Divergent Stereoselectivity in Phosphothreonine (pThr)-Catalyzed Reductive Aminations of 3-Amidocyclohexanones

Article abstract of DOI:10.1021/acs.joc.8b00207

Phosphothreonine (pThr)-embedded peptide catalysts are found to mediate the reductive amination of 3-amidocyclohexanones with divergent selectivity. The choice of peptide sequence can be used to alter the diastereoselectivity to favor either the cis-product or trans-product, which are obtained in up to 93:7 er. NMR studies and DFT calculations are reported and indicate that both pathways rely on secondary interactions between substrate and catalyst to achieve selectivity. Furthermore, catalysts appear to accomplish a parallel kinetic resolution of the substrates. The facility for phosphopeptides to tune reactivity and access multiple products in reductive aminations may translate to the diversification of complex substrates, such as natural products, at numerous reactive sites.

Full text of DOI:10.1021/acs.joc.8b00207

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Divergent Stereoselectivity in Phosphothreonine (pThr)-
Catalyzed Reductive Aminations of 3-Amidocyclohexanones
Christopher Ryan Shugrue, Aaron L. Featherston, Rachel M. Lackner, Angela Lin, and Scott J. Miller
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b00207 • Publication Date (Web): 16 Mar 2018
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Divergent Stereoselectivity in Phosphothreonine (pThr)-Catalyzed
Reductive Aminations of 3-Amidocyclohexanones
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Christopher R. Shugrue, Aaron L. Featherston, Rachel M. Lackner, Angela Lin, and Scott J. Miller*
Department of Chemistry, Yale University, P.O. Box 208107, New Haven, Connecticut 06520-8107, United States
Supporting Information Placeholder
We envisioned that Brønsted acid catalysis could be a useful format
for peptide-based catalysis, as non-C2-symmetric CPA
ABSTRACT: Phosphothreonine (pThr)-embedded peptide catalysts
are found to mediate the reductive amination of 3-amidocyclohexa-
nones with divergent selectivity. Choice of peptide sequence can be used
to alter the diastereoselectivity to favor either cis-product or trans-prod-
uct, which are obtained in up to 93:7 er. NMR studies and DFT calcula-
tions are reported and indicate that both pathways rely on secondary in-
teractions between substrate and catalyst to achieve selectivity. Further-
more, catalysts appear to accomplish a parallel kinetic resolution of the
substrates. The facility for phosphopeptides to tune reactivity and access
multiple products in reductive aminations may translate to the diversifi-
cation of complex substrates, such as natural products, at numerous re-
active sites.
Introduction
Synthetic chemists have often relied on “privileged scaffolds”, in
which one catalytic architecture can obtain high selectivities over diverse
sets of reactions.1 Chiral phosphoric acids (CPAs, Figure 1A) represent
a highly successful example of this paradigm, and CPAs relying on rigid,
C2-symmetric scaffolds, such as BINOL, have become one of the most
widespread and prodigious classes of asymmetric catalysts.2–5 Alterna-
tively, enzymes have evolved to overcome the challenges associated with
selective catalysis by retaining specific, catalytically active, and “privi-
leged” residues and three-dimensional “folds,” which are then effective
for a broad range of biochemical transformations.6–9 Changes to the vast
number of potential amino acid sequences can facilitate subtle or drastic
alterations to protein structure, molding enzymes to fit particular sub-
strate classes, tailored to achieve site-selective modifications of individ-
ual complex molecules, and even tuned to access different types of reac-
tivity from the same catalytic residues.10,11 Given the growing challenges
faced by synthetic chemists today, exploring the development of new
catalytic archetypes is advantageous, particularly ones that are tunable
to selectively catalyze reactions on specific, complex substrates.
