Regioselective synthesis of 13C1-labeled 2-deoxyribonolactones

Article abstract of DOI:10.1016/S0040-4020(00)01148-0

Syntheses of the five regioselectively ¹³C₁-labeled 5-O-benzyl-2-deoxyribonolactones are described. ¹³C₁-Labeled deoxyribonolactones were prepared by addition of KCN to epoxides 7 and subsequent lactonization of the resulting nitriles. Integration of the independent schemes leading to the five isotopomers of 9 results in an efficient and cost effective preparation of labeled mixtures of ¹³C mono-labeled deoxyribonolactones. These mixtures are the pivotal intermediates in the preparation of 'population labeled' ¹³C-labeled nucleoside phosphoramidites for solid-phase oligonucleotide synthesis.

Full text of DOI:10.1016/S0040-4020(00)01148-0

TETRAHEDRON
Pergamon
Tetrahedron 57 (2001) 1515±1524
13
Regioselective synthesis of C1-labeled 2-deoxyribonolactones
Michael P. Hayes, Paul J. Hatala, Brian A. Sherer, Xiaohong Tong, Nilo Zanatta,
Philip N. Borer and James Kallmertenp
Department of Chemistry, Syracuse University, 1-014 Center for Science and Technology, Syracuse, NY 13244-4100, USA
Received 6 July 2000; accepted 6 December 2000
AbstractÐSyntheses of the ®ve regioselectively 13C1-labeled 5-O-benzyl-2-deoxyribonolactones are described. 13C1-Labeled deoxyribo-
nolactones were prepared by addition of KCN to epoxides 7 and subsequent lactonization of the resulting nitriles. Integration of the
independent schemes leading to the ®ve isotopomers of 9 results in an ef®cient and cost effective preparation of labeled mixtures of 13C
mono-labeled deoxyribonolactones. These mixtures are the pivotal intermediates in the preparation of `population labeled' 13C-labeled
nucleoside phosphoramidites for solid-phase oligonucleotide synthesis. q 2001 Elsevier Science Ltd. All rights reserved.
1. Introduction
The advent of effective techniques for the solid-phase synth-
esis of oligonucleotides via the coupling of nucleoside phos-
phoramidites has fostered efforts focused on the
regioselective preparation of isotopically labeled nucleo-
sides for use as monomer building blocks in the synthesis
of small DNA and RNA molecules.5 Synthetic protocols
incorporating stable isotopes into the purine and pyrimidine
bases of nucleosides have emerged,6 however, the regiose-
lective incorporation of 13C labels into the deoxyribose
backbone of nucleosides has received less attention.
Serianni and co-workers have reported the preparation of
the 1-13C and 2-13C labeled deoxyriboses,7 and NMR studies
of deoxyoligonucleotides generated from regioselectively
labeled8 and synthetically derived, uniformly labeled9 deox-
yribose have appeared. Surprisingly, effective preparations
of each of the 13C1-labeled deoxyriboses and derived oligo-
nucleotides have not been described.
Multidimensional heteronuclear NMR spectroscopy has
emerged as a powerful tool for the investigation of the solu-
tion structure and dynamics of biological macromolecules.1
An intrinsic limitation of heteronuclear NMR analysis of
biopolymers is the low natural abundance of 13C and 15N,
and recent attention has focused on strategies for isotopic
enrichment of proteins and oligonucleotides. NMR analysis
of isotopically enriched biopolymers is facilitated by an
enhanced sensitivity over natural abundance spectra that
simplify assignments and spectral editing by heteronuclear
frequencies. The observed NMR sensitivity of enriched
systems additionally allows for sensitive measurement of
13C±1H, 13C±31P, and 13C±13C coupling constants, and
provides 13C±1H relaxation data for interpreting the
dynamic properties of macromolecular systems.
Proteins enriched in 13C- and/or 15N are readily obtained
from organisms grown in isotope exclusive media and
NMR studies of these uniformly labeled peptides have
been employed in the study of protein structure.2 The analo-
gous labeling of DNA and RNA using biosynthetic tech-
niques is more problematic. Microorganisms typically
produce signi®cantly more protein than DNA and there
are relatively few naturally occurring nucleic acids with
molecular weights in the range amenable to NMR study.
Uniformly labeled RNA oligonucleotides, prepared from
degradation of relatively abundant ribosomal RNA, have
been used to construct short oligonucleotides of de®ned
sequence.3 A similar degradation-reconstitution approach
has been employed to synthesize uniformly labeled DNA
oligomers.4
As part of our program to investigate the conformational
dynamics of DNA oligomers10 we sought a strategy for
the 13C enrichment of the carbohydrate backbone which
would broadly enhance the sensitivity of multidimensional
13C NMR experiments and at the same time isolate indivi-
dual 13C nuclei, simplifying interpretation of relaxation
properties. An attractive strategy for isotopic enrichment
of the carbohydrate backbone of deoxyribonucleotides
would be realized by the synthesis of labeled oligonucleo-
tide pl-111 from monomer reagents which were themselves
mixtures of the ®ve different 13C1-labeled phosphorami-
dite12 reagents pl-2, a labeling scheme we have designated
`population labeling.' As shown in Fig. 1, the preparation of
such population labeled oligonucleotides, pl-1 would
involve three distinct synthetic phases: (1) regioselective
synthesis of the ®ve 13C1-labeled deoxyriboses, (2) combi-
nation of suitably derivatized variants of these mono-
labeled sugars and conversion of the resulting mixture to
the corresponding nucleoside phosphoramidite pl-2, and (3)
Keywords: nucleic acids; labeling; NMR; regioselection.
Corresponding author. Tel.: 1315-443-2854; fax: 1315-443-4070;
e-mail: jkallmer@syr.edu
p
0040±4020/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(00)01148-0

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M. P. Hayes et al. / Tetrahedron 57 (2001) 1515±1524
Figure 1.
solid-phase synthesis of a oligonucleotide pl-1 obtained
from the above phosphoramidite reagents. Each deoxyri-
bose subunit of the oligonucleotide 1 obtained from such
a sequence would contain a single, randomly distributed 13C
atom. If equimolar amounts of all ®ve isotopomers were
used, this would result in a net 13C enrichment of up to
21% at each of the carbon atoms of the phosphodeoxyribose
backbone.13
issues be addressed. Oligonucleotide synthesis via the phos-
phoramidite method is intrinsically inef®cient with respect
to the phosphoramidite monomers. Typical experimental
procedures call for the use of up to a 10-fold excess of the
phosphoramidite for each coupling step, a stoichiometry
that is incompatible with the use of 13C-labeled monomers
prepared by multistep synthesis. Fortunately, for our
purposes, a recently described modi®cation of the phosphor-
amidite protocol allows for ef®cient recovery and recycling
of phosphoramidite reagents.15 A potentially more dif®cult
problem involves the formidable logistics of preparing
population labeled phosphoramidite reagents. Full imple-
mentation of a population-labeling scheme would require
the formal preparation of 20 different regioisomerically-
labeled monomer reagents (four bases each having ®ve
potential labeling sites). Our plan to prepare population
labeled oligonucleotides hinged on our development of an
economically practical and logistically manageable access
to the ®ve 13C1-deoxyribose isomers.
For a population labeled oligomer, only 13C±13C coupling
would occur at natural abundance ensuring that the domi-
nant NMR relaxation pathway involves the easily inter-
preted 13C±1H dipolar mechanism. Other advantages of
site-speci®cally labeled DNA include simple measurement
of one- and multiple-bond couplings between the 13C-label
and 1H, 13C, or 31P at natural abundance to determine chemi-
cal shifts, coupling constants, and estimate dihedral angles.
