Incorporation of deuterium-labelled analogs of isopentenyl diphosphate for the elucidation of the stereochemistry of rubber biosynthesis

Article abstract of DOI:10.1039/b515750a

A series of six deuterium-labelled analogs of isopententyl diphosphate (IPP) was prepared to investigate the detailed stereochemical course of addition of C₅ units during rubber biosynthesis in Hevea brasiliensis and Parthenium argentatum. These analogs were incorporated into the cis-polyisoprene chain by rubber transferase in rubber particles, and the stereochemistry was determined by ²H-NMR analysis of the polymer or of levulinic acid derivatives obtained from its ozonolytic degradation. Results indicate that rubber chain elongation occurs with loss of the pro-S hydrogen of IPP, addition of the allylic diphosphate to the si face of IPP and inversion of stereochemistry at the carbon bearing the diphosphate. The Royal Society of Chemistry.

Full text of DOI:10.1039/b515750a

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Incorporation of deuterium-labelled analogs of isopentenyl diphosphate for
the elucidation of the stereochemistry of rubber biosynthesis
Andrew A. Scholte and John C. Vederas*
Received 7th November 2005, Accepted 4th January 2006
First published as an Advance Article on the web 23rd January 2006
DOI: 10.1039/b515750a
A series of six deuterium-labelled analogs of isopententyl diphosphate (IPP) was prepared to investigate
the detailed stereochemical course of addition of C5 units during rubber biosynthesis in Hevea
brasiliensis and Parthenium argentatum. These analogs were incorporated into the cis-polyisoprene
chain by rubber transferase in rubber particles, and the stereochemistry was determined by 2H-NMR
analysis of the polymer or of levulinic acid derivatives obtained from its ozonolytic degradation.
Results indicate that rubber chain elongation occurs with loss of the pro-S hydrogen of IPP, addition of
the allylic diphosphate to the si face of IPP and inversion of stereochemistry at the carbon bearing the
diphosphate.
Introduction
Rubber is a vital raw material and is used in the production of
40 000 commercial products, including over 400 medical devices.1
Although there are more than 2500 known species of plants that
produce rubber, the primary commercial source of natural rubber
is the Brazilian rubber tree, Hevea brasiliensis.2 The H. brasilinesis
crop consists predominately of plantation-grown clonal trees.
This has resulted in a lack of genetic diversity making the crop
susceptible to pathogenic attack.3 Therefore, there is considerable
interest in developing an alternate commercial source of high
quality rubber, for example from the desert shrub, Parthenium
argentatum (guayule).4
Natural rubber (cis-1,4-polyisoprene) biosynthesis is catalyzed
by rubber transferase (EC 2.5.1.20), a membrane bound cis-
prenyltransferase.5,6 This enzyme controls the elongation of the
Scheme 1 Rubber biosynthesis.
terminal allylic diphosphate of the growing chain by isopentenyl
diphosphate 1 (IPP) to form rubber 3 (Scheme 1). The process
is usually initiated with a trans allylic diphosphate, for example
farnesyl diphosphate 2 (FPP), but all subsequent isoprene units
have cis geometry. Rubber transferase also requires a divalent
metal cofactor such as Mg2+ or Mn2+ for activity.7
Prenyltransferases, the enzymes that catalyze the formation
of linear prenyl chains, have been studied extensively. In 1966,
Cornforth et al. delineated the stereochemical outcome of the
Scheme 2 Stereochemical outcome of farnesyl diphosphate synthase.
trans-prenyltransferase, farnesyl diphosphate (FPP) synthase, the
enzyme that synthesizes the C15 isoprenoid farnesol skeleton.8,9
Cornforth and coworkers also investigated the stereochemistry
By using a series of deuterium labelled (4R)- and (4S)-mevalonic
of natural rubber biosynthesis. Incorporation of labelled (4R)
acid substrates, they were able to show that the reaction between
and (4S)-[4-3H1] mevalonate with crude plant homogenates from
IPP and dimethylallyl diphosphate 4 (DMAPP) occurs with
H. brasiliensis was consistent with the elimination of the pro-
overall inversion of stereochemistry of the allylic diphosphate
S hydrogen.10 However, as it has since been demonstrated that
plants biosynthesize IPP through both the cytosolic mevalonic
acid pathway and the plastidal methylerythritol 4-phosphate
pathway,11,12 the significance of Cornforth’s experiments with
with concomitant elimination of the pro-R proton of IPP to
form the trans-alkene of the intermediate geranyl diphosphate 5
(Scheme 2).
rubber is less clear.
Studies with undecaprenyl diphosphate (UPP) synthase (a cis-
Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada
prenyltransferase)13 and heptaprenyl disphosphate synthase (a
T6G 2G2. E-mail: john.vederas@ualberta.ca; Fax: 00 1 780 492 2134;
Tel: 00 1 780 492 5475
trans-prenyltransferase)14 led to the axiom that the pro-R hydrogen
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of IPP is eliminated during the biosynthesis of trans-prenyl chains
while the pro-S hydrogen is eliminated for cis-prenyl chains.
However, examination of the biosynthesis of polyprenols from
the plant Mallotus japonicus illustrated that, contrary to this
hypothesis, the pro-R hydrogen is eliminated during the biological
formation of the cis-prenyl chains of malloprenols.15
The stereochemical arrangement during the cis C–C bond
formation in the biosynthesis of UPP was reported by Ogura
and coworkers. Incorporation studies with deuterium-labelled
analogs of IPP revealed that reaction occurs on the si face of
the double bond in IPP.16,17 Related investigations with the trans-
prenyltransferases, heptaprenyl diphosphate synthase and FPP
synthase, indicate that the C–C bond formation also occurs at the si
face of IPP.17,18 In contrast, studies with FPP synthase from the pea
plant, Pisum sativum, indicate that addition occurs on the re face
of IPP, which is in disagreement with Cornforth’s stereochemical
picture of isoprenoid biosynthesis.19,20
Since there are differences between cis-prenyltransferases in the
stereochemistry of the hydrogen elimination from IPP, it cannot
be stated a priori that rubber transferase will proceed via loss of the
pro-R or pro-S proton. Moreover, in order to generate a detailed
picture of the stereochemical constraints exerted by rubber
transferase, two other stereochemical issues must be addressed,
namely which face (re or si) of the double bond in IPP participates
in its addition to the growing rubber chain, and whether this attack
on the terminal allylic diphosphate proceeds with retention or
inversion of stereochemistry. Generally, the displacement of the
allylic diphosphate by IPP has been shown to occur with inversion
of stereochemistry at the primary allylic carbon.9,21,22 Knowledge
of the three dimensional aspects of the rubber transferase reaction
can assist understanding of the mechanism of this enzyme that
is responsible for production of a major commodity. It also
allows comparison to other prenyltransferases and may help in
the development of new rubber producing systems, potentially
including new rubber crops.
In the present work, we elucidate the cryptic stereochemistry of
rubber biosynthesis by synthesis and incorporation of deuterium-
labelled analogs of IPP 6–11 (Fig. 1) followed by deuterium
NMR analysis of the resulting rubber or its degradation products.
Incorporations of compounds 6 and 7 into biosynthetically active
rubber particles from H. brasiliensis and P. argentatum can be
used to distinguish which face of the alkene participates in its
addition to the growing rubber chain, whereas utilisation of 8
and 9 determines the stereochemistry of hydrogen elimination.
Incorporation of labelled IPP analogs 10 and 11 verifies the
stereochemistry of displacement of the allylic diphosphate during
rubber biosynthesis.
Results and discussion
Substrates 6,23 7,23 and 824 were prepared as described previously.
The synthesis of (S)-[2-2H1]-IPP 9 is analogous to 8 and is
shown in Scheme 3. Addition of methylmagnesium bromide to L-
glyceraldehyde acetal25,26 12 and oxidation of the resulting alcohol
with TPAP and NMO provides the methyl ketone 13. Peterson
olefination of 13 and deprotection gives 14, which is tosylated in
pyridine at 0 ◦C to afford 15. Ring closure under basic conditions
generates the epoxide and treatment with sodium borodeuteride
in the presence of boron trifluoride–etherate produces the enan-
tiospecifically deuterium labelled alcohol, which is further reacted
with tosyl chloride to yield the tosylate 16. The enantiomeric purity
of 16 was determined to be >95% ee.27 Phosphorylation of 16
with tris(tetra-n-butyl)ammonium hydrogen pyrophosphate28 in
MeCN yields (S)-[2-2H1]-isopentenyl diphosphate 9.
The chiral diphosphates 10 and 11 can be prepared by phos-
phorylation of the corresponding chiral alcohols (Scheme 4).
Reaction of the Grignard reagent derived from allylic chloride
Scheme 3 Synthesis of (S)-[2-2H1]-IPP 9. Reagents and conditions:
˚
(i) MeMgBr, Et2O; (ii) TPAP, NMO, 4 A MS, 39%; (iii) LiCH2Si(Me)3
Et2O, −78 ◦C, 80%; (iv) HCl, EtOH, 75%; (v) TsCl, pyridine, 71%;
(vi) KOH; (vii) NaBD3CN, BF3·OEt2; (viii) TsCl, Et3N, DMAP, 26%;
(ix) (Bu4N)3HP2O7, MeCN, 87%.
Scheme 4 Synthesis of (S)- and (R)-[1-2H1]-IPP 10 and 11. Reagents and
conditions: (i) Mg, THF, D, 2 h; (ii) CO2, 39%; (iii) LiAlD4, Et2O, 70%;
(iv) IBX, DMSO; (v) (S)-Alpine borane, THF; (vi) TsCl, Et3N, DMAP,
10% for 21 and 11% for 22 (over 3 steps); (vii) (R)-Alpine borane, THF;
(viii) (Bu4N)3HP2O7, MeCN, 87% for 10 and 86% for 11.
Fig. 1 Synthetic target molecules: deuterium analogs of IPP 6–11.
