Synthesis and NMR characterization of (Z, Z, Z, Z, E, E,ω)- heptaprenol

Article abstract of DOI:10.1021/ja306184m

We describe a practical, multigram synthesis of (2Z,6Z,10Z,14Z,18E,22E)-3, 7,11,15,19,23,27-heptamethyl-2,6,10,14,18,22,26-octacosaheptaen-1-ol [(Z ₄,E₂,ω)-heptaprenol, 4] using the nerol-derived sulfone 8 as the key intermediate. Su

Full text of DOI:10.1021/ja306184m

Article
pubs.acs.org/JACS
Synthesis and NMR Characterization of (Z,Z,Z,Z,E,E,ω)-Heptaprenol
Dusan Hesek, Mijoon Lee, Jaroslav Zajícek, Jed F. Fisher, and Shahriar Mobashery*
̌
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States
S
* Supporting Information
ABSTRACT: We describe a practical, multigram synthesis of
(2Z,6Z,10Z,14Z,18E,22E)-3,7,11,15,19,23,27-heptamethyl-
2,6,10,14,18,22,26-octacosaheptaen-1-ol [(Z4,E2,ω)-heptapren-
ol, 4] using the nerol-derived sulfone 8 as the key
intermediate. Sulfone 8 is prepared by the literature route
and is converted in five additional steps (18% yield from 8) to
(Z4,E2,ω)-heptaprenol 4. The use of Eu(hfc)3 as an NMR shift
reagent not only enabled confirmation of the structure and
stereochemistry of 4, but further enabled the structural
assignment to a major side product from a failed synthetic
connection. The availability by this synthesis of (Z4,E2,ω)-heptaprenol 4 in gram quantities will enable preparative access to key
reagents for the study of the biosynthesis of the bacterial cell envelope.
several pivotal Lipid I/II structures (2 and 3 in Chart 1), such
as those having the meso-diaminopimelic substructure charac-
teristic of Gram-negative Lipid II, have yet to be completed.
Moreover, syntheses of prenol-containing structures often use
the plant-derived (Z7,E3,ω)-undecaprenol as the lipid compo-
nent. While the evidence thus far indicates that the (Z7,E3,ω)-
undecaprenol and (Z8,E2,ω)-undecaprenol are functionally
equivalent as the lipid component of Lipid II, these plant
polyprenols are themselves rare. Given the important role of
the lipid component in many aspects of biological recognition17
and the scarcity of the natural polyprenols, the development of
practical synthetic routes to these structures is important. The
chemical synthesis of oligo- and polyprenols for use in prenol-
containing structures is recent.23,24 Previous syntheses of
polyprenols include dolichol-20,25 (Z8,E2,ω)-undecaprenol,26
(Z7,E3,ω)-undecaprenol,27 and (Z4,E2,ω)-heptaprenol.24,26,28
Walker, Kahne, and colleagues19,20 showed that the Lipid II
analogue 6, having the shorter C35 (Z4,E2,ω)-heptaprenol
(Chart 1), is accepted in vitro as a component of the Lipid II
substrate of the penicillin-binding protein (PBP) enzymes.
Accordingly, a general synthesis of heptaprenolsand no less
importantly, a versatile spectroscopic means of confirming their
structurewould be especially useful. As a demonstration of
both of these objectives, we report the synthesis of
(2Z,6Z,10Z,14Z,18E,22E)-3,7,11,15,19,23,27-heptamethyl-
2,6,10,14,18,22,26-octacosaheptaen-1-ol (Z4,E2,ω)-heptaprenol
(4) and its complete 1H and 13C NMR assignment using chiral
shift reagent-facilitated analysis of its homo- and heteronuclear
2D spectra.
