Experimental lineage and functional analysis of a remotely directed peptide epoxidation catalyst

Article abstract of DOI:10.1021/ja410567a

We describe mechanistic investigations of a catalyst (1) that leads to selective epoxidation of farnesol at the 6,7-position, remote from the hydroxyl directing group. The experimental lineage of peptide 1 and a number of resin-bound peptide analogues were examined to reveal the importance of four N-terminal residues. We examined the selectivity of truncated analogues to find that a trimer is sufficient to furnish the remote selectivity. Both 1D and 2D ¹H NMR studies were used to determine possible catalyst conformations, culminating in proposed models showing possible interactions of farnesol with a protected Thr side chain and backbone NH. The models were used to rationalize the selectivity of a modified catalyst (17) for the 6,7-position relative to an ether moiety in two related substrates.

Full text of DOI:10.1021/ja410567a

Article
pubs.acs.org/JACS
Experimental Lineage and Functional Analysis of a Remotely
Directed Peptide Epoxidation Catalyst
Phillip A. Lichtor and Scott J. Miller*
Department of Chemistry, Yale University, P.O. Box 208107, New Haven, Connecticut 06520, United States
S
* Supporting Information
ABSTRACT: We describe mechanistic investigations of a catalyst
(1) that leads to selective epoxidation of farnesol at the 6,7-position,
remote from the hydroxyl directing group. The experimental lineage
of peptide 1 and a number of resin-bound peptide analogues were
examined to reveal the importance of four N-terminal residues. We
examined the selectivity of truncated analogues to find that a trimer
1
is sufficient to furnish the remote selectivity. Both 1D and 2D H
NMR studies were used to determine possible catalyst conforma-
tions, culminating in proposed models showing possible interactions of farnesol with a protected Thr side chain and backbone
NH. The models were used to rationalize the selectivity of a modified catalyst (17) for the 6,7-position relative to an ether
moiety in two related substrates.
(3). Critically, we had established previously that the reaction
catalyzed by 1 was predominantly hydroxyl-directed through the
observation of selective 6,7-epoxidation of both 2 and 3. In
addition, our preliminary experiments showed that catalysts of
this type deliver substantially lower selectivity for the 6,7-olefin of
farnesyl methyl ether, comparable to that exhibited by propionic
acid [Supporting Information (SI) Figure 9].
Other than the apparently fortuitous choice of the amino acids
and their position within our screening libraries, we developed 1
without any preconceived notion of how such a catalyst would
deliver selectivity. Therefore, an understanding of the mecha-
nism for the selectivity displayed by catalyst 1 demanded further
studies, which are presented herein. Simultaneously, a detailed
retrospective analysis of the arrival at the best peptide sequence
appeared to be inextricably related and has not been described
previously. The interplay of these studies is described below.
INTRODUCTION
■
Directing groups play an important role in complex molecule
synthesis.1 Catalytic reactions aided by directing groups are now
ubiquitous in the chemical literature. For example, great strides
have been made in the area of hydroxyl-directed epoxida-
tion, with enantioselective methods for allylic,2 homoallylic,3 and
even bis-homoallylic alcohols now recorded.4 More rare, however,
are catalytic reactions directed by a remote chemical moietythat
is, a functional group that is several bonds distal to the reactive site
of the substrate.5 Often, these general synthetic strategies rely on
conformational restriction or strong molecular interactions to
achieve selectivity.6 In contrast, we recently discovered a peptide-
based catalyst (1)7 capable of oxidizing an olefin positioned five
rotatable bonds (in addition to one C−C double bond) from its
directing group. Notably, the selectivity achieved by 1 differs
from that observed with farnesol (2) when m-chloroperoxybenzoic
acid (mCPBA) or, to our knowledge, any other previously known
site-selective oxidation catalyst is used. Despite the remarkable
site selectivity exhibited by catalyst 1 (∼1:8:1 for the 2,3-, 6,7-,
and 10,11-positions of 2, shown in green, blue, and red, respec-
tively, in Scheme 1A), the observed enantioselectivity for the 6,7-
position is low (∼10% ee). Given the peculiar selectivity imparted
by this remotely directed catalyst, we sought to understand the
molecular underpinnings of the catalyst−substrate interaction.