The increased sensitivity of population labeled oligomers
would allow segmental labeling of larger DNA molecules,
which are currently inaccessible due to spectral overlap.
Population labeled oligomers also represent excellent candi-
dates for measurement of residual dipolar couplings in
oriented systems to accurately determine long-range geome-
trical and dynamical information.14 Additionally, synthetic
access to selectively monolabeled deoxyribose subunits
presents opportunities for extension of the population label-
ing strategy to the preparation of DNA oligomers incorpor-
ating multiply-labeled deoxyribose units, further enhancing
spectroscopic sensitivity and economy of preparation.
2. Results and discussion
2.1. Synthesis of 13C1-labeled deoxyribonolactones
The ultimate objective of preparing mixtures of optically
active, 13C1-labeled 2-deoxypentose derivatives suggested
asymmetric synthesis as an attractive source for the indivi-
dual monolabeled sugars.16 To minimize the total number of
transformations required to prepare population labeled
phosphoramidite reagents pl-2, attention was focused on
the development of an integrated synthetic plan that
Successful implementation of our plan to prepare popula-
tion labeled deoxyoligonucleotides requires that two key
Figure 2.

M. P. Hayes et al. / Tetrahedron 57 (2001) 1515±1524
1517
deoxyribonolactone 1-13C-9 in 71% yield from 7. An enan-
tiomeric excess of 92% was indicated for 1-13C-9 by analy-
sis with (1)-Eu(hfc)3.19
With an effective protocol for the asymmetric construction
of the deoxyribonolactone system in hand, we turned our
attention to the regioselective synthesis of the 13C1-labeled
isomers of masked butenediol 3. As an initial approach to
13C1-labeled 3, we examined the addition of the anion
obtained from lithiation of O-benzyl propynol 1020 to
13C1-dimethylformamide (DMF), affording the sensitive
C1-labeled aldehyde 11 (Scheme 2). Reduction of 11 and
stereoselective transformation of the resulting propargyl
alcohol to the desired E-alkenol were accomplished in a
single step by treatment with LiAlH4, to give allylic alcohol
1-13C-12 in 30% overall yield, based on consumed 13C1-
DMF. We attribute the relative inef®ciency of this sequence
to the marginally effective addition of alkyne 10 to DMF, a
transformation typically accompanied by poor yields.21
Addition of alkenyl anions to DMF constitutes a more reli-
able synthetic entry to unsaturated aldehydes, and we there-
fore examined a more ef®cient route to 1-13C-12 based on
addition of a suitably functionalized propenyl nucleophile to
13C1-labeled DMF. Transmetalation of the readily available
stannane 1322 and addition of the resulting alkenyl lithium
reagent to 13C1-DMF gave aldehyde 14 which was reduced
without further puri®cation to afford the desired alcohol
1-13C-12 in 60% overall yield. Epoxidation of 1-13C-12
afforded epoxide 1-13C-7 (94% enantiomeric excess by
Mosher ester analysis23) which was subjected to
Scheme 1. Reagents: (a) K13CN, KI, EtOH±H2O, D, (b) HCl, H2O.
would accommodate the combination of mono-labeled
intermediates at early stages of our overall scheme. From
the dual perspective of convergence and conceptual simpli-
city, the well-studied nucleophilic homologation of opti-
cally active epoxy alcohols 4, prepared by Sharpless
asymmetric epoxidation,17 presented an attractive scenario
for the asymmetric construction of 13C1-deoxyribose deri-
vatives.18 Capture of 13C1-labeled epoxide 4 by a suitable
formate anion equivalent would establish the deoxyribose
system of 6, reducing the task of preparing ®ve 13C1-labeled
deoxyribonolactones to the more manageable challenge of
accessing 13C1-labeled E-1,4-butenediols, 3 (Fig. 2).
The ef®cacy of this scheme was demonstrated by the synth-
esis of C1-labeled lactone 1-13C-9. Epoxide (2R,3R)-7,
prepared in 94% enantiomeric excess by Sharpless epoxida-
tion of O-benzyl-1,4-butendiol,17 was treated with 13C-
labeled potassium cyanide using Ganem's general protocol
(Scheme 1) to effect Payne rearrangement and subsequent
incorporation of the cyano group.18 Hydrolysis of the result-
ing hydroxy nitrile 8 under acidic conditions was accompa-
nied by lactonization to yield the C1-labeled
Scheme 2. Reagents: (a) nBuLi, THF, 2788C, H13C(O)NMe2; (b) LiAlH4, Et2O, 08 to 258C; (c) MeLi, Et2O, 2788C, H13C(O)NMe2; (d) DIBAL, Et2O, 2788C;
(e) TBHP, Ti(OiPr)4, d-(2)-DIPT, DCM, 2208C; (f) KCN, KI, EtOH±H2O, D, then HCl, H2O.
Scheme 3. Reagents: (a) Bu3SnH, (1.3 equiv.), AIBN, 808C; (b) nBuLi, THF, 2788C, H13C(O)NMe2; (c) DIBAL, Et2O, 2788C; (d) KH, BnBr, DME; (e)
TBAF, H2O; (f) TBHP, Ti(OiPr)4, d-(2)-DIPT, DCM, 2208C; (g) KCN, KI, EtOH±H2O, D then HCl, H2O.

1518
M. P. Hayes et al. / Tetrahedron 57 (2001) 1515±1524
Scheme 4. Reagents: (a) P(OEt)3, re¯ux; (b) LDA, THF, BnOCH2CHO (20); (c) DIBAL, Et2O, 2788C; (d) TBHP, Ti(OiPr)4, d-(2)-DIPT, DCM, 2208C;
(e) KCN, KI, EtOH±H2O, D, then HCl, H2O.
Scheme 5. Reagents: (a) O3, DCM, 2788C; (b) LDA, THF, 19, 2788C; (c) DIBAL, Et2O, 2788C; (d) KH, BnBr, DME; (e) TBAF; (f) TBHP, Ti(OiPr)4,
d-(2)-DIPT, DCM, 2208C; (g) KCN, KI, EtOH±H2O, D, then HCl, H2O.
nucleophilic homologation with cyanide ion and subsequent
acid-catalyzed lactonization to give the C2-labeled deoxy-
ribonolactone 2-13C-9.
addition to 13C-DMF yielded 17, which was reduced without
further puri®cation to the silyl-protected alcohol 18. At this
juncture, the butenediol system of 18 was inverted by benzy-
lation and desilylation to form the C4-labeled alcohol 4-13C-
12. Sharpless epoxidation of 4-13C-12, followed bycyanation
and lactonizaion of the resulting oxirane 4-13C-7, afforded
the C5-labeled deoxyribonolactone 5-13C-9.
The latent symmetry of the 1,4-butenediol system suggested
a conceptually similar scheme for the preparation of the C5-
labeled lactone 5-13C-9 (Scheme 3) of 12. Hydrostannylation
of silyl-protected 15 produced a 7:1 mixture of 16 and the
regioisomeric 2-tributylstannylpropene. Lithiation of 16 and
Our strategy for introduction of 13C-label at C3 and C4 of
the deoxyribose system focused on the preparation of the C2
and C3-13C-labeled analogs of 3. Incorporation of 13C label
from phosphonate 19 (prepared in three steps from 2-13C
acetic acid24) was accomplished by Wadsworth±Emmons±
Horner addition of 19 to O-benzylglycaldehyde 2025 to give
the unsaturated ester 21 (Scheme 4). Further chemoselective
reduction with DIBAL yielded allylic alcohol 2-13C-12 in
49% overall yield from 19. Conversion of 2-13C-12 to
deoxyribonolactone 3-13C-5 was accomplished by our
standard sequence via epoxide 2-13C-7.