INTRODUCTION
■
The five-carbon isoprene segment is foundational to the
biosynthetic pathways of both primary and secondary
metabolism.1 One class of isoprenoids, the polyprenols, is
used catalytically in primary metabolism as a membrane-
integral carrier of hydrophilic structure: of the wall teichoic
acids in Gram-positive bacteria; of the saccharides destined for
the lipopolysaccharide of the outer membrane of Gram-
negative bacteria; of the peptidoglycan monomers used by
bacteria to assemble their cell wall; and of the saccharides
destined for post-translational modification of prokaryotic and
eukaryotic proteins.2,3 The discovery in 1964 that a lipid
intermediate was required for bacterial peptidoglycan syn-
thesis4,5 was followed in 1967 by its identification as a C55
undecaprenol containing 11 CC double bond functional
groups.6−8 As a result of the extensive overlap of resonances in
the polyprenols,9 the stereochemical assignment to the bacterial
u n d e c a p r e n o l a s t h e ( Z 8 , E 2 , ω ) - u n d e c a e n o l
[(2Z,6Z,10Z,14Z,18Z,22Z,26Z,30Z,34E,38E,42)-
3 , 7 , 1 1 , 1 5 , 1 9 , 2 3 , 2 7 , 3 1 , 3 5 , 3 9 , 4 3 - u n d e c a m e t h y l -
2,6,10,14,18,22,26,30,34,38,42-tetratetracontaundecaen-1-ol,
compound 1 in Chart 1] rests upon exquisite mechanistic
studies pertaining to its biosynthesis. These studies show that a
single enzyme, undecaprenol diphophate synthase, synthesizes
the diphosphate of 1 from farnesyl diphosphate by eight-fold
cis-prenyl homologation using isopentenyl diphosphate as the
prenyl donor.10−13
Nonetheless, the low pool levels of the bacterial lipid
intermediates,14 their limited accumulation in cell-free systems,
and the tedious effort involved in their isolation and purification
have restricted their availability for the study of the
peptidoglycan pathway.15 Several efforts toward the synthesis
of the Lipid I, II, and IV structures and their analogues, using
both chemical and chemoenzymatic methods, have been
reported.15−22 These syntheses are demanding, and indeed
Received: June 25, 2012
Published: August 4, 2012
© 2012 American Chemical Society
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Chart 1. Structures of the Undecaprenol-Containing Lipid I and Lipid II Used in Bacterial Peptidoglycan Biosynthesis and
Their Heptaprenol Analogues
Scheme 1. Retrosynthetic Disconnections Evaluated as a Practical Synthesis of (Z4,E2,ω)-Heptaprenol
desulfonylation reactions (see Scheme 3, 22 to 23 and 7c to
4C).
RESULTS AND DISCUSSION
■
Three different retrosynthetic disconnections toward 4 were
considered, corresponding to structures 7a, 7b, and 7c as key
intermediates (Scheme 1). These three intermediates coincide
with the reaction of different sulfone-stabilized carbanions (8,
11, and 12) with a diprenyl halide (9 or 10, X = Cl, Br).
Although the use of sulfone-stabilized carbanions is a standard
method for polyprenol synthesis, a systematic evaluation of the
most suitable reaction pairing in terms of yield and stereo-
chemical fidelity was absent from the literature. The most
frequently used route is A of Scheme 1. Our interest in the
alternative routes B and C emerged from our recognition, as we
examined these reactions, that the reagent pair 10 and 11 was
easier to prepare on scale than the reagent pair 8 and 9. While
routes A and B synthesize the Z4-tetraprenol for coupling with
the E,E-farnesol-derived sulfone 12, route C reacts 12 with the
nerol-derived halide 9, followed by a second addition of 9 to
complete the synthesis. Routes A and C have the advantage of
using the most readily prepared neryl reagent (9, compared to
the three other neryl derivatives 8, 10, and 11 shown in Scheme
2) as a starting material, but route C is disadvantaged relative to
routes A and B by its requirement for two separate
We converted (commercially available) nerol to the pivotal
intermediate 13 in 10 steps (including epoxide formation from
the bromohydrin, epoxide hydrolysis, oxidative cleavage,
reduction, iodination, triphenylphosphonium iodide formation,
and Wittig reaction with the hydroxyketone) using the
literature route.29 Although this route appears laborious, it is
quite amenable to scale (we have prepared 13 on a 50-g scale).
Compound 13 was separately transformed into the four neryl
derivatives (8−11) of Scheme 2. The syntheses of 9 and 11
were straightforward; the syntheses of 8 and 10 were not. The
synthesis of sulfone 8 ultimately involved comparison of two
different halides (10-Br and 10-Cl) for the sulfone alkylation
(see Figure 1, left side). Using 10-Br as a reagent, we isolated 8
as the major product. In contrast, the analogous reaction using
10-Cl as a reagent gave a 5:4 diastereomeric mixture of 8 and
1
15 as assessed by H NMR analysis. These structures were
assigned by extensive NMR analyses, done after removal of the
O-THP protecting group in order to both simplify the NMR
spectra and facilitate chromatographic purification.