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17 with CO2 affords the allylic acid 18. Reduction of the acid
with LiAlD4 provides the di-deuterio alcohol 19. Oxidation of 19
using PCC results in contamination of the desired product 20 by
the corresponding conjugated aldehyde. This problem is readily
circumvented by the using the mild IBX oxidant.29,30
R
Reaction of the labelled aldehyde 20 with (S)-Alpine-Boraneꢀ
(b-isopinocampheyl-9-boracyclo[3.3.1]nonane)31 gives (1R)-[1-
2H1]-3-methylbuten-1-ol. The use of this boron reagent has been
recently described for the preparation of chiral deuterium labelled
allylic diphosphates by Coates and coworkers.32 Stereochemical
analysis using Mosher’s ester33 indicates that the alcohol has an
ee of 90%. Treatment of this chiral alcohol with tosyl chloride
affords the tosylate 21, which is then converted to the desired (1S)-
[1-2H1]-IPP 10, by reaction with tris(tetra-n-butyl)ammonium
hydrogen pyrophosphate. Analogously, reduction of the aldehyde
Scheme 5 Stereochemistry of hydrogen loss during formation of rubber
by H. brasiliensis and P. argentatum.
diphosphate displacement (inversion or retention) during rubber
biosynthesis. Previously, the stereochemical analysis of farnesyl
diphosphate synthase has been accomplished by ozonolysis of the
labelled farnesol, obtained after incorporation of 4-deuterio IPPs,
to give (R)- or (S)-[3-2H1]-levulinic acid.18 The stereochemistry
of the levulinic acid was correlated with known optical rotations
values for stereospecifically deuterated levulinic acid.16 However,
this analysis relies on quite small optical rotation values to
differentiate the enantiomers. Since the enzyme preparations used
for the biosynthesis of rubber contain a large amount of existing
unlabelled rubber and only produce a small amount of new labelled
material, this particular procedure could not be employed for
stereochemical analysis.
R
20 with (R)-Alpine-Boraneꢀ and subsequent reaction with tosyl
chloride gives 22, which is then transformed into the enantiomeric
target, (1R)-[1-2H1]-IPP 11 (90% ee). The yields of 21 and 22 are
not optimized and are low due to volatility of the intermediate
aldehyde.
Stereochemistry of hydrogen loss during alkene formation
Initially the stereochemistry of the hydrogen elimination step
was investigated. Incorporations of specifically deuterated IPP
analogs 8 and 9 should result in retention of deuterium in one
case and in loss in the other. This can be easily detected by
2H-NMR analysis of the resulting rubber product. Thus, each
of the stereospecifically deuterated IPP’s, (R)-[2-2H1]-IPP 8 and
(S)-[2-2H1]-IPP 9 were independently incorporated into rubber
by incubation with rubber particles from H. brasiliensis and P.
argentatum containing rubber transferase. The resulting rubber
samples were isolated and analyzed by 2H-NMR spectroscopy.
Analyses of the rubber resulting from the incorporations of (S)-
[2-2H1]-IPP 9 show no increase in the intensity of the olefinic
signals. However, 2H-NMR analysis in CHCl3 of the rubber
isolated from incorporation of (R)-[2-2H1]-IPP 8 using either
species displays an increase in the intensity of the signal at 5.3 ppm
(Fig. 2). These results are consistent with pro-S hydrogen elimina-
tion and pro-R hydrogen retention during rubber biosynthesis in
both Hevea and Parthenium (Scheme 5). This outcome is also in
agreement with elimination of the pro-S hydrogen observed during
the biosynthesis of cis-prenyl chains by the bacterial enzyme UPP
synthase.13
An alternative solution would involve converting the deuterated
levulinic acids to their mandelate esters. H-NMR spectroscopy
2
could then be used to determine the stereochemistry of the
levulinic acid obtained by ozonolytic degradation of rubber
isolated from the enzymatic reactions. High resolution 500 MHz
NMR analysis of the mandelate ester of levulinic acid in benzene-
d6 reveals that all of the diastereotopic hydrogens at C-2 and at
C-3 can be differentiated. However, to assign the signals to the
pro-R or pro-S hydrogens at each carbon with certainty, synthesis
of labelled standards was necessary.
Chiral [2-2H]-levulinic acids appeared available by the hydrolysis
of the corresponding nitrile, which in turn, can be accessed by
displacement of a tosylate (Scheme 6). Ozonolysis of (1R)-tosylate
21 in CH2Cl2 at −78 ◦C followed by reduction with zinc and
acetic acid gives the methyl ketone 23. Reaction of 23 with sodium
cyanide in DMSO generates the volatile nitrile.34 Hydrolysis of the
deuterated nitrile with 6 M HCl produces the stereospecifically
labelled levulinic acid 25, which can be transformed into ester
27 by coupling with methyl (S)-(+)-mandelate. (2R)-Levulinate
Fig. 2 2H-NMR (76.5 MHz, CHCl3) spectrum of rubber from incorpo-
ration of (R)-[2-2H1]-IPP 8 by rubber particles from H. brasiliensis.
Scheme 6 Synthesis of deuterium labelled levulinate standards. Reagents
and conditions: (i) O3, −78 ◦C; (ii) Zn, AcOH, 97% for 23 and 90% for 24;
(iii) NaCN, DMSO; (iv) HCl, 34% for both 25 and 26 (over two steps);
(v) methyl (S)-(+)-mandelate, DCC, DMAP, 41% for both 27 and 28.
Stereochemical outcome of diphosphate displacement
Analogs of IPP 10 and 11 with stereospecific deuterium labels at
the 1-position were used to determine the stereochemistry of the
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28 can be made in an analogous manner from (1S)-tosylate 22.
2H NMR analysis in C6H6 shows that the deuterium in 27 has a
chemical shift of 2.48 ppm, whereas that in 28 is at 2.44 ppm. This
difference in chemical shifts is somewhat smaller than expected
based on precedents with unfunctionalised alkyl chains studied by
Parker35 or having remotely situated carbonyls examined by our
group.36,37
Incorporation of (1S)-[1-2H1] IPP 10 into rubber particles from
H. brasiliensis containing active rubber transferase-generated new
rubber. Ozonolysis of the rubber in CHCl3 gave levulinic acid,
which was then converted into its corresponding mandelate ester.
2H-NMR analysis in C6H6 reveals that the incorporated deuterium
has a chemical shift of 2.48 ppm. Correspondingly, incorporation
of (1R)-[1-2H1]-IPP 11 ultimately affords a mandelate ester whose
deuterium chemical shift in C6H6 is 2.44 ppm. These results
indicate that as expected displacement occurs with an overall
inversion of stereochemistry at the primary allylic carbon of
the growing rubber chain (Scheme 7). Analogous incorporation
studies with rubber particles from P. argentatum also show
inversion of stereochemistry at the primary allylic carbon.
Scheme 8 Synthesis of deuterium labelled levulinate standards. Reagents
and conditions: (i) LiAlD4, Et2O, 27%; (ii) IBX, DMSO, 91%;
(iii) (−)-DIP-Cl, THF; (iv) TsCl, Et3N, DCC, DMAP, 75% for 32 and 86%
for 33; (v) (+)-DIP-Cl, THF; (vi) lithium acetylide–diethylamine, DMSO;
(vii) Hg(OAc)2, AcOH; (viii) NaBH4, 42% for both 36 and 37 (over
two steps); NaIO4, RuCl3, CCl4–MeCN–H2O, 24% for both 38 and 39;
(x) methyl (S)-(+)-mandelate, DCC, DMAP, 41% for both 40 and 41.
oxidized with IBX to [1-2H1]-phenylethanal 31. Reduction with
(−)-b-chlorodiisopinocampheylborane39 affords the deuterated
alcohol and its subsequent reaction with tosyl chloride and Et3N
generates the tosylate 32. Displacement of the primary sulfonate
32 with lithium acetylide–diethylamine complex gives the terminal
acetylide 34. Reaction of the acetylide with a stoichiometric
amount of mercury (II) acetate followed by reduction of the
carbon–mercury bond with sodium borohydride and oxidation
of the secondary alcohol provides the desired methyl ketone 36
with no loss of the deuterium label.
Scheme 7 Use of 10 and 11 to determine stereochemistry of diphos-
phate displacement reaction. Reagents and conditions: (i) O3, CH2Cl2;
(ii) HCO2H, H2O2; (iii) methyl (S)-(+)-mandelate, DCC, DMAP.
Stereochemical direction of carbon–carbon bond formation
During the biosynthesis of rubber, a new carbon–carbon bond is
formed between the double bond of IPP and the allylic carbon of
the growing rubber chain. The face of the double bond (si or re)
that reacts can potentially be determined by incorporation of Z-
and E-[4-2H1]-IPP into rubber by the transferase. Ozonolysis of
the resulting rubber would give (3R)- or (3S)-levulinic acid, whose
methyl (S)-mandelate esters could be correlated with authentic
standards by 2H-NMR analysis.
The method described in Scheme 6 might initially seem
reasonable for the synthesis of the required standards, but the
rapid exchange of the ketone a-protons during hydrolysis of
the nitrile precludes this approach. Instead, mild hydration of
a terminal acetylene to its corresponding methyl ketone was
explored to circumvent this problem for construction of 38 and
39 (Scheme 8).38 Reduction of phenylacetic acid 29 with lithium
aluminum deuteride gives the deutero-alcohol 30, which can be
Oxidation of the phenyl group with ruthenium trichloride and
periodic acid gives the desired product, but with the loss of the
deuterium label. However, non-acidic oxidation of (3R)-butanone
36 with ruthenium trichloride and sodium periodate40 generates
the desired deuterium labelled acid, (3R)-levulinic acid 38, which is
converted to 40. Similar reaction conditions allow synthesis of the
corresponding ester of (3S)-levulinic acid 41 from (1R)-tosylate
33. 2H-NMR analysis in C6H6 shows that levulinate ester 40 has a
deuterium chemical shift of 2.01 ppm, whereas its diastereomer 41
has a chemical shift of 2.21 ppm. The difference in chemical shifts
of these diasterotopic hydrogens is unexpectedly large compared
to the smaller differences for deuteriums that are closer to the
chiral ester group in 27 and 28.
Incorporation of E-[4-2H1]-IPP 6 into rubber particles from
H. brasiliensis containing active transferase was followed by
ozonolytic degradation to levulinic acid and conversion to the
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(S)-mandelate ester. 2H-NMR analysis in C6H6 indicates the
incorporated deuterium has a chemical of 2.21 ppm, which
corresponds to (3S)-[3-2H1]-levulinate 41. Corresponding analysis
of the rubber isolated after incorporation of Z-[4-2H1]-IPP 7 shows
that the deuterium in the (S)-mandelate ester of levulinic acid has
an R configuration These observations demonstrate that si face
of the double bond in IPP reacts during the formation of rubber
(Scheme 9). Incorporation studies with rubber particles from P.
argentatum show the same stereochemical outcome.
