
a
(a) CH2(OMe)2, LiBr, TsOH; (b) (i) Mg, THF, (ii) 10; (c) (i) concentrated HCl (cat.), MeOH, 65 °C, (ii) PhSO3H/aq H2SO4 (cat.), toluene,
reflux; (d) MsCl, NEt3, CH2Cl2; (e) NaN3, DMF, 40 °C; (f) PPh3/H2O, THF; (g) 12, EDC, DMF; (h) NaOMe, MeOH; (i) glutaric anhydride,
DMAP, CH2Cl2.
and diverge about 60° away from one another, but both
bring the nucleoside analogue within close proximity of
some parts of the complementarity-determining regions
(CDRs). One conformation interacts almost exclusively
with the heavy-chain CDR3 (H-CDR3), and the other
interacts with H-CDR3, as well as H-CDR1 and H-CDR2.
Both conformations form hydrogen-bonding interactions
between the glutaric-amide linker and the antibody,
either to the side-chain amide nitrogen of the H96 Gln
in one mode, or to the backbone carbonyls of light-chain
91 (L91) Asn and L92 Leu in the other conformation. The
C-nucleoside fragment is also within hydrogen bonding
distance of antibody functional groups, but it is likely that
the mobility generates conformations in addition to what
is modeled. Although electron density exists for the
C-nucleoside in either conformation, the density is not
sufficient to confidently place the deoxyribose or phenyl
groups into an exact orientation and the B-factors in this
region are relatively high.
Similarly, in the 19G2-18 complex the linker and
C-nucleoside region can be modeled in at least two
different conformations placed almost 180° away from
one another (Figure 1B). One of the conformations
assumes a position near H-CDR3 as observed in 19G2-
9, whereas the other conformation interacts primarily
with the L-CDR3 loop, which was not observed in 19G2-
9. No hydrogen bonds are formed to the ligand prior to
the divergence of the conformations. However, the first
conformation forms a hydrogen bond between the last
ether oxygen of the linker and the backbone oxygen of
L92 Leu and both conformations bring the C-nucleoside
within hydrogen-bonding distance of the antibody. The
deoxyribose group can form a hydrogen bond to the
guanidinium group of H94 Arg and the backbone oxygen

1728 J . Org. Chem., Vol. 66, No. 5, 2001
Sch em e 2. Syn th esis of a Stilben e-Teth er ed C-Nu cleosid e Usin g a P olyeth er Lin k er a
Chen et al.
a
(a) CH2(OMe)2, LiBr, TsOH; (b) (i) t-BuLi, THF, -78 °C, (ii) 19, (iii) Et3SiH/BF3-Et2O, CH2Cl2, -78 °C; (c) TMSBr, CH2Cl2, -30 °C;
d) (i) TfO2, 2,4,6-collidine, CH2Cl2, -70 °C, (ii) 23; (e) TBAF, THF, 0 °C to room temperature; (f) MsCl, NEt3, CH2Cl2; (g) 26, NaH, THF,
60 °C; (h) concentrated HCl (cat.), MeOH, 65 °C; (i) CH2(OMe)2, LiBr, TsOH; (j) H2, 10% Pd/C, CHCl3.
Ta ble 1. X-r a y Da ta for An tibod y Com p lexes
of H96 Asn in one conformation, and in the other comes
parameter
molecular
space group
a (Å)
b (Å)
c (Å)
19G2-9
19G2-18
within hydrogen-bonding distance of the L92 Leu back-
bone oxygen. As was the case with 19G2-9, the high
B-factors for the C-nucleoside are probably indicative of
a wide range of possible conformations.
C2
C2
196.352
60.613
93.039
90
117.5
90
194.651
60.840
92.498
90
117.3
90
Our previous work on 19G2-12 suggested that the
linker lengths employed in 9 and 18 would allow complete
immersion of the stilbene moiety in the binding site,
while retaining the C-nucleoside portion at the precipice.
The X-ray structures show this to be the case. This now
serves as a guideline for our second-generation designs
in which the linker length will be increased in order to
ensure the complete emergence of trans-stilbene from the
∼8.5 Å deep major groove of double-helical DNA enabling
recognition by mAb 19G2. We anticipate that stilbene-
tethered C-nucleosides will be compatible with duplex
formation if the linker is of sufficient length and flex-
ibility to avoid steric congestion of the appended stilbene
with the DNA strands and/or the active site of a DNA-
utilizing enzyme. In this way, an appropriate linker
R (deg)
â (deg)
γ (deg)
data collection
resolution (Å)
20-2.45
20-2.2
(2.54-2.45)
(2.28-2.20)
observations
completeness (%)
I/σ
Rmerge (%)
refinement
Rwork
33485
47669
92.9 (90.8)
23.1 (4.6)
6.1 (26.4)
96.7 (92.5)
22.4 (2.8)
7.0 (39.7)
0.260
0.319
0.007
1.3
0.265
0.293
0.015
1.8
Rfree
bond lengths (Å)a
bond angles (deg)a
a
RMSD for both bond lengths and bond angles.

Stilbene-Tethered Nonnatural Nucleosides
J . Org. Chem., Vol. 66, No. 5, 2001 1729
C-nucleosides with a ribose,13 rather than deoxyribose
sugar, using a similar approach that would afford sub-
strates for RNA polymerases. The exo-nuclease-deficient
Klenow fragment of E. coli DNA polymerase I is able to
efficiently recognize a large number of nonnatural hy-
drophobic bases and incorporate them into DNA. In this
way, mapping and chain-termination sequencing could
be used in a fashion similar to current protocols.14
Hybridization, widely used in high-throughput genomics
strategies, would also be feasible. Yet, a significant
advance will come from polymerase-mediated extension
of DNA containing the nonnatural base, at present a
hurdle in most cases, for synthesis of read-through or
runoff transcripts/reverse transcripts. Ultimately, with
regard to both nonnatural DNA and genomes in the years
to come, a sequencing methodology will be needed
comparable to what is now routine with natural DNA.
