Synthesis and spectroscopic analysis of a stereoisomer library of the phytophthora mating hormone α1 and derived bis-Mosher esters

Article abstract of DOI:10.1021/ja2082679

Fluorous mixture synthesis provided all eight diastereomers of the phytophthora hormone α1 with the R configuration at C11 as individual samples after demixing and detagging. The library of all possible bis-Mosher esters (16) was then made by esterification. Complete sets of ¹H, ¹³C, and (for the Mosher esters) ¹⁹F NMR spectra were recorded, assigned, and compared with each other and with published spectra. Not all of the spectra are unique, and the ¹H NMR spectra of the Mosher esters provided the most information. The previous assignment of the natural sample as an "all-R" stereoisomer mixed with its 3S-epimer was confirmed.

Full text of DOI:10.1021/ja2082679

ARTICLE
pubs.acs.org/JACS
Synthesis and Spectroscopic Analysis of a Stereoisomer Library of the
Phytophthora Mating Hormone α1 and Derived Bis-Mosher Esters
Reena Bajpai and Dennis P. Curran*
Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, United States
S Supporting Information
b
ABSTRACT: Fluorous mixture synthesis provided all eight
diastereomers of the phytophthora hormone α1 with the R
configuration at C11 as individual samples after demixing and
detagging. The library of all possible bis-Mosher esters (16) was
1
then made by esterification. Complete sets of H, 13C, and (for
the Mosher esters) 19F NMR spectra were recorded, assigned,
and compared with each other and with published spectra. Not all
of the spectra are unique, and the 1H NMR spectra of the Mosher
esters provided the most information. The previous assignment of
the natural sample as an “all-R” stereoisomer mixed with its 3S-epimer was confirmed.
’ INTRODUCTION
The power of modern NMR spectroscopy to characterize
small organic molecules has increased to such a level that Saielli
and Bagno dared to ask the provocative question in a 2009 article
entitled: “Can two molecules have the same NMR spectrum?” 1
Their short answer was “no”.2 Saielli and Bagno were comparing
isomers, and perhaps they were thinking primarily about con-
stitutional isomers. In fact, there are plenty of diastereoisomers
that have the same NMR spectra.
We have been making libraries of natural product stereoi-
somers by fluorous mixture synthesis3 with the immediate goals
of structure assignment and structureÀactivity relationship. In
the larger picture, the various libraries of spectra pose questions
of how to decide when spectra are the same. They also begin to
give a feeling for when spectra of diastereomers are likely to be
the same and when they are not. At one extreme, natural pro-
Figure 1. Examples of natural product stereoisomer libraries with
remote (murisolin) and nearby (passifloricin) stereocenters.
ducts like murisolins (Figure 1) with remote stereocenters pose
big problems because many diastereomers have substantially
identical spectra.4 For example, each of the 32 diastereomers of
murisolin exhibits one of only six different 1H NMR spectra. There
separated by three carbon atoms each, the disposition in 1 falls
roughly in between the remote stereocenters of murisolin and
the nearby ones of passifloricin. We challenge readers with the
question of Saielli and Bagno: can any two of the eight diaster-
eomers of 1 have the same 1H or 13C NMR spectrum? Further, a
bis-Mosher ester forms at C1 and C16 of 1. Can any of the
resulting 16 diastereomers have the same 1H, 13C, or 19F NMR
spectrum? Pause to make your assessment, then read the rest of
this work.
are 64 Mosher ester diastereomers derived from the murisolins,
but even here only 16 of these exhibit a unique spectrum. The
other 48 diastereomers come in 24 pairs with identical spectra.
With murisolins, having the same spectrum is the rule, not the
exception.
At the other extreme, natural products like passifloricin with
nearby stereocenters can be analyzed straightforwardly. Each of
1
the eight diastereomers of passifloricin has a unique H and
13C NMR spectrum.5 This means that spectra of synthetic and
natural samples can be compared directly to give an unambig-
uous yes/no answer about identity.
Hormone α1 is the universal mating hormone of heterothalic
(sexually reproducing) species of phytophthora.6 These hardy,
fungi-like species are among the most destructive plant parasites,
Here we describe the synthesis and analysis of the complete
stereoisomer library of the phytophthora α1 mating hormone 1
(Figure 2) and the derived Mosher esters. With four stereocenters
Received: September 7, 2011
Published: November 03, 2011
r
2011 American Chemical Society
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Figure 2. Structures of the phytophthora α1 mating hormone 1, open
form (major) and hemiacetal form (minor, two stereoisomers).
causing diseases of worldwide importance.7 For example, the late
blight of potato, caused by Phytophthora infestans, resulted in the
Irish potato famine during the mid-19th century.