Scheme 10 Stereochemistry of rubber transferase.
(0.25, 0.5, 1.0 mm) with silica gel (EM Science, Kieselgel 60
F254). Analytical TLC was performed on glass plates (5 × 1.5
cm) pre-coated (0.25 mm) with silica gel (EM Science, Kieselgel
60 F254). Compounds were visualized by exposure to UV light or
by staining with a 1% Ce(SO4)2·4H2O 2.5% (NH4)Mo7O24·4H2O
in 10% H2SO4 followed by heating on a hot plate. Flash column
chromatography was performed with Silicycle silica gel (60, 230–
400 mesh).
Scheme 9 Analysis of stereochemical direction of carbon–carbon bond
formation on the IPP double bond. Reagents and conditions: (i) O3,
CH2Cl2; (ii) HCO2H, H2O2; (iii) methyl (S)-(+)-mandelate, DCC, DMAP.
High-performance liquid chromatography (HPLC) was per-
formed on a Beckman System Gold instrument equipped with
a model 166 variable wavelength UV detector and an Altex 210A
injector with a 500 or 1000 lL sample loop. The columns used
were Grace-Vydac. All HPLC solvents were prepared fresh daily
and filtered with a Millipore filtration system under vacuum before
use.
NMR spectra were recorded on a Varian Inova 600, Inova 400,
Inova 300 or Unity 500 spectrometer. For 1H (300, 400, 500 or 600
MHz) values are referenced to 7.24 ppm (CHCl3), to 7.15 ppm
(C6H6) and for 13C (100 or 125 MHz) referenced to 77.0 ppm
(CDCl3).
Optical rotations were measured on a Perkin-Elmer 241 po-
larimeter with a micro cell (100 mm path length, 1 mL) and the
values are given in 10−1 deg cm2 g−1. IR spectra were recorded
on a Nicolet Magna-IR 750 with a Nic-Plan microscope FT-IR
spectrometer. Mass spectra were recorded on a Krator AEI MS-50
(HREIMS) and a ZabSpec Isomass VG (HRESMS).
Conclusion
In summary, six deuterium-labelled analogs of IPP were prepared
to study the stereochemical outcome of rubber biosynthesis in two
plant species, H. brasiliensis and P. argentatum. Incorporation of
these IPP analogs into rubber particles and 2H-NMR analysis of
the rubber and its degradation products indicate that the stere-
ochemistry of rubber transferase in both species is identical and
similar to that of the related cis-prenyltransferase, undecaprenyl
diphosphate synthase (Scheme 10). Thus, the pro-S hydrogen (Hs)
is preferentially cleaved during the polymerization, and si face
addition to the allylic diphosphate occurs with overall inversion
of stereochemistry. This reveals the 3-dimensional arrangement of
substrates within the active site of rubber transferase, and gives a
better mechanistic understanding of how rubber is biosynthesized.
Experimental
All chemicals were purchased from Aldrich Chemical company
(Madison, WI) or Sigma Chemicals (St. Louis, MO). All sol-
vents, unless otherwise indicated, were HPLC grade and used
as such. Tetrahydrofuran (THF) and diethyl ether (Et2O) were
distilled over sodium under and an argon atmosphere. Acetonitrile
(MeCN), dichloromethane (CH2Cl2), triethylamine and pyridine
were distilled from calcium hydride. Methanol (MeOH) and
ethanol (EtOH) were distilled over magnesium turnings and a
catalytic amount of iodine. Evaporation refers to removal of
solvent under reduced pressure on a rotary evaporator. Preparative
TLC was performed on glass plates (20 × 20 cm) pre-coated
E-[4-2H1]-Isopentenyl diphosphate (6)17,23
A solution of E-[4-2H1]-3-methyl-3-buten-1-yl tosylate (85 mg,
0.35 mmol) in MeCN (10 mL) was treated with tris-n-
butylammonium hydrogen diphosphate and the resulting reaction
mixture was stirred for 18 h at room temperature. The solvent was
evaporated and the residue was dissolved in a minimal volume of
IPA–NH4HCO3 (25 mM) 1 : 49. The solution was passed through
an ion exchange column containing DOWEX AG 50W X8 (100–
200 mesh, 15 equiv.) cation exchange resin (ammonium form).
The clear eluent was lyopholized to dryness to give a white solid.
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1H NMR analysis indicated that tetra-n-butyl ammonium ion had
been exchanged for ammonium ion. The residue was dissolved in
100 mM NH4HCO3 (3 mL) and transferred to a centrifuge tube.
A solution of MeCN–IPA 1 : 1 was added, and the contents of the
tube were vortexed followed by centrifugation of the suspension
for 5 min at 2000 rpm. The supernatant was removed and the
process was repeated 3 times. The combined supernatants were
concentrated in vacuo and lyopholized. Purification of the crude
product using HPLC (Vydac 259VHP reverse phase polymer
column; 4.6 × 150 mm; 10% MeCN, 90% 100 mM NH4HCO3
20 min, 10–50% MeCN over 5 min, 50% MeCN 10 min, 50–90%
MeCN over 1 min, 90% MeCN 20 min, tR 44.0 min) afforded the
title compound 6 as a white solid (88 mg, 85%). IR (lscope) 3141
−5.10 (m, 1P); HRMS (ES −ve) calcd for [M − H]− C5H10DO7P2
246.0037, found 246.0032.
(2S)-1,2,3-Butanetriol-1,2-acetonide
Methylmagnesium bromide (11.5 mL, 3.0 M solution in Et2O,
34.5 mmol) was added dropwise to a stirring solution of (S)-
glyceraldehyde acetonide25,26 (12) (22.9 mmol) in Et2O (15 mL)
at 0 ◦C. The ice bath was removed and stirring was continued
at room temperature overnight. The reaction was quenched by
pouring the reaction mixture onto ice/saturated NH4Cl solution
and the aqueous layer was extracted with Et2O (3 × 15 mL). The
combined ethereal extracts were washed with water, brine, dried
over Na2SO4 and concentrated in vacuo. Purification of the crude
product by flash column chromatography (SiO2, pentanes–Et2O,
1 : 1) afforded the alcohol as a 1 : 1 mixture of diastereomers
(1.3 g, 39%). IR (CH2Cl2, cast) 3455, 2986, 2886, 1669, 1456,
1
(b), 3045, 2919, 1405 cm−1; H NMR (D2O–ND4OD, 500 MHz)
d the vinylic proton was obscured by solvent, 4.04 (dt, 2H, J =
6.7 Hz, 6.7 Hz CH2OP), 2.38 (t, 2H, J = 6.7 Hz, CH2CH2), 1.76
(s, 3H, C CCH3); 13C NMR (D2O–ND4OD, 125 MHz) d 144.6,
=
1
1214 cm−1; H NMR (CDCl3, 300 MHz) d 3.88–4.05 (m, 3H,
1
2
111.6 (t, JC–D = 23.6 Hz), 64.7 (d, JC–P = 5.6 Hz), 38.5, 22.1;
31P NMR (D2O–ND4OD, 162 MHz) d −8.80 (m, 1P), −5.10 (m,
1P,); HRMS (ES −ve) calcd for [M − H]− C5H10DO7P2 246.0037,
found 246.0035.
CH2CH, CHOH), 3.70 (dd, 1H, J = 6.2 Hz, 7.9 Hz, CHOC), 2.01
(b, 1H, OH) 1.43 (s, 3H, C(CH3)2), 1.37 (s, 3H, C(CH3)2), 1.16
(d, 3H, J = 6.5 Hz, CHCH3), 1.15 (d, 3H, J = 6.4 Hz, CHCH3);
13C NMR (CDCl3, 125 MHz) d 109.5, 109.1, 80.4, 79.4, 68.8, 66.8,
66.1, 64.5, 26.7, 26.5, 25.3, 25.2, 18.9, 18.3; HRMS (ES +ve) calcd
for [M + Na]+ C7H14O3Na 169.0835, found 169.0837.
Z-[4-2H1]-Isopentenyl diphosphate (7)17,23
A similar procedure was employed as that described for the
preparation of 6. The reaction of Z-[4-2H1]-3-methyl-3-buten-1-
yl tosylate (10 mg, 0.041 mmol) in MeCN (10 mL) with tris-n-
butylammonium hydrogen diphosphate (111 mg, 0.123 mmol)
gave the crude labelled IPP. Purification of the crude product
using HPLC (Vydac 259VHP reverse phase polymer column; 4.6 ×
150 mm; 10% MeCN, 90% 100 mM NH4HCO3 20 min, 10–50%
MeCN over 5 min, 50% MeCN 10 min, 50–90% MeCN over 1 min,
90% MeCN 20 min, tR 44.0 min) afforded the title compound 7
as a white solid (11 mg, 87%). IR (lscope) 3020 (b), 1608, 1559,
(3S)-3,4-Dihydroxybutanoneacetonide (13)
A solution of NMO (1.57 g, 13.4 mmol) in CH2Cl2 (20 mL)
was treated with MgSO4 and stirred at room temperature for
20 min. After removal of the drying agent by gravity filtration, 4 A
˚
molecular sieves were added followed by a solution of (2S)-1,2,3-
butanetriol-1,2-acetonide (1.29 g, 8.95 mmol) in CH2Cl2 (10 mL).