Finally, targeting DNA with a macromolecular marker
has unique advantages associated with the ability to
apply immobilization technology for fragment isolation
and recovery. Continued developments in nonnatural
nucleobase design and the protein engineering of poly-
merase substrate specificity and activity will eventually
provide a unique set of tools for the investigation of
genetic material.
Exp er im en ta l Section
Gen er a l Meth od s. 1H and 13C NMR spectra were measured
on a Brucker AMX-400 or Brucker AMX-500 spectrometer as
indicated. Chemical sifts (ppm) were reported relative to
internal CDCl3 (1H, 7.26 ppm and 13C, 77.0 ppm), CD3OD (1H,
3.30 ppm and 13C, 49.2 ppm), and DMSO-d6 (1H, 2.49 ppm and
13C, 39.0 ppm). HRMS spectra were recorded using electro-
spray ionization (ESI) or MALDI techniques. Glassware and
solvents were dried by standard methods. Flash chromatog-
raphy was performed on silica gel 60 (230-400 mesh) and thin-
layer chromatography on glass plates coated with a 0.02 mm
layer of silica gel 60 F-254. All chemical reagents and solvents
were from Aldrich Chem. Co., unless otherwise noted, and used
without further purification.
4-[2-(Meth oxym eth oxy)eth yl]br om oben zen e (2). To a
solution of 4-bromophenethyl alcohol 1 (1.0 g, 5.0 mmol) in
dimethoxymethane (10 mL) were added LiBr (87 mg, 1.0
mmol) and p-TsOH-H2O (95 mg, 0.50 mmol) with stirring.
The white suspension was stirred at room temperature for 2
h or until completion of the reaction (TLC; hexane/EtOAc, 4/1).
Brine was added, and the mixture was extracted with ether.
After evaporation of the solvent, the crude product was purified
using flash chromatography (FC) (hexane/EtOAc, 4/1) to afford
1.17 g (96%) of 2 as a colorless oil. 1H NMR (CDCl3, 500 MHz)
δ 7.41 (d, 2H, J ) 8.0 Hz), 7.12 (d, 2H, J ) 8.0 Hz), 4.60 (s,
2H), 3.74 (t, 2H, J ) 7.0 Hz), 3.29 (s, 3H), 2.86 (t, 2H, J ) 7.0
Hz). 13C NMR (CDCl3, 125 MHz) δ 138.0, 131.4, 130.6, 120.0,
96.4, 68.0, 55.2, 35.7.
1,4-An h yd r o-2-d eoxy-1-C-[4-[2-(m eth oxym eth oxy)eth -
yl]p h en yl]-D-er yth r o-p en titol 3,5-Bis(4-m eth ylben zoa te)
(3). A solution of 2 (0.967 g, 3.95 mmol) in THF (4 mL) was
added into a flask charged with Mg powder and a few crystals
of iodine at room temperature under nitrogen. The mixture
was stirred at 50 °C for 2 h to complete the formation of the
Grignard reagent. A solution of chlorosugar 10 (1.23 g, 3.16
F igu r e 1. (A) A ribbon diagram of the 19G2-9 complex with
the Fo-Fc electron density for 9 contoured at 1.3σ. Residues
that pack against the stilbene molecule, such as H103 Trp are
shown explicitly. (B) A ribbon diagram of the 19G2-18 complex
with the Fo-Fc electron density for 18 contoured at 1.3σ. The
primary conformational models for 9 and 18 are shown in blue,
a second conformation is shown in pink.
might allow for base pairing and nucleoside ligation
during DNA synthesis.
In the work described, we have provided a proof-of-
principle for construction of a new class of compounds of
potential value in both current and future DNA and
genomic studies. The structures as presented are readily
amenable to activation as the 2′-OH phosphoramidite for
oligonucleotide synthesis or formation of the 5′-OH
triphosphate for use as DNA polymerase or reverse
transcriptase substrates using well-established meth-
ods.11,12 Furthermore, it should be possible to prepare the
(13) (a) Krohn, K.; Heins, H.; Wielckens, K. J . Med. Chem. 1992,
35, 511-517. (b) Hamamichi, N.; Miyasaka, T. J . Org. Chem. 1991,
56, 3731-3734.
(14) (a) Hunkapiller, M. W. Curr. Opin. Gen. Dev. 1991, 1, 88-92.
(b) Bina-Stein, M.; Thoren, M.; Salzman, N.; Thompson, J . A. Proc.
Natl. Acad. Sci. U. S.A. 1979, 76, 731-735. (c) Zimmern, D.; Kaesberg,
P. Proc. Natl. Acad. Sci. U.S.A. 1978, 75, 4257-4261. (d) Sanger, F.;
Nicklen, S.; Coulsen, A. R. Proc. Natl. Acad. Sci. U.S.A. 1977, 74,
5463-5467.
(11) Schweitzer, B. A.; Kool, E. T. J . Am. Chem. Soc. 1995, 117,
1863-1872.
(12) Kova´cs, T.; O¨ tvo¨s, L. Tetrahedron Lett. 1988, 29, 4525-4528.

