Combinatorial evolution of site- and enantioselective catalysts for polyene epoxidation

Article abstract of DOI:10.1038/nchem.1469

Selectivity in the catalytic functionalization of complex molecules is a major challenge in chemical synthesis. The problem is magnified when there are several possible stereochemical outcomes and when similar functional groups occur repeatedly within the same molecule. Selective polyene oxidation provides an archetypical example of this challenge. Historically, enzymatic catalysis has provided the only precedents. Although non-enzymatic catalysts that meet some of these challenges became known, a comprehensive solution has remained elusive. Here, we describe low molecular weight peptide-based catalysts, discovered through a combinatorial synthesis and screening protocol, that exhibit site- and enantioselective oxidation of certain positions of various isoprenols. This diversity-based approach, which exhibits features reminiscent of the directed evolution of enzymes, delivers catalysts that compare favourably to the state-of-the-art for the asymmetric oxidation of these compounds. Moreover, the approach culminated in catalysts that exhibit alternative-site selectivity in comparison to oxidation catalysts previously described.

Full text of DOI:10.1038/nchem.1469

ARTICLES
PUBLISHED ONLINE: 14 OCTOBER 2012 | DOI: 10.1038/NCHEM.1469
Combinatorial evolution of site- and
enantioselective catalysts for polyene epoxidation
Phillip A. Lichtor and Scott J. Miller
*
Selectivity in the catalytic functionalization of complex molecules is a major challenge in chemical synthesis. The problem
is magnified when there are several possible stereochemical outcomes and when similar functional groups occur
repeatedly within the same molecule. Selective polyene oxidation provides an archetypical example of this challenge.
Historically, enzymatic catalysis has provided the only precedents. Although non-enzymatic catalysts that meet some of
these challenges became known, a comprehensive solution has remained elusive. Here, we describe low molecular weight
peptide-based catalysts, discovered through a combinatorial synthesis and screening protocol, that exhibit site- and
enantioselective oxidation of certain positions of various isoprenols. This diversity-based approach, which exhibits features
reminiscent of the directed evolution of enzymes, delivers catalysts that compare favourably to the state-of-the-art for the
asymmetric oxidation of these compounds. Moreover, the approach culminated in catalysts that exhibit alternative-site
selectivity in comparison to oxidation catalysts previously described.
nalysis of biosynthetic pathways reveals that functional group asymmetric oxidation of allylic urethanes17 and indoles18. During
selectivity in enzymatic reactions is a persistent issue. For the course of the catalytic cycle, the carboxylic acid side chain of
A
example, olefins may undergo oxidation in favour of C–H aspartic acid is activated with a carbodiimide, which generates a tran-
bonds, or instead of alcohols, and different enzymes are engaged sient peracid on reaction with hydrogen peroxide (Fig. 1a). Catalytic
to reverse the order of events. Similarly, the derivation of strategies turnover of this cycle is enhanced through the addition of common
for the chemical synthesis of complex structures requires constant nucleophilic co-catalysts such as 1-hydroxybenzotriazole (HOBt)
consideration of selectivity. The complexity of the situation is ampli- and N,N-dimethylaminopyridine (DMAP), an approach distinct
fied when identical functional groups occur repeatedly within the from enzymatic ‘epoxidases’19, which often exploit cofactors. When
same molecule. Now, for example, catalysts may be called on to applied to farnesol (1), this catalytic cycle, which employs propionic
oxidize a singular olefin in the presence of others, and different cat- acid (2) as the catalyst, furnishes the same product selectivity as the
alysts may be needed to target alternative sites. Even so, selectivity textbook-level reagent meta-chloroperbenzoic acid (m-CPBA),
for one of the multiple sites represents but one aspect of the chemi- which is used as a stoichiometric reagent (Fig. 1b).
cal challenge, because many functional groups may also react in
The work began with an assessment of peptide-embedded
ways that yield new isomers, which adds stereochemical diversity aspartates to realize the site-selective oxidation of farnesol (1).
to the positional selectivity outcomes. Thus, the selective oxidation Could catalysts based on aspartic acid be found that would
of polyenes presents a profound challenge.
oxidize farnesol with high levels of site selectivity and ultimately
For many years, the selective functionalization of similarly reac- enantiocontrol? Could catalysts be found that would functionalize
tive olefins within polyenes was observed primarily through the the allylic alkene20 or other olefins of 1? Oxidation of 1 with (þ)-
study of enzymes1. Today, small-molecule catalysts are known that or (2)-DIPT/Ti(O-i-Pr)4 (DIPT ¼ diisopropyl tartrate) under the
also meet some of these challenges, yet catalyst types that deliver conditions of Sharpless delivers 3 with excellent selectivity
substantially different site selectivity are not well established, and (82–95% enantiomeric excess (e.e.), Fig. 1c)20–26. However, we
neither are generalizable protocols for their discovery. Solutions to lacked a clear structure-based hypothesis for how peptide-based
these problems provided by venerable catalysts enable allylic olefin catalysts might achieve our goals. Therefore, we applied an
epoxidation2–5, oxidation of subterminal olefins of polyenes6,7 and on-bead screening protocol to discover peptide oxidation catalysts27
selective oxidation of conjugated systems8,9. Some strategies for the selective oxidation of 1 (Fig. 1d).