scaffolds have been underexplored in this regime and could be broadly
applicable, given the breadth of CPA-catalyzed chemistry. As phosphor-
ylated amino acids, such as phosphothreonine, are ubiquitous in na-
ture,14–18 we wondered whether these could be adopted in phosphoric
acid catalyzed reactions. The incorporation of the pThr-monomer into
the peptide chain is achieved using a HATU-mediated peptide coupling
of commercially available Fmoc-pThr(Bn)-OH to various sequences,
resulting in phosphopeptides with high yields and purity (Figure 1A).19
One such approach to asymmetric catalysis involves mimicking the
breadth and selectivity of enzymes by appending minimal and distinct
peptide sequences (normally <4 residues) to a conserved catalytic resi-
due.12 The diverse peptide sequences fold in different ways, resulting in
a wide variety of accessible conformations, which allow for tuning of the
catalytically active residue’s reactivity and selectivity.13 Simultaneously,
the functional group-rich nature of peptides chains can also offer sec-
ondary points of contact—in a sense, outer sphere interactions—to
bind to substrates, differentiating diastereomeric transition state ener-
gies, and leading to good levels of selectivity.15
Our studies in phosphopeptide-mediated transformations began with
transfer hydrogenation. BINOL-derived CPAs have previously been
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used in the reductions of numerous imines,20–24 enamines,25 and hetero-
cycles,26–29 normally in the presence of Hantzsch ester (HEH) as the hy-
dride source.30–33 Dihydropyridine HEH mimics the activity of
NAD(P)H, one of nature’s most prolific hydride sources,34,35 and it was
particularly compelling to combine this reductant with the bioinspired
pThr-residue. As such, the transfer hydrogenation of 8-aminoquinolines
(2) was pursued, and enantioselectivities as high as 94:6 er are achieved
(Figure 1B).36 Notably, selectivities are equivalent to BINOL-derived
CPAs, despite the increased flexibility of the phosphopeptides. The ef-
fectiveness of a non-C2-symmetric CPA-scaffold represents a fundamen-
tal departure in strategy for CPA catalyst development, as the pThr-em-
bedded peptides appear to achieve selectivity via secondary interactions
between catalyst and substrates, as opposed to forming a small, sterically
hindered binding pocket. This success not only offers credence to the
idea that phosphopeptides and BINOL-derived catalysts could serve as
a complementary system, but also could spark curiosity in previously un-
derexplored in vivo functions of pThr and its potential to serve as a
Brønsted acid in nature. As we have expanded our purview into this area,
we wished to assess the generality of pThr-containing peptides for con-
fronting an unusual, stereochemically complicated and functional
group-rich scaffold that would differ greatly from 2, and perhaps be rep-
resentative of functionality found in natural products, as a model for site-
selective modifications.37 In order to address this challenge, we assem-
bled >200 unique phosphopeptides for evaluation.
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Results and Discussion
Catalyst optimization. Ketones are a ubiquitous in natural prod-
ucts,38 and reductive aminations have previously been reported using
BINOL-derived CPAs on a range of ketones.32,39,40 In a parallel line of
research, our group has pursued the aspartyl-peptide-catalyzed Baeyer–
Villiger oxidation (BVO) of 3-amidocyclohexanones (4, Figure 1C),
with a similar goal of achieving peptide-based, divergent stereocontrol.41
The presence of a directing group on this substrate class enhances its po-
tential to engage in secondary interactions with peptide catalysts. Ac-
cordingly, we wondered if these 3-amidocyclohexanones could be ame-
nable to selective reductive aminations, catalyzed by the pThr/HEH
system (Figure 1D). Reactions of 4 have the potential to produce both
trans- and cis-products, offering the possibility for pThr-embedded cat-
alysts and BINOL-derived CPAs to exhibit divergent selectivities and
access to different products. As such, our studies on the reductive ami-
nation of 3-amidocyclohexanones are presented herein.
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The intrinsic diastereoselectivity for the reductive amination of sub-
strate 4a with p-anisidine, in the presence of HEH, was assessed with di-
phenyl phosphate, a simple achiral phosphoric acid. A 69:31 dr was ob-
served in favor of trans-7a (Table 1, entry 1), establishing this value as
an intrinsic dr to which all subsequent results could be compared. Re-
ductive amination in the presence of P1, which achieves up to 94:6 er in
the transfer hydrogenation of 8-aminoquinolines, resulted in racemic
product (Table 1, entry 2). Continued screening with the phosphopep-
tide library revealed thatP2 induces a 79:21 er for cis-7a (Table 1, entry
3), suggestive of a significant opportunity to enhance catalyst-substrate
interactions. Additional screening indicated that 1,1-disubstitued resi-
dues at the i+2 position (see Figure 1A for “i+n” nomenclature)42 were
unsuccessful at achieving er, and mono-substituted amino acids were
next pursued. The incorporation of residues with heteroatoms for addi-
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tional points of contact with substrates resulted in diminished enanti-
as the i+3 residue was found to slightly tune enantioselectivities for cis-
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oselectivity for cis-7a (Table 1, entries 4,5; er = 61:39 and 66:34) Vary-
ing the i+2 position revealed a subtle preference for larger side chains.