In 2005, Ojika and co-workers first isolated 1.2 mg of hormone
α1 containing“unknown impurities” from 1830 L of cultural broth
of the A1 mating type of Phytophthora nicotianae.6 The two-
dimensional structure of the hormone α1 was shown to be 1,11,16-
trihydroxy-3,7,11,15-tetramethylhexadecan-4-one (1, Figure 2).
Yajima and co-workers quickly confirmed the 2D structure of
1 by intentionally synthesizing a mixture containing all 16
possible stereoisomers.8 Yajima commented that this mixture
Figure 3. Retrosynthetic plan and tagging strategy.
hormone are highly precious, but copies of its 1H and 13C NMR
spectra are available along with 1H NMR spectra of its bis-(R)-
and bis-(S)-Mosher esters.6 These are the basis for comparison
and assignment.
Here we report the fluorous mixture synthesis of all diaster-
eomers of 1 with the R configuration at C11. In turn, each of
these was converted to its bis-(R)- and bis-(S)-Mosher esters.14
The comparison of the spectra of these two complete stereoiso-
mer libraries (eight isomers of 1 and 16 bis-Mosher esters) is an
interesting exercise in deciding whether similar spectra are the
same or different. We confirm that the natural sample of 1 is a 3/2
mixture of (3R,7R,11R,15R)-1 and its (3S) epimer (along with
some hemiacetals). Taking into account Ojika’s reasonable
assertion that isomerization occurred at C3 during isolation,10
the hormone α1 is (3R,7R,11R,15R)-1 as proposed by Yajima.
1
looked like a single compound by H and 13C NMR spectros-
copy. In turn, this spectrum matched that of the natural sample.
This suggests that all eight isomers have the same spectra, which
turns out not to be the case.
In 2007, we made isomers of 1 and learned that the extraneous
peaks in the spectra of the natural sample derived not from
impurities per se, but from minor hemiacetal forms in equilibri-
um with 1.9 Also, we and Ojika both showed by different means
that the isolated sample was a mixture of two epimers at C3. By
using Mosher esters, Ojika proposed that hormone α1 was (15R)
and a 3/2 mixture of (3R/3S).10
By testing a series of biased stereoisomer mixtures on phy-
tophthora oospores (spores) in 2008, Yajima was able to show
that the natural product had the (11R) configuration.11 The first
phase of this work was complicated because epimerization
occurred at several stereocenters during the synthesis, so the
stereoisomer composition of the tested samples could only be
estimated. However, Yajima then made all four possible iso-
mers with the (11R) configuration and showed that only one,
(3R,7R,11R,15R)-1, was active in the oospore assay. Loh and co-
workers have also recently reported a synthesis of the “all-R”
isomer of 1.12
At the same time as Yajima, Feringa made two stereo-
isomers of 1, (3S,7S,11S,15S)-1 (“all-S”-1), and its C7 epimer,
(3S,7R,11S,15S)-1, by using his asymmetric conjugate addition
method. He observed that both samples were active in carefully
controlled oospores assays.13 According to Yajima’s work, these
isomers should not have been active. However, it is difficult to
compare the results of such functional assays across laboratories.
Because of the discrepancies in assays and problems with
epimerization, the stereostructure of 1 should be confirmed
by spectroscopic or analytical means. Samples of the natural
’ RESULTS AND DISCUSSION
Synthetic Plan. Our first generation synthesis of isomers of 1
capitalized on the latent symmetry of the fragment spanning
carbons C6ÀC16.9 Strategically, this led to problems in control-
ling the configuration at C11. Tactically, we learned that epimer-
ization at C3 was facile. We designed the revised synthetic plan
shown in Figure 3 both to address these issues and to accom-
modate the needed fluorous tags. Yajima’s testing of stereoiso-
mer mixtures and individual samples pointed firmly to the 11R
configuration,11 so we fixed that stereocenter and made all eight
possible diastereomers by varying the other three stereocenters.
We expected that reduction, demixing and detagging of M215
would provide the eight target isomers. A KocienskiÀJulia olefina-
tion16 couples M3 and M4 to provide M2. In the tagging plan, left
fragment M3 will be made as two mixtures of two quasiisomers17
with either the 7S or 7R configuration fixed. The configuration at
C3 is encoded by the fluorous PMB (FPMB) group on O1, as
indicated at the bottom of Figure 3. Right fragment M4 will be
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Scheme 1. Synthesis of the Left Fragment, a Pair of Two
Quasiisomers (7S)-M3a/b and (7R)-M3a/b
Scheme 2. Synthesis of Tagged Quasiracemate M13b/c
Bearing the C15 Stereocenter
Scheme 3. Synthesis of the Right Fragment, Quasiracemate
M4b/c
made as one mixture of two quasiisomers with a fluorous PMB
group encoding the configuration at C15 and with the R con-
figuration at C11. The pairwise coupling of (7S)-M3 and
(7R)-M3 with M4 then provides two corresponding mixtures of
M2, now of four quasiisomers each, ready for reduction, demixing,
and detagging.