provide access to internal epoxides of polyenes using stoichiometric
auxiliary-based ‘template-directed’ strategies10–12. In this article, we Results and discussion
describe catalysts based on low molecular weight peptides13,14, dis- Our approach employs the one-bead-one-compound (OBOC)
covered through a combinatorial synthesis and screening protocol, approach to library design28, which capitalizes on split-pool combi-
that exhibit site- and enantioselective oxidation of various isopre- natorial synthesis29,30. This method also allows for an evolutionary
nols. The diversity-based approach15,16 delivers catalysts that development of catalyst optimization31, in analogy to the strategy
compare favourably to the state-of-the-art for the asymmetric oxi- employed in the directed evolution of enzymes31–34
.
dation of certain isoprenols, such as geraniol, farnesol and geranyl-
For our initial screening, we made a number of strategic
geraniol. Moreover, the approach leads to catalysts that offer decisions. For example, we elected to run reactions to a low conver-
alternative site selectivities in ways that are not known for oxidation sion so as to allow a preliminary assessment of catalyst krel values. We
catalysts described until now.
define krel as the relative rate of epoxidation at each olefinic site of 1
Previously, we developed a catalytic cycle that employed an aspar- (2,3-position to give 3; 6,7-position, 4; 10,11-position, 5). It is also
tic acid-based carboxylic acid and applied this methodology to the appreciated that reaction conversion (that is, the extent of overoxidation)
Department of Chemistry, Yale University, PO Box 208107, New Haven, Connecticut 06520-8107, USA. e-mail: scott.miller@yale.edu
*
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.1469
ARTICLES
a
b
O
CH3
CH3
CH3
2,3
†
R3HN
*
Conditions
R1
NHR4
O
R2
R5N
H3C
OH
O
10,11
6,7
C
NR5
O
Farnesol (1)
Farnesol epoxides
Monoepoxide ratio‡
OH
Relative
Entry
Oxidant/acid
conversion‡
10,11-
6,7-
2,3-
R3HN
NHR4
O
O
R1
R2
*
67%
2.6
2.2
1.0
1
H3C
O
O
OH
2
R3HN
O
NR5
NHR4
H
O
O
O
Cl
NHR5
2†
H2O2
65%
2.7
2.2
1.0
O
H
0.1 equiv.
O
i-PrO2C
CO2i-Pr
c
CH3
CH3
CH3
O
CH3
CH3
CH3
Six reports (1993–2010)
82%–95% e.e.
(references 21–26)
HO
OH
H3C
OH
H3C
OH
Ti-(O-i-Pr)4
4 Å molecular sieves
t-BuOOH
–50 to –20 ºC
Goal
CH3
CH3
CH3
CH3
CH3
CH3
CH3
d
Peptide A
2,3-epoxyfarnesol (3)
6,7-epoxyfarnesol (4)
10,11-epoxyfarnesol (5)
H3C
OH
H3C
OH
O
CH3
CH3
Peptide B
Peptide C
H3C
OH
OH
This work found peptide A
O
and peptide B
CH3
O
CH3
CH3
H3C
Figure 1 | Precedent and goal of catalytic oxidation of farnesol. a, Catalytic cycle of aspartic acid-mediated epoxidations. b, Site selectivity of m-CPBA and
epoxidation based on propionic acid. c, Precedent of the Sharpless epoxidation of 1. d, The goal of the present study. *Entry 1, 10 mol% acid, HOBt
(10 mol%), DMAP (10 mol%), 1.0 equiv. DIC, 2.0 equiv. H2O2. †Entry 2, 1.0 equiv. m-CPBA, Na2HPO4 (2.0 equiv.), DCM, H2O. ‡Determined by uncalibrated
GC integrations (see Supplementary Information for details).
can influence product distributions and muddle the extraction of (n ¼ 7 × 7 × 8 × 8, see the Supplementary Information). On com-
meaningful krel comparisons among catalysts35. Thus, we planned pletion, the theoretical distribution of peptides in this library
to validate the best ‘hit’ catalysts at a later stage under synthetically follows the histogram shown in Fig. 2b: 6% of beads contain the
meaningful conditions, with sufficient stoichiometry of the terminal parent sequence (6), 25% contain a single substitution, 38%
oxidant to achieve good yields of products (see below).
contain two substitutions and so on.