Furthermore, an additional methylene spacer between the bulky group
and the backbone results in lower er for cis-7a [Chg to Cha (Table 1,
entries 6,7; er = 76:24 and 72:28), Phg to Phe (Table 1, entries 8,9; er =
74:26 and 70:30), and Ile to Leu (Table 1, entries 11,12; er = 79:21 and
77:23)]. Overall, catalysts with Tle (P2) and Val (P12) exhibited the
highest selectivities (up to 83:17 er, Table 1, entry 13) and thus catalysts
with these residues at the i+2 position were chosen as sequences to ex-
amine further. Of note, this catalyst proved to shift the dr in favor of the
cis-product, as trans-7a/cis-7a now reached 58:42.
7a to 93:7 (Table 1, entry 31). This selectivity represented the maxi-
mum enantioselectivity we had observed to this point, peptide P32 was
identified as the hit catalyst to study additional aspects of the chemistry.
We also note some results with catalysts of dramatically different
structure. These include peptide sequences that are vastly different from
the hit catalyst, and also a comparative result with a venerable C2-sym-
metric CPA (S)-TRIP (1), which is utilized in a myriad of transfor-
mations.2 In order to target divergent selectivity with alternative pep-
tide-based catalysts, we examined a pThr-based catalyst with the ap-
pended sequence of peptide 6, previously utilized in the BVO of 3-ami-
docyclohexanones (4),41 an analogous substrate to the corresponding
imine undergoing reduction in the present work. As such, peptide P33
was synthesized, wherein pThr replaces the aspartic acid catalytic resi-
due of 6. P33 results in an entirely different selectivity profile, instead
favoring trans-7a with 74:26 dr and 75:25 er (Table 1, entry 34). This
result highlights how the diversity of peptide sequences appended to a
distinct catalytic residue enables access to multiple products with com-
plementary selectivity. Minimal optimization of P33 resulted in the ob-
servation of i+4 Phe-containing P34 as a slightly better performing cat-
alyst (Table 1, entry 35, trans-7a er = 82:18, cis-7a er = 78:22). Finally,
the results with catalysts P32 and P34 were compared to (S)-TRIP (1).
In this case, catalyst 1a favors the formation of trans-7a with 79:21 dr
and 71:29 er (Table 1, entry 34) under the conditions we employed in
our study, which were not further optimized. P32 and 1 are complemen-
tary catalysts, allowing access to both diastereomers of 7a with good lev-
els of selectivity.
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Varying the stereochemistry at the i+1 and i+2 positions resulted in
diminished er. L-stereochemistry at the i+1 position reduced the er for
cis-7a to 53:47 (Table 1, entry 15). Additionally, P13, which contains
D-stereochemistry at the i+2 position, retained some enantioselectivity
for cis-7a (72:28), but not trans-7a (59:41, Table 1, entry 14).
Given these results, and in light of the fact that further evaluation of
the i+2 and i+3 positions did not improve selectivities, we hypothesized
that the catalytic residue itself is critical for binding to imine 8a. Hence,
alterations closer to the N-terminus might induce enhanced selectivities.
As such, the stereochemistry of the pThr residue was examined. Inter-
estingly, only the naturally occurring Thr-stereochemistry was compati-
ble with good selectivity, as variation of the Thr b-stereocenter (i.e., use
of allo-pThr or DpThr) resulted in low enantioselectivty (Table 1, en-
tries 16,17; er = 58:42 and 57:43). The importance of a b-methyl group
was demonstrated by replacing pThr with pSer, resulting in 59:41 er for
cis-7a (Table 1, entry 18).
Tuning of reaction conditions. While enantioselectivities as high as
93:7 can be achieved with this substrate class, conversions were nor-
mally low with 24 h reaction times. Product 7a could be obtained in
higher conversion with longer reaction times, as 96 h resulted in approx-
imately twofold increase in conversion (Table 2). The lower reactivity
of this system is ascribed both to difficulties associated formation of
imine 8a, in addition to catalyst inhibition induced by the basicity of the
secondary amine product 7a and the pyridine byproduct of HEH.
Furthermore, substituted amido-proline catalysts P18 and P19 were
examined due to the potential for offering additional points of contact
for substrates binding adjacent to the pThr-residue43. Intriguingly, in-
corporation of a trans-amidoproline directing group overturns the enan-
tioselectivity to favor the opposite enantiomer of cis-7a in 39:61 er (Ta-
ble 1, entry 19). Alternatively, the cis-amidoproline directing group
simply yields cis-7a with slightly diminished er (Table 1, entry 20; er =
71:29).