Syntheses of the Fragments M3 and M4. The preparation
of phenyltetrazolyl sulfone quasiisomers (7S)/(7R)-M3 is shown
in Scheme 1. Myers reagent (R,R)-518 was alkylated with iodo
PMB ether 6a bearing a CF3 group to give (3S)-7a, while its
quasienantiomer (3R)-7b (not shown) was made from (S,S)-5 and
the reagent 6b bearing a C4F9 group.19 Mixing of 7a and 7b and
reduction with lithium amidoborohydride20 gave alcohol M8a/b
in 87% yield, then Swern oxidation21 provided aldehyde M9a/b
(72%). As expected,22 these and other quasienantiomers be-
haved like true enantiomers on silica gel and chromatography by
1H or 13C NMR analyses. But they were readily separated or
analyzed by fluorous HPLC or 19F NMR spectroscopy.
To assess stereopurity prior to the upcoming addition reaction,
a small sample M9a/b was reduced back to the corresponding
alcohol. The resulting quasienantiomers M8a/b were easily de-
mixed, then converted to the Mosher esters (see Supporting Infor-
mation (SI)). Analysis of the 1H NMR spectra as usual14 showed
that each sample had about a 94/6 enantiomer ratio. This ratio
presumably reflects the diastereoselectivity in the Myersalkylation.
Next, dibromides (R)-10 and (S)-1023 were prepared in three
steps from the Roche ester (see SI). That these fragments were
enantiopure was later confirmed by analysis of the final products.
Metalation of (R)-10 and addition of fragment M9a/b provided
alcohol (7R)-M11a/b in 100% crude yield.24 The sample
appeared to be a 1/1 mixture of isomers at C4 (by H NMR
1
analysis), but this is inconsequential since this center will be
oxidized at the end. Removal of the TBS group and installation
of the phenylsulfonyl tetrazole under standard conditions25
gave (7R)-M3a/b in three steps. Likewise, the complementary
quasiisomer mixture (7S)-M3a/b was made starting from (S)-10 in
comparable yields.
Scheme 2 shows the synthesis of tagged quasienantiomers
M13b/c bearing the C15 stereocenter. (2S)-Methyl 3-hydroxy-
2-methylpropanoate (Roche ester) was tagged with a fluorous
PMB group bearing a C4F9 label to provide (S)-12b. Its quasi-
enantiomer (R)-12c was prepared analogously from (S)-Roche
ester and the C6F13-labeled PMB reagent. Mixing to make a
quasiracemate, then reduction with DiBAL provided a primary
alcohol in 98% yield. Standard Mitsunobu coupling with 1-phenyl-
5-thiotetrazole (91% yield) followed by molybdate-catalyzed
peroxide oxidation provided phenyl sulfonyltetrazole M13b/c
in 90% yield.
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Scheme 4. Fragment Coupling and Completion of the
Synthesis Illustrated in the 7S Series
Figure 4. Typical chromatogram from a semipreparative demixing run
with 40 mg of (7S)-M20a/b,b/c. Conditions: 80:20 CH3CN:H2O to
100% CH3CN in 30 min, then 100% CH3CN for 70 min at a flow rate of
7 mL/min; FluoroFlash PF8 column.
The synthesis of fragment M4b/c is summarized in Scheme 3.
Alkene 14, readily available in four steps from butyn-4-ol (see
SI), was subjected to Sharpless asymmetric epoxidation26 to give
(S,R)-15 in an enantiomer ratio of about 92/8 according to
Mosher ester analysis. Reduction of 15 by LiAlH4 provided a diol
in 65% yield. This was bis-silylated with TESOTf (90%), then
direct Swern oxidation provided aldehyde (R)-16 in 77% yield.
To complete the right fragment M4b/c, KocienskiÀJulia
coupling of the quasienantiomers M13b/c with the single enan-
tiomer (R)-16 provided alkene M17b/c as an 80/20 mixture of
E/Z isomers in 83% yield after flash chromatography. The TBS
and TES groups were removed with TBAF, then resilylation with
TESOTf provided a bis-silyl ether that was directly oxidized by
the Swern method (77%) to give aldehyde M4b/c.
Figure 5. Partial 1H NMR spectra (600 MHz, CDCl3) of (3S,7S,-
11R,15R)-20a,b, (3S,7S,11R,15S)-20a,c, (3R,7S,11R,15R)-20b,b, and
(3R,7S,11R,15S)-20b,c (top to bottom). Most parts of the spectra are
very similar, but notice the differences in the region 2.4À2.5 ppm.
reduction to saturate the alkenes and alkyne (89% yield), then
DMP oxidation of the C4 alcohol to give ketone M20 (92%).