We began with the evaluation of a small library of resin-bound
Approximately 228 beads from this library were tested, including
peptide catalysts we had synthesized in a parallel fashion, as well catalyst 7, which favoured epoxide 3 with an approximate doubling
as a diverse split-and-pool library for the oxidation of 1. (The of selectivity (5:4:3 ¼ 1.3:1.0:8.2, Table 1, entry 2). This result rep-
data from the initial screen are given in the Supplementary resented a further departure of the performance of a peptide-
Information.) Both the parallel synthesis library and many based catalyst from that of the control catalyst 2, data that stimulated
members of the unbiased split-pool library displayed some selectiv- the evaluation of a second biased library.
ity, mostly towards the 2,3-position of 1 (Table 1, selected results).
For the new OBOC library, now biased towards 7, we fixed the
These observations were auspicious, given that many catalysts i þ 1 residue as Pro and i þ 2 position as Asn(Trt), where Trt
exhibited significant differences from the product distribution indicates trityl, with the hypothesis that each was important for
observed with catalytic propionic acid (5:4:3 ¼ 2.6:2.2:1.0, Fig. 1b). selectivity. Then the identities of three adjacent residues (i þ 3,
From the initial screening, catalyst
6
displayed the most i þ 4 and i þ 5) were varied. Biased library 2 (Table 1, entries 3
significant deviation from the distribution afforded by propionic and 4) produced catalysts 8a and 9a, which exhibited an even
greater site selectivity, again nearly double the preference for 3.
acid (5:4:3 ¼ 1.0:1.0:3.9, Table 1, entry 1).
To enhance the selectivity for 3, a small OBOC combinatorial Catalyst 8a demonstrated a 5:4:3 ratio of 1.2:1.0:18.0. Catalyst 9a
library was synthesized and biased towards peptide 6, with four vari- produced a ratio of 1.2:1.0:14.6. In contrast, the truncated analogue
able positions adjacent to the catalytic aspartic acid (Fig. 2a). To bias 10 exhibited a reduced selectivity of 1.3:1.0:5.0 (entry 5).
the library, at each variable position approximately 50% of the func-
Next we assessed the performance of the catalysts on resynth-
tionalized polystyrene beads were coupled to the parent residue esis and evaluation in solution under synthetically meaningful
(which corresponds to catalyst 6) at each position, and the remain- conditions (for example, 1.0 theoretical equiv. oxidant, controlled
ing 50% of the beads were distributed evenly for coupling to either by the stoichiometry of N,N′-diisopropylcarbodiimide (DIC)).
seven different residues (positions i þ 1 and i þ 2) or six different As discussed below, we observed that catalysts provide better
residues (positions i þ 3 and i þ 4, Fig. 2). The resulting library pos- results when they are dissolved in solution, as opposed to when
sesses a theoretical size of about 3,000 unique peptide sequences they are immobilized on beads. Perhaps of greater importance
2
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ARTICLES
Table1 | Results from on-bead screening.
CH3
CH3
CH3
H3C
OH
CH3
O
CH3
CH3
CH3
CH3
CH3
2,3
Resin-bound peptide
HOBt, DMAP, DIC (0.3 equiv.)
5
H3C
OH
CH3
H3C
OH
O
10,11
6,7
CH3
CH3
4
H2O2 (1 equiv.)
DCM
H3C
OH
O
Farnesol (1)
Run to low conversions
3
Entry
i
i 11 i 12
i 13
i 14
i 15
Catalyst Approximate
10,11- 6,7- 2,3- Standard
Library
conversion (%)
(5)
(4) (3)
deviation*
1
Boc-Asp Pro
Boc-Asp Pro
D-Thr(O-
t-Bu)
Asn(Trt)
Asn(Trt) Leu
-
-
6
7
13
1.0
1.0
3.9 0.1
Initial
screening
Directed
2
D-Phe
Thr(OBn)
13
1.3
1.0
8.2 0.1
library No. 1
Directed
library No. 2
3
4
5
6
Boc-Asp Pro
Boc-Asp Pro
Boc-Asp Pro
Asn(Trt)
Asn(Trt)
Asn(Trt)
D-Phe
D-Phe
D-Phe
Thr(OBn) Asn(Trt) 8a
15
12
14
14
1.2
1.2
1.3
1.5
1.0
1.0
1.0
1.9
18.0 0.5
14.6 0.7
5.0 0.0
1.0 0.0
Pro
Asn(Trt) 9a
-
-
-
-
10
11
Boc-Asp D-Pro Thr(OBn) Leu
Truncated
directed
library No. 2
Directed
7
Boc-Asp D-Pro Thr(OBn) Asn(Trt) Tyr(O-
-
12a
16
1.5
2.9
1.0 0.0
t-Bu)
library No. 3
Variations of selective peptide catalysts
OtBu
O
O
H3C
OBn
NHTrt
O
O
H3C
OBn
O
O
O
NHTrt
H
N
O
O
O
H
N
H
H
R
N
N
N
N
N
N
H
N
H
N
R
O
N
H
N
O
N
H
H
O
O
BocHN
NHTr
O
O
O
BocHN
O
t
BocHN
O
O
O
O
NH
O
NHTrt
12a, R = NH-resin
12b, R = OCH3
12c, R = NH-n-Bu
TrtHN
O
OH
OH
OH
O
R
8a, R = NH-resin
8b, R = OCH3
9a, R = NH-resin
9b, R = OCH3
12d, R = NH-Gly-OCH3
*Reactions for 2,3-selective catalysts were run in triplicate from trials run at the same time with the same batch of reagents, and conversions and monoepoxide ratios were estimated by GC (see the
Supplementary Information for details).