An analogous scheme was employed for the preparation of
lactone 4-13C-9. Addition of 19 to silyl-protected aldehyde
23 yielded E-unsaturated ester 24, which upon reduction
furnished alcohol 3-13C-12. Conversion to 4-13C-9 was
accomplished as described above (Scheme 5).
A representative 2D-spectrum of 5-13C-9 (Fig. 3) illustrates
the ,90-fold increase in sensitivity compared to natural
abundance for a regioselectively 13C-labeled ribonolactone.
The upper panel shows a region from the HSQC acquired
with 4 scans per increment (the minimum number for the
pulse program), and shows only 1H±13C-5. The lower
1
Figure 3. H-13C HSQC of 5-13C-9.

M. P. Hayes et al. / Tetrahedron 57 (2001) 1515±1524
1519
Figure 4. (a) The complete 13C NMR spectra of pl-9. Non-enriched peaks are as follows: benzyl group-137.2, 128.5, 128.0, 127.6, 73.7; DC offset glitch-
100.0; CDCl3-triplet at 77.0. (b) Quantitative 13C of pl-9. Each panel is a 3 ppm portion of the entire spectrum.
Scheme 6. Reagents: (a) KH, BnBr, DME; (b) TBAF; (c) TBHP, Ti(OiPr)4, d-(2)-DIPT, DCM, 2208C; (d) KCN, KI, EtOH±H2O, D, then HCl, H2O.

1520
M. P. Hayes et al. / Tetrahedron 57 (2001) 1515±1524
HSQC spectrum required 64 scans per increment. The
natural abundance signals are shown with only one or two
contours; the labeled peak is so intense that its t2-and t1-
noise ridges dominate the spectrum.
300 MHz spectrometers unless otherwise noted. Proton
chemical shifts are reported in d, using the residual
CHCl3, as internal reference (7.26 ppm), unless otherwise
noted. J values are given in Hz. Carbon chemical shifts are
reported in d, using CDCl3, as an internal reference
(77.0 ppm), unless otherwise noted. Optical rotations were
measured on either Jasco DIP-1000 or a Perkin±Elmer 241
spectrometer. Enantiomeric excess was determined by
either Mosher ester derivatization (ME),23 or by chiral
shift analysis (CSA) with (1)-Eu(hfc)3.19 THF and DME
were distilled from sodium benzophenone ketyl under a
nitrogen atmosphere. Dichloromethane, benzene and pyri-
dine were distilled from calcium hydride before use.
Hexanes, ethyl acetate, ether, and anhydrous ether were
used from the supplier without further puri®cation. Silica
gel (230±400 mesh) was used for ¯ash column chromato-
graphy. TLC analysis was performed on Whatman K6F
silica gel 60 plates and were stained with one of the follow-
ing: anisaldehyde (method A), phosphomolybdic acid
(PMA) (method B). Reactions were performed in ¯ame or
oven dried glassware under a nitrogen or argon atmosphere
where appropriate. Reagents were used from suppliers with-
out puri®cation unless otherwise indicated.
Combination of equimolar quantities of the ®ve 13C1-labeled
lactones affords the corresponding population-labeled
mixture, pl-9. The 13C NMR spectrum of this mixture,
shown in Fig. 4a, demonstrates the 20-fold enhanced 13C
sensitivity of at each position of the lactone ring in compar-
ison to the unlabeled 5-O-benzyl group. Relative values for
the degree of isotopic substitution at each labeled position
were obtained by quantitative 13C NMR (Fig. 4b). The
chemical shift, integration, and position of each carbon
are indicated. An integrated scheme, in which labeled inter-
mediates are strategically combined at key stages of the
synthesis, offers an alternative entry to pl-9 with signi®cant
gains in ef®ciency and economy (Scheme 6). Thus, 13C1-
labeled alcohols 18 and 25 can be combined and the result-
ing mixture converted to the mixture of alcohols 3-13C-12
and 4-13C-12. Further combination with stoichiometric
quantities of alcohols 1-13C-12 and 2-13C-12, followed by
our standard sequence, yields a mixture of the 4 mono
labeled lactones 9. Final addition of a stoichiometric amount
of 1-13C-9 yields the population-labeled mixture pl-9. Over-
all, this integrated scheme results in the savings of 8
synthetic operations over the independent syntheses and
combination of the individual 13C1-labeled lactones, and
provides an ef®cient route to population-labeled mixtures
of 9, where each 13C is present at ,20%.
HSQC spectra were acquired on a Bruker Avance 500 MHz
spectrometer, using standard pulse programs with gradients.
HSQC spectra were collected with 4 scans per increment, or
64 scans per increment (depending on whether the natural
abundance peaks were desired), into 2048£512 points with
no sample spinning and no zero-®lling. The sweep width
was 3255 Hz in F2 and 12500 Hz in F1. A squared sine
window function was applied in F2 and F1.
3. Conclusions
In contrast to quantitative 13C NMR acquisition parameters
typically employed for unlabeled compounds,26 we have
found the following parameters practical for 13C-labeled
compounds: an inverse gated pulse program collected 32 k
points per scan with a 60 second delay time and a sweep
width of 22675 Hz. Data so acquired was zero-®lled to 64 k
and transformed with no window function.
In conclusion, we have successfully prepared a series of
regioselectively labeled 13C1-deoxyribonolactones, com-
pounds that represent key intermediates for the preparation
of 13C-labeled nucleotides and carbohydrate-backbone-
labeled DNA oligomers. Simple modi®cations of our
general synthetic scheme can be used to prepare all possible
13C-isotopomers of deoxyribose, and will provide access to
multiply-labeled deoxyribose derivatives and a wide range
of alternative population-labeling scenarios. For example,
4.1.1. [1-13C]-(E)-4-Benzyloxy-but-2-en-1-ol. Route A
(1-13C-12). To a solution of benzyl ether 10 (1.02 g,
7.02 mmol) in 5 mL THF at 2788C was added dropwise
nBuLi (2.37 mL, 2.5 M in hexanes). The resulting solution
was stirred at 2788C for 30 min, at which time 13C-DMF
(0.4 g, 5.4 mmol) was added and the mixture was allowed to
warm to ambient temperature over 3 h. The mixture was
diluted with 10 mL ether and washed successively with
5 mL portions of 10% HCl, sat. aq. NaHCO3, and brine.
The organic layer was dried with MgSO4, ®ltered and
concentrated to give the crude aldehyde 11 as a dark oil.
The crude reaction product was typically subjected directly
to LiAlH4 reduction, but could be further puri®ed by ¯ash
chromatography (5:1 hexanes/EtOAc) to give 11 as a pale
doubly
labeled
13C1,13Cx-deoxyribose
derivatives
(xˆ2,3,4,5) are readily available from the four 13C-7 isoto-
pomers by using K13CN in the ®nal homologation/ring-
closure step of our sequence. Similar modi®cations to the
general synthetic scheme described herein will yield other
multiply-labeled schemes of increased ef®ciency and econ-
omy and which represent useful tools for the NMR spectro-
scopy of nucleic acids. Efforts in these areas are the subject
of ongoing investigations and will be the subject of future
reports from these laboratories.