Additional optimization of conditions was performed using catalyst
P32, and it was found that an aqueous wash with a saturated NaHCO3
solution prior to flash chromatography modestly perturbed the dr for
these reductive aminations. With diphenyl phosphate as catalyst, the in-
trinsic diastereoselectivity was still found to favor trans-7a with 55:45 dr
with these new conditions (Table 2, entry 5). P32 and P34 gave 33:67
and 54:46 dr respectively, increased amounts of the cis diastereomer,
while maintaining similar er levels for both cis-4a and trans-4a (Table 2,
entries 6–7). Alternatively, (S)-TRIP produced a similar dr value
(70:30) with increased er values for both cis and trans product (82:18
and 70:30 respectively, Table 2, entry 8). We presume that catalysts may
induce a secondary resolution of products through selective protona-
tion. Peptides appear to be matched to protonate cis product preferen-
tially, enhancing dr for cis-4a.44 Alternatively, BINOL-derived 1 instead
selectively protonates the favored enantiomers of cis-4a and trans-4a, re-
sulting in an increase in observed er for both diastereomers with an ad-
ditional base wash to breakup this salt.
Encouraged by these variations in observed enantioselectivity with al-
terations near to the pThr residue, the N-terminal protecting group of
the peptide was varied (Table 1, entries 21–26). Utilization of electron
rich benzamide protecting groups, such as p-MeO-Bz (P24) results in
selectivities as high as 91:9 er for cis-7a, and with a dr of 51:49 (Table 1,
entry 25).
With these results in hand, given the lack of substantial variation in
selectivities with alterations to the i+2 and i+3 positions, a truncation
study on the peptide was performed, in which each residue is sequen-
tially deleted (Table 1, entries 27–31). The C-terminus was capped
both as a methyl ester, in analogy to the hit sequence, and also as a me-
thyl amide. Moderate levels of er were maintained upon truncation, with
even a minimally protected pThr-monomer yielding 75:25 er of cis-7a
(Table 1, entry 31). While Fmoc-protected P27 results in even higher
er (Table 1, entry 28; er = 85:15) than Fmoc-protected tetramer P12
(Table 1, entry 13; er = 83:17), these catalysts are both less selective
than the elaborated tetramers. In all cases, the methyl esters performed
better than the methyl amides. This indicates that the peptide conformer
that leads to high levels of selectivity in this reaction does not rely on the
i to i+3 b-turn, which requires an NH bond on the i+2/i+3 amide and is
generally enhanced by the presence of a C-terminal amide.
Based on these observations, we designed a catalyst incorporated all
of the key attributes identified in the above studies. A minimal screen
was performed to establish the best pairing of the N-terminal functional
group within a truncated tripeptide catalyst. Thus, P31 resulted in the
highest cis-7a-selective dr, 45:55 (Table 1, entry 32). Finally, Ser(tBu)
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Therefore, in sum, the selectivities of these reactions can be reduced to
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three ratios: catalyst selectivity for (1) the reaction of (R)-4a versus (S)-
4a [(kR1+kR2)/(kS1+kS2)], (2) the formation of (R)-trans-7a versus (R)-
cis-7a, and (3) the formation of (S)-trans-7a versus (S)-cis-7a.
Analysis of these different rates revealed a difference in the capacity
of the pThr peptide-based catalysts and either diphenyl phosphate 9 or
(S)-TRIP (1) to process functionalized substrates. The crude product
ratios (out of 100) are shown in Figure 2A and the relevant rates for
each catalyst are shown in Figure 2B. While peptides P32 and P34 ex-
hibit characteristics of a parallel kinetic resolution, reacting with both
enantiomers of 4a at similar rates and converting each to a different dia-
stereomer of 7a, (S)-TRIP (1) reveals a preference to mediate the re-
ductive amination of (S)-4a operating closer to a kinetic resolution par-
adigm. Furthermore, while 1 only yields good selectivity for (S)-4a (1 :
3.59 ratio), which is matched with the catalyst, it is notable that P32 is
able to carry out the reductive amination of both (R)-4a and (S)-4a at
high levels of selectivity (1 : 6.00 and 1 : 7.12 ratio respectively). These
results show the substantially different mechanisms of actions of these
phosphopeptides and the BINOL-derived CPAs, and encourages the in-
vestigation of these catalytic systems as complementary scaffolds.