1730 J . Org. Chem., Vol. 66, No. 5, 2001
Chen et al.
mmol) in THF (8 mL) was added at 0 °C, and the reaction
mixture was stirred at room temperature for 12 h. The mixture
was concentrated to a small volume. The residue was purified
by FC (hexane/EtOAc, 4/1) to give 1.0 g (60%) of 3 as an oil
that was a mixture of isomers (R/â ) 78/22). 1H NMR (CDCl3,
500 MHz) δ 7.93-7.71 (m, 4H), 7.36-7.32 (m, 2H), 7.28-7.17
(m, 6H), 5.61-5.58 (m, 1 H, R-isomer and â-isomer 3′-H), 5.34
(t, 1H, R-isomer 1′-H, J ) 6.6 Hz), 5.23 (dd, 1H, â-isomer 1′-
H, J ) 11.4, 5.5 Hz), 4.69-4.52 (m, 5H), 3.77-3.73 (m, 2H),
3.30 (s, 3H), 2.96-2.88 (m, 3H, benzylic, R-isomer 2′-Hâ), 2.51
(dd, 1H, â-isomer 2′-HR, J ) 13.2, 4.4 Hz), 2.44-2.40 (m, 6H),
2.32-2.20 (m, 1H, R-isomer 2′-HR, â-isomer 2′-Hâ). 13C NMR
(CDCl3, 125 MHz) δ 166.3, 166.2, 166.1, 166.0, 165.9, 144.0,
143.8, 143.7, 143.6, 140.1, 138.8, 138.5, 138.4, 138.0, 138.8,
130.0, 129.6, 129.5, 129.1, 129.0, 128.9, 128.8, 128.7, 127.0,
126.9, 126.8, 125.9, 125.7, 125.6, 96.3, 82. 8, 81.9, 80.6, 80.0,
77.2, 76.3, 68.4, 68.2, 64.7, 64.5, 55.0, 41.6, 40.2, 35.8, 21.6,
21.5. MALDI-FTMS: calcd for M + Na+ 541.2197, found
541.2211.
1,4-An h yd r o-2-d eoxy-1-C-[4-(2-h yd r oxyeth yl)p h en yl]-
D-er yth r o-p en titol 3,5-Bis(4-m eth ylben zoa te) (4). Com-
pound 3 (1.0 g, 1.9 mmol) was dissolved in MeOH (25 mL) with
one drop of 37% HCl. The mixture was stirred at 65 °C. After
completion of the reaction in 6-8 h, the solvent was evapo-
rated. The residue was purified by FC (hexane/EtOAc, 2:1) to
afford 0.82 g (90%) of a colorless syrup (R/â) 73/27). The
compound (0.82 g, 1.7 mmol) was epimerized in toluene (50
mL) with benzenesulfonic acid (30 mg, 0.17 mmol), concen-
trated H2SO4 (1 drop), and water (3 drops). The mixture was
stirred vigorously and refluxed for 4 h. After concentration,
the crude product was purified by FC (hexane/EtOAc, 2/1) to
afford 0.39 g (48%) of 4 as an oil (R/â ) 42/58). 1H NMR (CDCl3,
400 MHz) δ 8.00-7.71 (m, 4H), 7.39-7.34 (m, 2H), 7.29-7.34
(m, 2H), 7.29-7.17 (m, 6H), 5.62-5.58 (m, 1H), 5.34 (t, 1H,
R-isomer 1′-H, J ) 6.8 Hz), 5.23 (dd, 1H, â-isomer 1′-H, J )
11.2, 5.0 Hz), 4.70-4.52 (m, 3H), 3.87-3.82 (m, 2H), 2.97-
2.84 (m, 3H), 2.52 (dd, 1H, â-isomer 2′-HR, J ) 13.8, 5.0 Hz),
2.44-2.40 (m, 6H), 2.34-2.20 (m, 1H). 13C NMR (CDCl3, 125
MHz) δ 166.4, 166.1, 166.0, 144.1, 144.0, 143.8, 140.4, 138.8,
138.2, 137.7, 129.8, 129.7, 129.6, 129.2, 129.1, 129.0, 128.9,
127.1, 127.0, 126.8, 126.2, 126.0, 125.9, 81.9, 80.0, 76.4, 64.8,
64.6, 63.6, 63.5, 40.3, 38.8, 21.7, 21.6. MALDI-FTMS: calcd
for M + Na+ 497.1934, found 497.1932.
1,4-An h yd r o-2-d eoxy-1-C-[4-[2-[(m eth ylsu lfon yl)oxy]-
et h yl]p h en yl]-D-er yth r o-p en t it ol 3,5-Bis(4-m et h ylb en -
zoa te) (5). Compound 4 (0.387 g, 0.82 mmol) was dissolved
in CH2Cl2 (10 mL). Methanesulfonyl chloride (0.126 mL, 1.63
mmol) and then NEt3 (0.262 mL, 1.88 mmol) were added at 0
°C under nitrogen. The mixture was stirred overnight while
the temperature was allowed to rise to room temperature. The
CH2Cl2 layer was washed with water and brine and then dried
over Na2SO4. After evaporation of solvent, 0.435 g (96%) of
the product 5 was obtained as a yellow oil and used in the
next step without further purification.1H NMR (CDCl3, 500
MHz) δ 7.97-7.72 (m, 4H), 7.40-7.18 (m, 8H), 5.61-5.58 (m,
1H), 5.34 (t, 1H, R-isomer 1′-H, J ) 7.0 Hz), 5.24 (dd, 1H,
â-isomer 1′-H, J ) 11.0, 5.2 Hz), 4.70-4.52 (m, 3H), 4.42-
4.37 (m, 2H), 3.08-3.97 (m, 2H), 2.84 (s, 3H), 2.23 (dd, 1H,
â-isomer 2′-HR, J ) 14.0, 5.2 Hz), 2.44-2.40 (m, 6H), 2.31-
2.19 (m, 1H). 13C NMR (CDCl3, 125 MHz) δ 166.3, 166.0, 144.1,
143.9, 143.8, 141.2, 140.0, 135.9, 135.4, 131.5, 129.6, 129.5,
129.1, 129.0, 128.9, 127.0, 126.9, 126.7, 126.2, 126.0, 82.9, 82.0,
80.4, 80.0, 77.1, 76.3, 70.0, 64.6, 64.5, 53.4, 41.6, 40.3, 37.2,
35.2, 31.4, 21.7, 21.6, 21.5. MALDI-FTMS: calcd for M + Na+
575.1710, found 575.1711.