The solution was stirred for 15 min prior to the addition of TPAP
(50 mg, 0.14 mmol). The reaction was stirred for 6 h at room
temperature at which point the solution was filtered through a
plug of silica gel to remove the ruthenium catalyst. The eluent
was washed with a saturated solution of CuSO4, water, brine and
water. The organic layer was dried over MgSO4 and evaporation
of the solvent in vacuo gave the ketone 13 as a colorless oil (1.28 g,
quantitative). [a]D20 = −57.0◦ (c 1.4, CH2Cl2); IR (CH2Cl2, cast)
2990, 2938, 1720, 1420; cm−1; 1H NMR (CDCl3, 300 MHz) d 4.39
(dd, 1H, J = 7.7 Hz, 5.6 Hz, CHOC), 4.18 (dd, 1H, J = 8.6 Hz,
7.8 Hz, CH2OC), 3.98 (dd, 1H, J = 8.6 Hz, 5.6 Hz, CH2OC), 2.23
(3H, s, CH3CO), 1.48 (s, 3H, C(CH3)2), 1.38 (s, 3H, C(CH3)2);
13C NMR (CDCl3, 125 MHz) d 209.2, 111.1, 80.5, 66.5, 26.3,
1
1405 cm−1; H NMR (D2O–ND4OD, 76.5 MHz) d 4.84 (s, 1H,
=
C CH, 4.04 (dt, 2H, J = 6.7 Hz, 6.7 Hz, CH2OP), 2.38 (t, 2H,
J = 6.7 Hz, CH2CH2), 1.76 (s, 3H, C CCH3); 13C NMR (D2O–
=
1
ND4OD, 125 MHz) d 144.3, 111.6 (t, JC–D = 23.6 Hz), 66.4 (d,
2JC–P = 5.6 Hz), 38.3, 22.1; 31P NMR (D2O–ND4OD, 162 MHz) d
−9.04 (m, 1P), −5.01 (m, 1P,); HRMS (ES −ve) calcd for [M −
H]− C5H10DO7P2 246.0037, found 246.0037.
(2R)-[2-2H1]-Isopentenyl diphosphate (8)24,41
A similar procedure was employed as that described for the
preparation of 6. The reaction of (R)-[2-2H1]-isopentenyl tosylate
(10 mg, 41 lmol) in MeCN (2 mL) with tris-n-butylammonium
hydrogen diphosphate (111 mg, 0.123 mmol) gave the crude
labelled IPP. Purification of the crude product using HPLC (Vydac
259VHP reverse phase polymer column; 4.6 × 150 mm; 10%
MeCN, 90% 100 mM NH4HCO3 20 min, 10–50% MeCN over
5 min, 50% MeCN 10 min, 50–90% MeCN over 1 min, 90% MeCN
20 min, tR 44.0 min) afforded the title compound 8 as a white solid
•
26.1, 25.1; HRMS (EI) calcd for [M+ ] C7H12O3 144.0786, found
144.0789, 129 (68%), 101 (100%), 83 (18%), 73 (40%), 61 (60%).
(2S)-3-(Trimethylsilyl)methyl-1,2,3-butanetriol-1,2-acetonide
To a vigorously stirring solution of acetonide 13 (1.3 g, 9.14 mmol)
in Et2O (50 mL) was added (trimethylsilyl)methyllithium (15.5 mL,
1.0 M solution in pentanes, 15.5 mmol) dropwise at −78 ◦C. The
resulting solution was stirred at −78 ◦C for 1 h at which point
a solution of NH4Cl–NaHCO3 (20 mL) was carefully added
to quench the reaction. The aqueous layer was separated and
extracted with Et2O (3 × 15 mL). The combined ethereal extracts
were washed with water and brine, dried over MgSO4, and
concentrated in vacuo. Purification of the crude alcohol using flash
column chromatography (SiO2, pentanes–Et2O, 10 : 1) afforded
1
(11 mg, 87%). IR (lscope) 2722 (b), 1685, 1431 cm−1; H NMR
=
(D2O–ND4OD, 300 MHz) d 4.79 (m, 1H, C CH), the other vinylic
proton was obscured by solvent, 3.97 (m, 2H, CH2OP), 2.30 (m,
1H, CHDCH2), 1.68 (s, 3H, C CCH3); 13C NMR (D2O–ND4OD,
=
100 MHz) d 145.2, 112.8, 67.1 (d, 2JP–P = 5.8 Hz), 39.2 (1JC–D = 22
Hz), 23.1; 31P NMR (D2O–ND4OD, 162 MHz) d −9.00 (m, 1P),
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=
=
the alcohol as a 1 : 1 mixture of diastereomers (2.13 g, 80%). IR
(CH2Cl2, cast) 3490, 2986, 2953, 2892, 1456, 1418 cm−1; 1H NMR
(CDCl3, 300 MHz) d 3.81–3.95 (3H, m, CH2OC + CHOC), 1.44
(3H, s, C(CH3)2), 1.38 (3H, s, C(CH3)2), 1.14 (3H, s, CH3COH)
1.07 (1H, d, J = 14.7 Hz, CH2Si), 0.93 (1H, d, J = 14.7 Hz, CH2Si),
0.87 (1H, d, J = 14.7 Hz, CH2Si), 0.72 (1H, d, J = 14.7 Hz, CH2Si),
0.1 (9H, s, Si(CH3)3), 0.07 (9H, s, Si(CH3)3); 13C NMR (CDCl3,
100 MHz) d 109.4, 109.3, 83.3, 83.2, 72.2, 65.3 (2C), 30.2, 27.4,
26.7, 26.5, 25.5, 25.4, 24.5, 0.6 (3C), 0.5 (3C); HRMS (ES +ve)
calcd for [M + Na]+ C11H24O3SiNa 255.1387, found 255.1386.
300 MHz) d 5.18 (m, 1H, C CH2), 5.03 (m, 1H, C CH2), 2.88
(t, 1H, J = 4.2 Hz, CHCH2), 2.73 (dd, 1H, J = 5.3 Hz, 2.7 Hz,
13
=
CHCH2), 2.47 (m, 1H, CHCH2), 1.64 (s, 3H, C CCH3); C NMR
(CDCl3, 125 MHz) d 141.5, 114.6, 54.5, 46.8, 16.2; HRMS (EI)
calcd for [M+ ] C5H8O 84.0575, found 84.0563, 69 (35%).
•
(2S)-[2-2H1]-Isopentenol43
To a stirring solution of (R)-2-isopropenyl oxirane, sodium
cyanoborodeuteride (167 mg, 2.54 mmol) and a small amount
bromocresol green (5 mg) in Et2O (5 ml) was added dropwise a
BF3–etherate solution until the color of the solution was observed
to be yellow. The resulting mixture was stirred at room temperature
for 5 h and additional amounts of BF3–etherate were added
periodically to keep the solution acidic. The solution was diluted
with brine and extracted with Et2O (3 × 15 mL). The combined
ethereal extracts were◦dried over MgSO4 and evaporation of the
solvent in vacuo at 0 C gave the deuterio alcohol as a colorless
liquid. The volatile alcohol was partially characterized and used
(2R)-3-Methyl-3-butene-1,2-diol (14)
A
stirring solution of (2S)-3-(trimethylsilyl)methyl-1,2,3-
butanetriol-1,2-acetonide (1.19 g, 5.12 mmol) in EtOH (16 mL)
was treated with 3 M HCl (0.5 mL) and the mixture was refluxed
for 2 h. After cooling to room temperature the solution was
neutralized with the slow addition of solid NaHCO3. The
precipitated solids were filtered, washed with EtOH, and the
filtrate was concentrated in vacuo. The resulting residue was
re-dissolved in MeOH and treated with Et2O (50 mL). The
precipitated solids were filtered and the solvent was evaporated
in vacuo which afforded the title compound 14 as a colorless oil
(390 mg, 75%). [a]D20 = −15.7◦ (c 1.4, CH2Cl2); IR (CH2Cl2, cast)
3373, 3075, 2970, 2879, 1652, 1444; cm−1; 1H NMR (CDCl3,
1
in the next step without further purification. H NMR (CDCl3,
500 MHz) d 4.87 (m, 1H, C CH2), 4.79 (m, 1H, C CH2), 3.72 (m,
2H, CH2OH), 2.29 (m, 1H, CHDCH2), 1.76 (m, 3H, C CCH3).
=
=
=
(2S)-[2-2H1]-Isopentenyl tosylate (16)41
A stirring solution of (2S)-[2-2H1]-isopentenol and tosyl chloride
(67 mg, 1.91 mmol) in CH2Cl2 (5 mL) was treated with Et3N
(0.27 mL, 1.91 mmol) and DMAP (10 mg (136 mg, 1.11 mmol).
The resulting mixture was stirred at room temperature for 12 h,
at which time Et2O (50 mL) was added. Precipitated solids were
removed by filtration, and the filtrate was concentrated in vacuo.
Purification of the crude tosylate by flash column chromatography
(SiO2, hexane–EtOAc, 10 : 1) afforded the tosylate 16 as an
oil (80 mg, 26% for three steps). [a]2D0 = −0.4◦ (c 1.0, CH2Cl2);
=
=
300 MHz) d 5.06 (m, 1H, C CH2), 4.96 (m, 1H, C CH2), 4.18
(dd, 1H, J = 7.2 Hz, 3.4 Hz, CHOH), 3.70 (dd, 1H, J = 11.2 Hz,
3.5 Hz, CH2OH), 3.55 (dd, 1H, J = 11.2 Hz, 7.3 Hz, CH2OH),
2.58 (b, 2H, OH), 1.75 (m, 3H, C CCH3); 13C NMR (CDCl3,
125 MHz) d 144.1, 112.0, 75.7, 65.2, 18.9; HRMS (EI) calcd for
C5H10O2 [M+ ] 102.0681, found 102.0679, 84 (44%), 71 (100%).
=
•
(2R)-3-Methyl-3-butene-1,2-diol-1-yl tosylate (15)
1
To a stirring solution of diol 14 (225 mg, 2.20 mmol) in pyridine
IR (CH2Cl2, cast) 3078, 2971, 1652, 1495, 1177 cm−1; H NMR
(5 mL) at 0 ◦C was added freshly recrystallized tosyl chloride
(CDCl3, 300 MHz) d 7.79 (m, 2H, Ar), 7.34 (m, 2H, Ar), 4.79 (m,
◦
=
=
(462 mg, 2.42 mmol) in pyridine (10 mL) and stirred at 0 C for
1H, C CCH2), 4.68 (m, 1H, C CCH2), 4.12 (m, 2H, CH2OS),
2.45 (s, 3H, ArCH3), 2.34 (m, 1H, CHDCH2), 1.66 (m, 3H,
18 h. The solution was then poured onto ice-water and extracted
with Et2O (3 × 15 mL). The combined ethereal extracts were
washed with 1 M HCl followed by a saturated NaHCO3 solution,
water and brine, and dried over MgSO4. Evaporation of the solvent
C CCH3); 2H NMR (CHCl3, 61.4 MHz) d 2.36; 13C NMR
=
(CDCl3, 125 MHz) d 144.8, 140.1. 133.2, 129.8, 127.9, 113.2, 66.8,
•
36.4 (t, J = 19.5 Hz), 22.3, 21.7; HRMS (EI) calcd for [M+ ]
afforded the tosylate 15 as a colorless oil (564 mg, 71%). [a]D20
=
C12H15DO3S 241.0883, found 241.0877, 155 (52%), 91 (79%).