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Assessment of directing group scope. Under optimized condi-
tions, catalyst P32 tolerates alterations of the N-protecting group of 4
(Table 3). In addition to the previously utilized phenyl acetamide (4a,
Table 3, entries 1–2), P32 was able to process benzamide-protected 4b
to cis-7b with an er of 82:18, with slight perturbation of dr favoring the
cis product (Table 3, entries 3–4, dr changes from 62:38 with 9 to 51:49
with P32). Cbz-protected 4c was also tolerated, and cis-7c could be ob-
tained with an er of 86:14 and a dr of 35:65, which is more cis-selective
in comparison to reactions employing (PhO)2P(O)OH (9) as catalyst
(Table 3, entries 5–6, dr = 56:44). Finally, sulfonamide 4d provides the
most drastic change from 4a, and is converted to cis-7d with 89:11 er
and 49:51 dr (Table 3, entry 8). The intrinsic dr with achiral catalyst 9
was 68:32, once again revealing that the peptides induce significant per-
turbations (Table 3, entry 7).
Consideration of the mechanistic basis of stereochemical out-
comes. We next sought to understand the catalytically active confor-
mation of P32, and how the peptide interacts with the imines en route
to reaction products. The absolute configuration of enantiomerically
pure product cis-7d was determined by X-ray crystallography, and crys-
tal structures of (±)-cis-7d and (±)-trans-7d were also solved (see Sup-
porting Information for more details), which is critical for
understanding possible catalyst-substrate interactions. However, iden-
tifying either the lowest energy, or reactive conformation of the reacting
cycloheximine 8 is non-trivial. Thus, we sought to assess computation-
ally the relative energies of both the E- and Z-imines, with the 3-amido
group in both the axial and equatorial orientation. DFT calculations
were performed on the B3LYP/6-311+G(d,p) level to optimize all
structures, and single point energy calculations were performed with
M06-2x/6-311++G(2d,3p).547–49 These calculations were performed in
conjunction with the IEF-PCM solvation model.50,51 As previously de-
scribed for cyclohexane 10,52 as a control, the amido group was favored
in the equatorial position by DG298K = 1.06 kcal/mol as expected (Fig-
ure 3A). Addition of the ketone moiety (4a), however, the favorability
of the equatorial orientation was negligible, indicating both confor-
mations are accessible in solution (Figure 3B). This could be the result
of removing one 1,3-diaxial interaction between the 3-amido group and
an axial C–H, in addition to 4a-eq having a substantially larger dipole
moment than 4a-ax (D = 3.31 debye).
Mechanism-driven experiments. The observed diastereo- and en-
antioselectivity for reactions of substrates like 4 offer convenient metrics
to gauge the effectiveness of the catalysts in a complex parallel kinetic
resolution (see Figure 2).45.46 As ketone 4a is racemic, the catalyst must
react with two enantiomers [(R)-4a and (S)-4a]. The reductive amina-
tion of each of these enantiomers therefore results in both a cis-product
and a trans-product with conserved absolute stereochemistry; the over-
all transformation thus consists of two diastereoselective reactions.
We next applied this computation technique to the four potential
conformations of 8a, and for ease of calculation, it was assumed that P32
does not selectively catalyze the imine formation, and that all imine iso-
mers are in equilibrium. E-8a-ax was found to be the most stable imine
by a small margin; however, all imines are energetically accessible in the
reaction mixture (Figure 3C). We hypothesized that the directing
group in an equatorial position could be more favorable with regard to
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steric considerations. Due to the orientation of the benzamide group, it
Upon assignment of the full proton NMR spectrum of P25, 2D
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NOESY experiments were conducted. At similar concentration to pep-
tide-mediated reactions (6.0 mM), 14 inter-residue NOE correlations
were observed and are shown in Figure 5A. NOEs, shown in green, or-
ange, and blue, point to a preferred orientation for the pThr residue. The
three magenta NOE contacts in Figure 5B suggest contacts between the
NHVal and the protons of the a-C–H of Thr, and C–H on the bottom
face of Pro. These contacts are possible if this N–H bond has rotated
underneath the Pro residue. This places NHVal in close proximity to both
C=OThr (green), and the C=OPG (blue), and could indicate competitive
is possible that an attractive CH–p interaction with the anisidine arene
results in some stabilization (Figure 4A). E-8a-eq, alternatively, cannot
geometrically access this CH–p interaction (Figure 4B). Peptide cata-
lysts are therefore able to navigate a complex mixture of equilibrating
imines (both E and Z, and axial and equatorial isomers, for both enanti-
omers of 4a; potentially producing eight imines), yielding orthogonally
protected 1,3-diamine products with high selectivities.