was our observation of significant enantioselectivity in these formed with 92% e.e. (entry 4). Catalyst 9b showed no selectivity
experiments. We had hypothesized that catalyst-dependent site for the 2,3-olefin of farnesyl methyl ether, which supports a
selectivity might also be coupled to the observation of enantio- hydroxyl-directed mechanism.
selectivity for a given monoepoxide isomer. The notion that
The initial evaluation of the libraries also revealed catalyst 11,
the same intrinsic transition-state organization associated with a which exhibited a surprising, albeit modest, selectivity for the 6,7-
specific site selectivity might also translate into the organization position of 1 to favour 4 (Table 1, entry 6; 5:4:3 ¼ 1.5:1.9:1.0).
required for enantioselectivity finds analogy in our previous rate- Catalyst 11, with D-Pro in the i þ 1 position, was unique in that pre-
based selections for catalysts that also turned out to be enantiose- viously we had not observed even this modest level of alternative
lective29. With these considerations in mind, we synthesized 8a regioselectivity. We therefore prepared a new library, biased
and 9a as their corresponding C-terminal methyl esters, 8b and towards the structure of 11, wherein the i þ 1 D-Pro was fixed and
9b. When examined under conditions designed to lead to full con- the adjacent three residues were varied. Therein, we found catalyst
version to monoepoxide (1.0 theoretical equiv. oxidant (1.0 equiv. 12a, which displayed an improved selectivity for 4 (Table 1, entry
DIC, 2.0 equiv. H2O2)), methyl esters 8b and 9b both provided 7; 5:4:3 ¼ 1.5:2.9:1.0). When resynthesized as the C-terminal
excellent overall selectivity. Catalyst 8b delivered 3 with essentially methyl ester 12b and evaluated in CHCl3, this catalyst yielded a
total site selectivity and in 82% e.e. (Table 2, entry 1a). Catalyst 9b product distribution of up to 1.0:4.1:1.3 (5:4:3). Next, we evaluated
also exhibited high site selectivity, and 3 showed a slightly a second-generation library to target 6,7-selective catalysts deliber-
improved 86% e.e. (entry 1b). Strikingly, these peptide-based ately. Intriguingly, this study did not reveal catalysts that exhibited
catalysts exhibited site selectivities and enantioselectivities that dramatically improved selectivity for 4, although several comparable
compare favourably to the Sharpless catalyst for the oxidation catalysts were found (see Supplementary Table S1). The sequences
of 1 (Fig. 1c).
of the peptides unveiled by this library prompted us to prepare ana-
Given the analogy, we also evaluated catalyst 9b for the asym- logues of catalyst 12a in a hypothesis-driven manner. We projected
metric epoxidation of other terpenes to assess its generality that a C-terminal amide might better resemble the catalysts in their
towards allylic alcohols. Peptide 9b maintained excellent site selec- on-bead screening form (wherein peptides are linked to the resin as
tivity for geraniol (.100:1 site selectivity, 87% e.e., Table 2, entry 2). the amide). Catalysts 12c (with a C-terminal n-Bu amide) and 12d
Nerol showed somewhat better enantioselectivity (entry 3, 93% e.e.) (with C-terminal Gly–OMe) were examined. Although both cata-
and retained site selectivity for the 2,3-cis-allylic position. Prenol lysts favoured production of epoxide 4, catalyst 12d exhibited
also gave a favourable result, with the corresponding epoxide greater selectivity (5:4:3 ¼ 1.2:8.0:1.0) under optimized conditions
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.1469
ARTICLES
The appreciable, catalyst-dependent selectivity for the 6,7-position
of farnesol is quite unusual. As in the case of the 2,3-selective cata-
lysts, experiments with farnesyl methyl ether exhibited little to no
selectivity (data not shown). Thus, the observed 6,7-selectivity may
be largely the result of a hydroxyl-directed mechanism, despite the
distance of the hydroxyl, which is six bonds away from the reacting
olefin. Yet, unlike the results obtained with the best 2,3-selective cat-
alysts, the production of 4 through the action of catalyst 12d occurred
with only a modest, but measurable, 10% e.e. The observation that
both high site selectivity and enantioselectivity can track together
qualitatively with 2,3-selective catalysts, but do not appear as tightly
coupled with 6,7-selective catalysts, is intriguing. This may result
from the higher number of rotatable bonds between the directing
hydroxyl group and the preferred site of oxidation. In our screening
studies (in corresponding non-optimized experiments) we observed
catalysts that gave a somewhat lower site selectivity (Supplementary
Table S2, 5:4:3, ꢀ1:3–4:1), but slightly higher enantioselectivity
(ꢀ20–30% e.e.). As our data sets are not large, this concept requires
further study for a full assessment. Even so, to our knowledge the
observed 6,7-site selectivity exhibited by catalyst 12d greatly contrasts
with the product distribution induced by m-CPBA and that of any
other reported catalyst (Fig. 3b).