1,4-An h yd r o-2-d eoxy-1-C-[4-(2-a zid oet h yl)p h en yl]-D-
er yth r o-p en titol 3,5-Bis(4-m eth ylben zoa te) (6). Compound
5 (0.78 g, 1.43 mmol) was dissolved in anhydrous DMF (15
mL) under nitrogen, and then NaN3 (0.186 g, 2.86 mmol) was
added. The mixture was stirred at 40 °C and followed by TLC
which showed completion in 4 h. After dilution with EtOAc,
aqueous workup, and solvent evaporation, the residue was
purified by FC (hexane/EtOAc, 3:1). The desired â-isomer of 6
(0.30 g, 42%) eluted first and was obtained as a syrup. 1H NMR
(CDCl3, 500 MHz) δ 7.98 (d, 2H, J ) 8.0 Hz), 7.94 (d, 2H, J )
8.0 Hz), 7.35 (d, 2H, J ) 8.0 Hz), 7.27 (d, 2H, J ) 8.0 Hz),
7.22 (d, 2H, J ) 7.7 Hz), 7.18 (d, 2H, J ) 7.7 Hz), 5.61 (d, 1H,
J ) 5.5 Hz), 5.24 (dd, 1H, J ) 10.6, 4.8 Hz), 4.65-4.64 (m,
2H), 4.54-4.52 (m, 1H), 3.49 (t, 2H, J ) 7.4 Hz), 2.88 (t, 2H,
J ) 7.4 Hz), 2.52 (dd, 1H, J ) 14.0, 5.2 Hz), 2.44 (s, 3H), 2.41
(s, 3H), 2.27-2.20 (m, 1H). 13C NMR (CDCl3, 100 MHz) δ 166.3,
166.1 144.1, 143.8, 139.1, 137.6, 129.7, 129.6, 129.2, 129.1,
128.8, 127.0, 126.9, 126.2, 82.9, 80.6, 77.2, 64.7, 52.3, 41.6, 34.9,
21.7, 21.6. MALDI-FTMS: calcd for M + Na+ 522.1999, found
522.1997.
1,4-An h yd r o-2-d eoxy-1-C-[4-(2-a m in oet h yl)p h en yl]-D-
er yth r o-p en titol 3,5-Bis(4-m eth ylben zoa te) (7). Compound
6 (0.357 g, 0.71 mmol) was dissolved in THF (10 mL). Ph3P
(0.28 g, 1.06 mmol) and water (0.1 mL) were added. The
reaction mixture was stirred at room temperature under
nitrogen for 36 h until TLC showed the disappearance of
starting material. The mixture was concentrated and the
residue purified by FC (hexane/EtOAc, 1/2) to remove Ph3P,
Ph3PO and byproducts, and then (CH2Cl2/MeOH, 3/1) to give
0.30 g (83%) of 7 as a yellow syrup. 1H NMR (CDCl3, 400 MHz)
δ 7.98 (d, 2H, J ) 8.2 Hz), 7.94 (d, 2H, J ) 8.2 Hz), 7.33 (d,
2H, J ) 8.2 Hz), 7.27 (d, 2H, J ) 8.2 Hz), 7.22 (d, 2H, J ) 7.9
Hz), 7.16 (d, 2H, J ) 7.9 Hz), 5.62-5.60 (m, 1H), 5.23 (dd, 1H,
J ) 10.8, 5.0 Hz), 4.65-4.64 (m, 2H), 4.53 (brs, 1H), 2.95 (brs,
2H), 2.74 (t, 2H, J ) 7.0 Hz), 2.51 (dd, 1H, J ) 13.8, 5.0 Hz),
2.43 (s, 3H), 2.40 (s, 3H), 2.28-2.20 (m, 1H). 13C NMR (CDCl3,
100 MHz) δ 166.3, 166.1, 144.1, 143.8, 139.4, 138.4, 129.6,
129.1, 129.0, 128.9, 127.0, 126.9, 126.1, 125.9, 82.8, 80.7, 77.2,
64.7, 43.3, 41.6, 39.4, 21.7, 21.6. MALDI-FTMS: calcd for M
+ Na+ 496.2094, found 496.2100.