Catalyst 1 was developed using an on-bead screening
protocol8 used to evolve combinatorial libraries for site selectivity
in the epoxidation of farnesol (Scheme 1A). Each peptide
belonging to the initial screening libraries contained a Boc-
protected aspartic acid residue at the N-terminus, which
possesses a catalytic carboxylic acid moiety in the side chain. In
the catalytic cycle (abbreviated in Scheme 1B),9 the carboxylic
acid of 1 is activated with N,N′-diisopropylcarbodiimide (DIC)
and, in the presence of H2O2, forms a peptidyl peracid that
selectively epoxidizes the 6,7-olefin of either 2 or geranylgeraniol
RESULTS AND DISCUSSION
■
6,7-Selective Catalyst Lineage. A limited understanding of
the importance of the residues within peptide 1 was initially
derived from studies of the libraries leading to the discovery of
1. The observation of a peptide that exhibited a modicum of
selectivity for the 6,7-position of 2 (relative to the 2,3- or 10,
11-positions) was unexpected. In fact, the library from which this
initial 6,7-selective “hit” derived was generated in order to study
2,3-selectivity. This library had been designed such that two
internal residues had been omitted from the original hexameric
sequences.7 This shortened library of tetramers was biased to
contain a choice of D-amino acids adjacent to the catalytic
N-terminal aspartic acid residue and two additional C-terminal
variable residues. The first peptide that was found to exhibit the
Received: October 17, 2013
Published: April 1, 2014
© 2014 American Chemical Society
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Finally, a third-generation library was studied wherein the i + 2
Thr(Bn) was fixed and the i + 3 position was biased toward
sequences containing Asn(Trt). A number of peptides from this
library were found to yield favorable selectivity (Figure 1C), and
we subjected many to sequencing. While it is difficult to draw
conclusions from any individual peptide sequenced from these
libraries without validation, all of the high-performing sequences
contained an i + 3 Asn(Trt), suggesting that the Asn(Trt)
conferred advantageous selectivity. However, a variety of other
residues was found in the i + 4 and i + 5 positions in these high-
performing sequences (Figure 1D).
Scheme 1. Previously Reported 6,7-Oxidation and Proposed
Peptide-Catalyzed Epoxidaton Catalytic Cycle
a
It is interesting to note that the best catalysts from each library
generation seem to yield similar positional selectivity when
examined on solid supports and under the initial screening
conditions (Figure 1A−C). While many of the catalysts found
from the later library generations performed similarly, these on-
bead screens yielded several leads for peptide validation and
optimization in solution, which ultimately led to 1. Nonetheless,
the on-bead screening data suggest that a tetramer is sufficient to
achieve 6,7-selectivity on-bead (from the first-generation library,
observed 4:5:6 = 1.2:2.1:1.0) and that the best sequenced
peptides from each on-bead generation contained common
elements: an i + 2 Thr(Bn) and an i + 3 Asn(Trt).
With no other structural data but with knowledge of the amino
acid sequence of peptide 1, and no expectation that the full
diversity of each peptide library had been sampled, we
hypothesized that the embedded dipeptide D-Pro-Thr(Bn)
might enforce a β-turn. Notably, D-Pro adjacent to a β-branched
amino acid is suggestive of a type II′ β-turn,15 an oft-studied
motif in a number of peptide-based catalysts from our lab16 and
others.17 To examine the existence of this structural element
within 1, we resorted to the examination of further altered
sequences within on-bead peptide libraries.
a
DIU = N,N′-diisopropylurea.