1
oil. IR (neat): 1632; H NMR: 4.37 (s, 2H), 4.63 (s, 2H),
7.36 (s, 5H), 9.25 (d, Jˆ194.8 Hz, 1H); 13C NMR: 176.2.
4. Experimental
4.1. General
To a 08C solution of aldehyde 11 in 8 mL ether was slowly
added LiAlH4 (0.204 g, 5.4 mmol) and the resulting mixture
was allowed to warm to ambient temperature and stirred for
12 h. The reaction mixture was re-cooled to 08C and
quenched by sequential addition of 1 mL of H2O, 2 mL
IR spectra were recorded on a Perkin Elmer Paragon 1000
FT spectrometer, with sodium chloride plates, and are
1
reported in wavenumbers (cm21). H- and 13C NMR were
taken on Bruker Avance 300, and General Electric QE-

M. P. Hayes et al. / Tetrahedron 57 (2001) 1515±1524
1521
20% aq. NaOH, and 1 mL brine. The resulting mixture was
diluted with 30 mL ether, ®ltered, and concentrated to give a
pale oil. Flash chromatography (5:1 hexanes/EtOAc)
afforded (288 mg, 30% based on consumed 13C DMF) of
alcohol 1-13C-12 as a pale oil. Rfˆ0.29 (1:1 hexanes/EtOAc)
Jˆ2.15 Hz, 2H); 13C NMR: 3.4, 13.7, 26.0, 29.1, 29.7, 61.4,
104.9, 148.5.
4.1.4. [1-13C]-(E)-4-(tert-Butyl-dimethyl-silanyloxy)-but-
2-en-1-ol (18). To a stirred solution of vinyltin reagent
(1.806 g, 4.05 mmol) in 6 mL THF was added n-BuLi
(1.62 mL, 2.5 M in hexanes) at 2788C for 1 h. 13C-DMF,
which was prestirred in BaO for 2 h at 2788C, in 2 mL THF
was added to the vinyltin solution and the resulting mixture
was stirred at 2788C for 2 h. The reaction was quenched by
pouring into a separatory funnel containing 10 mL, 10%
HCl, and diluted with 50 mL ether. The organic layer was
washed with another portion of 10 mL 10% HCl. The
organic layer was then separated and dried with MgSO4,
®ltered, and concentrated to give a yellow oil, which was
puri®ed by ¯ash chromatography to give a yellow oil
1
method A; IR (neat): 3427; H NMR: 1.82 (br s, 1H), 4.04
(d, Jˆ4.8 Hz, 1H), 4.15 (dd, Jˆ142.4, 4.5 Hz, 2H), 5.89 (m,
2H), 7.34 (m, 5H); 13C NMR: 62.9; HRMS (m/z): [M]1
calcd for [13C]C10H14O2, 179.1027; found, 179.0998.
4.1.2. [1-13C]-(E)-4-Benzyloxy-but-2-en-1-ol. Route B
(1-13C-12). To a solution of stannane 13 (1.765 g,
4.09 mmol) in 5 mL THF at 2788C was added nBuLi
(1.84 mL, 2.5 M in hexanes). The mixture was stirred for
2 h at 2788C, and was then added to a 08C solution of 13C-
DMF (200 mg, 2.70 mmol) in 1 mL THF. The reaction
mixture was stirred for 2.5 h and poured into a separatory
funnel containing 5 mL 10% HCl and 10 mL ether. The
organic layer was separated and washed with two additional
5 mL portions of HCl. The aqueous layer was extracted with
25 mL of ether, and the combined organic layers were dried
over MgSO4, ®ltered, and concentrated to give crude alde-
hyde 14 as a yellow oil. The crude reaction product was
typically subjected directly to DIBAL reduction, but could
be further puri®ed by ¯ash chromatography (4:1 hexanes/
EtOAc) to give (344 mg, 72%) of 14 as a pale oil. IR (neat):
1
(374 mg, 69% from 13C-DMF). IR (neat): 1659; H NMR:
0.08 (s, 6H), 0.90 (s, 9H), 4.44 (m, 2H), 6.42 (m, 1H), 6.89
(m, 1H), 9.60 (dd, Jˆ8.0 and 171.3 Hz, 1H); 13C NMR:
193.2.
The aldehyde was taken up in 9 mL CH2Cl2, and treated
with DIBAL (2 eq, 3.72 mL, 1.0 M in hexanes) at 2788C.
After 35 min, the reaction was quenched by addition of
3 mL MeOH. The quenched reaction mixture was diluted
with 40 mL of CH2Cl2, 3 mL of 3.0 M Rochelle salt, and
stirred overnight. The layers were separated and the aqueous
layer was extracted with two 35 mL portions of CH2Cl2. The
combined organic layers were dried over MgSO4, ®ltered,
and concentrated to give a yellow oil which was puri®ed by
¯ash chromatography (2:1 hexanes/EtOAc) to provide a
yellow oil (339 mg, 90%). Overall yield from two steps:
62%. Rfˆ0.48 (2:1 hexanes/EtOAc) method A; IR (neat):
3365; 1H NMR: 0.08 (s, 6H), 0.90 (s, 9H), 4.16 (dd, Jˆ4.8
and 142.2 Hz, 2H), 4.39 (d, Jˆ4.8 Hz, 2H), 5.84 (m, 2H);
13C NMR: 63.2; HRMS (m/z): [M1H]1 calcd for
[13C]C10H15O2, 204.1600; found, 204.1493.
1
1652; H NMR: 4.30 (dd, Jˆ1.45, 5.54 Hz, 2H), 4.60 (s,
2H), 6.45 (m, 2H), 6.89 (m, 2H), 9.59 (dd, Jˆ7.85,
172.5 Hz, 1H); 13C NMR: 193.2.
Crude aldehyde 14 was taken up in 5 mL anhydrous ether,
cooled to 2788C and treated with 3.9 mL DIBAL (2 equiv.,
1.0 M in hexanes). The reaction mixture was stirred for 2 h
at 2788C and quenched by careful addition of 2 mL MeOH
and 1 mL H2O. The resulting mixture was diluted with
20 mL ether and treated with 3 mL aq. Rochelle salt.
After transferring to a separatory funnel the aqueous layer
was extracted with 10 mL ether and the combined organic
layers were dried over MgSO4, ®ltered, and concentrated.
The residual oil was puri®ed by ¯ash chromatography (1:1
hexanes/EtOAc) to give (288 mg, 60% based on consumed
4.1.5. [4-13C]-(E)-4-Benzyloxy-but-2-en-1-ol (4-13C-12).
To a stirred solution of alcohol 17 (205 mg, 1.0 mmol) in
5 mL DME was added NaH (27 mg, 1.1 mmol) at 08C. The
reaction was stirred for 10 min, at which time benzyl bro-
mide (207 mg, 1.2 mmol) was added and the mixture
allowed to warm to ambient temperature. After 3 h, the
mixture was diluted with ether and transferred to a separa-
tory funnel. The organic layer was washed with 10 mL H2O,
and then dried with MgSO4, ®ltered, and concentrated to
give a yellow oil which was puri®ed by ¯ash chromato-
graphy (10:1 hexanes/EtOAc) to give of the benzyl ether
as a yellow oil. IR (neat): 3395; 1H NMR: 014 (s, 6H), 0.98
(s, 9H), 4.14 (dd, Jˆ142.3 and 4.8 Hz, 2H), 4.33 (d,
Jˆ4.8 Hz, 2H), 4.56 (d, Jˆ5.3 Hz, 2H), 5.89 (m, 2H),
7.36 (m, 5H); 13C NMR: 70.2.