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isomerization under one set of standard halogenation
conditions, but not under another, set the requirement for
full verification of structure and stereochemistry, following each
key step of our synthesis.
Scheme 2. Syntheses of the Four Neryl Units 8−11
From 8, three routes toward heptaprenol 4 were compared
(Scheme 3). Routes A and B used different neryl derivatives to
give the nerylneryl derivative 18 (route A) and nerylneryl
derivative 20 (route B), respectively. Each was separately
coupled with farnesylsulfone 12. Route C used repetitive
coupling of neryl derivative 9. In contrast to the final prenol
structures, the penultimate synthetic intermediates give well-
dispersed and easily interpretable NMR spectra, as exemplified
by the complete assignment of 18-OH, 20-OH, and 7b (18-
OH was obtained after removal of O-THP in 18, Supporting
Information). Last, intermediates 7a, 7b, and 7c were
deprotected by sequential reductive desulfonylation and
reductive debenzylation to give 4 (identified in Scheme 3 by
their routes of synthesis: 4A, 4B, and 4C).
1
The H and 13C spectra of alcohols 17 (from 8) and 16
(from 15) are too similar to allow definitive structural
assignment by themselves. Following resonance assignments
from 2D homo- and heteronuclear correlation spectra, analysis
of the corresponding ROESY spectra (Figure 1) identified the
difference. Compound 17 (arising from reaction with 10-Br)
had the desired (2Z,6Z) stereochemistry, whereas 16 (from
reaction with 10-Cl) was the (2Z,6E)-stereoisomer. While E,Z
scrambling is well known to occur during the synthesis of many
allyl halides from allyl alcohols, we are at a loss to suggest the
critical experimental difference accounting for the contrast
between 10-Br and 10-Cl, and as well as to account for the
unanticipated regiochemistry of the isomerization. Nonetheless,
the discovery of this unexpectedly facile (and reproducible)
1
The final products 4A and 4C gave identical H NMR
spectra, characterized by eight methyl resonances in a 4:2:1 Z/
E/ω pattern (Figure 2). Further NMR analysis (see below)
verified the product stereochemistry as the desired (Z4,E2,ω)-
heptaprenol 4. The purity of the heptaprenol 4 isolated by silica
flash chromatography (following the reductive desulfonylation
step) exceeded 90% by 1H NMR analysis (Supporting
Information). The analytical data for 4 matched the published
data given by Chang et al.28 In contrast, the heptaprenol from
route B was a 1:3 diastereomeric mixture of 4 and an isomer
designated 4B2. The mixture was separated by column
Figure 1. Key correlations in the 1H ROESY NMR spectra used to assign the alcohol diastereomers 16 (top spectrum) and 17 (bottom spectrum).
Empty boxes in top spectrum indicate the absence of correlations from 6Z (H-6,H-9) and (H-5,H-8). Instead, the ROESY spectrum for 16 shows
correlation between H-6 and H-8 (colored in blue), demonstrating 6E configuration. Relevant synthetic transformations are summarized on the left.
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Scheme 3. Synthetic Routes Evaluated as a Practical Synthesis of the (Z4,E2,ω)-Heptaprenol
was best. The use of chiral shifts to disperse the resonances of
terpenoids for the purpose of structure assignment31,32 or for
enantiomeric ratio determination33−36 is known, but infre-
quent. Optimization of the molar ratio of Eu(hcf)3 to 4 for the
purpose of structure assignment is shown in Figure 3. The
Figure 2. Comparison of 1H NMR spectra (500 MHz) of the
heptaprenol isomers 4A, 4B2, and 4C in CDCl3.
1
chromatography. Although the H NMR spectra of 4 and 4B2
are very similar (and show extensive overlap of signals), the 1H
spectrum of 4B2 has two distinguishing resonances (Figure 2,
red arrows). These new resonances are diagnostic of an
undesirable isomerization.