If peptide 1 were to exhibit a turn, then perhaps on-bead
libraries with different turn-promoting residues at the i + 2
position, such as Val or Aib, might also work at this position. Such
substitutions were thought to be a potential avenue for the
identification of more selective catalysts.
alternate 6,7-selectivity, Boc-Asp-D-Pro-Thr(Bn)-Leu (Boc = tert-
butoxycarbonyl, Bn = benzyl), exhibited only modest selectivity
(4:5:6 = 1.3:1.5:1.0, point shown in Figure 1A).10 A subsequent
library biased toward this initial hit was immediately prepared.7
Split and pool11,12 libraries biased13 for this study were pre-
pared such that at each varied position in the peptide design,
roughly half the library members possess the amino acid of the
parent sequence while the remaining library members would
have one of six or seven alternative residues. Additionally, each
successive library was designed to possess a longer sequence
length than the previous generation, with an additional variable
residue added to the C-terminal side.7
At the same time that the first directed library was being pre-
pared (second generation), further screening of the initial library
(first generation) ultimately led to another more 6,7-selective
peptide with an i + 3 trityl (Trt)-protected asparagine [Asn(Trt)]
(4:5:6 = 1.2:2.1:1.0). The data from the first-generation library are
presented graphically in Figure 1A and discussed in more detail below.
Figure 1 contains ternary plots that illustrate the percentage
distribution of the monoepoxides formed by catalysts during
on-bead screening experiments. In these plots, the horizontal lines
mark the gradations of 6,7-selectivity. Thus, points appearing higher
on the graph represent product mixtures with a higher proportion of
product 5. Points that appear closer to the other vertices represent
peptides that produce a higher proportion of 4 or 6.
Four distinct biased split-and-pool libraries of on-bead
peptides derived from libraries that initially led to 1 were
therefore compared for site selectivity in the epoxidation of 2
(compare panels C and E of Figure 1). Each library possessed
either Val, Aib, Thr(tBu), or Thr(Bn) in the i + 2 position.
Although the data in Figure 1E represent only a limited sampling
of three libraries, the testing of different i + 2 residues while
simultaneously evaluating other residues at adjacent positions
can potentially reveal epistatic interactions. That is, in theory, this
experiment allows for the possibility of finding better peptides
that might only be found through multiple amino acid sub-
stitutions, where the better selectivity results from some com-
bination of changes. Nonetheless, peptides containing protected
Thr derivatives [Thr(Bn) or Thr(tBu)] were more 6,7-selective
than those containing either Val or Aib. These findings suggest that
even if the i + 2 Thr(Bn) is turn-promoting, it likely plays an
additional role in delivering selectivity for the 6,7-olefin of 2.
Peptides with either an i + 2 Thr(Bn) or Thr(tBu) demon-
strated favorable 6,7-selectivity on-bead and in solution. During
the optimization studies of the 6,7-selective catalysts in solution,
we generally observed that the site and enantioselectivity
afforded by the catalysts for the 6,7-position were uncorrelated.
For example, i + 2 Thr(Bn)-containing peptide 7 gave 4:5:6 =
1.0:4.1:1.3 with 5 in 1.6:1.0 er, yet the Thr(tBu)-containing
analogue of 7 gave 4:5:6 = 1.0:3.7:1.1 with 5 in 1.9:1.0 er. These
Screening of the second generation of catalysts biased toward
sequences containing an i + 3 Leu revealed two additional se-
quences that performed better than the others (4:5:6 = 1.0:1.7:1.1
and 1.5:2.6:1.014). Both of these second-generation hits contained
an i + 2 Thr(Bn) and an i +3 Asn(Trt) (Figure 1B).
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Figure 1. (A−C) Ternary plots showing an overlay of peptide selectivities from successive generations of 6,7-selective peptides, where each axis
represents the fraction of the total monoepoxide: (A) the first library demonstrating 6,7-selectivity (first generation, red); (B) the first biased library for
6,7-selectivity (second generation, green); (C) second biased library (third generation, blue). Points that are higher (further away from the triangle base)
are more 6,7-selective. The highlighted generation from each plot is in the solid color in the foreground atop the other library generations in the
background. Solid markers indicate peptides that were sequenced. (D) List of a portion of sequenced peptides from each library generation (shown with
solid markers in A−C). Residues shown in blue indicate were picked from the pool of variable residues from their particular library. (E) Selectivity with
amino acids at the i + 2 position, which can be compared to the library with i + 2 Thr(Bn) directly to the left (C, third generation, blue). Product ratios
were measured by GC. In all of the on-bead screening studies, the formation of diepoxides was intentionally limited through the use of 0.3 equiv of DIC,
resulting in analysis of low-conversion reaction mixtures.
findings underline the potentially complicated relationship
between the different rates of epoxidation leading to either site
and/or enantioselectivity.