−13.3◦ (c 1.2, CH2Cl2); IR (CH2Cl2, cast) 3533, 3069, 2951, 2921,
1
1652, 1495, 1190 cm−1; H NMR (CDCl3, 500 MHz) d 7.81 (m,
(2S)-[2-2H1]-Isopentenyl diphosphate (9)41
=
2H, Ar), 7.36 (m, 2H, Ar), 5.06 (m, 1H, C CH2), 4.97 (m, 1H,
A similar procedure was employed as that described for the prepa-
ration of 6. The reaction of (2S)-[2-2H1]-isopentenyl tosylate (16)
(10 mg, 0.041 mmol) in MeCN (2 mL) with tris-n-butylammonium
hydrogen diphosphate gave the crude labelled IPP. The reaction
was worked up as previously described. Purification of the crude
product using HPLC (Vydac 259VHP reverse phase polymer
column; 4.6 × 150 mm; 10% MeCN, 90% 100 mM NH4HCO3
20 min, 10–50% MeCN over 5 min, 50% MeCN 10 min, 50–90%
MeCN over 1 min, 90% MeCN 20 min, tR 44.0 min) afforded the
title compound 9 as a white solid (11 mg, 87%). IR (lscope) 2707
(b), 1685, 1430 cm−1; 1H NMR (D2O–ND4OD, 400 MHz) d 4.99
=
C CH2), 4.31 (dd, 1H, J = 7.6 Hz, 3.2 Hz, CHOH), 4.12 (dd, 1H,
J = 10.6 Hz, 3.3 Hz, CH2OS), 3.97 (dd, 1H, J = 10.3 Hz, 7.7 Hz,
CH2OS), 2.46 (s, 3H, ArCH3), 2.06 (b, 1H, OH) 1.70 (m, 3H,
C CCH3); 13C NMR (CDCl3, 100 MHz) d 145.1, 142.0, 132.8,
=
129.9 (2C), 128.0 (2C), 113.7, 73.0, 72.4, 21.7, 18.6; HRMS (ES
+ve) calcd for [M + Na]+ C12H16O4SNa 279.0662, found 279.0660.
(R)-Isopropenyl oxirane42
Finely powdered KOH (1.0 g, 17.8 mmol) was added to tosylate
15 (325 mg, 1.27 mmol) and heated to 60 ◦C for 1 h. After
cooling to room temperature, the residue was dissolved in Et2O and
filtered through a mini-pad of Celiteꢀ to remove solid impurities.
This compound was partially characterized and used without
further purification for the subsequent reaction. 1H NMR (CDCl3,
=
(m, 2H, C CH), 4.18 (m, 2H, CH2OP), 2.53 (m, 1H, CHDCH2),
R
1.91 (s, 3H, C CCH3); 13C NMR (D2O–ND4OD, 100 MHz) d
=
145.2, 112.8, 67.1 (d, 2JP–P = 5.8 Hz), 39.2 (1JC–D = 22 Hz), 23.1;
31P NMR (D2O–ND4OD, 162 MHz) d −9.05 (m, 1P), −5.05 (m,
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1P); HRMS (ES −ve) calcd for [M − H]− C5H10DO7P2 246.0037,
to afford the aldehyde 20 as colorless oil. The product was used
without further purification as the aldehyde is very unstable to
heat or base, and isomerization to the conjugated aldehyde is
observed during storage. IR (CH2Cl2, cast) 3077, 2953, 1732,
found 246.0036.
3-Methyl-3-butenoic acid (18)44
1
1651, 1436 cm−1; H NMR (CDCl3, 500 MHz) d 5.00 (m, 1H,
In a flame dried, argon flushed round-bottom flask was added
freshly ground magnesium turnings (4.98 g, 205 mmol) and THF
(75 mL). To this suspension, a solution of 3-chloro-2-methyl-
propene (17) (10.0 g, 221 mmol) in THF (25 mL) was then added
dropwise, followed by a crystal of iodine. After the addition was
complete the reaction mixture was refluxed for 2 h and cooled
to room temperature. The reaction was stirred for 30 min and
then cooled to −78 ◦C with a dry ice–acetone bath. At this point,
CO2 gas was bubbled though the solution for 1 h and then the
temperature was slowly allowed to increase to 0 ◦C by removal
of the ice bath. Once the reaction reached 0 ◦C, it was cooled
back down to −78 ◦C with bubbling of CO2 through solution for
10 min and then the reaction mixture was allowed to warm up to
10 ◦C. The solution was basified to pH 10 with cold 2 M NaOH
and washed with Et2O (3 × 50 mL). The aqueous layer was then
acidified with cold 4 M HCl to pH 2 and extracted with Et2O
(3 × 75 mL). The combined ethereal extracts were washed with
brine, dried over MgSO4 and concentrated in vacuo to give the acid
18 as a colorless oil (4.0 g, 39%). IR (CH2Cl2, cast) 3083, 2978,
1711, 1651, 1413 cm−1; IR (CH2Cl2, cast) 3083, 2978, 1711, 1651,
=
=
C CH2), 4.84 (m, 1H, C CH2), 3.06 (s, 2H, CH2CDO), 1.78 (m,
=
3H, C CCH3).
(1R)-[1-2H1]-Isopentenyl tosylate (21)
To a stirring solution of [1-2H1]-3-methylbut-3-en-1-al (20)
(5.67 mmol) in Et2O at 0 C was added a solution of (S)-Alpine
borane (13.6 mL, 0.5 M solution in THF, 6.80 mmol) dropwise
over 20 min. After addition, the reaction was warmed to room tem-
perature and stirred for 18 h. Acetaldehyde (0.38 mL, 0.79 mmol)
was added and stirred for 5 min at which time the volatiles were
◦
◦
removed by evaporation (40 C, 0.05 mm Hg). The residue was
dissolved in Et2O and was treated with ethanolamine (0.41 mL,
6.80 mmol) and the solids were removed by filtration. The filtrate
was concentrated in vacuo and purification of the crude product by
flash column chromatography (SiO2, pentanes–Et2O, 4 : 1) affor-
ded the alcohol. Tosyl chloride (1.19 g, 6.23 mmol), Et3N (0.96 mL,
6.85 mmol) and DMAP (10 mg, 0.08 mmol) was added to a solu-
tion of the crude alcohol in CH2Cl2 (10 mL). The resulting reaction
mixture was stirred at room temperature for 18 h at which time it
was diluted with water (10 mL) and extracted with CH2Cl2. The
combined organic extracts were washed with water and brine, dried
over MgSO4 and concentrated in vacuo. The crude tosylate was pu-
rified using flash column chromatography (SiO2, hexane–EtOAc,
10 : 1) to afford the title compound 21 as a colorless oil (131 mg,
10% for three steps). [a]2D0 = +0.9◦ (c 1.5, CH2Cl2); IR (CH2Cl2,
1
1413 cm−1; H NMR (CDCl3, 300 MHz) d 10.0 (b, 1H, CO2H),
=
=
4.97 (m, 1H, C CH2), 4.90 (m, 1H, C CH2), 3.09 (d, 2H, J =
1.0 Hz, CH2), 1.85 (m, 3H, C CCH3); 13C NMR (CDCl3, 100
=
•
MHz) d 177.7, 137.9, 115.3, 43.1, 22.4; HRMS (EI) calcd for [M+ ]
C5H8O2 100.0524, found 100.0521, 85 (17%), 82 (22%), 72 (32%),
59 (37%), 55 (76%).
cast) 2927, 1649, 1364, 1177 cm−1; H NMR (CDCl3, 400 MHz)
d 7.76 (m, 2H, Ar), 7.32 (m, 2H, Ar), 4.76 (m, 1H, C CH2), 4.65
1
[1,1-2H2]-3-Methyl-3-buten-1-ol (19)22
=
=
(m, 1H, C CH2), 4.11 (tt, 1H, J = 6.9 Hz, 1.2 Hz CHDOS), 2.45
A solution of lithium aluminum deuteride (5.0 ml, 1.0 M solution
in Et2O, 5.0 mmol) was added dropwise to a stirring solution of
3-methyl-3-butenoic acid (18) (1.0 g, 10.0 mmol) in Et2O (10 mL)
at 0 ◦C. The ice bath was removed and stirring was continued for
3 h at room temperature. The reaction mixture was then cooled to
0 ◦C and quenched with slow addition of solid Na2SO4·10H2O and
stirred for 30 min. The precipitated aluminum salts were removed
by filtration and the filtrate was concentrated in vacuo to give the
alcohol 19 as a colorless liquid (0.88 g, 70%). IR (CH2Cl2, cast)
3346, 3076, 2917, 2849, 2208, 1651, 1455 cm−1; 1H NMR (CDCl3,
(s, 3H, ArCH3), 2.32 (d, 2H, J = 6.9 Hz, CH2CHD), 1.63 (m,
3H, C CCH3); H NMR (CHCl3, 76.5 MHz) d 4.11; 13C NMR
2
=
(CDCl3, 100 MHz) d 144.7, 140.2, 133.3, 129.9 (2C), 128.0 (2C),
113.1, 68.3 (t, J = 22.9 Hz), 36.7, 22.4, 21.7; HRMS (ES +ve)
calcd for [M + Na]+ C12H15DO3SNa 264.0775, found 264.0773.
(1S)-[1-2H1]-Isopentenyl tosylate (22)
A similar procedure was employed as that described for the
preparation of 21. Substitution of (R)-Alpine borane for the (S)-
Alpine borane in the procedure used to prepare the enantiomeric
tosylate 21 gave the desired compound 22. [a]2D0 = −1.8◦ (c 0.5,
CH2Cl2); IR (CH2Cl2, cast) 3078, 2926, 2200, 1653, 1598,
1365 cm−1; H NMR (CDCl3, 300 MHz) d 7.76 (2H, m, Ar),
7.32 (2H, m, Ar), 4.76 (m, 1H, C CH2), 4.65 (m, 1H, C CH2),
=
=
300 MHz) d 4.86 (m, 1H, C CCH2), 4.78 (m, 1H, C CCH2),
2
=
2.28 (s, 2H, CH2CD2), 1.75 (m, 3H, C CCH3); H NMR (CHCl3,
61.4 MHz) d 3.68; 13C NMR (CDCl3, 100 MHz) d 144.2, 112.7,
•
59.5 (qn, JC–D = 22 Hz), 40.7, 22.2; HRMS (EI) calcd for [M+ ]
1
1
=
=
C5H8D2O 88.0857, found 88.0854, 70 (98%), 56 (100%).