Figure 3. 1H NMR spectra (500 MHz) of 4 in the presence of
different molar ratios of Eu(hcf)3 (from bottom to top, molar ratios of
0, 0.04, 0.09, 0.13, and 0.17 equiv of shift agent to 4). The optimal
balance between resonance dispersion and line broadening occurs at
0.13 equiv of Eu(hcf)3 (3 mg of Eu(hfc)3 to 10 mg of 4 in CDCl3).
The extensive resonance overlaps for 4 and 4B2, and the
requirement for a definitive verification of the structure of 4,
prompted a search for a general NMR method to facilitate the
structural assignment of prenol derivatives. Proof of structure
by NMR of a polyprenol (and of the synthetic intermediates
leading to the polyprenol) is rare. Indeed, there is a 45-year gap
between the discovery of polyprenol involvement in bacterial
primary metabolism and a full NMR-based structural assign-
ment of a polyprenol, that of the plant-derived (Z6,E3,ω)-
decaprenol [(2Z,6Z,10Z,14Z,18Z,22Z,26E,30E,34E,38)-
3 , 7 , 1 1 , 1 5 , 1 9 , 2 3 , 2 7 , 3 1 , 3 5 , 3 9 - d e c a m e t h y l -
2,6,10,14,18,22,26,30,34,38-tetracontadecaen-1-ol] accom-
plished in 2009 using 3D NMR spectral analyses.30 The
absence of this characterization is understandable, due to the
extensive overlap of resonances even at high magnetic field
(∼18.79 T). Nonetheless (and to our very pleasant surprise),
an exploration of several common chiral shift reagents
(including Eu(tcf)3, Eu(hcf)3, Pr(hcf)3, Yt(hcf)3, and Resolve-
Al AgFOD) demonstrated promise. Of these reagents, Eu(hcf)3
structure of heptaprenol 4 was verified by analysis of
homonuclear (DQF-COSY, TOCSY, ROESY) and hetero-
nuclear (1H−13C HSQC, HSQC-TOCSY, HMBC) spectra
1
obtained using 0.13 equiv of Eu(hcf)3 to disperse the H
1
resonances. H connectivities were established by analysis of
the DQF-COSY and TOCSY spectra.
Resonances of all carbons with directly attached protons
were assigned using the HSQC and HSQC-TOCSY spectra.
For example, Figure 4A shows the correlation of the vinyl
hydrogen and carbon resonances in the HSQC-TOCSY
spectrum. The HMBC spectrum was then employed to assign
the quaternary carbon resonances and to verify the correctness
of the connectivities established by the analysis of the other
spectra. Figure 4B shows the connectivities of the first two
prenyl units (colored orange and turquoise, respectively)
deduced from the HMBC spectrum. The H-5 resonance is
identified in the HMBC spectrum by its correlation with the
three carbon resonances (C-4, C-6, and C-7) of its prenyl unit
(colored in turquoise), and by its additional correlation with
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Figure 4. (A) Use of the HSQC-TOCSY spectrum for 13C (CH3, CH2, and CH) resonance correlation. Each prenyl unit is shown in a different
color. (B) Interpretive use of the HBMC exemplified by the correlations for the two terminal prenyl units (colored orange and turquoise). (C)
Pictorial summary of the 1H resonance assignments for 4 (800.13 MHz spectrum, obtained using a solution of 10 mg of 4 and 3 mg of Eu(hcf)3 in
600 μL of CDCl3).
1
the C-3 signal of the neighboring prenyl unit (colored orange).
The H-4 resonance also correlates with the C-2 and C-3
resonances from the neighboring prenyl unit (colored orange).
The signal of H-34 correlates with the signal of C-8 (colored
purple). In this way, connectivity among the prenyl units was
established. Finally, the assignment to each CC double bond
of an E or Z configuration was determined by analyzing the
identities for 4. The complete H and 13C NMR resonance
assignments for 4 are given in Table S2 in the Supporting
Information.
Eu(hcf)3 likewise facilitated the NMR assignment of
structure to 4B2 (derived from 7b). While the dispersion
effect of Eu(hcf)3 was less than that seen for 4, it was
nonetheless sufficient for the NMR assignment of structure to
4B2.