N-[2-[4-[2-Deoxy-3,5-bis-O-(4-m eth ylben zoyl)-D-er yth r o-
p en tofu r a n osyl]p h en yl]eth yl]-N′-[4-[(1E)-2-p h en yleth en -
yl]p h en yl]p en ta n ed ia m id e (8). Into a mixture of 7 (163 mg,
0.345 mmol) and 12 (117 mg, 0.379 mmol) in DMF (3.5 mL)
was added EDC-HCl (88 mg, 0.448 mmol) at room tempera-
ture. The mixture was stirred under nitrogen for 4 h. After
concentration, the residue was purified by FC (EtOAC) to
afford 172 mg (65%) of 8 as a syrup. 1H NMR (CDCl3, 400
MHz) δ 8.50 (s, 1H), 7.97 (d, 2H, J ) 8.2 Hz), 7.94 (d, 2H, J )
8.2 Hz), 7.56 (d, 2H, J ) 8.5 Hz), 7.49-7.42 (m, 4H), 7.36-
7.12 (m, 11H), 7.02 (dd, 2H, J ) 20.0, 16.4 Hz), 5.91 (t, 1H, J
) 6.2 Hz), 5.59 (d, 1H, J ) 6.8 Hz), 5.20 (dd, 1H, J ) 10.9, 5.0
Hz), 4.67-4.59 (m, 2H), 4.52-4.52 (m, 1H), 3.52-3.51 (m, 2H),
2.80 (t, 2H, J ) 7.0 Hz), 2.50 (dd, 1H, J ) 14.1, 5.9 Hz), 2.42
(s, 3H), 2.40 (s, 3H), 2.35-2.31 (m, 2H), 2.62-2.23 (m, 2H),
1.98-1.92 (m, 2H). 13C NMR (CDCl3, 100 MHz) δ 172.8, 171.1,
166.4, 166.1, 144.2, 143.9, 138.7, 138.5, 137.6, 137.3, 133.0,
129.7, 129.2, 128.9, 128.6, 127.6, 127.4, 127.0, 126.9, 126.4,
126.3, 119.8, 82.9, 80.6, 77.1, 64.7, 41.4, 40.4, 36.1, 35.2, 35.0,
21.8, 21.6. MALDI-FTMS: calcd for M + Na+ 787.3354, found
787.3334.
N-[2-[4-[2-Deoxy-D-er yth r o-p en tofu r a n osyl]p h en yl]eth -
y l]-N ′-[4-[(1E )-2-p h e n y le t h e n y l]p h e n y l]p e n t a n e d i-
a m id e (9). Compound 8 (172 mg, 0.225 mmol) was dissolved
in MeOH/CH2Cl2 (3 mL/2 mL) at room temperature under
nitrogen. A solution of 25% MeONa in MeOH (0.154 mL, 0.675
mmol) was added with stirring. After 30 min, a suspension
developed and TLC indicated the disappearance of starting
material. After stirring for an additional 1.5 h, solid NH4Cl
was added to quench the reaction followed by water (1 mL).
The solid was collected by filtration, washed with water, and
dried under vacuum in a desiccator to afford 94 mg (76%) of 9
as a white powder. 1H NMR (DMSO-d6, 500 MHz) δ 9.92 (brs,
1H), 7.86 (brs, 1H), 7.61 (d, 2H, J ) 8.4 Hz), 7.56 (d, 2H, J )
7.4 Hz), 7.52 (d, 2H, J ) 8.4 Hz), 7.35 (t, 2H, J ) 7.7 Hz),
7.26-7.22 (m, 3H), 7.19-7.11 (m, 4H), 4.96-4.94 (m, 2H), 4.69
(brs, 1H), 4.16 (brs, 1H), 4.76-4.75 (m, 1H), 3.49-3.38 (m, 2H),
3.26-3.23 (m, 2H), 2.68 (t, 2H, J ) 7.4 Hz), 2.31 (t, 2H, J )
7.4 Hz), 2.11 (t, 2H, J ) 7.0 Hz), 2.03 (dd, 1H, J ) 12.4, 5.5
Hz), 1.83-1.72 (m, 3H). 13C NMR (DMSO-d6, 125 MHz) δ
171.5, 170.8, 140.3, 138.8,138.4, 137.2, 131.7, 128.6, 128.3,
128.0, 127.3, 126.8, 126.2, 126.1, 119.1, 87.7, 79.0, 72.4, 62.5,
43.4, 35.7, 34.9, 34.6, 21.1. MALDI-FTMS: calcd for M + Na+
551.2516, found 551.2524.

Stilbene-Tethered Nonnatural Nucleosides
J . Org. Chem., Vol. 66, No. 5, 2001 1731
5-Oxo-5-[[4-[(1E)-2-p h en yleth en yl]p h en yl]a m in o]p en -
ta n oic Acid (12). A solution of trans-4-aminostilbene 11 (0.53
g, 2.7 mmol) (TCI Chem. Co.) in CH2Cl2 (10 mL) was stirred
at room temperature, and triethylamine (1.13 mL, 7.1 mmol)
was added followed by glutaric anhydride (464 mg, 4.05 mmol)
and 4-(dimethylamino)pyridine (DMAP) (2 mg). The solution
was stirred at room temperature for 18 h, poured into water/
EtOAc, shaken, and filtered, and the solid was washed with
water, EtOAc, hexane, and then dried that afforded 12 as a
white solid (340 mg, 41%).1H NMR (DMSO-d6, 500 MHz): δ
9.96 (1H, s), 7.60 (2H, d, J ) 8.8 Hz), 7.56 (2H, d, J ) 7.4 Hz),
7.52 (2H, d, J ) 8.8 Hz), 7.35 (2H, t, J ) 7.4 Hz), 7.23 (1H, t,
J ) 7.4 Hz), 7.18 (1H, d, J ) 16.5 Hz), 7.13 (1H, d, J ) 16.5
Hz), 2.36 (2H, t, J ) 7.3 Hz), 2.27 (2H, t, J ) 7.3 Hz), 1.81
(2H, quin, J ) 7.3 Hz). 13C NMR (DMSO-d6, 125 MHz): δ
174.12, 170.69, 138.80, 137.22, 131.78, 128.65, 128.03, 127.30,
126.88, 126.84, 126.23, 119.10, 35.39, 32.96, 20.39. MALDI-
FTMS: calcd for C19H19NO3 332.1263 (M + Na+), found:
332.1256.