That catalyst 12d shows a preference for the 6,7-position of 1
stimulated us to assess it for the oxidation of a more complex
polyene, geranylgeraniol (13), which presents four competing
olefins for functionalization. Figure 3 shows the raw data for the oxi-
dation of 1 (Fig. 3b) and 13 (Fig. 3c) with m-CPBA and with the
catalysts described above. With geranylgeraniol (13, Fig. 3c), the
results are quite striking. With m-CPBA, the four monoxides were
formed essentially without control. Catalyst 9b, as with the oxidation
of 1, exhibited a high site selectivity to give the 2,3-monoxide
of geranylgeraniol (14) in 85% yield, 86% e.e. Catalyst 12d again
exhibited a striking site selectivity for an internal alkene, which
we assigned as the 6,7-monoxide 15. The monoepoxide 14 and
the other monoepoxides (that is, the 10,11- and 14,15-epoxides,
Boc-Asp-Pro-D-Thr(O-t-Bu)-Asn(Trt)-Leu-
Initial hit: 6
a
Split-and-pool
library synthesis
Boc-Asp-Xaa-Xaa-Xaa-Xaa-linker-
First generation library
Bias
i + 1
i + 2
i + 3
i + 4
~50%
Pro
D-Thr(O-t-Bu)
Asn(Trt)
Leu
Hyp(O-t-Bu)
Asn(Trt)
Lys(Ac)
Ile
Asn(Trt)
Tyr(O-t-Bu)
D-Phe
Pro
Tyr(O-t-Bu)
D-Phe
Phe
Tyr(O-t-Bu)
Lys(Ac)
D-Ala
~50%
Tyr(O-t-Bu)
D-Phe
Thr(OBn)
D-Thr(O-t-Bu)
Val
Thr(OBn)
D-Val
Thr(OBn)
Val
Thr(OBn)
D-Val
Pro
40
30
20
10
0
b
0
1
2
3
4
Number of residues different from parent
Figure 2 | Catalyst library design by split-and-pool synthesis via the OBOC
library method. a, Design of the first directed library. b, Histogram of
theoretical library composition.
(Fig. 3a,b). Furthermore, this product ratio simplified an otherwise see the Supplementary Information) were also produced in the
difficult separation of products by chromatography as it allowed the reaction, but as minor products. To our knowledge, the catalytic
isolation of 4 in 43% yield as a .94:6 mixture of 4:5 isomers.
site-selective epoxidation of geranylgeraniol (or farnesol) at a
Table 2 | Selectivity of peptide 9b with different linear terpenoid alcohols.
R1
O
R1
Peptide (10 mol%)
HOBt, DMAP, DIC (1 equiv.)
R2
OH
R2
OH
H2O2 (2 equiv.)
DCM, 4 ºC, 7 h
Site selectivity
Catalyst 10,11- 6,7- 2,3-
Entry
Substrate
Yield (%) e.e. (%)
Product*
CH3
CH3
CH3
CH3
CH3
CH3
O
1a
1b
8b
9b
1.0
1.0
1.0
1.0
.100 n.d.
.100 81
82
86
H3C
OH
H3C
OH
3
1
CH3
CH3
CH3
CH3
2
3
9b
9b
1.0
1.0
.100 80
87
93
H3C
OH
H3C
OH
O
CH3
CH3
CH3
CH3
O
.100 79
H3C
H3C
OH
OH
CH3
CH3
O
4
9b
75†
92
H3C
OH
H3C
OH
*See Supplementary Information for details of absolute configuration assignment. †GC yield.
4
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ARTICLES
a
Farnesol (1)
Geranylgeraniol (13)
CH3 CH3
Peptide 9b
Conditions in Table 2
CH3
CH3
CH3
CH3
CH3
H3C
OH
H3C
Peptide 12d (10 mol%)
OH
CH3
CH3
CH3
CH3
14
HOBt (10 mol%), DMAP (10 mol%)
1.1 equiv. DIC, 1.5 equiv. H2O2,
CHCl3, –12 ºC to –18 ºC
85% yield
86% e.e.