On-Bead Analogue Studies. Substitutions of other residues
within 1 were studied by examining specific analogues on-resin. These
peptide analogues were examined on-resin for convenience of
synthesis and ease of screening, and they yielded reduced selectivity in
comparison with their solution-phase counterparts. Additionally, the
on-bead reactions were performed under unoptimized conditions
with substoichiometric oxidant, restricting the conversion.
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a b
,
Table 1. Product Distributions Resulting from Oxidation of 2 with a Number of On-Bead Peptide Analogues of 1
entry
i
i + 1
i + 2
i + 3
i + 4
10,11- (4)
6,7- (5)
2,3- (6)
1
2
Boc-Asp
Boc-Asp
Boc-Asp
Boc-Asp
Boc-Asp
Boc-Asp
Boc-Asp
Boc-Asp
Boc-Asp
Boc-Asp
Boc-Asp
Boc-Asp
D-Pro
D-Pro
D-Pro
D-Pro
D-Pro
D-Pro
D-Pro
D-Pro
D-Pro
D-Pro
D-Pro
D-Pro
Thr(Bn)
Thr(Trt)
Ser(Bn)
Ile
Asn(Trt)
Asn(Trt)
Asn(Trt)
Asn(Trt)
Asp(tBu)
Hse(Trt)
Ser(Trt)
Leu
Tyr(tBu)
Tyr(tBu)
Tyr(tBu)
Tyr(tBu)
Tyr(tBu)
Tyr(tBu)
Tyr(tBu)
Tyr(tBu)
Phe
1.2
1.1
1.1
1.0
1.0
1.1
1.1
1.2
1.2
1.2
1.2
1.2
2.3
1.5
1.7
1.5
1.2
1.1
1.1
1.5
2.2
2.1
2.3
2.2
1.0
1.0
1.0
1.1
1.1
1.0
1.0
1.0
1.0
1.0
1.0
1.0
3
4
5
Thr(Bn)
Thr(Bn)
Thr(Bn)
Thr(Bn)
Thr(Bn)
Thr(Bn)
Thr(Bn)
Thr(Bn)
6
7
8
9
Asn(Trt)
Asn(Trt)
Asn(Trt)
Asn(Trt)
10
11
12
hPhe
2-Nal
Cha
a
Reactions were conducted with 2 (1 μmol, 1.0 equiv), DIC (0.3 equiv), 1-hydroxybenzotriazole (HOBt) (0.1 equiv), 4-dimethylaminopyridine
b
(DMAP) (0.1 equiv), H2O2 (1.0 equiv), dichloromethane (DCM) (0.2 M), and a single bead bound to peptide. 2-Nal = 2-naphthylalanine, Cha =
cyclohexylalanine, hPhe = homophenylalanine, Hse = homoserine.
Figure 2. (A) Selectivities observed with truncated peptides. (B) Abbreviated 1D 1H NMR spectra of 1 and its truncated analogues acquired at −20 °C
at 20 mM.
An on-bead analogue of 1 furnished a 4:5:6 product mixture of
1.2:2.3:1.0 (Table 1, entry 1). Modifications to the i + 2 Thr side
chains, such as replacement of the benzyl protecting group with
a trityl group (entry 2) or removal of the γ-methyl (entry 3)
resulted in diminished selectivity. Replacement of the ethereal
O-benzyl with an ethyl group (Ile; entry 4) still furnished some
selectivity for 5, although it was attenuated.