1
The H NMR spectrum of this mixture was now easily inter-
pretable, and the 19F spectrum showed resonances characteristic
of the expected 1/1/1/1 mixture of the four quasiisomers.
Likewise, starting from (7R)-M3a/b, the mixture of the four
quasiisomers M20 bearing the (R) configuration at C7 was
produced.
Synthesis and Separation of the Stereoisomer Library.
The completion of the synthesis is illustrated in Scheme 4 start-
ing with the 7S series quasiisomers M3a/b. Kocienski-Julia
coupling of this free alcohol and M4b/c mediated by 2 equiv
NaHMDS provided the full carbon skeleton M18a/b,b/c but
only in an unacceptable 35% isolated yield. In contrast, silylation
of M3a/b with TESOTf followed by coupling with M4b/c and
1 equiv of NaHMDS provided M19a/b,b/c in 87% yield. The
C8ÀC9 alkene is again presumably formed as a mixture of
Demixing of the quasiisomer mixtures by preparative HPLC
over FluoroFlash silica gel proceeded smoothly. About 200 mg of
each quasiisomer was produced, and each of these samples was
demixed in 40 mg injections. A typical preparative chromatogram
is shown in Figure 4. The quasiisomer eluted in order of in-
creasing fluorine content was confirmed by MS and 19F NMR
spectroscopic analysis of the pure samples. The overall mass
recovery of the preparative HPLC purifications exceeded 80%.
On the basis of the enantiomer purities of the precursors,
quasiisomers 20 should be >80% isomerically pure, with small
amounts of epimers at C3, C11, and C15. Careful inspection of
the 1H NMR spectra of the eight pure fluorous-tagged samples
yielded only one tidbit of information about isomeric purity.
Expansions of the 1H NMR spectra of the four quasiisomers 20
in the (7S) series are overlaid in Figure 5. Like those of the
1
isomers, but overlapping in the alkene region of the H NMR
spectrum of M19 prevented assessment of the ratio. It is again
remarkable that M19 exhibits a single spot on TLC analysis
and can be readily purified by flash chromatography. Consider
that this sample is probably a mixture of 32 compounds: four
quasiisomers, plus two stereoisomers at C4, plus E/Z isomers at
both alkenes.
Following desilylation with 2 N HCl (98%), all of the true-
isomer features of the mixture were removed by diimide
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Scheme 5. Representative Detagging Reaction
1
Figure 6. Expansion of the H5 region of the H NMR spectra (700
MHz, CD3OD) of two representative isomers of 1. Top spectrum,
(3S,7R,11R,15S)-1; C3/C7-syn; bottom spectrum (3S,7S,11R,15S)-1,
C3/C7-anti. Note the small resonances from the syn contaminant in
the bottom spectrum of the anti isomer on either side of the triplet at
2.54 ppm.
Table 1. Results of Hydrogenolysis Reactions of PMBF
Ethers 20, the Amount of Products Isolated, and the Per-
centage Yields
precursor PMBF at C1 PMBF at C16
product
yield (%)
the same, and therefore what firm conclusions can be drawn
about structure.
1
The complete set of H (700 MHz) and 13C (175 MHz)
From the Mixture (7S,11R)-20
spectra of all eight isomers of 1 in CD3OD tabulated in the SI,
20a,b
20a,c
20b,b
20b,c
CF3
C4F9
C6F13
C4F9
C6F13
(3S,7S,11R,15R)-1
(3S,7S,11R,15S)-1
(3R,7S,11R,15R)-1
(3R,7S,11R,15S)-1
62
69
69
86
1
Tables S1ÀS3, and copies are also provided. The H NMR
CF3
spectra (Figure S4 of the SI) group into two pairs of four
compounds with substantially identical spectra. The only differ-
ence between these groups is the resonances for H5. Figure 6
shows the H5 resonances for representative isomers with C3,C7-
anti and C3,C7-syn configurations. In the anti isomer, the two
protons resonate together as a triplet at 2.55 ppm, while the syn
isomer exhibits a broader, more complex multiplet (presumably
two ddd’s) from 2.60À2.48 ppm. Again, epimerization at C3 is
evident and was estimated at 15À20% for the anti isomers.
Presumably it is similar for the syn isomers, but overlapping
prevents estimation in that series.