H3C
OH
O
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H3C
OH
H3C
OH
O
O
4, 43% yield
15, 42% yield
13
b
c
1
GC trace
GC trace
Catalyst or
oxidant
Selectivity
10,11- 6,7-
Catalyst or
oxidant
Selectivity
14,15- 10,11- 6,7-
5
4
2,3-
1.0
2,3-
1.0
3
15
14
m-CPBA
2.7
1.0
2.2
1.0
m-CPBA
3.1
3.1
2.5
9b
>100
9b
1.0
1.0
1.0
>100
12d
1.2
8.2
1.0
12d
1.0
1.8
13.2 1.5*
48
50
52
54 min
50
52.5
55
57.5 min
Figure 3 | Results and raw data for the site-selective oxidation of farnesol and geranylgeraniol with m-CPBA, 2,3-selective catalyst 9b and 6,7-selective
catalyst 12d. a, Optimized oxidation conditions with 12d. b,c, Comparison of product ratios and GC spectra from crude reaction mixtures with m-CPBA,
catalyst 9b and catalyst 12d, with the relevant area of GC spectra for farnesol (b) and geranylgeraniol (c). *The peak has a shoulder that is integrated
and so the area is overestimated. Conditions for m-CPBA reactions are given in Fig. 1b, for reactions with catalyst 9b in Table 2 and for reactions with
catalyst 12d in (a).
brought to a cold room (4 8C), where the reaction was stirred vigorously. Seven
hours after the addition of DIC, the reaction was quenched with a saturated aqueous
solution of Na2SO3 and stirred for a few moments, sitting in ice, before it was allowed
to warm to room temperature. Saturated aqueous NaHCO3 and hexane or EtOAc
were added, the mixture was vortexed, allowed to settle and then the organic layer
was removed, followed by 2–3 additional hexane or EtOAc extracts, performed
similarly. If the site selectivity was to be determined, an aliquot of the combined
organics was removed, diluted with additional hexanes and analysed by gas
chromatography (GC). The GC sample was returned to the organics, which were
then concentrated in vacuo or under a stream of N2 and purified by flash silica-gel
column chromatography.
position other than the 2,3-olefin has not been reported previously.
The challenge of the task is manifest in the nearly equivalent reac-
tivity of the 14,15-, 10,11- and 6,7-olefins. Thus, the observations
shown in Fig. 3 may represent an important step forwards,
enabled by the iterative peptide-based catalyst screening approach.
Conclusions
In summary, we discovered different peptide catalysts capable of
furnishing different isomers of epoxyfarnesol and related com-
pounds that are minor products of generic peracid-based olefin
epoxidation. Peptide 9b appears to operate via a hydroxyl-directing
mechanism, analogous to the Sharpless asymmetric epoxidation
and with comparable selectivity. Peptide 12d provides unprece-
dented selectivity for a catalyst for the internal olefin of farnesol,
with substantial site selectivity but modest enantioselectivity.
Discovery of catalysts for complex selectivity situations, wherein a
myriad of possible products may be formed as a function of a cata-
lytic mechanism of bond formation, is a state-of-the-art challenge
for asymmetric catalysis endeavours. The application of diversity-
based approaches, such as that described herein, may prove fruitful,
and may also offer some analogy to the directed evolution strategies
employed by natural and engineered enzymatic systems.
General procedure for the epoxidation of 6,7-olefin in polyisoprenols with
peptide 12d. To a test tube with stir bar and Teflon-lined screw cap was added
peptide 12d (0.1 equiv.), substrate (1.0 equiv.), about two-thirds of a freshly
.
prepared/sonicated solution that contained HOBt H2O (0.1 equiv.) and DMAP (0.1
equiv.) in CHCl3 (the CHCl3 was passed through basic alumina prior to mixing with
DMAP/HOBt), and then 30% aqueous H2O2 (2.0 equiv.). The test tube was placed
in ice and allowed to chill before DIC (1.0 equiv.) was added, followed by the
remaining CHCl3 solution (to a concentration of 0.2 M of substrate) rinsed down the
sides of the reaction tube. The reaction tube was then sealed with a screw cap under
ambient conditions (without the exclusion of air) and placed in an i-PrOH bath
maintained at 212 8C to 218 8C by a cryostat. The reaction was then stirred
vigorously. About 25–48 hours after the addition of DIC, the reaction was quenched
with a saturated aqueous solution of Na2SO3 and stirred for a few moments, still at a
low temperature, before it was allowed to warm to room temperature. Saturated
aqueous NaHCO3 and EtOAc were added and the mixture was vortexed and allowed
to settle, after which the organic layer was removed, along with three additional
EtOAc extracts performed similarly. An aliquot of the combined organics was
removed, diluted with additional EtOAc and analysed by GC. The organics were
concentrated in vacuo and then purified by flash column chromatography.