Modifications to the i + 3 Asn(Trt) also resulted in reduced
selectivity. When other trityl-containing amino acids [Hse(Trt)
and Ser(Trt); Table 1, entries 6 and 7] or Asp(tBu) (entry 5)
were studied in this position, the selectivity was nearly entirely
lost. In comparison with other substitutions, Leu yielded a decent
level of selectivity (entry 8), though less than the parent peptide,
which is potentially meaningful because Leu is sometimes
thought to be an Asn isostere,18 though without a trityl pro-
tecting group. These observations are consistent with those made
during the screening of on-bead libraries: both the i + 2 Thr(Bn)
and i + 3 Asn(Trt) are important and sensitive to substitution.
Substitution of the i + 4 Tyr(tBu) side chain with other aryl-
(Table 1, entries 9−11) and alkyl-containing (entry 12) side
chains did not seem to lead to drastic changes in selectivity.
These observations largely agree with those made from studies of
substitutions of the i + 4 and i + 5 residues with peptides under
more homogeneous conditions (i.e., not resin-bound).19
Studies with Truncated Peptides. To explore the
necessity of each amino acid, the selectivities of a number of
truncated analogues of 1 (7−12) were evaluated in solution. The
results, presented in Figure 2, underline the complicated function
that each amino acid of 1 plays in delivering optimal selectivity
for the 6,7-olefin. Notably, these reactions were performed at ca.
5 °C lower than the previously reported epoxidation conditions.7
At −20 °C, 1 furnishes slightly higher selectivity (4:5:6 =
1.1:8.6:1.0; Figure 2A).
The truncated peptides reveal the interesting roles played by
the i + 3 Asn(Trt) and i + 4 Tyr(tBu). Replacement of the
C-terminal Gly-OCH3 of 1 with a methyl ester, as in 7, results in
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Figure 3. (A) Selected 1H−1H ROESY correlations found in 1 at 25 °C and truncated peptide 7 at −20 °C. (B) Two structural ensembles from the
20 CNS-generated structures of 1 computed using 25 °C ROESY data. (C) One structure chosen from the 20 structures shown with side chains.
roughly the same 6,7:10,11 selectivity but decreases the 6,7:2,3
selectivity by about half. Deletion of each sequential amino acid
from the C-terminus seems to decrease the overall selectivity for
the 6,7-olefin relative to the 10,11-olefin. Moreover, through
each truncation, the selectivity for 6 increases relative to 5, yet
peptides 7, 8, and 9 display roughly the same 5:6 selectivity of 3:1
to 4:1.
Consistent with the hypothesis of an interaction of the
Thr(Bn) region of the peptide with farnesol, tripeptide 9 with a
C-terminal Thr(Bn)-NHCH3 (entry 4) is sufficient to furnish
some 6,7-selectivity, though reduced from the parent peptide
(entry 1). However, tripeptide 10 with a C-terminal methyl ester
lacks selectivity entirely. Additionally, the selectivity actually
decreases substantially and begins to favor the 2,3-olefin within
farnesol in the case of 11 and 12, where the Thr(Bn) is lacking.
We were surprised to find that the simple aspartic acid derivative
12 slightly favors 6 given that simpler peracids (e.g., propionic
acid) favor oxidation of the most electron-rich olefin to make 4.
Perhaps these observations underscore the surprising frequency
with which catalysts selective for the olefin most proximal to
a directing group are found. Indeed, our experience explor-
ing catalysts for the oxidation of 2 has been rife with allylic
epoxidation catalysts.
protons that would participate in the turn, while the NH-Thr
signal shifts upfield.
While the NH-Asn signals shift back upfield in the spectra of
pentamer 7 and hexamer 1 (relative to 8), the observed Boc-NH
shifts appear further downfield for both. Indeed, the spectra of
the longer peptides are difficult to interpret given the broadening
of some peaks at this low temperature. Nonetheless, 2D 1H−1H
rotating-frame nuclear Overhauser effect spectroscopy (ROESY)
data corroborate the interpretation of a β-turn (Figure 3A).