C4F9
C4F9
From the Mixture (7R,11R)-20
20a,b
20a,c
20b,b
20b,c
CF3
C4F9
C6F13
C4F9
C6F13
(3S,7R,11R,15R)-1
64
66
85
63
CF3
(3S,7R,11R,15S)-1
(3R,7R,11R,15R)-1
(3R,7R,11R,15S)-1
C4F9
C4F9
natural product isomers (see below), these spectra reveal the
C3/C7 syn/anti ratio. The diastereotopic methylene group at C5
appears as a 2H triplet at about 2.45 ppm for the syn isomers (top
two spectra), but as two doublets of doublets of doublets at about
2.48 and 2.70 ppm (bottom two spectra) for the anti iso-
mers. Careful expansion and integration suggested each sample
contained 10À12% of its epimer at this pair of centers. Only
about 5% was expected based on the enantiomeric purity of the
relevant precursors. This suggests that despite our best efforts,
some epimerization still occurred at C3. This suggestion was
soon confirmed by the Mosher analysis.
After surveying several conditions, we settled on hydrogeno-
lysis for the removal of the PMB groups of 20. In a typical
experiment (Scheme 5), (3R,7S,11R,15S)-20b,c was dissolved in
EtOAc and exposed to palladium on carbon under a balloon of
hydrogen. After 2 days, the sample was filtered through diato-
maceous earth, then the product was carefully purified by
automated flash chromatography to give 8 mg of the correspond-
ing stereoisomer of 1 in 62% yield.
Likewise, the other seven samples were processed to give 4À
9 mg of the corresponding final samples of 1 (62À85% yields after
careful purification). These results are summarized in Table 1,
which is also a handy reference for the tag-encoding pattern of the
final products before detagging. The complete stereostructures of
all eight diastereomers of 1 are shown in Figure S1 of the SI.
Spectral Comparison of Samples of 1 and Derived Mosher
Esters. With the eight isomers of 1 in hand, we first compared the
spectra of the samples to each other, then to the natural sample.
In this way, we know which spectra are different and which are
The 13C NMR spectra of all eight isomers are very similar.
Most resonances fall in a range of about 0.01À0.02 ppm,
although some (notably C10/12 and C18) show somewhat
larger differences in some pairs of isomers. However, even
though we know that the samples are not isomerically pure, we
did not observe the clear doubling of any resonance in any of the
spectra. Accordingly, comparison of 13C NMR resonances is not
a reliable tool to differentiate isomers.
With these spectra, we can now understand why Yajima’s
spectra of the 16-isomer mixture match the spectra of the natural
product. The 13C NMR spectra are so similar that the spectrum
of the natural sample with two isomers looks the same as the
spectrum of a sample with all the isomers. For the 1H NMR case,
the natural sample contains one C3,C7-syn isomer and one anti
isomer. Thus it has one representative of each of the two possible
1H NMR spectra from the stereoisomer mixture, and therefore
looks like the spectrum of the complete mixture.
Each of the eight isomers of 1 was converted to both bis-R
and bis-S-Mosher esters to obtain a 16-stereoisomer library of the
bis-Mosher esters of hormone α1. In the typical esterification
reaction (Scheme 6), a solution of (3S,7S,11R,15R)-1 in DCM
was treated with DCC and (S)-MTPA acid. The product was
purified by flash column chromatography to obtain 72% of the
bis-MTPA ester (3S,7S,11R,15R)-21S. The other 15 bis-Mosher
1
esters were made similarly, and their structures and H NMR
spectra are shown in the SI.
The 1H NMR spectra of the Mosher esters were much more
informative than the spectra of the starting compounds.
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Regarding isomer purity, the H5 protons are now separate
ddd’s in each isomer, so both the identity and the amount of
isomer contamination could be assessed. Because we have all
diastereomers of the Mosher esters, minor resonances in one
sample must always match the major resonances in another.
Indeed, each Mosher ester had a significant set of minor
resonances that matched those of its C3 epimer. The amount
of impurity (18À34%) was more than expected (15À20%) for
most isomers, suggesting that additional small amounts of
epimerization may have occurred during Mosher ester forma-
tion. Table 2 summarizes the yields and purities of the Mosher
ester library.
Mosher esters are usually used for spectroscopic analysis and
not diastereomeric separation. Nonetheless, we subjected the
bis-S-MTPA ester (3S,7S,11R,15S)-21S to purification by semi-
prep HPLC with a chiral (S,S)-Whelk-O column eluting with
97:3 hexanes/2-propanol. The HPLC chromatogram from a
5 mg injection is shown in Figure 7. The major isomer eluted at
52.2 min and the minor C3 epimer eluted at about 52.5 min as a
shoulder to the major peak. Because, of the considerable overlap,
peak shaving was conducted to obtain several fractions.