1
13C-DMF) of 1-13C-12 as a yellow oil. IR (neat): 3427; H
NMR: 1.82 (br s, 1H), 4.04 (d, Jˆ4.8 Hz, 1H), 4.15 (dd,
Jˆ4.5 and 142.4 Hz, 2H), 5.89 (m, 2H), 7.34 (m, 5H); 13C
NMR: 62.9; HRMS (m/z): [M]1 calcd for [13C]C10H14O2,
179.1027; found, 179.0998.
4.1.3. tert-Butyl-dimethyl-(3-tributylstannanyl-allyloxy)-
silane (16). To a round bottom ¯ask containing silyl ether
15 (3.15 g, 18.5 mmol) was added Bu3SnH (7.01 g,
24.1 mmol) and AIBN (300 mg, 1.85 mmol). The resulting
mixture was heated to 808C for 2 h and then cooled to
ambient temperature resulting in a 7:1 mixture of products,
as determined by GC. Preparative GC (20% SE-30 on 60/80
Chromosorb W) gave two clear oils. Major product:
Rfˆ0.90 (2:1 hexanes/EtOAc) method B; IR (neat):
To a solution of benzyl ether (400 mg, 1.52 mmol) in 10 mL
THF at 08C was added 3.0 mL TBAF (1.0 M, 3.0 mmol).
After stirring for 30 min, the reaction mixture was diluted
with ether and transferred to a separatory funnel where the
organic layer was washed with H2O. The aqueous layer was
extracted with ether and the combined organic layers were
dried over MgSO4, ®ltered, and concentrated to give a pale
oil. Puri®cation by ¯ash chromatography (1:1 hexanes/
EtOAc) afforded (280 mg, 63%) of allylic alcohol, 4-13C-
1
2989.5; HNMR: 0.07 (s, 6H), 0.88 (m, 6H), 0.91 (s, 9H),
0.96 (s, 9H), 1.29 (m, 6H), 1.54 (m, 6H), 4.21 (d, Jˆ3.7 Hz,
2H), 6.07 (m, 1H), 6.15 (d, Jˆ19.1 Hz, 1H); 13C NMR: 9.4,
13.7, 26.0, 27.3, 29.1, 66.7, 126.9, 147.3. Minor product: IR
1
(neat): 2989.5; H NMR: 0.01 (s, 3H), 0.60 (s, 3H), 0.884
(m, 6H), 0.912 (s, 9H), 0.930 (s, 9H), 1.297 (m, 6H), 1.552
(m, 6H), 4.281 (m, 2H), 5.171 (d, Jˆ2.15 Hz, 2H), 5.86 (d,

1522
M. P. Hayes et al. / Tetrahedron 57 (2001) 1515±1524
12 as a pale yellow oil. IR (neat): 3380; 1H NMR: 1.69 (br s,
1H), 4.04 (dd, Jˆ5.1, 141.4 Hz, 2H), 4.16 (d, Jˆ4.4 Hz,
2H), 4.54 (s, 2H), 5.89 (m, 2H), 7.34 (m, 5H); 13C NMR:
70.2; HRMS (m/z): [M]1 calcd for [13C]C10H14O2,
179.1027; found, 179.1007.
en-1-ol (1.327 mg, 6.5 mmol) was dissolved in 25 mL
DME and cooled to 08C and then treated with washed
NaH (334 mg, 14.3 mmol). The mixture was stirred at 08C
for 5 min and benzyl bromide (1.34 g, 7.85 mmol) was
added in 2 mL DME. The reaction was then stirred at ambi-
ent temperature for 10 h. The reaction was quenched with
H2O, separated and the organic layer extracted with ether,
dried over MgSO4, ®ltered, and concentrated. The crude
product was dissolved in 70 mL THF and then treated
with TBAF (20 mL, 1.0 M in THF). The mixture was stirred
at ambient temperature for 5 h. The reaction was then trans-
ferred to a separatory funnel and the organic layer was
washed with brine, dried over MgSO4, ®ltered, and concen-
trated. The crude product was puri®ed by ¯ash chromato-
graphy to afford (920 mg, 79%) of a yellow oil. IR (neat):
3537.0; 1NMR: 1.54 (br s, 1H), 4.04 (m, 2H), 4.18 (m, 2H),
4.53 (s, 2H), 5.86 (dm, 155.5, 1H), 5.92 (m, 1H), 7.34 (m,
5H); 13C NMR: 127.8; HRMS (m/z): [M]1 calcd for
[13C]C10H14O2, 179.1027; found, 179.1010.
4.1.6. [2-13C]-(E)-4-Benzyloxy-but-2-en-1-ol (2-13C-12).
[2-13C]-(E)-4-Benzyloxy-but-2-enoic acid methyl ester
(100 mg, 0.49 mmol) was dissolved in 5 mL ether, cooled
to 08C and then treated with DIBAL (1.3 mL, 1.0 M in
hexanes). The reaction was stirred at 08C for about
10 min. The reaction was quenched with H2O, and treated
with Rochelle salt (satd. soln., 5 mL) and stirred for another
1 h. The organic phase was then separated, dried, and
concentrated. The crude product was puri®ed by ¯ash chro-
matography (10:1 hexanes/EtOAc) to afford the allylic alco-
1
hol (55 mg, 76%). IR (neat): 3536.2; H NMR: 1.61 (br s,
1H), 4.04 (m, 2H), 4.16 (m, 2H), 4.53 (s, 2H), 5.85 (m, 1H),
5.92 (dm, Jˆ149.0 Hz, 1H), 7.34 (m, 5H); 13C NMR: 132.2;
HRMS (m/z): [M]1 calcd for [13C]C10H14O2, 179.1027;
found, 179.1001.
4.2. Representative procedure for Sharpless epoxidation
of 4-benzyloxy-but-2-en-1-ols
4.1.7. [2-13C]-(E)-4-(tert-Butyl-dimethyl-silanyloxy)-but-
2-enoic acid methyl ester (24). To a suspension of washed
NaH (90 mg, 3.75 mmol) in 8 mL DME at ambient
temperature was added dropwise a solution of [2-13C]-
(diethoxy-phosphoryl)-acetic acid methyl ester (784 mg,
3.7 mmol) in 2 mL DME. The resulting solution was stirred
for 1 h, at which time a solution of (tert-butyl-dimethyl-
silanyloxy)-acetaldehyde (974 mg, 5.6 mmol) in 2 mL
DME was added dropwise. The reaction mixture was stirred
at ambient temperature for 1 h and then quenched by addi-
tion of 1 mL H2O. The mixture was transferred to a separa-
tory funnel and extracted with two 10 mL portions of ether
and then combined organic phases washed with H2O and
brine, dried over MgSO4, ®ltered, and concentrated. The
residual dark oil was puri®ed by ¯ash chromatography to
afford (550 mg, 64%) as an oil. Rfˆ0.58 (3:1 hexanes/
4.2.1.
[1-13C]-(2R,3R)-(3-Benzyloxymethyl-oxiranyl)-
methanol (1-13C-7). To a round bottom ¯ask was added
Ê
powdered, activated 4 A molecular sieves (120 mg), and a
stir bar. The round bottom ¯ask was then ¯ame dried and
allowed to cool to ambient temperature under nitrogen.