The spectra suggest a through-space correlation between the
β-protons of the i Asp and i + 3 Asn(Trt) in spectra of 1 and 7
acquired at 25 and −20 °C, respectively. Additionally, these data
suggest that the Hα-D-Pro is close to the amide protons of both
the i + 2 Thr(Bn) and i + 3 Asn(Trt). Notably, many of the
correlations found in 1 at 25 °C are also found in truncated
peptide 7 at −20 °C, indicating that the room-temperature data
for 1 are likely relevant.
To determine whether the NMR data fit our qualitative
inferences about secondary structure, we estimated and binned
distance restraints from the ROESY data for 1 acquired at 25 °C
for structure calculations using the program CNS.20 The inter-
pretation of these structures is accompanied by the usual caveats.
For example, as with any structural study of a catalyst, observa-
tions of the resting or intermediate state may differ completely
from those of the active catalyst.21 Additionally, calibration of
distance restraints is usually performed by comparison to an ex-
perimentally determined distance (e.g., two ortho aryl protons),
but we were unable to find pairs of protons that could confidently
be used for this purpose, so we looked at two different calibra-
tion sets between Hα-Asp and Hδ-D-Pro (see the Supporting
Information). From each calibrated distance, the other
correlations were assigned to different bins as appropriate, and
10 structures that best fit the data were analyzed from each data
set. An overlay of the combined 20 structures suggested the
existence of two structural ensembles, each of which contained
members derived from both of the distance restraint sets. The
two ensembles are shown in Figure 3B.
1H NMR-Assisted Analysis of Catalyst Structure. The
intriguing transition from the observed 6,7-selectivity to an
absence of selectivity with the change of an amide to an ester in
the trimer may result from a variety of scenarios. The NH of the
methyl amide in 9 may interact with farnesol to deliver selectivity.
Alternatively, the change from amide to ester (9 to 10) may
result in a number of conformational changes, which are manifest
1
in the H NMR spectrum. Compared with the other peptides,
some proton signals in the spectra of 10 are doubled, whereas the
longer truncated variants seem to display less conformational
heterogeneity. One possible conformation available to 9 but not
10 is a β-turn with a 10-membered ring formed through hydrogen
bonding of the methyl amide.
The selectivity data from the truncated analogues combined
with the 1D 1H NMR spectra (Figure 2B) suggest that a β-turn
may form in trimer 9 and the longer peptides. In particular, the
1H NMR spectrum of tetramer 8 reveals significant downfield
shifts of the Boc-NH and NH-Asn signals, corresponding to
The calculated structures agree with other experimental ob-
servations presented herein and provide insight into some
possible structural features. Many of the structures that best fit
the data are suggestive of a β-turn characterized by an apparent
hydrogen bond between the i Asp-CO and the i + 3 NH-
Asn(Trt) (the distance between the backbone i CO and i + 3 Nα
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ranged from 2.8 to 3.7 Å across the 20 structures). Indeed, these
observations are not surprising given the correlation between the
β-protons of those residues in the NMR spectra. A smaller set of
the calculated structures may also contain a hydrogen bond
between the i + 2 NH-Thr(Bn) and the i CO, indicative of a
γ-turn.22 Additionally, the data suggest that the i + 5 Tyr(tBu)
side chain is oriented underneath the turn, as is evident from a
representative structure picked from the ensembles (Figure 3C).
On the basis of the aforementioned experimental evidence, we
hypothesized that the interactions of 1 and 2 defined in Figure 4
are consistent with the selective oxidation of the 6,7-olefin. We
consider on the basis of analogue and structural studies that the
NH of the i + 2 Thr(Bn) could interact with the hydroxyl (as
shown). The data suggest that a type II′ β-turn can form, which
orients this NH of the i + 2 Thr(Bn) in the same direction as the
aspartic acid side chain (upward as in Figure 4A,B). A γ-turn may
loss of 6,7-selectivity (Table 1, entry 4; see also Table 2, entry 4).