4.11 (tt, 1H, J = 7.0 Hz, 1.2 Hz, CHDOS), 2.45 (s, 3H, ArCH3),
2
[1-2H1]-3-Methylbut-3-en-1-al (20)22
=
2.32 (d, 2H, J = 6.8 Hz, CHDCH2), 1.63 (m, 3H, C CCH3); H
NMR (CHCl3, 76.5 MHz) d 4.11; 13C NMR (CDCl3, 100 MHz) d
To a stirring solution of IBX (4.88 g, 17.4 mmol) in DMSO
(40 mL) was added [1,1-2H2]-3-methyl-3-buten-1-ol (19) (500 mg,
5.67 mmol) and stirred at room temperature for 18 h. The solution
was cooled to 0 ◦C and then H2O (5 mL) was added the resulting
mixture was stirred for an additional 5 min at 0 ◦C. The mixture
144.8, 140.1, 133.3, 129.9 (2C), 128.0 (2C), 133.1, 68.3 (t, 1JC–D
=
23.0 Hz), 36.7, 22.4, 21.7; HRMS (ES +ve) calcd for [M + Na]+
C12H15DO3SNa 264.0775, found 264.0773.
(1S)-[1-2H1]-Isopentenyl diphosphate (10)
R
was filtered through a pad of Celiteꢀ and the filtrate was extracted
exhaustively with Et2O. The combined ethereal extracts were
washed with brine, dried over MgSO4 and concentrated in vacuo
A similar procedure was employed as that described for the prepa-
ration of 6. The reaction of (1R)-[1-2H1]-isopentenyl tosylate (21)
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(10.5 mg, 44 lmol) in MeCN (2 mL) with tris-n-butylammonium
hydrogen diphosphate (118 mg, 0.131 mmol) gave the crude
labelled IPP. Purification of the crude product using HPLC (Vydac
259VHP reverse phase polymer column; 4.6 × 150 mm; 10%
MeCN, 90% 100 mM NH4HCO3 20 min, 10–50% MeCN over
5 min, 50% MeCN 10 min, 50–90% MeCN over 1 min, 90%
MeCN 20 min, tR 44.0 min) afforded the title compound 10 as
NMR (CDCl3, 300 MHz) d 7.77 (m, 2H, Ar), 7.33 (m, 2H, Ar),
4.24 (m, 1H, CHDOS), 2.82 (d, 2H, J = 6.3 Hz, CH2CHD), 2.43
(s, 3H, ArCH3) 2.13 (s, 3H, CH3CO); 2H NMR (C6H6, 76.5 MHz)
d 4.24.
(4S)-[4-2H1]-2-Butanone-4-yl tosylate (24)
Substitution of (1S)-[1-2H]-isopentenyl tosylate (22) for (1R)-[1-
2H]-isopentenyl tosylate (21) in the procedure used to prepare the
enantiomeric tosylate 23 gave the title compound 24. Chromato-
graphic properties and spectral data were identical.
a white solid (10 mg, 87%). IR (lscope) 2722 (b), 1685, 1431 cm−1;
1
=
H NMR (D2O–ND4OD, 600 MHz) d 5.10 (m, 1H, C CH), 5.08
=
(m, 1H, C CH) 4.27 (dt, 1H, J = 6.8 Hz, 6.8 Hz, CHDOP),
2.62 (d, 2H, J = 6.8 Hz, CH2CHD), 2.02 (m, 3H, C CCH3); 13C
=
NMR (D2O–ND4OD, 125 MHz) d 144.6, 112.2, 64.4 (dt, 1JC–D
=
(2S)-[2-2H1]-Levulinic acid (25)
22.9 Hz, 2JC–P = 5.5 Hz), 38.4, 22.3; 31P NMR (D2O–ND4OD, 162
MHz) d −9.00 (m, 1P), −5.10 (m, 1P); HRMS (ES −ve) calcd for
[M − H]− C5H10DO7P2 246.0037, found 246.0035.
A stirring solution of (4R)-[4-2H1]-2-butanone-4-yl tosylate (23)
(92 mg, 0.38 mmol) in DMSO (5 mL) was treated with sodium
cyanide (56 mg, 1.14 mmol) and stirred at 60 ◦C for 12 h. The
reaction mixture was cooled to room temperature and extracted
with CH2Cl2. The combined organic extracts were washed with
brine, dried over MgSO4 and concentrated in vacuo. To the
crude nitrile was added 6 M HCl (1.5 mL, 1.5 mmol) and
heated to reflux for 5 h. The reaction mixture was cooled to
room temperature and extracted with CH2Cl2. The combined
organic extracts were washed with brine, dried over MgSO4
and concentrated in vacuo to give the crude acid 25 (15 mg,
34%). This compound was used without further purification for
the preparation of (S)-(+)-(methoxycarbonyl)benzyl (2S)-[2-2H1]-
levulinate (27). For unlabelled material: IR (CH2Cl2, cast) 3111,
1716, 1402, 1369, 1165 cm−1; 1H NMR (CDCl3, 300 MHz) d 2.73
(m, 2H, CH2CO), 2.61 (m, 2H, CH2COOH), 2.17 (s, 3H, CH3CO);
13C NMR (CDCl3, 100 MHz) d 206.9, 178.4, 37.7, 29.7, 27.8;
(1R)-[1-2H1]-Isopentenyl diphosphate (11)
A similar procedure was employed as that described for the
preparation of 6. The reaction of (1S)-[2-2H]-isopentenyl tosylate
(22) (11.0 mg, 0.044 mmol) in MeCN (2 mL) with tris-n-
butylammonium hydrogen diphosphate (123 mg, 0.137 mmol)
gave the crude labelled IPP. Purification of the crude product
using HPLC HPLC (Vydac 259VHP reverse phase polymer; 4.6 ×
150 mm; 10% MeCN, 90% 100 mM NH4HCO3 20 min, 10–50%
MeCN over 5 min, 50% MeCN 10 min, 50–90% MeCN over 1 min,
90% MeCN 20 min, tR 44.0 min) afforded the title compound 11 as
a white solid (12 mg, 86%). IR (lscope) 2846 (b), 1652, 1436 cm−1;
1
=
H NMR (D2O–ND4OD, 500 MHz) d 5.10 (m, 1H, C CH) 5.08
=
(m, 1H, C CH), 4.26 (m, 1H, CHDOP), 2.61 (d, 2H, J = 6.8 Hz,
CH2CHD), 2.00 (m, 3H, C CCH3); d 13C NMR (D2O–ND4OD,
=
•
HRMS (EI) calcd for [M+ ] C5H8O3 116.0474, found 116.0472, 101
125 MHz) d 144.6, 112.2, 64.4 (dt, 1JC–D = 22.9 Hz, 2JC–P = 5.5 Hz),
38.4, 22.6; 31P NMR (D2O–ND4OD, 162 MHz) d −9.00 (m, 1P),
−5.10 (m, 1P); HRMS (ES −ve) calcd for [M − H]− C5H10DO7P2
246.0037, found 246.0038.
(22%), 73 (47%), 56 (100%). The (2S)-[2-2H1]-levulinic acid (25)
showed identical chromatographic and spectral properties except
for the following: 2H NMR (C6H6, 61.4 MHz) d 2.61.
(2R)-[2-2H1]-Levulinic acid (26)
(4R)-[4-2H1]-2-butanone-4-yl tosylate (23)
Substitution of (4S)-[4-2H1]-2-butanone-4-yl tosylate (24) into the
described procedure used to prepare the enantiomeric acid 25 gave
the title compound 26. Chromatographic properties and spectral
data were identical to those of 25.
(1R)-[1-2H1]-Isopentenyl tosylate (21) (200 mg, 0.832 mmol) was
dissolved in CH2Cl2 (10 mL) and cooled to −78 ◦C in a dry ice–
acetone bath. To this solution was bubbled O3 for 20 min at which
time the solution was purple in color. Excess O3 was purged by
bubbling O2 through the solution for 5 min. The solution was then
warmed to room temperature and zinc (109 mg, 1.67 mmol) was
added, followed by acetic acid (0.26 mL, 4.50 mmol) and stirred at
room temperature for 2 h. The reaction mixture was diluted with
H2O and extracted with Et2O. The combined ethereal extracts were
washed with a saturated solution of NaHCO3, water and brine,
dried over MgSO4 and concentrated in vacuo to give the tosylate
23 (195 mg, 97%). For unlabelled material: IR (CH2Cl2, cast) 2921,
(S)-(+)-(Methoxycarbonyl)benzyl (2S)-[2-2H1]-levulinate (27)
To a stirring solution of (2S)-[2-2H1]-levulinic acid (25) (15 mg,
0.13 mmol) in CH2Cl2 (5 mL) was added (S)-(+)-mandelic acid
methyl ester (24 mg, 0.14 mmol), DCC (32 mg, 0.16 mmol)
and DMAP (2 mg, 0.02 mmol). The resulting reaction mixture
was stirred at room temperature for 18 h and filtered through
a sintered glass funnel to remove solid impurities. The crude
ester was purified by flash column (SiO2, hexane–EtOAc, 4 : 1)
to afford the title compound 27 (14 mg, 41%) as a colorless oil.