With some understanding of the substrate conformational profile in
hand, we turned our attention to studying the conformational issues as-
sociated with the catalysts by NMR spectroscopy. One challenge associ-
ated with NMR analysis of the phosphopeptides is their propensity to
exhibit multiple conformations and tautomers.40 Indeed, the pThr resi-
due itself can theoretically adopt a number of intramolecular H-bonding
interactions between the phosphoric acid proton, the phosphate O- at-
oms, and any of the amides on the peptides, in addition to the number
of secondary structures the peptide backbone can adopt. Catalyst P25,
which shows comparable selectivities to hit catalyst P32 (see Table 1,
9
formation of a NHVal–C=OThr g-turn and a b-turn involving the NHVal
–
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C=OPG. (This possible turn is notable in that it is one residue shifted
from the proverbial C=OThr–NHLeu b-turn). This hypothesis is further
validated with the observation of NOE contacts between a furanoyl C–
H bond and the Val methine C–H (Figure 5B, red).This possible con-
formation is consistent with results of the catalytic reactions involving
truncation or changes to the i+3 position. This residue, in the observed
solution conformation, may only partially assist to orient the NHi+2. The
incorporation of more electron-rich N-terminal protecting groups, such
as p-MeO-Bz or furanoyl-groups, could enforce this turn by either favor-
ing the more Lewis basic C=OPG to act as an intramolecular H-bond ac-
ceptor, or by disfavoring NHThr from serving as an intramolecular H-
bond donor.
1
entries 26 and 33) was chosen for this study as the H NMR stretches
were well-resolved.
(A)
(B)
E-8a-ax (calculated)
E-8a-eq (calculated)
Figure 4. Calculated, lowest energy structure of (A) E-8a-ax and (B) E-
8a-eq.
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1H NMR spectra were recorded on 400 MHz, 500 MHz, or 600 MHz Agilent
With these considerations in mind, mechanistic hypotheses for for-
mation of both trans-product and cis-product may be advanced, and are
presented in Figures 5C and 5D. First, a b-turn between NHVal–C=OPG
is proposed as the secondary structure for peptide catalysts, in accord
with our experimental observations. As DFT calculations indicated the
accessibility of multiple chair conformations of 8a in solution, we envi-
sion that E-8a-eq could interact most productively, in a Curtin-Hammett
fashion, with P32 as shown. Given the importance of the 3-amido di-
recting group, it is likely that the basic imine lone pair is oriented on the
same side as the directing group, implying that E-imines may be favored
over Z-imines, facilitating secondary points of contact. While E-8a-eq is
not the lowest energy conformation of 8a, it is energetically accessible,
and presents a sterically accessible environment upon protonation by
P32. E-8a-ax, of course, could also be processed, but our analysis in-
cludes the intuitive assumption that reactions of the axial conformations
should be highly cis-selective, which we do not observe. Thus, returning
to models involving E-8a-eq, the NHThr is oriented directly towards the
phenyl acetamide’s C=O to engage in an H-Bond (Figures 5C and 5D).
Intriguingly, this interaction is possible for both enantiomers of 8a, al-
beit presenting the opposite imine face towards the bifunctionally acti-
vated HEH. This results in one enantiomer of 8a being processed to the
trans-product, and one to cis-product, in accord with results under the
optimized conditions. Hence, the high selectivities in this parallel kinetic
resolution could be the result of flexibility in the peptide structure to ac-
commodate two dissimilar substrates in the same binding pocket.