Analysis of the other libraries is also internally consistent
(Figure 1E; Val and Aib libraries).
The hypothetical transition-state interactions between the
catalyst and substrate in Figure 4 are consistent with the observed
selectivity with trimer 9 and are supported by the NMR studies of
the longer peptides. Interestingly, the minimal enantioselectivity
in the formation of 5 can be explained by the accessibility of
either face of the olefin through a number of possible σ-bond
rotations in the substrate (different rotational configurations
are shown in Figure 4A′,B′).
It is not clear from these data what the additional amino acids
that are potentially disposed farther away from the Thr(Bn) and
catalytic Asp residue are doing to improve the 6,7-selectivity of 1.
We wonder whether there may be a number of peptide−farnesol
interactions that lead to allylic alcohol epoxidation, as the 2,3-
position is most proximal to the putative directing group. Our
studies have suggested that the mode by which these catalysts
achieve selectivity is quite delicate, which is perhaps underlined
by the limited solvent compatibility. Whereas other peptidyl
peracids that our group has studied have generally yielded some
selectivity in a range of solvents, we have observed substantial
selectivity of 1 toward 5 only in halogenated solvents. Moreover,
our data are consistent with several possible transition-state
models (e.g., Figure 4) that cannot be resolved at this time.
Experimental Interrogation of the Proposed Models.
To test our hypothetical models, we re-examined a substrate that
we had studied earlier, farnesyl methyl ether (13). As noted in the
Introduction, we found that a 6,7-selective pentamer affords no
appreciable selectivity under a previous set of reaction con-
ditions, commensurate with loss of a hydroxyl-group-directed
mechanism. Under the updated reaction conditions of Table 2,
Table 2. Correlation of the i + 3 Position with Hydroxyl and
Methoxyl Directing Groups
Figure 4. Hypothesized models for selectivity toward 5.
also form in addition to or in combination with the β-turn
(Figure 4C). Furthermore, the i + 2 Thr(Bn) side chain Oγ and
the backbone NH may serve as hydrogen-bond acceptor and
donor, respectively, with the hydroxyl of farnesol, leading to the
proper proximal position of the 6,7-olefin relative to the modeled
peracid (Figure 4A−C). While the NMR experiments suggest a
β-turn, which is often accompanied by the discussed i → i + 3
hydrogen bond, it is possible that under the reaction conditions,
the NH of the i + 3 Asn(Trt) is oriented such that a hydrogen
bond forms with the substrate hydroxyl as well (Figure 4B). It is
not clear whether the intramolecular i → i + 3 hydrogen bond is
beneficial to the selectivity or whether the i + 3 NH is important
for binding with the substrate, or both, a detail that is difficult to
discern experimentally. Regardless, it appears that this NH is
important for one or both hydrogen-bonding interactions. Of
note, our data support models wherein at least two loci con-
tribute to the hydrogen-bonding network between the catalyst
and substrate. That is, ablation of the ethereal hydrogen-bond
acceptor in the i + 2 position is not enough to induce a complete
we found once again that peptide 1 affords no selectivity between
the 10,11-epoxide 14 and the 6,7-epoxide 15 (entry 1). More-
over, when the terminus is functionalized (e.g., 13), we observe
little formation of the 2,3-epoxide (e.g., 16); the reactivity of the
allylic olefin in the electrophilic epoxidation is attenuated by the
electron-withdrawing oxygen-containing substituent, and there is
no compensatory hydroxyl direction.23 However, the lack of
selectivity between the 10,11-epoxide 14 and the 6,7-epoxide 15
observed with catalyst 1 allows a test of our models with an
opportunity to enhance selectivity for one isomer.
The models in Figure 4 are consistent with the methyl group of
farnesyl methyl ether (13) obstructing the highly orchestrated
hydrogen-bonding array that accommodates the hydroxyl of
farnesol (one scenario is depicted in Figure 5A). Notably, each of
the arrays in Figure 4 is buttressed by an attractive interaction
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between the Thr(Bn) γ-oxygen atom and the OH group.