1
ROESY spectrum. Figure 4C summarizes the H resonance
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Figure 5. (A,B) HSQC-TOCSY and (C,D) HBMC spectra of two atypical prenyl units of 4B2 (800.13 MHz, obtained using a solution of 10 mg of
4B2 and 3 mg of Eu(hcf)3 in 600 μL of CDCl3).
The structural difference occurs from C-5 to C-9 in 4B2,
giving two distinct and atypical units: one having three
continuous CH2 carbons (C-4, C-5, and C-6) and the other
having a single CH2 between two vinyl units (C-9 between C-8
and C-10). The two distinguishing resonances in the 1H
spectrum of 4B2 (Figure 2) are those of H-5 and H-9. As a
prenyl segment has a single vinyl hydrogen, there is always only
one crosspeak in the vinyl 13C region of HSQC-TOCSY
between vinyl proton and carbon resonances, which corre-
sponds to a given vinyl CH group (see for example the
crosspeak for the CH-6 vinyl group shown in Figure 4A). In
contrast, the vinyl H-8 resonance of 4B2 shows in the same
type of spectrum two crosspeaks: one with the vinyl C-8
resonance and the other with vinyl the C-10 resonance (Figure
5A). Similarly, the vinyl H-10 resonance also shows two
crosspeaks: one with the vinyl C-10 resonance and the other
with the vinyl C-8 resonance. Both H-8 and H-10 resonances
then exhibit a crosspeak with the C-9 resonance at δ = 27.19
ppm, defining the first atypical unit. The second unit contains
three consecutive CH2’s (correlations are shown in Figure 5B).
The HMBC spectrum confirmed the proposed connectivities of
the two atypical units: both connected to each other and
between the first and fourth prenyl units (Figure 5C,D). Hence
the terminal prenyl (colored orange) is connected to the
−(CH2)3− segment (colored turquoise) as evidenced by the
HMBC correlation of the H-35 resonance with the C-4
resonance. Correlations of the H-6 and H-34 resonances with
the C-8 resonance confirmed connection of the −(CH2)3−
segment to the prenyl segment colored in purple. Finally, the
crosspeak between the H-33 (purple unit) and C-12 resonances
confirmed connectivity to the prenyl unit colored in green. The
1H and 13C resonance assignments for 4B2 are given in Table
S2 in the Supporting Information.
An overall 30% yield of the 3:1 mixture of 4B2 and 4 (from
prenyl sulfone 11) characterized the failed route B. The two
successful routes A and C to 4 gave comparable overall yields of
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HSQC-TOCSY, and HMBC NMR spectra. Proton connectivities were
derived from the DQF-COSY and TOCSY spectra. 13C resonances
corresponding to carbons with directly attached protons were assigned
using HSQC and HSQC-TOCSY spectra. HMBC spectra were used
to assign resonances of the quaternary carbons and to validate the
connectivities established by the other spectra. ROESY spectra were
utilized to establish the E or Z configurations for each CC double
bond. Experiments were performed at 25 °C using a Bruker AVANCE
II spectrometer equipped with a TCI cryoprobe and operating at a 1H
resonance frequency of 800.13 MHz. Standard pulse sequences were
used.45−50 Time domain data (t2 and t1) for 2D experiments were
recorded as 2048 × 1024 complex matrices with 16 and 32 scans per
t1 increment for homonuclear and heteronuclear spectra, respectively.