For unlabelled material: IR (CH2Cl2, cast) 3034, 2954, 1745, 1719,
1
1719, 1359, 1176 cm−1; H NMR (CDCl3, 300 MHz) d 7.77 (m,
2H, Ar), 7.33 (m, 2H, Ar), 4.24 (t, 2H, J = 6.4 Hz, CH2OS),
2.81 (t, 2H, J = 6.4 Hz, CH2CO), 2.43 (s, 3H, ArCH3) 2.13 (s,
3H, CH3CO); 13C NMR (CDCl3, 125 MHz) d 204.3, 144.9, 132.7,
129.9 (2C), 128.0 (2C), 64.9, 42.2, 30.3, 21.7; HRMS (EI) calcd for
1
1587, 1364, 1152 cm−1; H NMR (C6D6, 300 MHz) d 7.42 (m,
2H, Ar), 7.04 (m, 3H, Ar), 6.05 (s, 1H, CHCO2Me), 3.18 (s, 3H,
OCH3), 2.50 (m, 2H, J = 7.0 Hz, CH2CO2R), 2.21 (td, 1H, J =
6.3 Hz, 18.2 Hz, CH2CO), 2.01 (td, 1H, J = 6.3 Hz, 18.2 Hz,
CH2CO), 2.01 (s, 3H, CH3CO); 13C NMR (CDCl3, 125 MHz) d
206.1, 172.1, 169.2, 133.7, 129.3, 128.8 (2C), 127.6 (2C), 74.6, 52.6,
•
[M+ ] C11H14O4S 242.0613, found 242.0619, 214 (5%), 172 (100%),
155 (27%), 108 (42%), 91 (100%). The (4R)-[4-2H1]-2-butanone-
4-yl-tosylate showed identical chromatographic properties and
1
displayed similar spectral properties except for the following: H
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•
37.8, 29.8, 27.8; HRMS (EI) calcd for [M+ ] C14H16O5 264.0998,
7.36 (m, 2H, Ar), 7.21–7.27 (m, 3H, Ar), 3.86 (dt, 1H, J = 6.6 Hz,
1.5 Hz, CHDCH2), 2.88 (d, 2H, J = 6.3 Hz, CH2CHD); 13C NMR
(CDCl3, 125 MHz) d 138.5, 129.0 (2C), 128.6 (2C), 126.5, 63.3
found 264.0989, 232 (5%), 205 (4%), 166 (7%), 149 (6%), 121
(10%), 99 (100%). The (S)-(+)-(methoxycarbonyl)benzyl (2S)-[2-
2H1]-levulinate (27) showed identical chromatographic properties
and displayed similar spectral properties except for the following:
2H NMR (C6H6, 76.5 MHz) d 2.48.
•
(t, JC–D = 22 Hz), 39.1; HRMS (EI) calcd for [M+ ] C8H9DO
123.0793, found 123.0795, 106 (3%), 91 (100%), 65 (14%). A
stirring solution of the alcohol (107 mg, 0.88 mmol) and tosyl
chloride (202 mg, 1.06 mmol) in CH2Cl2 (5 mL) was treated with
Et3N (0.16 mL, 1.16 mmol) and DMAP (10 mg 0.08 mmol). The
resulting reaction mixture was stirred at room temperature for 18 h,
at which time it was diluted with water (10 mL) and extracted
with CH2Cl2. The combined organic extracts were washed with
water and brine, dried over MgSO4 and concentrated in vacuo.
The crude tosylate was purified by flash column chromatography
(SiO2, hexane–EtOAc, 10 : 1) to afford the tosylate 32 as an oil
(184 mg, 75%). IR (CH2Cl2, cast) 3029, 2924, 2193, 1597, 1362,
1178 cm−1; 1H NMR (CDCl3, 300 MHz) d 7.68 (m, 2H, Ar), 7.19–
7.29 (m, 5H, Ar), 7.09 (m, 2H, Ar), 4.18 (m, 1H, CHDCH2), 2.93
1
(S)-(+)-(Methoxycarbonyl)benzyl (2R)-[2-2H1]-levulinate (28)
A procedure similar to that used for the preparation of (S)-
(+)-(methoxycarbonyl)benzyl (2S)-[3-2H1]-levulinate (27) was em-
ployed except that (2R)-[2-2H1]-levulinic acid (26) was used as the
starting material. Spectral data were similar to those of 27 except
for 2H NMR (C6H6, 76.5 MHz) d 2.44
[1,1-2H2]-2-Phenylethanol (30)45
A similar procedure was employed as that described for the
preparation of 19. The reaction of phenylacetic acid (29) (5.0 g,
36.7 mmol) with a solution of lithium aluminum deuteride (12.3 ml
of 1.0 M solution in Et2O) afforded the alcohol 30 (1.25 g, 27%) IR
(CH2Cl2, cast) 3311, 3086, 3062, 3028, 2932, 2213, 1603, 1496 cm−1;
1H NMR (CDCl3, 300 MHz) d 7.36–7.21 (5H, m, Ar), 2.87 (2H, s,
2
(d, 2H, J = 7.1 Hz, CH2CHD), 2.41 (s, 3H, ArCH3); H NMR
(CH2Cl2, 76.5 MHz) d 4.23; 13C NMR (CDCl3, 125 MHz) d 144.6,
136.2, 133.0, 129.8 (2C), 128.8 (2C), 128.6 (2C), 126.8 (2C), 126.9,
•
1
70.3, (t, JC–D = 23 Hz), 35.3, 21.6; HRMS (EI) calcd for [M+ ]
C15H15DO3S 277.0883, found 277.0884, 172 (5%), 155 (15%), 105
(100%), 91 (83%).
CH2); H NMR (CHCl3, 76.5 MHz) d 3.86; 13C NMR (CDCl3,
2
100 MHz) d 138.4, 129.0 (2C), 128.6 (2C), 126.5, 63.0 (1JC–D = 23
(1R)-[1-2H1]-2-Phenylethyl tosylate (33)
•
Hz), 39.0; HRMS (EI) calcd for [M+ ] C8H8D2O 124.0855, found
124.0857, 106 (7%), 91 (100%).
A similar procedure was employed as that described for the prepa-
ration of 32. Treatment of [1-2H]-2-phenylethanal (31) (4.03 mmol)
in THF (10 mL) with (+)-b-chlorodiisopinocampheylborane
(1.55 g, 4.84 mmol) gave the crude alcohol. Purification using flash
column chromatography (SiO2, hexane–EtOAc, 4 : 1) afforded
(1R)-[1-2H]-phenylethanol as a colorless oil (200 mg, 40%). IR
(CH2Cl2, cast) 3335, 3085, 3062, 2932, 2158, 1603, 1496 cm−1;
1H NMR (CDCl3, 300 MHz) d 7.29–7.36 (m, 2H, Ar), 7.21–7.27
(m, 3H, Ar), 3.86 (dt, 1H, J = 6.6 Hz, 1.5 Hz, CHDCH2), 2.88
(d, 2H, J = 6.3 Hz, CH2CHD); 13C NMR (CDCl3, 125 MHz) d
138.5, 129.0 (2C), 128.6 (2C), 126.5, 63.3 (t, 1JC–D = 22 Hz), 39.1;
[1-2H1]-2-Phenylethanal (31)46
A similar procedure was employed as that described for the
preparation of 20. Thus, reaction of [1,1-2H2]-phenylethanol (30)
with IBX (1.70 g, 6.14 mmol) in DMSO (15 mL) gave the aldehyde
31 (450 mg, 91%) after work-up. The crude aldehyde was used
without further purification as the aldehyde is unstable at room
temperature. Data for unlabelled material: IR (CH2Cl2, cast) 3086,
3062, 3028, 1723, 1603, 1453 cm−1; 1H NMR (CDCl3, 300 MHz) d
9.76 (t, 1H, CHO, J = 2.4 Hz), 7.42–7.21 (m, 5H, Ar), 3.70 (d, 2H,
•
HRMS (EI) calcd for [M+ ] C8H9DO 123.0793, found 123.0795.
•
CH2 J = 2.4 Hz); HRMS (EI) calcd for [M+ ] C8H8O 120.0575,
106 (3%), 91 (100%). Reaction of the alcohol (250 mg, 2.06 mmol)
in CH2Cl2 (15 mL) with p-toluenesulfonyl chloride (471 mg,
2.47 mmol), triethylamine (0.38 mL, 2.72 mmol) and DMAP
(10 mg, 0.08 mmol) provided the crude tosylate. Purification
by flash column chromatography (SiO2, hexane–EtOAc, 10 : 1)
afforded 33 as an oil (490 mg, 86%). IR (CH2Cl2, cast) 3030, 2925,
2195, 1653, 1598, 1362, 1176 cm−1; 1H NMR (CDCl3, 300 MHz) d
7.67 (m, 2H, Ar), 7.19–7.29 (m, 5H, Ar), 7.09 (m, 2H, Ar), 4.18 (m,
1H, CHDCH2), 2.93 (d, 2H, J = 7.1 Hz, CH2CHD), 2.41 (s, 3H,
ArCH3); 2H NMR (CH2Cl2, 76.5 MHz) d 4.23; 13C NMR (CDCl3,
125 MHz) d 144.6, 136.2, 133.0, 129.8 (2C), 128.8 (2C), 128.6 (2C),
127.8 (2C), 126.9, 70.3 (t, 1JC–D = 23 Hz), 35.3, 21.6; HRMS (EI)
found 120.0574, 106 (7%), 91 (100%). The labelled aldehyde 31
had similar data to that of the unlabelled material except for the
following: 1H NMR (CDCl3, 300 MHz) d 7.21–7.42 (5H, m, Ar),
3.70 (2H, s, CH2).
(1S)-[1-2H1]-2-Phenylethyl tosylate (32)
An oven dried 100 mL round bottom flask was charged with (−)-
b-chlorodiisopinocampheylborane (1.55 g, 4.84 mmol) in an inert
atmosphere and dissolved in THF (10 mL). The solution was
◦
cooled to −78 C and to this cold solution was added dropwise
a solution of [1-2H1]-phenylethanal (31) (4.03 mmol) and stirred
with warming to room temperature for 18 h. The solvent was
removed in vacuo and a-pinene was removed by high vacuum for
8 h. The residue was dissolved in Et2O and to this stirring solution
was added diethanolamine (1.02 mL, 10.65 mmol). The solids
•
calcd for [M+ ] C15H15DO3S 277.0883, found 277.0884, 172 (5%),
155 (15%), 105 (100%), 91 (83%).
(3R)-[3-2H1]-4-Phenyl-1-butyne (34)
R
were removed by filtration through a pad of Celiteꢀ and filtrate
was concentrated in vacuo. Purification of the crude alcohol by
flash column chromatography (4 : 1 hexanes–EtOAc) gave the
alcohol. Data for alcohol: IR (CH2Cl2, cast) 3335, 3085, 3062,
2932, 2158, 1603, 1496 cm−1; 1H NMR (CDCl3, 300 MHz) d 7.29–
To a stirring solution of (1S)-[1-2H]-2-phenylethyl tosylate (32)
(200 mg, 0.721 mmol) in DMSO (5 mL) was added lithium
acetylide–ethylene diamine complex (199 mg, 2.16 mmol). The
resulting reaction mixture was stirred at room temperature for
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2 h and then poured into a solution of cold 1 M HCl to quench
the reaction. The insoluble material was removed by filtration
the mercuric salt. Addition of NaBH4 (202 mg, 5.34 mmol)
and oxidation by PDC (220 mg, 0.59 mmol) afforded (3S)-
[3-2H1]-4-phenyl-2-butanone (37). Purification by flash column
chromatography (SiO2, pentanes–Et2O) provided the ketone 37.