Additional experimental procedures, descriptions of compounds and analytical
methods are given in the Supplementary Information.
Methods
General procedure for the epoxidation of allylic alcohols with peptide 9b. To a
test tube with a stir bar and Teflon-lined screw cap was added peptide 9b (0.1 equiv.),
allylic alcohol (1.0 equiv.), a freshly prepared/sonicated solution that contained
.
HOBt H2O (0.1 equiv.) and DMAP (0.1 equiv.) in dichloromethane (DCM, to a
concentration of 0.2 M of substrate), and then 30% aqueous H2O2 (2.0 equiv.). The
test tube was placed in ice (if running the reaction below room temperature) and
allowed to chill before adding DIC (1.0 equiv.). The reaction tube was then sealed
with a screw cap under ambient conditions (without the exclusion of air) and
Received 24 May 2012; accepted 28 August 2012;
published online 14 October 2012
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.1469
ARTICLES
23. Tanuwidjaja, J., Ng, S-S. & Jamison, T. F. Total synthesis of ent-
dioxepandehydrothyrsiferol via a bromonium-initiated epoxide-opening
cascade. J. Am. Chem. Soc. 131, 12084–12085 (2009).
References
1. van Tamelen, E. E. & Heys, J. R. Enzymic epoxidation of squalene variants.
J. Am. Chem. Soc. 97, 1252–1253 (1974).
24. Uyanik, M., Ishibashi, H., Ishihara, K. & Yamamoto, H. Biomimetic synthesis of
acid-sensitive (–)-caparrapi oxide and (þ)-8-epicaparrapi oxide induced by
artificial cyclases. Org. Lett. 7, 1601–1604 (2005).
25. Marshall, J. A. & Hann, R. K. A cascade cyclization route to adjacent
bistetrahydrofurans from chiral triepoxyfarnesyl bromides. J. Org. Chem.
73, 6753–6757 (2008).
26. Dittmer, D. C. et al. A tellurium transposition route to allylic alcohols:
overcoming some limitations of the Sharpless–Katsuki asymmetric epoxidation.
J. Org. Chem. 58, 718–731 (1993).
27. Lichtor, P. A. & Miller, S. J. One-bead-one-catalyst approach to aspartic acid-
based oxidation catalyst discovery. ACS Comb. Sci. 13, 321–326 (2011).
2. Katsuki, T. & Martin, V. S. Asymmetric epoxidation of allylic alcohols: the
Katsuki–Sharpless epoxidation reaction. Org. React. 48, 1–299 (1996).
3. Zhang, W., Basak, A., Kosugi, Y., Hoshino, Y. & Yamamoto, H. Enantioselective
epoxidation of allylic alcohols by a chiral complex of vanadium: an effective
controller system and a rational mechanistic model. Angew. Chem. Int. Ed. 44,
4389–4391 (2005).
4. Malkov, A. V., Czemerys, L. & Malyshev, D. A. Vanadium-catalyzed asymmetric
epoxidation of allylic alcohols in water. J.Org. Chem. 74, 3350–3355 (2009).
5. Egami, H., Oguma, T. & Katsuki, T. Oxidation catalysis of Nb(salan) complexes:
asymmetric epoxidation of allylic alcohols using aqueous hydrogen peroxide.
J. Am. Chem. Soc. 132, 5886–5895 (2010).
6. Barlan, A. U., Basak, A. & Yamamoto, H. Enantioselective oxidation of olefins
catalyzed by a chiral bishydroxamic acid complex of molybdenum. Angew.
Chem. Int. Ed. 45, 5849–5852 (2006).
7. Corey, E. J. & Zhang, J. Highly effective transition structure designed catalyst
for the enantio- and position-selective dihydroxylation of polyisoprenoids.
Org. Lett. 3, 3211–3214 (2001).
8. Chang, S., Lee, N. H. & Jacobsen, E. N. Regio- and enantioselective catalytic
epoxidation of conjugated polyenes. Formal synthesis of LTA4 methyl ester.
J.Org. Chem. 58, 6939–6941 (1993).
9. Burke, C. P. & Shi, Y. Regio- and enantioselective epoxidation of dienes by a
chiral dioxirane: synthesis of optically active vinyl cis-epoxides. Angew. Chem.
Int. Ed. 45, 4475–4478 (2006).
10. Breslow, R. & Maresca, L. M. Template-directed epoxidation of farnesol and
geranylgeraniol as conformational probes. Tetrahedron Lett. 10, 887–890 (1978).