spectrometers at ambient temperature. Samples were prepared in chloroform-d
(CDCl3), dimethyl sulfoxide-d6 (d6-DMSO), methanol-d4 (CD3OD), or ben-
zene-d6 (C6D6). 1H NMR data are reported as chemical shifts with multiplicity,
coupling constants (J) in Hz, and integrations. Proton chemical shifts are re-
ported in ppm (d) and referenced to tetramethylsilane (TMS) or residual solvent
(CHCl3, d 7.26 ppm, DMSO d 2.50 ppm, CD3OH d 3.31 ppm, C6H6 d 7.16
ppm).57 Multiplicity is reported as follows: singlet (s), broad singlet (bs), doublet
(d), broad doublet (bd), doublet of doublets (dd), broad triplet (bt), doublet of
doublet of doublets (ddd), doublet of doublet of doublet of doublets (dddd),
doublet of doublet of doublet of triplets (dddt), doublet of doublet of triplets
(ddt), doublet of triplets (dt), doublet of triplet of doublets (dtd), dou-blet of
triplets of triplets (dtt), doublet of pentets (dp), triplet (t), tri-plet of doublets
(td), triplet of triplets (tt), triplet of doublet of doublets (tdd), triplet of doublet
of triplets (tdt), triplet of triplet of dou-blets (ttd), quartet (q), multiplet (m), and
overlapping multiplets (comp). 13C NMR spectra were recorded on 500 (126)
MHz or 600 (151) MHz Agilent spectrometers with complete proton decoupling
at ambient temperature, unless otherwise noted. Carbon chemical shifts are re-
ported in ppm (d) and referenced to tetramethylsilane (TMS) or solvent
(CDCl3, d 77.16 ppm, d6-DMSO d 39.52 ppm, CD3OD d 49.00 ppm, C6D6 d
128.06 ppm).53 31P NMR spectra were recorded on 500 MHz Agilent spectrom-
eters with complete proton decoupling at ambient temperature.
Low resolution mass spectrometry (MS) was acquired on a Waters SQD2
UPLC-MS equipped with an electrospray ionization (ESI) detector, a QToF
mass spectrometer, and a photodiode array detector, with Waters ACQUITY
UPLC BEH C18 (1.7 µm, 2.1 x 50 mm) and Waters CORTECS UPLC C18 (1.6
µm, 3.0 x 50 mm) columns. High resolution mass spectrometry (HRMS) was
conducted by the Mass Spectrometry Laboratory at the University of Illinois at
Urbana-Champaign using a Waters Synapt G2-Si instrument equipped with a
QToF mass spectrometer, an ESI detector. Elemental Analysis (EA) was con-
ducted by Robinson Microlit Laboratories (Ledgewood, NJ).
Conclusion
Infrared spectra were obtained using a Nicolet ATR/FT-IR spectrometer, and
In conclusion, we have reported the application of pThr-embedded
peptides to the reductive amination of 3-amidocyclohexanones. The
phosphopeptides tolerate a range of N-protecting groups on the sub-
strates and are able to overturn the inherent trans selectivity of this reac-
tion to achieve up to 93:7 er and 35:65 dr for cis-product. Hit catalyst,
P32, adopts an intriguing secondary structure and is believed to engage
in secondary interactions with both enantiomers of starting material,
and processes each to a different diastereomer of product through a par-
allel kinetic resolution. We are hopeful that the study of the pThr-cata-
lyzed reductive aminations of small molecules will translate to advances
in the site-selective modification of ketone-containing natural products.
ν
max (cm–1) were partially recorded in accordance with convention. Optical rota-
tions were recorded on a Perkin Elmer Polarimeter 341 at the sodium D line (1.0
dm path length) at 20 °C. Analytical thin-layer chromatography (TLC) was per-
formed using EMD Millipore silica gel 60 F254 precoated plates (0.25 mm thick-
ness). The developed plates were visualized by a UV lamp and/or potassium per-
manganate (KMnO4) stain.
Normal-phase column chromatography was performed with either silica gel
60 Å (32-63 microns) or with a Biotage Isolera One flash purification system
equipped with 15 g, 30 g, 60 g, or 120 g SNAP Ultra HP-Sphere 25 μm columns
using an appropriate linear gradient of EtOAc/Hexanes. Reversed-phase column
chromatography as performed with a Biotage Isolera One flash purification sys-
tem equipped with 30 g, 60 g, or 120 g SNAP KP-C18-HS or SNAP Ultra-C18
columns with an appropriate gradient of MeCN/H2O.
Enantiomeric ratio (er) values were acquired using an Agilent 1100 series an-
alytical chiral HPLC equipped with a photodiode array detector (210 nm, 230
nm, 250 nm, and 254 nm) with Chiralpak IC, IB, OD-H, and AD-H columns (5
μm particle size, 4.5 x 250 mm).
Representative Synthesis and Characterization of Peptide P12 (For P1–
P31, General Procedure #1). See Figure SI-1. Peptide Coupling #1: To a flame
EXPERIMENTAL SECTION
General Experimental Methods. Room temperature is defined as 21–23 °C.