Methylene chloride (3.5 mL), d-(2)-diisopropyl tartrate
(34 mg, 0.146 mmol), and Ti(OiPr)4 (32 mg, 0.114 mmol)
were added sequentially, and the resulting heterogeneous
solution was cooled to 2258C (CO2 (s), CCl4) and stirred
for 30 min. TBHP was then added at 2258C, and the solu-
tion was aged for 30 min. Meanwhile, the allylic alcohol
(100 mg, 0.558 mmol) was diluted in 2.5 mL CH2Cl2 and
Ê
stirred over 4 A molecular sieves while the catalyst was
aging. After the aging period the allylic alcohol was
added to the catalyst and the reaction mixture was stirred
at 2258C for 1 h before being placed in a 2208C freezer
overnight. The reaction mixture was quenched by addition
of dimethyl sul®de at 2208C and allowing the solution to
warm to ambient temperature over 2 h. The quenched
mixture was ®ltered through a frit and concentrated to
yield a yellow crude product that was puri®ed by ¯ash chro-
matography to afford (86.6 mg, 79%) as a colorless oil.
1
EtOAc) method A; IR (neat): 1723; NMR: 0.10 (s, 6H),
0.94 (s, 9H), 3.76 (s, 3H), 4.35 (m, 2H), 6.12 (ddt,
Jˆ165.33, 15.5, 2.27 Hz, 1H), 7.03 (ddd, Jˆ15.5, 3.24,
3.12 Hz, 1H); 13C NMR: 119.2; HRMS (m/z): [M1H]1
calcd for [13C]C10H22O3Si, 232.1450; found, 232.1449.
4.1.8. [2-13C]-(E)-4-(tert-Butyl-dimethyl-silanyloxy)-but-
2-en-1-ol (17). The [2-13C]-(E)-4-(tert-Butyl-dimethyl-sila-
nyloxy)-but-2-enoic acid methyl ester 19 (200 mg,
0.87 mmol) was dissolved in 10 mL ether, cooled to 08C
and then treated with DIBAL (1.74 mL, 1.0 M in hexanes).
The reaction was stirred at 08C for about 30 min. The reac-
tion was then quenched with H2O, treated with Rochelle salt
(sat. soln., 5 mL) and stirred for 2 h. The organic phase was
separated, dried over MgSO4, ®ltered, and concentrated.
The crude product was puri®ed by ¯ash chromatography
(10:1 hexanes/EtOAc) to afford (126 mg, 72%). IR (neat):
Rfˆ0.25
(1:1
hexanes/EtOAc);
eeˆ94%
(ME);
[a]D ˆ119.627 (c 0.24, CHCl3); IR (neat): 3427.9,
25
1
2862.7, 1496.9, 1103.4; H NMR: 1.75 (br s, 1H), 3.10
(m, 1H), 3.25 (m, 1H), 3.52 (dd, Jˆ5.5 Hz, 11.5, 1H),
3.66 (dm, Jˆ142.7 Hz, 1H), 3.77 (dd, Jˆ2.9, 11.5 Hz,
1H), 3.95 (m, Jˆ143.3 Hz, 1H), 4.57 (ABq, Jˆ12.2 Hz,
2H), 7.32 (m, 5H); 13C NMR: 61.1; HRMS (m/z): [M]1
calcd for [13C]C10H14O3, 195.0976; found, 195.1014.
4.2.2.
[2-13C]-(2R,3R)-(3-Benzyloxymethyl-oxiranyl)-
1
methanol (2-13C-7). Eeˆ90%(ME); [a]D ˆ119.0 (c
0.31, CHCl3); IR (neat): 3427.9, 2862.7, 1496.9, 1103.4;
1H NMR: 1.75 (br t, 1H), 3.10 (dm, Jˆ173.4 Hz, 1H),
3.25 (m, 1H), 3.52 (ddd, Jˆ2.5,5.5, 11.5 Hz, 1H), 3.66
(m, 1H), 3.77 (dt, Jˆ2.7, 11.5 Hz, 1H), 3.95 (m, 1H), 4.57
(ABq, Jˆ12.2 Hz, 2H), 7.32 (m, 5H); 13C NMR: 55.7;
HRMS (m/z): [M]1 calcd for [13C]C10H14O3, 195.0976;
found, 195.1011.
25
3346.7, 2919.8, 2848.6, 1463.7, 1250.2; H NMR: 0.09 (s,
6H), 0.92 (s, 9H), 4.20 (m, 4H), 5.80 (m, 1H), 5.62 and 6.15
(dm, Jˆ154.8 Hz, 1H); 13C NMR: 128.9; HRMS (m/z):
[M]1 calcd for [13C]C10H22O2Si, 203.1423; found,
203.1424.
4.1.9. [3-13C]-(E)-4-Benzyloxy-but-2-en-1-ol (3-13C-12).
The [2-13C]-(E)-4-(tert-butyl-dimethyl-silanyloxy)-but-2-

M. P. Hayes et al. / Tetrahedron 57 (2001) 1515±1524
1523
4.2.3.
[3-13C]-(2R,3R)-(3-Benzyloxymethyl-oxiranyl)-
HRMS (m/z): [M1H]1 calcd for [13C]C11H15O4, 224.1004;
found, 224.1008.
methanol (3-13C-7). Eeˆ93%(ME); [a]D ˆ118.6 (c
0.42, CHCl3); IR (neat): 3427.9, 2862.7, 1496.9, 1103.4;
1H NMR: 1.75 (br s, 1H), 3.10 (m, 1H), 3.25 (m,
Jˆ226.5 Hz, 1H), 3.52 (m, 1H), 3.66 (m, 1H), 3.77 (m,
1H), 3.95 (dm, Jˆ143.3 Hz, 1H), 4.57 (ABq, Jˆ12.2 Hz,
2H), 7.32 (m, 5H); 13C NMR: 54.7; HRMS (m/z): [M]1
calcd for [13C]C10H14O3, 195.0976; found, 195.0994.
25
4.3.4.
dihydro-furan-2-one
[4-13C]-(4S,5R)-5-Benzyloxymethyl-4-hydroxy-
(4-13C-9).
Eeˆ93%(CSA);
25
[a]D ˆ12.3 (c 0.21, CHCl3); IR (neat): 3433.7, 1776.7;
1NMR: 2.29 (br s, 1H), 2.48 (td, Jˆ2.7, 18.0 Hz, 1H),
2.98 (ddd, Jˆ2.2, 6.7, 18.0 Hz, 1H), 3.68 (dd, Jˆ3.7,
10.5 Hz, 1H), 3.74 (m, 1H), 4.49 (m, 1H), 4.56 (ABq,
Jˆ11.8 Hz, 2H), 4.59 (dm, Jˆ153.4 Hz, 1H), 7.34 (m,
5H); 13C NMR: 86.4; HRMS (m/z): [M1H]1 calcd for
[13C]C11H15O4, 224.1004; found, 224.1008.
4.2.4.
[4-13C]-(2R,3R)-(3-Benzyloxymethyl-oxiranyl)-
methanol (4-13C-7). Eeˆ95%(ME); [a]D ˆ119.0 (c
0.28, CHCl3); IR (neat): 3427.9, 2862.7, 1496.9, 1103.4;
1H NMR: 1.75 (br t, 1H), 3.10 (m, 1H), 3.25 (m, 1H),
3.52 (ddd, Jˆ5.3, 11.5, 141.4 Hz, 1H), 3.66 (m, 1H), 3.77
(ddd, Jˆ2.6, 11.2, 126.8 Hz, 1H), 3.95 (m, 1H), 4.57
(dABq, Jˆ4.4, 12.2 Hz, 2H), 7.32 (m, 5H); 13C NMR:
69.6; HRMS (m/z): [M]1 calcd for [13C]C10H14O3,
195.0976; found, 195.1015.