The enantiomeric ketone had identical spectral properties to that
of (3R)-[3-2H1]-4-phenyl-2-butanone (36).
R
through a pad of Celiteꢀ and the filtrate was extracted with
Et2O. The combined ethereal extracts were washed with water
and brine, dried over MgSO4 and evaporation gave the crude
alkyne 34. The crude oil was used without any further purification
for the preparation of (3R)-[3-2H1]-4-phenyl-2-butanone (36). IR
(CH2Cl2, cast) 3304, 3078, 3025, 2976, 2258, 1601, 1494, 1079 cm−1;
1H NMR (CD2Cl2, 400 MHz) d 7.37–7.40 (m, 2H, Ar), 7.20–7.33
(m, 3H, Ar), 2.83 (d, 2H, J = 7.1 Hz, CHDCH2), 2.48 (m, 1H,
CHDCH2), 1.98 (d, 1H, J = 2.6 Hz, C≡CH); 2H NMR (CH2Cl2,
76.5 MHz) d 2.47; 13C NMR (CD2Cl2, 125 MHz) d 136.8, 128.5
(2C), 127.8 (2C), 126.3, 83.8, 68.9, 34.8, 20.3 (t, 1JC–D = 20.0 Hz);
(3R)-[3-2H1]-Levulinic acid (38)
A solution of (3R)-[3-2H1]-4-phenyl-2-butanone (36) (106 mg,
0.721 mmol) in CCl4 (4 mL), MeCN (4 mL) and H2O (6 mL) was
treated with sodium periodate (1.54 g, 7.21 mmol) and ruthenium
trichloride (3 mg, 14 lmol). The resulting reaction mixture was
stirred at room temperature for 18 h. The solution was diluted with
water and extracted with CH2Cl2. The combined organic extracts
were dried with MgSO4 and concentrated in vacuo to give the crude
acid 38 (20 mg, 24%). The acid was used for the preparation of (S)-
(+)-(methoxycarbonyl)benzyl (3R)-[3-2H1]-levulinate (40) without
further purification. For unlabelled material: IR (CH2Cl2, cast)
3111, 1716, 1402, 1369, 1165 cm−1; 1H NMR (CDCl3, 300 MHz) d
2.73 (m, 2H, CH2CO), 2.61 (m, 2H, CH2COOH), 2.17 (s, 3H,
CH3CO); 13C NMR (CDCl3, 100 MHz) d 206.9, 178.4, 37.7,
•
HRMS (EI) calcd for [M+ ] C10H9D 131.0845, found 131.0846,
105 (43%), 91 (100%).
(3S)-[3-2H1]-4-Phenyl-1-butyne (35)
A similar procedure was employed as that described for the prepa-
ration of 34. The reaction of (1R)-[1-2H]-2-phenylethyl tosylate
(33) (200 mg, 0.721) in DMSO (5 mL) with lithium acetylide–
ethylene diamine complex (199 mg, 2.16 mmol) gave the crude
(3S)-[3-2H1]-4-phenylbutyne (35). The crude oil was used without
any further purification for the preparation of (3S)-[3-2H1]-4-
phenyl-2-butanone (37). The enantiomeric alkyne had identical
spectral properties to that of (3R)-[3-2H1]-4-phenylbutyne (34).
•
29.7, 27.8; HRMS (EI) calcd for [M+ ] C5H8O3 116.0472, found
116.0474, 101 (22%), 73 (47%), 56 (100%). The (3R)-[3-2H1]-
levulinic acid (38) showed identical chromatographic and spectral
properties except for the following: 2H NMR (C6H6, 61.4 MHz) d
2.73.
(3R)-[3-2H1]-4-Phenyl-2-butanone (36)
(3S)-[3-2H1]-Levulinic acid (39)
To
a
stirring solution of (3R)-[3-2H1]-4-phenylbutyne (34)
(0.721 mmol) in acetic acid (6.0 mL) and water (1.0 mL) was added
mercuric acetate (919 mg, 2.88 mmol). The resulting reaction
mixture was stirred at 70 ◦C for 3 h at which time water (30 mL) was
added. The pH of the solution was adjusted to 7 with the addition
of sodium acetate. The mixture was cooled to 0 ◦C in an ice-water
bath and sodium borohydride was added carefully. The reaction
mixture was then stirred for 30 min and filtered through a pad of
A similar procedure was employed as that described for the
preparation of 38. Treatment of (3S)-[3-2H1]-4-phenyl-2-butanone
(37) (106 mg, 0.721 mmol) in CCl4 (4 mL), MeCN (4 mL) and H2O
(6 mL) with sodium periodate (1.54 g, 7.21 mmol) and ruthenium
trichloride (3 mg, 0.014 mmol) gave (3S)-[3-2H1]-levulinic acid (39)
as a crude mixture. The acid was used without further purification
for the preparation of (S)-(+)-(methoxycarbonyl)benzyl (3S)-[3-
2H1]-levulinate (41). Spectral data was identical to that reported
for 38.
R
Celite,ꢀ and the filtrate was extracted with Et2O. The combined
ethereal extracts were washed with water and brine, and dried over
MgSO4 and concentrated in vacuo. The crude residue was dissolved
in CH2Cl2 and treated with PDC (298 mg, 0.79 mmol) and stirred
for 3 h at room temperature. The suspension was filtered through
(S)-(+)-(Methoxycarbonyl)benzyl (3R)-[3-2H1]-levulinate (40)
R
a pad of Celiteꢀ and the filtrate was concentrated in vacuo to
To a stirring solution of (3R)-[2-2H1]-levulinic acid (38) (18 mg,
0.154 mmol) in CH2Cl2 (5 mL) was added (S)-(+)-mandelic acid
methyl ester (28 mg, 0.17 mmol), DCC (39 mg, 0.19 mmol)
and DMAP (2 mg, 0.02 mmol). The resulting reaction mixture
was stirred at room temperature for 18 h and filtered through a
sintered glass funnel to remove the solid impurities. The crude
ester was purified by flash column (SiO2, hexane–EtOAc, 4 : 1)
to afford the title compound 40 (16 mg, 41%) as a colorless oil.
For unlabelled material: IR (CH2Cl2, cast) 3034, 2954, 1745, 1719,
give the crude ketone. Purification by flash chromatography (SiO2,
pentanes–Et2O, 10 : 1) to afford the ketone 36 (45 mg, 42%) as a
colorless liquid. IR (CH2Cl2, cast) 3027, 2956, 2871, 1717, 1602,
1497, 1162 cm−1; 1H NMR (CDCl3, 300 MHz) d 7.15–7.29 (m, 5H,
Ar), 2.86 (d, 2H, J = 7.0 Hz, CH2CHD), 2.74 (1H, m, CH2CHD),
2
2.12 (3H, s, CH3CO); H NMR (CHCl3, 76.5 MHz) d 2.74; 13C
NMR (CDCl3, 125 MHz) d 207.8, 141.0, 128.5 (2C), 128.3 (2C),
1
126.1, 45.2, 30.1 (t, JC–D = 29 Hz), 29.8; HRMS (EI) calcd for
•
[M+ ] C10H11DO 149.0951, found 149.0944, 134 (30%), 106 (98%),
1
1587, 1364, 1152 cm−1; H NMR (C6D6, 300 MHz) d 7.42 (m,
91 (100%).
2H, Ar), 7.04 (m, 3H, Ar), 6.05 (s, 1H, CHCO2Me), 3.18 (s, 3H,
OCH3), 2.50 (m, 2H, J = 7.0 Hz, CH2CO2R), 2.21 (td, 1H, J =
6.3 Hz, 18.2 Hz, CH2CO), 2.01 (td, 1H, J = 6.3 Hz, 18.2 Hz,
CH2CO), 2.01 (s, 3H, CH3CO); 13C NMR (CDCl3, 125 MHz) d
206.1, 172.1, 169.2, 133.7, 129.3, 128.8 (2C), 127.6 (2C), 74.6, 52.6,
(3S)-[3-2H1]-4-Phenyl-2-butanone (37)
A similar procedure was employed as that described for the
preparation of 36. Treatment of a solution of (3S)-[3-2H1]-4-
phenylbutyne (35) (0.721 mmol) in acetic acid (6.0 mL) and
water (1.0 mL) with mercuric acetate (919 mg, 2.88 mmol) gave
•
37.8, 29.8, 27.8; HRMS (EI) calcd for [M+ ] C14H16O5 264.0998,
found 264.0989, 232 (5%), 205 (4%), 166 (7%), 149 (6%), 121
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(10%), 99 (100%). The (S)-(+)-(methoxycarbonyl)benzyl (3R)-[3-
2H1]-levulinate (40) showed identical chromatographic properties
and displayed similar spectral properties except for the following:
2H NMR (C6H6, 76.5 MHz) d 2.01.
Acknowledgements
The authors would like to acknowledge Dr Deborah Scott, Dr
Colleen McMahan and Dr Katrina Cornish (US Department of
Agriculture, Agricultural Research Station (USDA-ARS), Albany
CA) for the preparation of rubber particles from H. brasiliensis
and P. argentatum. This work was supported by the Advanced
Food and Biomaterials Network (AFMNet), the Natural Sciences
and Engineering Research Council of Canada, the Alberta
Heritage Foundation for Medical Research, the Canada Research
Chair in Biooorganic Chemistry and Medicinal Chemistry and
the USDA.
(S)-(+)-(Methoxycarbonyl)benzyl (3S)-[3-2H1]-levulinate (41)
A procedure similar to that used for the preparation of (S)-(+)-
(methoxycarbonyl)benzyl (3R)-[3-2H1]-levulinate (40) was em-
ployed except that (3S)-[2-2H1] levulinic acid (39) was used as the
starting material. Spectral data were similar to those of 40 except
for 2H NMR (C6H6, 76.5 MHz) d 2.21.
References
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The total reaction volume was typically 250 lL (100 mM Tris–HCl
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◦
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