The first fraction contained 1.2 mg of essentially pure
(3S,7S,11R,15S)-21S (>95%) as assessed by 1H NMR spectros-
copy. Likewise, we subjected the remaining 15 Mosher ester
samples to semipreparative HPLC purification with peak shaving
to sacrifice quantity for quality (isomeric purity). The results of
these experiments are also summarized in Table 2.
Remarkably, we obtained highly isomerically enriched frac-
tions (>95%) of the major bis-Mosher ester from 13 of the 16
samples. In two of the three other cases, partial enrichment was
observed. These imperfections may have been due to peak
shaving problems; however, because we now had the spectrum
of the pure minor isomer in each of the three contaminated
samples (it was the major isomer of another sample), it was now
easy to subtract away the minor resonances and assign the
remaining ones. In this way, the resonances of all 16 1H Mosher
1
NMR spectra (700 MHz) were assigned with the aid of 1HÀ H
COSY experiments. Likewise, we recorded the complete set of 16
19F NMR spectra at 282 MHz.
The full set of 19F NMR spectra are shown in the Figure S7 of
the SI. Each of the 16 isomers exhibited one of two principal
types of 19F NMR spectra. In the eight isomers with the absolute
configurations of the Mosher ester at C1 and C3 “matched”
(both R or both S), there were two peaks of equal intensity at
about À72.47 and À72.53 ppm. In the other eight isomers where
these configurations were “mismatched” (one R, the other S),
there was a single peak at about À72.53 ppm. The chemical shifts
are not identical in all of the isomers, but the variations are small
(<0.03 ppm).
Apparently then, the chemical shift of the Mosher ester CF3
group on C16 is about the same (À72.53 ppm) in all isomers. In
half of the cases, this resonance overlaps with the resonance of
Scheme 6. Synthesis of One Representative of the 16-Mem-
ber Mosher Ester Stereoisomer Library
Figure 7. Typical chromatogram from semipreparative HPLC separa-
tion of the Mosher ester (3S,7S,11R,15S)-21S. The shoulder on the tail
of the main peak is the C3 epimer (3R,7S,11R,15S)-21S.
Table 2. Summary of the Synthesis and Purity of the Mosher Ester Library
starting material
MTPA acid
product
yield (%)
C3 epimer before/after HPLC (%)
(3S,7S,11R,15R)-1
(3S,7S,11R,15R)-1
(3S,7S,11R,15S)-1
(3S,7S,11R,15S)-1
(3R,7S,11R,15R)-1
(3R,7S,11R,15R)-1
(3R,7S,11R,15S)-1
(3R,7S,11R,15S)-1
(3S,7R,11R,15R)-1
(3S,7R,11R,15R)-1
(3S,7R,11R,15S)-1
(3S,7R,11R,15S)-1
(3R,7R,11R,15R)-1
(3R,7R,11R,15R)-1
(3R,7R,11R,15S)-1
(3R,7R,11R,15S)-1
R
S
(3S,7S,11R,15R)-21R
(3S,7S,11R,15R)-21S
(3S,7S,11R,15S)-21R
(3S,7S,11R,15S)-21S
(3R,7S,11R,15R)-21R
(3R,7S,11R,15R)-21S
(3R,7S,11R,15S)-21R
(3R,7S,11R,15S)-21S
(3S,7R,11R,15R)-21R
(3S,7R,11R,15R)-21S
(3S,7R,11R,15S)-21R
(3S,7R,11R,15S)-21S
(3R,7R,11R,15R)-21R
(3R,7R,11R,15R)-21S
(3R,7R,11R,15S)-21R
(3R,7R,11R,15S)-21S
72
73
63
61
82
64
68
77
79
78
77
73
64
80
77
63
18/<5
19/<5
33/<5
34/<5
23/<5
21/<5
26/11
26/<5
28/<5
28/28
17/<5
16/<5
24 /<5
25/17
19/<5
20/<5
R
S
R
S
R
S
R
S
R
S
R
S
R
S
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configurations are mismatched, the Δδ of the two H16 is 0.03
ppm (spectra 1 and 3).
With hindsight, we can see that these features (C3 and C15
absolute configuration, C3/C7 relative configuration) are largely
mutually independent, meaning that it would have been possible
to make as few as four Mosher esters. “Cutting and pasting” of the
relevant sections of these four spectra would have made good
approximations of the missing 12 spectra.
Unfortunately, the Mosher ester spectra do not provide
information about the configuration at C11. In other words,
the 16 1H NMR spectra of isomers 21 come in eight substantially
identical pairs. The members of each pair have the same con-
figurations at the Mosher ester, C3, C7, and C15, but the
opposite configuration at C11.
1
Figure 8. Two examples of the H5 region of the H NMR spectra
of the Mosher esters. (3S,7S,11R,15S)-21S (top, C3,C7-anti) and
(3S,7R,11R,15S)-21S (bottom, C3,C7-syn).