25
4.3.5.
dihydro-furan-2-one
[5-13C]-(4S,5R)-5-Benzyloxymethyl-4-hydroxy-
(5-13C-9).
Eeˆ95%(CSA);
[a]D2512.4 (c 0.16, CHCl3); IR (neat): 3433.7, 1776.7;
1NMR: 1.07 (br s, 1H), 2.48 (dd, Jˆ2.8, 18.0, 1H), 2.98
(dd, Jˆ6.8, 18.0, 1H), 3.68 (dd, Jˆ10.6, 143.2H), 3.74
(dd, Jˆ2.9, 10.5 Hz, 1H), 4.49 (m, 1H), 4.56 (ABq,
Jˆ11.9, 2H), 4.59 (m, 1H), 7.34 (m, 5H); 13C NMR: 69.4;
HRMS (m/z): [M1H]1 calcd for [13C]C11H15O4, 224.1004;
found, 224.1005.
4.3. Representative procedure for preparation of [13C]-5-
O-benzyl-2-deoxyribonolactones
4.3.1.
[1-13C]-(4S,5R)-5-Benzyloxymethyl-4-hydroxy-
dihydro-furan-2-one (1-13C-9). To a solution of epoxide
7 (2.94 g, 15 mmol) in 120 mL of 2:1 EtOH/H2O, was
added K13CN (1.5 g, 22.5 mmol) and KI (24 mg,
0.15 mmol). The reaction mixture was heated at re¯ux for
48 h, cooled to ambient temperature and carefully acidi®ed
to pH 2 with 10% HCl. The resulting mixture was warmed
to 608C and stirred for 16 h, at which time the reaction
mixture was cooled and transferred to a separatory funnel
and the aqueous layer was extracted with three 50 mL
portions of Et2O. The combined ether extracts were dried
over MgSO4, ®ltered, and concentrated. The crude product
was puri®ed by ¯ash chromatography (2:1 hexanes/EtOAc)
to give (2.19 g, 71%) of lactone 1-13C-9 as a colorless oil.
Acknowledgements
Support for this work in the form of research grants from the
National Institutes of Health (GM-35069, GM-32691). A
generous gift of 2-13C acetic acid from Cambridge Isotope
Laboratories, through their Grants Program is gratefully
acknowledged.
References
1. (a) Ikura, M.; Kay, L. E.; Bax, A. Biochemistry 1990, 29,
4659. (b) Kay, L. E.; Clore, G. M.; Bax, A.; Gronenborn,
A. M. Science 1990, 249, 411. (c) Robertson, A. D.; Rhyu,
G. I.; Westler, W. M.; Markley, J. L. Biopolymers 1990, 29,
461. (d) Cavanagh, J.; Fairbrother, W. J.; Palmer III, A. G.;
Skelton, N. J. Protein NMR Spectroscopy: Principles and
Practice, Academic: San Diego, CA, 1996. (e) Clore, G. M.;
Gronenborn, A. M. NMR of Proteins, CRC: Boca Raton, FL,
1993.
25
eeˆ92% (CSA); [a]D ˆ12.3 (c 0.23, CHCl3); Rfˆ0.27
1
(1:1 hexanes/EtOAc); IR (neat): 3434, 1777; NMR: 2.09
(br d, 1H), 2.48 (ddd, Jˆ2.6, 5.1, 17.9 Hz, 1H), 2.98 (td,
Jˆ6.6, 18.0 Hz, 1H), 3.68 (dd, Jˆ3.5, 10.5 Hz, 1H), 3.74
(dd, Jˆ3.0, 10.5 Hz, 1H), 4.49 (m, 1H), 4.56 (ABq,
Jˆ11.8 Hz, 2H), 4.59 (m, 1H), 7.34 (m, 5H); 13C NMR:
176.0; HRMS (m/z): [M1H]1 calcd for [13C]C11H15O4,
224.1004; found, 224.1005.
2. (a) Reilly, D.; Fairbrother, W. J. J. Biomol. NMR 1994, 4, 459.
(b) Hansen, A. P.; Petros, A. M.; Mazer, A. P.; Pederson,
T. M.; Rueter, A.; Fesik, S. W. Biochemistry 1992, 31, 12713.
3. (a) Batey, R. T.; Battiste, J. L.; Williamson, J. R. Methods
Enzymol. 1995, 261, 300. (b) King, G. C.; Harper, J. W.; Xi,
Z. Methods Enzymol. 1995, 261, 436. (c) Nikonowicz, E. P.;
Sirr, A.; Legault, P.; Jucker, F. M.; Baer, L. M.; Pardi, A.
Nucleic Acid Res. 1992, 20, 4507.
4.3.2.
dihydro-furan-2-one
[2-13C]-(4S,5R)-5-Benzyloxymethyl-4-hydroxy-
(2-13C-9).
Eeˆ94%(CSA);
25
[a]D ˆ12.3 (c 0.18, CHCl3); IR (neat): 3433.7, 1776.7;
1NMR: 2.11 (br s, 1H), 2.48 (ddd, Jˆ2.7, 18.0, 136.1 Hz,
1H), 2.98 (ddd, Jˆ6.8, 18.0, 133.7 Hz, 1H), 3.68 (dd, Jˆ3.7,
10.5 Hz, 1H), 3.74 (dd, Jˆ3.7, 10.5 Hz, 1H), 4.49 (m, 1H),
4.56 (ABq, Jˆ11.8 Hz, 2H), 4.59 (m, 1H), 7.34 (m, 5H); 13C
NMR: 38.4; HRMS (m/z): [M1H]1 calcd for
[13C]C11H15O4, 224.1004; found, 224.1004.
4. (a) Masse, J. E.; Bortmann, P.; Dieckmann, T.; Feigon, J.
Nucleic Acids Res. 1998, 26, 2618. (b) Louis, J. M.; Martin,
R. G.; Clore, G. M.; Gronenborn, A. M. J. Biol. Chem. 1998,
273, 2374. (c) Smith, D. E.; Su, J. Y.; Jucker, F. M. J. Biomol.
NMR 1997, 10, 245. (d) Zimmer, D. P.; Crothers, D. M. Proc.
Natl. Acad. Sci. USA 1995, 92, 3091.
4.3.3.
dihydro-furan-2-one
[3-13C]-(4S,5R)-5-Benzyloxymethyl-4-hydroxy-
(3-13C-9).
Eeˆ90%(CSA);
25
[a]D ˆ12.2 (c 0.14, CHCl3); IR (neat): 3433.7, 1776.7;
1NMR: 2.09 (br s, 1H), 2.48 (ddd, Jˆ2.4, 5.3, 18.0 Hz,
1H), 2.98 (ddd, Jˆ0.9, 6.6, 18.0 Hz, 1H), 3.68 (m, 1H),
3.74 (m, 1H), 4.49 (m, 1H), 4.56 (ABq, Jˆ11.8 Hz, 2H),
4.59 (dm, Jˆ153.4 Hz, 1H), 7.34 (m, 5H); 13C NMR: 69.6;
5. For a review see: Serianni, A. S.; Barker, R. 11. In Isotopes in
the Physical and Biomedical Sciences, Buncel, E., Jones, J. R.,
Eds.; Elsevier: Amsterdam, 1987; pp 211.