1,4-An h ydr o-2-deoxy-1-C-[4-[[2-[2-[[4-[(1E)-2-ph en yleth -
en yl]p h en yl]m eth oxy]eth oxy]eth oxy]m eth yl]] p h en yl]-
3,5-O-[1,1,3,3-tetr a k is(1-m eth yleth yl)-1,3-d isiloxa n ed iyl]-
D-er yth r o-p en titol (17). Triflic anhydride (0.0176 mL, 0.105
mmol) was added to dry CH2Cl2 (0.5 mL) at -70 °C under N2
followed by a solution of 16 (46.7 mg, 0.10 mmol) and 2,4,6-
collidine (0.0139 mL, 0.105 mmol) in CH2Cl2 (1 mL). After 30
min, a solution of 23 (29.8 mg, 0.10 mmol) and 2,4,6-collidine
(0.0264 mL, 0.20 mmol) in CH2Cl2 (1 mL) was added with
stirring. After 30 min, the mixture was allowed to warm to
room temperature for an additional 3 h. The reaction mixture
was concentrated and purified by PTLC (hexane/EtOAc, 2/1)
to give 17 (17.9 mg, 24%) as a colorless oil.1H NMR (CDCl3,
400 MHz) δ 7.53-7.48 (m, 4H), 7.38-7.24 (m, 9H), 7.10 (s,
2H), 5.08 (t, 2H, J ) 8.0 Hz), 4.58 (s, 2H), 4.56 (s, 2H), 4.53-
4.50 (m, 1H), 4.13 (d, 2H, J ) 8.0 Hz), 3.91-3.85 (m, 2H),
3.71-3.62 (m, 8H), 2.38-2.32 (m, 1H), 2.09-2.02 (m, 1H),
1.11-1.01 (m, 28H). 13C NMR (CDCl3, 100 MHz) δ 141.4, 137.7,
137.5, 137.3, 128.6, 128.5, 128.3, 128.1, 127.8, 127.6, 126.5,
125.9, 86.4, 78.9, 73.3, 73.0, 70.7, 69.4, 69.3, 63.7, 43.1, 17.6,
17.4, 17.4, 17.2, 17.1, 17.0, 13.5, 13.4, 13.0, 12.5. MALDI-
FTMS: calcd for M + Na+ 769.3926, 769.3914.
1,4-An h ydr o-2-deoxy-1-C-[4-[[2-[2-[[4-[(1E)-2-ph en yleth -
en yl]p h en yl]m eth oxy]eth oxy]eth oxy]m eth yl]] p h en yl]-
D-er yth r o-p en titol (18). To a solution of 17 (17.9 mg, 0.024
mmol) in THF (0.3 mL) at 0 °C under N2 was added TBAF
(1.0 M in THF, 0.072 mL, 0.05 mmol). The mixture was stirred
for 2 h while the reaction temperature was allowed to warm
to room temperature. After concentration, the crude oil was
purified by PTLC (EtOAc/MeOH, 40/1) to give 18 (11.8 mg,
98%) as a white syrup. 1H NMR (CDCl3, 400 MHz) δ 7.53-
7.48 (m, 4H), 7.38-7.24 (m, 9H), 7.10 (s, 2H), 5.16 (dd, 1H, J
) 10.0, 5.0 Hz), 4.58(s, 2H), 4.57 (s, 2H), 4.42-4.40 (m, 1H),
4.01-3.99 (m, 1H), 3.81 (dd, 1H, J ) 12.0, 4.0 Hz), 3.73-3.63
(m, 9H), 2.23 (ddd, 1H, J ) 13.2, 5.5, 1.8 Hz), 2.05-1.98 (m,
1H), 1.93 (brs, 2H). 13C NMR (CDCl3, 100 MHz) δ 140.9, 138.3,
138.1, 137.7, 137.1, 129.1, 129.0, 128.8, 128.6, 128.4, 128.0,
126.9, 126.5, 87.6, 80.3, 74.2, 73.4, 73.3, 71.1, 69.9, 63.8, 44.5.
MALDI-FTMS: calcd for M + Na+ 527.2404, found 527.2402.
4-[(Meth oxym eth oxy)m eth yl]br om oben zen e (14). To a
solution of 4-bromobenzyl alcohol 13 (4.0 g, 21.4 mmol) in
dimethoxymethane (40 mL) were added LiBr (0.37 g, 4.28
mmol) and p-TsOH-H2O (0.41 g, 2.14 mmol). The white
suspension was stirred at room temperature for 2 h and then
quenched by addition of brine, followed by extraction of the
mixture with ether. After evaporation, the residue was purified
by FC (hexane/EtOAc, 4/1) to give 14 (4.0 g, 81%) as a colorless
oil.1H NMR (CDCl3, 400 MHz) δ 7.47 (d, 2H, J ) 8.0 Hz), 7.23
(d, 2H, J ) 8.0 Hz), 4.69 (s, 2H), 4.54 (s, 2H), 3.40 (s, 3H). 13C
NMR (CDCl3, 100 MHz) δ 136.8, 131.4, 129.4, 121.4, 95.6, 68.3,
55.3.