To learn if there were any meaningful differences in the 13C
NMR spectra of the bis-Mosher esters, we recorded and com-
1
13
pared 1D 13C and 2D HÀ C HMQC spectra of (3R,7R,-
11R,15R)-21R and (3S,7S,11R,15S)-21S. These spectra, shown
in the SI, were also substantially identical and no differences were
seen in the nonoverlapping peaks or cross-peaks.
1
We also recorded H NMR spectra of several pairs of C11
epimeric Mosher esters in C6D6, but again could not find clear
differences. Thus, it is not currently possible to differentiate C11
epimers of either the natural product or any of its bis-Mosher
esters by 1H, 13C, or 19F NMR spectroscopy.
With the detailed understanding of the Mosher esters pro-
vided by the complete library of spectra, we are now in position to
assess the published spectra of other synthetic and natural
samples, and to confirm the structure of the natural product.
Figure 9. Four examples of the H1/H16 region of the 1H NMR spectra
of the Mosher esters. Protons H1 are downfield from H16 in all spectra.
Spectra in order from top to bottom are as follows: (1) (3S,7R,11R,15S)-
21R; (2) (3R,7R,11R,15R)-21R; (3) (3S,7S,11R,15R)-21S; and (4)
(3R,7S,11R,15S)-21S.
1
Dr. Ojika kindly provided the original FID of his H NMR
spectrum of the bis-(R)-Mosher ester of the natural hormone,
and the overlay of this spectrum with two members of the
Mosher ester library is shown in Figure S11 of the SI. The
resonances of the major isomer in this spectrum overlay with
(3R,7R,11R,15R-21R (and the 11S epimer), while the minor
isomer resonances overlay with the C3 epimer (3S,7R,11R,15R)-
21R (and the 11S epimer).
This confirms the assignment of the R configuration to the C3,
C7, and C15 stereocenters in the natural hormone. The C11
stereocenter cannot be assigned from the spectra because there
are no substantive differences. Fortunately, it is clear from Yajima’s
testing results that C11 has the R configuration. Accordingly, we
confirm Yajima’s assignment of the natural hormone as “all-R”,
(3S,7R,11R,15R).
In the process of assigning hormone configuration, Yajima
made all four individual epimers of 1 and published copies of
their bis-(R)-Mosher esters as Supporting Information. Two
pairs of these compounds differed only in configuration at C11.
Like ours, these pairs of spectra are identical. Further, after
accounting for differences in spectrometer frequency, we can
show by comparison with our spectra that Yajima’s stereochem-
ical assignments are all correct, and more importantly, that the
Mosher ester samples are all of good quality and relatively free
from isomer impurities.27
Feringa and co-workers reported the synthesis and testing of
two isomers of 1, and they commented that the NMR spectra of
both of their isomers were identical to the spectra of the natural
product. One of Feringa’s isomers is the enantiomer of the
natural product (all-S-1), while the other is the C7 epimer of the
enantiomer. Because these are C3/C7 syn/anti isomers, their
spectra should not match each other, nor should they match the
natural sample.
the C1Mosher ester (one peak is seen) and in the other half it
does not (two peaks are seen). Surprisingly, it is the Mosher ester
that is further from its stereocenter (C1/C3) that exhibits
different 19F chemical shift, not the Mosher ester that is closer
to its stereocenter (C16/C15).
1
The H NMR spectra of the Mosher esters provide much
information, including the absolute configurations at C3 and C15
and the relative configuration of C3/C7. At 700 MHz, the C5
protons in all of the Mosher esters appeared as two well resolved
ddd in all cases. The relative configuration of C3/C7 could be
read from the difference in chemical shift between the two C5
protons. In the anti isomers these differed by 0.08À0.09 ppm,
while in the syn isomers the difference was 0.15À0.16 ppm.
Figure 8 shows one representative example of each case. Table S4
of the SI lists chemical shifts of H5 for all of the isomers.
The region 4.3À4.1 ppm contains the protons adjacent to the
Mosher esters, and is very diagnostic. The configuration at C3
can be read from the protons on C1, while the configuration at
C15 can be read from the proton resonances on C16. One
spectrum each of the four possibilities here is shown in Figure 9,
while the full set of 16 expansions are in Figure S9 of the SI. When
the Mosher ester and C3 configuration are matched, the H1
signals are well resolved as a tdd and a ddd (see spectra 2 and 3).
When the configurations are mis-matched, these resonances are
coincident and a simple 2H triplet results (see spectra 1 and 4).