6. (a) SantaLucia, Jr., J.; Shen, L. X.; Cai, Z.; Lewis, H.; Tinoco,

1524
M. P. Hayes et al. / Tetrahedron 57 (2001) 1515±1524
Jr., I. Nucleic Acids Res. 1995, 23, 4913. (b). Williamson, J. R.;
Boxer, S. G. Nucleic Acid Res. 1988, 16, 1529.
Nucleic Acids Res. 1984, 12, 6827. (f) Hubbard, A. J.;
Jones, A. S.; Walker, R. T. Nucleic Acids Res. 1984, 12, 6827.
13. Unlabeled sites still contribute 1.1% natural abundance.
Therefore, if 1/5 of the mixture is a 3-13C1-labeled lactone,
and the other 4/5 of the mixture is 1.1% then (1/5)p99%1(4/
5)p1.1<21%.
14. (a) Tjandra, N.; Bax, A. Science 1997, 278, 1111. (b) Hansen,
M. R.; Hanson, P.; Pardi, A. Methods Enzymol. 2000, 317,
220.
7. (a) Synder, J. R.; Serianni, A. S. Carbohydr. Res. 1987, 163,
169. (b) Hayes, M. L.; Pennings, N. J.; Serianni, A. S.; Barker,
R. J. Am. Chem. Soc. 1982, 104, 6764. (c) Kline, P. C.;
Serianni, A. S. J. Am. Chem. Soc. 1990, 112, 7373. (d) Wu,
J.; Serianni, A. S. Biopolymers 1994, 34, 1175. (e) In addition,
Serianni and Bondo have described the syntheses of 26 of the
32 13C-labeled isotopomers of d-ribose. See: Serianni, A. S.;
Bondo, P. B. J. Biomol. Struct. Dyn. 1994, 11, 1133.
15. Scremin, C. L.; Zhou, L.; Srinivasacher, K.; Beaucage, S. L. J.
Org. Chem. 1994, 59, 1963.
8. (a) Gaudin, R.; Paquet, F.; Chanteloup, L.; Beau, J. -M.;
Thuong, N. T.; Lancelot, G. J Biomol. NMR 1995, 5, 49. (b)
Bornet, O.; Lancelot, G.; Chanteloup, L.; Nguyen, T. T.; Beau,
J. M. J. Biomol. NMR 1994, 4, 575.
16. McGarvey, G. J.; Kimura, M.; Oh, T.; Williams, J. M. J.
Carbohydr. Chem. 1984, 3, 125.
17. (a) Katsuki, T.; Lee, A. W. M.; Ma, P.; Martin, V. S.; Masa-
mune, S.; Sharpless, K. B.; Tuddenham, D.; Walker, F. J. J.
Org. Chem. 1982, 47, 1373. (b) Ma, P.; Martin, V. S.; Masa-
mune, S.; Sharpless, K. B.; Viti, S. M. J. Org. Chem. 1982, 47,
1378.
9. (a) Ono, A.; Tate, S.; Ishide, Y.; Kainosho, M. J. Biomol. NMR
1994, 4, 581. (b) Tate, S.; Ono, A.; Kainosho, M. J. Magn.
Reson. B 1995, 106, 89. (c) Tolbert, T. J.; Williamson, J. R. J.
Am. Chem. Soc. 1997, 119, 12100.
18. Wrobel, J. E.; Ganem, B. J. Org. Chem. 1983, 48, 3761. A
conceptually similar approach was employed for the synthesis
of a perdeuterated deoxyribose derivative: see Goering, B. K.;
Lee, K.; An, B.; Cha, J. K.; J. Org. Chem. 1993, 58, 1100.
19. Fraser, R. R.; Petit, M. A.; Saunders, J. K. J. Chem. Soc.,
Chem. Commun. 1971, 1450.
10. (a) Borer, P. N.; LaPlante, S. R.; Hakkinen, A.; Kumar, A.;
Levy, G. C.; Martin, A.; Zanatta, N. Biochemistry 1994, 33,
2441. (b) LaPlante, S. R.; Hakkinen, A.; Levy, G. C.; van der
Marel, J.; Wang, A. H. -J.; Zanatta, N.; Borer, P. N. Biochem-
istry 1994, 33, 2430. (c) Ashcroft, J.; LaPlante, S. R.; Borer,
P. N.; Cowburn, D. J. Am. Chem. Soc. 1989, 111, 363. (d)
LaPlante, S. R.; Ashcroft, J.; Cowburn, D.; Levy, G. C.; Borer,
P. N. J. Biomol. Struct. Dyn. 1988, 5, 1089. (e) Borer, P. N.;
LaPlante, S. R.; Zanatta, N.; Levy, G. C. Nucleic Acids Res.
1988, 16, 2323. (f) Stone, M. P.; Winkle, S. A.; Borer, P. N. J.
Biomol. Struct. Dyn. 1986, 3, 767. (g) Borer, P. N.; Zanatta,
N.; Holak, T. A.; Levy, G. C.; van Boom, J. H.; Wang, A. H. -
J. J. Biomol. Struct. Dyn. 1984, 1, 1373.
20. Benzyl ether 10 and silyl ether 15 were synthesized from
propargyl alcohol and the appropriate protecting group using
common conditions.
21. (a) Jones, E. R. H.; Skattebol, L.; Whiting, M. C. J. Chem. Soc.
1958, 1054. (b) Journet, M.; Dongwei, C.; DiMichele, L. M.;
Larsen, R. D. Tetrahedron Lett. 1998, 39, 6427.
22. Jung, M. E.; Light, L. A. Tetrahedron Lett. 1982, 3851.
23. (a) Dale, J. A.; Mosher, H. S. J. Am. Chem. Soc. 1973, 95, 512.
(b) Sullivan, G. R.; Dale, J. A.; Mosher, H. S. J. Org. Chem.
1973, 38, 2143.
11. Labels for compounds are given two designations: (1) either
the position of the 13C-label, or pl for population labeled (e.g.
pl-1), and (2) a number for the class of compound. Therefore,
all epoxides are 7, but 4-13C-7 is 13C labeled at the 4 position.
24. Samuel, S. P.; Niu, T.-Q.; Erickson, K. L. J. Am. Chem. Soc.
1989, 111, 1429.
p
In ®gures the 13C-label is indicated by .
25. Both 20 and 23 were synthesized by the following procedure:
(1) di-protection of 2-butene-1,4-diol with either TBSCl or
BnBr and (2) ozonolysis of the double bond.
12. (a) Beaucage, S. L.; Caruthers, M. H. Tetrahedron Lett. 1981,
22, 1859. (b) McBride, L. J.; Caruthers, M. H. Tetrahedron
Lett. 1983, 24, 245. (c) Beaucage, S. L.; Iyer, R. P.
Tetrahedron 1992, 48, 2223. (d) Caruthers, M. H.; Barone,
A. D.; Beaucage, S. L.; Dodds, D. R.; Fisher, E. F.; McBride,
L. J.; Matteucci, M.; Stabinsky, Z.; Tang, J. Y. Methods
Enzymol. 1987, 154, 287. (e) Tanaka, T.; Letsinger, R. L.
26. Braun, S.; Kalinowski, H.-O.; Berger, S. 150 and More Basic
NMR Experiments. Wiley-VCH: New York, 1998; pp 298.
27. The literature value for (2S,3S)-(3-benzyloxymethyl-oxira-
24
nyl)-methanol: (aD ˆ221 see Ref. 15b).