1,4-An h yd r o-2-d eoxy-1-C-[4-[(m eth oxym eth oxy)m eth -
yl]p h en yl]-3,5-O-[1,1,3,3-tetr a k is(1-m eth yleth yl)-1,3-d isi-
loxa n ed iyl]-D-er yth r o-p en titol (15). To a solution of com-
pound 14 (0.23 g, 1.0 mmol) in dry THF (2.5 mL) at -78 °C
under N2 was added t-BuLi (1.7 M in pentane, 1.17 mL, 2.0
mmol). The mixture was stirred for 30 min and then trans-
ferred to a solution of 19 (0.224 g, 0.60 mmol) in dry THF (2.5
mL) at -78 °C. After 1 h, the reaction was quenched with
saturated aqueous NH4Cl and the mixture extracted with
ether. The organic layer was washed with water and brine and
dried over Na2SO4, and the solvent was evaporated to give a
crude oil. To a solution of the oil at -78 °C in CH2Cl2 (5 mL)
under N2 were added Et3SiH (0.288 mL, 1.8 mmol) and BF3‚
Et2O (0.227 mL, 1.8 mmol). The mixture was stirred at -78
°C for 6 h and then quenched by addition of sat. NaHCO3 at
-78 °C. The mixture was extracted with ether, and the ether
layer was washed with water and brine and dried over Na2-
SO4. After evaporation, the crude oil was purified by FC
(hexane/EtOAc, 8/1) to give product 15 (0.13 g, 42%) as a
colorless oil. 1H NMR (CDCl3, 500 MHz) δ 7.33 (s, 4H), 5.10
(t, 1H, J ) 7.3 Hz), 4.70 (s, 2H), 4.58 (s, 2H), 4.56-4.52 (m,
1H), 4.15 (d, 1H, J ) 8.4 Hz), 3.94-3.87 (m, 2H), 3.41 (s, 3H),
2.40-2.35 (m, 1H), 2.09-2.03 (m, 1H), 1.14-0.95 (m, 28H).
13C NMR (CDCl3, 125 MHz) δ 141.6, 137.0, 127.9, 125.8, 95.5,
86.3, 78.8, 73.2, 68.8, 63.6, 55.2, 43.1, 17.5, 17.4, 17.3, 17.2,
17.0, 16.9, 13.4, 13.3, 12.9, 12.5. MALDI-FTMS: calcd for M
+ Na+ 533.2725, found 533.2725.
4-Ch lor om eth yl-tr a n s-stilben e (21). To a solution of
4-hydroxymethyl-trans-stilbene 20 (0.557 g, 2.65 mmol) and
triethylamine (0.85 mL, 6.1 mmol) in CH2Cl2 (30 mL) at 0 °C
was added MsCl (0.41 mL, 5.3 mmol) dropwise with stirring.
The reaction mixture was stirred at room-temperature over-
night. After workup, 21 (0.602 g, 99%) was obtained as a white
1
solid. H NMR (CDCl3, 400 MHz) δ 7.54-7.50 (m, 4H), 7.40-
7.35 (m, 4H), 7.30-7.28 (m, 1H), 7.12 (d, 2H, J ) 2.3 Hz), 4.61
(s, 2H).
11-[4-[(1E)-2-P h en yleth en yl] p h en yl]-2,4,7,10-tetr a oxa -
u n d eca n e (22). The alcohol 26 (0.40 g, 2.65 mmol) was treated
with 60% NaH (0.19 g, 4.75 mmol) in dry THF (10 mL) at room
temperature for 10 min. To this mixture, a solution of 21 (0.602
g, 2.65 mmol) in THF (10 mL) and cat. NaI was added. The
mixture was stirred at 60 °C overnight. The reaction was
quenched by addition of water and the mixture extracted with
ether. After evaporation, the crude oil was purified by FC
1
(hexane/EtOAc, 2/1) to give 22 (0.7 g, 77%) as a yellow oil. H
NMR (CDCl3, 500 MHz) δ 753-7.49 (m, 4H), 7.38-7.34 (m,
4H), 7.28-7.25 (m, 1H), 7.11 (s, 2H), 4.68 (s, 2H), 4.58 (s, 2H),
3.74-3.66 (m, 8H), 3.38 (s, 3H). 13C NMR (CDCl3, 100 MHz) δ
137.6, 137.2, 136.6, 128.6, 128.5, 128.3, 128.1, 127.6, 126.4,
96.5, 72.9, 70.6, 70.5, 69.4, 66.7, 55.1. MALDI-FTMS: calcd
for M + Na+ 365.1723, found 365.1716.