The C16 methylene protons always appear as doublets of
doublets, but with different Δδ. When the Mosher ester con-
figuration and the C15 configuration are matched, the Δδ of
the two H16 is 0.17 ppm (spectra 2 and 4), while when the
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We compared the key H5 resonances in Feringa’s two spectra
from the Supporting Information,27 and indeed these regions are
very similar. In addition, we agree that both spectra match that of
the natural sample reasonably well. This means that neither of
Feringa’s samples is pure. Furthermore, while estimating ratios
from pdf spectra is difficult, it seems clear that the H5 triplet
resonance predominates in both spectra. This means that the
C3/C7 anti isomer is the major component in both samples,
even though one of the samples should have been the syn isomer.
Apparently, epimerization at C3 occurred at the late stages of
Feringa’s synthesis.
then compared in a spreadsheet. Our work points out a problem
with this approach; direct comparison of the spectra of the
candidates to those of the natural product is out of order. First,
the spectra of the candidates have to be compared with each
other. Unless these can be reliably differentiated, there is no point
in comparing them to the natural product. In addition, the results
suggest caution in ad hoc assumptions that compounds with
stereocenters separated by as few as three atoms will reliably have
different spectra.
The 19F and 13C NMR spectra of the Mosher esters provide
limited information, but the 1H NMR spectra are by far the most
informative. Even so, and despite the presence of not one but two
Finally, we also reviewed spectra in the Supporting Informa-
tion of Loh's paper for synthetic all R-1. The H5 resonance here is
indeed a clean triplet for the C3/C7 anti isomer, with no
evidence of contamination of the syn isomer. Recall that this
1
Mosher esters (on O1 and O16), the appearance of the H
Mosher NMR spectra still did not depend on the configuration of
at C11. Impressive long-range effects of Mosher esters have been
observed,28 but assumptions that such effects will translate to
very different kinds of compounds can be perilous.
1
syn/anti ratio is the only information provided by H NMR
spectra of the hormones. The presence of minor epimers at other
stereocenters cannot be assessed because of the identical spectra.
Here is where the strengths of fluorous mixture synthesis
come to the fore. If all of the relevant isomers can be made
together, then no assumptions need to be taken at the outset. You
may not be able to predict whether spectra will be identical or
not, but in the end you will know with certainty which are and
which are not. And if you do have to make Mosher or other chiral
derivatives to differentiate isomers, then it does not matter
whether the advanced Mosher (or any other) rule works or
not. You are matching actual candidate spectra; you do not need
any models or associated rules or guidelines derived therefrom.
Either the spectra match, or they do not.
’ CONCLUSIONS
Fluorous mixture synthesis has provided all eight diastereo-
mers of the phytophthora hormone α1 with the R configuration
at C11 as individual samples after demixing and detagging. The
samples were not isomerically pure because some epimerization
had occurred at C3. This could be assessed by 1H NMR analysis,
but that feature (relative configuration between C3 and C7)
proved to be the only difference. In other words, each of the eight
1
isomers exhibited one of only two different H NMR spectra.
The 13C NMR spectra provided no differentiating information;
all eight spectra were very similar.
’ ASSOCIATED CONTENT
S
The library of all possible bis-Mosher esters (16) was then
made by esterification. Surprisingly, in most of the cases, it was
possible to substantially enrich the major isomer by chromatog-
Supporting Information. Contains complete experi-
b
mental details, tabular NMR data, supplemental figures, and
copies of NMR spectra of the hormone and Mosher ester
libraries. This material is available free of charge via the
Internet at http://pubs.acs.org.
1
raphy with a chiral HPLC column. The complete set of H
and 19F NMR spectra were recorded and assigned along with a
partial set of 13C spectra. Analysis of this data identified several
convenient, redundant features to assign the configurations at
C3, C7, and C15.
’ AUTHOR INFORMATION
The 16 1H NMR spectra of the Mosher ester library fell into
eight identical pairs; no information was provided about the C11
configuration. Fortunately, it was clear from Yajima’s prior work
that C11 must have the R configuration. Knowing this and having
access to Ojika’s Mosher spectra of the natural sample, we
confirmed Yajima’s assignment of the hormone as “all-R”.
Did you make predictions about whether the spectra of the
hormone and Mosher ester library members would be the same
or different? If so, then how well did you do? We find it surprising
that the 1H and especially the 13C NMR spectra of the hormone
isomers are so similar. The tabulated 13C NMR resonances
exhibit small differences for some isomers, but it is not clear that
any of these differences is reliable for assignment. For example,
we could not uniquely match any of the published 13C NMR
spectra of natural or synthetic samples of the hormone to one of
the eight spectra in Table S3 of the SI. Try it yourself.
Corresponding Author
curran@pitt.edu
’ ACKNOWLEDGMENT
We thank the National Institutes of Health, National Institute
of General Medical Sciences, for funding of this work. We thank
Dr. M. Ojika for copies of NMR spectra and original FID data.
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