Substitution of the i + 2 Thr(Bn) of 1 with an Ile, as in peptide 17,
eradicates this attractive interaction. Its absence could then make
space available for the methyl group while retaining a less
elaborate but still functional group-directing array for ether-
directed epoxidation (Figure 5B). Consistent with this notion,
distribution of epoxides observed for squalene itself under the
various conditions we have examined. Taken together, these re-
sults represent a self-consistent data set for the models presented
in Figures 4 and 5, ultimately derived from NMR structural data,
extensive SAR, and challenging of the model with a new substrate
type.
Concluding Perspectives. We close by noting a potential
irony emerging from this study. Although we employed a
diversity-based approach to catalyst discovery, we found that the
catalyst that provides selectivity for the 6,7-position of farnesol
exhibits a propensity for β-turn formation, a motif that our lab has
studied on numerous occasions. However, the structural analysis
of 1 and its large extended family of variants led to new insights.
Notably, an interaction between the backbone NH and the side-
chain oxygen of Thr is thought to form part of the oxyanion hole
of lipases.24 Indeed, the comparative behavior of catalysts 1 and
17 provides some analogy for this feature.25 Indeed, since our
experiments with catalysts 1 and 17 provide evidence for remote
directing effects with different substrate types, the observations
highlight the tunability of catalyst hydrogen-bond arrays to
accommodate different substrate-directing groups.
Figure 5. Hypothesized interactions of 13 with i + 3 side-chain variants
of 1 and 17. One of the several scenarios based on Figure 4 is depicted.
catalyst 17 delivers an increase in the product ratio to nearly 1:4
for the 6,7-position (14:15; Table 2, entry 2), instead of the
1:1 ratio observed with catalyst 1 (entry 1). As emphasized in
Figure 5A, it is possible that with 1, unfavorable steric or
electronic interactions exclude ether-directed chemistry. How-
ever, with the alkyl side chain of Ile in 17, the peptide is still able
to accommodate hydrogen-bonding interactions with the
ethereal oxygen of the substrate (Figure 5B), albeit with a
perhaps reduced efficiency as one of several hydrogen-bond loci
is removed. In this vein, catalyst 17 is indeed quite a bit less
selective than catalyst 1 for farnesol itself (4:5:6 = 1.1:8.6:1.0
for 1 and 1.7:2.1:1.1 for 17; Table 2, entries 3 and 4). Taken
together, the picture that emerges as farnesol (2) and its derived
methyl ether (13) are evaluated with catalysts 1 and 17 is one that
links the models in Figures 4 and 5 in a self-consistent manner.
A thought-provoking and also consistent experiment was
performed with difarnesyl ether (18). Of note, this compound is
an oxide of the venerable natural product squalene, with a sole
oxygen atom inserted within its central C−C bond. When
catalyst 17 was examined with substrate 18, once again we ob-
served that the catalytic formation of monoxides was appreciably
enhanced in the anticipated internal olefin 19 (19:20 > 3:1;
Scheme 2 inset), with minimal allylic oxidation detected. This
Substrates like farnesol, its derived ethers, and many other
polyenes remain great challenges for total control of site selec-
tivity through catalysis. Nevertheless, the mechanistic hypotheses
derived from the experimental data presented herein have
suggested several opportunities for further catalyst development.
Future efforts in our lab will be focused on developing analogues
of catalyst 1 (and 17) for remote oxidation of other substrates
and other positions.
ASSOCIATED CONTENT
* Supporting Information
■
S
Additional figures, experimental details, characterization data,
and calculated structures. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
scott.miller@yale.edu
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank Dr. Michael Giuliano and Dr. Eric Paulson
for discussions about NMR structure elucidation and Prof.
Matthew Sigman and Elizabeth Bess (University of Utah) for
helpful discussions. This work was supported by the National
Institutes of Health (NIH) (R01-GM096403 to S.J.M).
Additionally, P.A.L. was partially supported by the NIH (CBI-
TG-GM-067543).
Scheme 2. Comparison of Reactions with Difarnesyl Ether
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