2-[2-[[4-[(1E)-2-P h en yleth en yl]ph en yl]m eth oxy]eth oxy]-
eth a n ol (23). A solution of 22 (0.7 g, 2.05 mmol) in MeOH
(10 mL) was treated with a catalytic amount of concentrated
HCl at 65 °C. The reaction was followed by TLC until starting
material disappeared (∼8 h). The mixture was concentrated
and purified by FC (hexane/EtOAc, 1/1) to give 23 (0.524 g,
86%) as a pale white solid. 1H NMR (CDCl3, 400 MHz) δ 7.54-
7.50 (m, 4H), 7.39-7.34 (m, 4H), 7.29-7.26 (m,1H), 7.12 (s,
2H), 4.57 (s, 2H), 3.75-3.73 (m, 2H), 3.71-3.68 (m, 2H), 3.66-
3.64 (m, 2H), 3.62-3.60 (m, 2H), 3.02 (brs, 1H). 13C NMR
(CDCl3, 100 MHz) δ 137.2, 137.0, 136.6, 128.5, 128.0, 127.9,
1,4-An h yd r o-2-d eoxy-1-C-[4-(h yd r oxym eth yl)p h en yl]-
3,5-O-[1,1,3,3-tetr a k is(1-m eth yleth yl)-1,3-d isiloxa n ed iyl]-
D-er yth r o-p en titol (16). To a solution of compound 15 (0.121
g, 0.237 mmol) in CH2Cl2 (5 mL) at -30 °C under N2 was added
TMSBr (0.125 mL, 0.949 mmol). After stirring at -30 °C for
1 h, the reaction was quenched by addition of sat. NaHCO3
and the mixture extracted with ether. After evaporation, the
crude oil was purified by PTLC (hexanes/EtOAc, 1/1) to give
1
16 (43 mg, 39%) as a colorless oil. H NMR (CDCl3, 400 MHz)
δ 7.34 (s, 4H), 5.09 (t, 1H, J ) 7.0 Hz), 4.68 (s, 2H), 4.55-4.51
(m, 1H), 4.13 (dd, 1H, J ) 10.3, 2.1 Hz), 4.93-3.86 (m, 2H),
2.37-2.34 (m, 1H), 2.06 (dt, 1H, J ) 12.9, 7.6 Hz), 1.12-0.94
(m, 28H). 13C NMR (CDCl3, 125 MHz) δ 141.6, 127.1, 126.1,
86.4, 78.8, 73.2, 65.2, 63.6, 43.1, 17.6, 17.4, 17.3, 17.2, 17.1,
17.0, 13.5, 13.4, 13.0, 12.5. MALDI-FTMS: calcd for M + Na+
489.2463, found 489.2457.

1732 J . Org. Chem., Vol. 66, No. 5, 2001
Chen et al.
127.4, 126.3, 72.8, 72.3, 70.2, 69.2, 61.5. MALDI-FTMS: calcd
for M + Na+ 321.1461, found 321.1454.
held UV lamp at 312 nm (Spectronics Corp.; Westbury, NY).
The crystals were soaked in a cryobuffer consisting of 20%
glycerol, 0.25 mM 9 or 18, mother liquor, and 5% DMF and
flash frozen in liquid nitrogen. X-ray diffraction was collected
in-house with an FRD X-ray generator and a RAXISIV2+
detector. Data were processed and scaled using the HKL
software package.15 The previously determined structure of the
antibody (PDB ID CODE 1FL3) was used as a starting model
for refinement. Multiple rounds of rigid body refinement,
B-factor refinement, Powell minimization, simulated annealing
Di(eth ylen e glycol)ben zyl Meth oxym eth yl Eth er (25).
To a solution of di(ethylene glycol) benzyl ether 24 (2.0 g, 10.0
mmol) in dimethoxymethane (20 mL) were added LiBr (0.17
g, 2.0 mmol) and p-TsOH-H2O (0.19 g, 1.0 mmol). The white
suspension was stirred at room temperature for 3 h, and then
the reaction was quenched by addition of brine and the mixture
extracted with ether. After evaporation, the residue was
purified by FC (hexanes/AcOEt 3/1) to give 25 (1.92 g, 80%)
1
17
as a colorless oil. H NMR (CDCl3, 400 MHz) δ 7.35-7.26 (m,
in CNS and manual rebuilding in O were performed.16,
5H), 4.67 (s, 2H), 4.58 (s, 2H), 3.72-3.64 (m, 8H), 3.37 (s, 3H).
13C NMR (CDCl3, 125 MHz) δ 138.2, 128.3, 127.6, 127.5, 96.5,
73.2, 70.6, 70.5, 69.4, 66.8, 55.1.
Ack n ow led gm en t. This work was supported by
funding from the Skaggs Institute for Chemical Biology.
K.D.J . is an Arthur C. Cope Scholar.
Di(eth ylen e glycol) Meth oxym eth yl Eth er (26). The
benzyl ether 25 (1.92 g, 8.0 mmol) was dissolved in chloroform
(10 mL) with 10% Pd/C (0.85 g, 0.1 mmol) and stirred under
a hydrogen atmosphere provided by a balloon. The reaction
was followed by TLC and was complete in 1 h. The mixture
was filtered and the filtrate concentrated to give 26 (1.12 g,
Su p p or tin g In for m a tion Ava ila ble: Copies of the 1H
NMR and 13C NMR spectra for all prepared compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
1
93%) as a colorless oil. H NMR (CDCl3, 400 MHz) δ 4.68 (s,
2H), 3.76-3.62 (m, 8H), 3.38 (s, 3H). 13C NMR (CDCl3, 125
MHz) δ 96.5, 72.4, 70.4, 66.9, 61.7, 55.2.
J O001676F
X-r a y Cr ysta llogr a p h y. Crystals of mAb 19G2 were
soaked overnight with a 0.25 mM solution of either 9 or 18
(DMF stock solutions) in mother liquor from the crystal growth
(12% poly(ethylene glycol), 0.1 M sodium acetate pH 4.75, 0.3
M magnesium chloride) containing 5% DMF. Formation of the
complex in the crystal was assayed by the appearance of blue
fluorescence from soaked crystals when illuminated by a hand-
(15) Otwinowski, Z.; Minor, W. Methods Enzymol. 1997, 277, 307-
236.
(16) Brunger, A. T.; Adams, P. D.; Clore, G. M.; Delano, W. L.; Gros,
P.; Grosse-Kunstleve, R. W.; J iang, J .-S.; Kuszewski, J .; Nilges, M.;
Pannu, N. S.; Read, R. J .; Rice, L. M.; Simonson, T.; Warren, G. L.
Acta Crystallogr. 1998, D54, 905-921.
(17) J ones, T. A.; Kjeldgaard, M. O. Methods Enzymol. 1997, 277,
173-208.