Relaxation delay between individual scans and spin-lock time for
TOCSY experiments was 1.4 s and 60 ms, respectively. For ROESY
experiments, a relaxation delay of 4 s and a spin-lock time of 400 ms
gave 2048 × 512 complex time domain points. Linear prediction to
1024 complex points was applied in the t1 domain. The data were zero-
filled to obtain final 2048 × 2048 complex matrices. In all other
homonuclear 2D experiments, zero-filling was used only in the t1
domain to obtain final 2048 × 2048 complex time domain data. In
heteronuclear 2D experiments, linear prediction to 2048 complex data
points was employed in the t1 domain, which was zero-filled to 4096 to
get final 2048 × 4096 complex time domain data. Shifted sine bell
weighting functions were applied in both domains prior to double
Fourier transformation. In the 3D 1H−13C HSQC-TOCSY experi-
ments, 1024 × 48 × 128 complex time domain points in t3, t2, and t1
domains were collected with 16 scans per time increment, relaxation
delay of 1.3 s, and 60 ms spin-lock. Linear predictions to 128 and 256
complex points were utilized in the t2 and t1 domains. Zero-filling gave
2048 × 256 × 512 complex matrices. Shifted sine bell weighting
functions were applied in all three domains prior to triple Fourier
transformation. Spectra were processed using Bruker TopSpin 2.1
20% for route A and 18% for route C relative to their respective
prenyl sulfones (8 and 12) as starting materials. In terms of
heptaprenol yield the two routes are equivalent. A clear choice
between the two routes emerges for the synthesis of longer
polyprenols. After the first coupling reaction, route A requires
transformation of the coupling product 18 to halide 19 via O-
THP deprotection, followed by bromination. Route C requires
transformation of the coupling product 22 to sulfone 24 via
debenzylation/detosylation, followed by bromination and
subsequent tosylation. Overall, these three steps of route C
are lower yielding (39%), as compared to the overall yield
(69%) for the comparable steps of route A. For longer prenols
(polyprenols) route C is uncompetitive compared to route A, as
route C will require iterative repetition of the lower-yielding
steps.
CONCLUSION
■
Polyprenols are the membrane-bound component of such a
diversity of biosynthetic intermediates that they have been
termed “superlipids”.37 This diversity is exemplified by the
recent syntheses, using both chemical and chemoenzymatic
means, of the GlcNAc-UDP intermediate of bacterial cell wall
biosynthesis;38 of the glycosyl intermediates of bacterial N-
linked glycosylation;27,39 of bacterial teichoic acid biosyn-
thesis;40 of the Gram-positive bacterial S-layer glycan;41 of the
bacterial polysialic capsule;42 and of the mycobacterial arabinan
glyconjugates.43,44 In all of these syntheses the prenol is a mass-
limiting reagent.
Herein we report a practical, multigram synthesis of the
(Z4,E2,ω)-heptaprenol 4 as a key enabling material for the
synthesis of these biosynthetic intermediates, and also including
the Lipid II intermediate for peptidoglycan biosynthesis. The
purity of 4 we obtain (>90%) is directly comparable to the
purity of 4 that may be obtained by solid-phase synthesis.28 The
key advantage of our solution synthesis is its potential for scale.
The pivotal intermediate 8 is prepared easily on a 30-g scale,
and may be transformed to secure 4 on a >2-g scale.
Our optimized synthesis uses the classical retrosynthetic
route (route A) for Z-prenyl homologation. Notwithstanding
the precedence for route A, an unexpectedly facile and unusual
rearrangement was encountered during allylic chlorination of a
key intermediate, but not seen during allylic bromination of this
same intermediate under otherwise identical experimental
conditions. Our optimism that a better synthesis of 4 might
be attained using a more accessible reagent pair in an alternate
route (route B) was dashed by a facile isomerization that
occurred during the reductive desulfonylation of a late-stage
intermediate. These observations emphasize the necessity for
rigorous proof of structure in prenol synthesis, even following
seemingly straightforward synthetic transformations. Here,
proof of the latter isomerization and verification of the final
(Z4,E2,ω)-heptaprenol structure of 4 used extensive two-
dimensional NMR correlations in the presence of the chiral
shift reagent Eu(hfc)3 for resonance dispersion. The advanta-
geous use of chiral shift reagents and high magnetic field
strength as seen here for these prenols suggests a general value
to this approach for NMR structural assignment.
1
1
software. H spectra, the H dimension in 2D heteronuclear spectra,
and the 1D 13C{1H} spectra were referenced to solvent (DMSO, δH
2.5 and δC 39.5 ppm; CDCl3, δH 7.27 and δC 77.23 ppm). The 13C
dimension in the 2D heteronuclear spectra was referenced indirectly.51
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental procedures and NMR data for all new
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
mobashery@nd.edu
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Prof. Po-Huang Liang (Academia Sinica) for the
clarification of the stereochemistry of the endogenous bacterial
undecaprenol. This work was supported by a grant from the
National Institutes of Health. The Mass Spectrometry &
Proteomics Facility of the University of Notre Dame is
supported by grant CHE-0741793 from the National Science
Foundation.
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