11. Saito, I., Mano, T., Nagata, R. & Matsuura, T. Inter- and intramolecular
epoxidation utilizing silyl-protected peroxy esters and copper salt.
Tetrahedron Lett. 28, 1909–1912 (1987).
´
28. Lam, K. S., Lebl, M. & Krchnak, V. The ‘one-bead-one-compound’
combinatorial library method. Chem. Rev. 97, 411–448 (1997).
29. Furka, A., Sebestyen, F., Asgedom, M. & Dibo, G. General method for rapid
synthesis of multicomponent peptide mixtures. Int. J. Pept. Protein Res. 37,
487–493 (1991).
30. Lam, K. S. et al. A new type of synthetic peptide library for identifying ligand-
binding activity. Nature 354, 82–84 (1991).
31. Singh, J. et al. Application of genetic algorithms to combinatorial synthesis: a
computational approach to lead identification and lead optimization. J. Am.
Chem. Soc. 118, 1669–1676 (1996).
32. Reetz, M. T. Laboratory evolution of stereoselective enzymes: a prolific source of
catalysts for asymmetric reactions. Angew. Chem. Int. Ed. 50, 138–174 (2011).
33. Brustad, E. M. & Arnold, F. H. Optimizing non-natural protein function with
directed evolution. Curr. Opin. Chem. Biol. 15, 201–210 (2011).
34. Copeland, G. T. & Miller, S. J. Selection of enantioselective acyl transfer catalysts
from a pooled peptide library through a fluorescence-based activity assay: an
approach to kinetic resolution of secondary alcohols of broad substrate scope.
J. Am. Chem. Soc. 123, 6496–6502 (2001).
35. Schreiber, S. L., Schreiber, T. S. & Smith, D. B. Reactions that proceed with a
combination of enantiotopic group and diastereotopic face selectivity can deliver
products with very high enantiomeric excess: experimental support of a
mathematical model. J. Am. Chem. Soc. 109, 1525–1529 (1987).
12. Gnanadesikan, V. & Corey, E. J. A strategy for position-selective epoxidation of
polyprenols. J. Am. Chem. Soc. 130, 8089–8093 (2008).
13. Colby Davie, E. A., Mennen, S. M., Xu, Y. & Miller, S. J. Asymmetric catalysis
mediated by synthetic peptides. Chem. Rev. 107, 5759–5812 (2007).
14. Wennemers, H. Asymmetric catalysis with peptides. Chem. Commun. 47,
12036–12041 (2011).
15. Francis, M. B., Jamison, T. F. & Jacobsen, E. N. Combinatorial libraries of
transition-metal complexes, catalysts and materials. Curr. Opin. Chem. Biol. 2,
422–428 (1998).
16. Kuntz, K. W., Snapper, M. L. & Hoveyda, A. H. Combinatorial catalyst discovery.
Curr. Opin. Chem. Biol. 3, 313–319 (1999).
Acknowledgements
This work is supported by National Institutes of Health (NIH R01-GM096403) to S.J.M.,
and P.A.L. was partially supported by NIH CBI-TG-GM-067543. P.A.L. thanks B. Fowler
for collaborating to build the library that resulted in catalyst 6, and S. Alexander and the
Schepartz laboratory for assistance.
17. Peris, G., Jakobsche, C. E. & Miller, S. J. Aspartate-catalyzed asymmetric
epoxidation reactions. J. Am. Chem. Soc. 129, 8710–8711 (2007).
18. Kolundzic, F., Noshi, M. N., Tjandra, M., Movassaghi, M. & Miller, S. J.
Chemoselective and enantioselective oxidation of indoles employing aspartyl
peptide catalysts. J. Am. Chem. Soc. 133, 9104–9111 (2011).
19. Thibodeaux, C. J., Chang, W-C. & Liu, H-W. Enzymatic chemistry of
cyclopropane, epoxide and aziridine biosynthesis. Chem. Rev. 112,
1681–1709 (2012).
20. Sharpless, K. B. Searching for new reactivity (Nobel Lecture). Angew. Chem.
Int. Ed. 41, 2024–2032 (2002).
21. Kotaki, T., Shinada, T. & Kaihara, K. Structure determination of a new juvenile
hormone from a Heteropteran insect. Org. Lett. 11, 5234–5237 (2009).
Author contributions
P.A.L. designed and performed the experiments and S.J.M. oversaw the project. Both
authors analysed data and co-wrote the manuscript.
Additional information
Supplementary information and chemical compound information are available in the
online version of the paper. Reprints and permission information is available online at
http://www.nature.com/reprints. Correspondence and requests for materials should be
addressed to S.J.M.
22. Koohang, A. et al. Enantioselective inhibition of squalene synthase by aziridine Competing financial interests
analogues of presqualene diphosphate. J. Org. Chem. 75, 4769–4777 (2010).
The authors declare no competing financial interests.
6
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