All reagents were purchased from commercial sources and used as received unless
otherwise noted. Solvents used for reactions and for azeotropic drying, such as
toluene (PhMe), methylene chloride (CH2Cl2), N,N-dimethylformamide
(DMF), and tetrahydrofuran (THF), were obtained from a Seca Solvent Purifi-
cation System by Glass Countour, in which the solvents were dried over alumina
and dispended under an atmosphere of argon. For all other purposes, solvents
were used as received from commercial sources unless otherwise noted.
dried 100 mL round bottom flask equipped with a stir bar was added HCl•Leu-
OMe (1.36 g, 7.5 mmol, 1.0 equiv.), Boc-Val-OH (1.63 g, 7.5 mmol, 1.0 equiv.),
EDC•HCl (1.63 g, 8.3 mmol, 1.1 equiv.), and HOBt•H2O (1.63 g, 8.3 mmol, 1.1
equiv.). To the solid mixture was added CH2Cl2 (30 mL), followed by Hunig’s
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Base (958 μL, 16.5 mmol, 2.2 equiv.). The resulting solution was stirred overnight
mmol, 5.0 equiv.), Cl-HOBt (930 mg, 5.50 mmol, 5.0 equiv.) followed by 17 mL
NMP and then NMM (1.20 mL, 11.0 mmol, 10.0 equiv.) was next added. This
solution was added to the resin (rinsing the vial with 1 mL NMP). The vessel was
rotated to homogeneity for 5 h. Upon completion of the reaction, the vessel was
drained and washed with DMF (20 mL x 3) and CH2Cl2 (20 mL x 3), agitating
to homogeneity each time. Upon draining the final wash, the resin was taken onto
the next step without any further purification.
for 12–18 h, upon which the reaction was transferred to a separatory funnel and
the organics were washed with NaHCO3 (30 mL, saturated aqueous), 10% citric
acid (50 mL, aqueous), and brine (50 mL, saturated, aqueous). The organics
were collected, dried over Na2SO4, filtered, and concentrated in vacuo to yield
2.63 g of Boc-Val-Leu-OMe as a white solid (98% yield), the identity of which
was confirmed by UPLC-MS. MS (ESI-QToF) m/z: [M+Na]+ Calcd for
1
2
3
4
5
6
7
8
C
17H32N2O5Na 367.22; Found 367.22.
Deprotection #1: A 250 mL round bottom flask was charged with Boc-Val-Leu-
Deprotection #1: The resin was suspended in 20% v/v piperidine/DMF and
rotated for 20 min. Upon draining the reaction vessel, the resin was washed with
DMF (20 mL x 3) and CH2Cl2 (20 mL x 3), agitating to homogeneity each time.
Upon draining the final wash, the resin was taken onto the next step without any
further purification.
Peptide Coupling #2: The same reaction conditions as in Peptide Coupling #1
were utilized, except using Fmoc-DPro-OH (1.86 g, 5.50 mmol, 5.0 equiv.) as the
amino acid. The reaction was also rotated overnight for 12 h.
OMe (2.36 g, 7.5 mmol, 1.0 equiv.) and a stirbar, and then capped with a septum
pierced with a Teflon cannula leading to a solution of NaHCO3 (saturated, aque-
ous). HCl (7.5 mL, 4N in 1,4-dioxane) was then added, and the resulting solution
was stirred vigorously for 1 h. After completion of the reaction, Nitrogen gas was
vigorously bubbled through the solution using a needle until all solvent was re-
moved, revealing HCl•H-Val-Leu-OMe as a crude white solid. The solid was
dried under vacuum for a 1 h and was used in the next step without further char-
acterization or purification.
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Cleavage: The resin was suspended in 20 mL 4:1:1
CH2Cl2/trifluroethanol/AcOH and rotated for 30 min. Upon completion of the
reaction, the vessel was drained and the supernatant collected. The resin was fur-
ther washed with CH2Cl2 (20 mL x 3) agitating to homogeneity each time, and
the combined organics were concentrated in vacuo to yield 287 mg Fmoc-DPro-
Val-Ser(tBu)-OH as a white solid (45% yield, 4 steps). The cleaved peptide was
taken onto the next step without any further purification
Peptide Coupling #2: To the 250 mL round bottom flask containing HCl•H-
Val-Leu-OMe was added Boc-DPro-OH (1.60 g, 7.5 mmol, 1.0 equiv.),
EDC•HCl (1.63 g, 8.3 mmol, 1.1 equiv.), and HOBt•H2O (1.63 g, 8.3 mmol, 1.1
equiv.). To the solid mixture was added CH2Cl2 (30 mL), followed by Hunig’s
Base (958 μL, 16.5 mmol, 2.2 equiv.). The resulting solution was stirred overnight
for 12–18 h, upon which the reaction was transferred to a separatory funnel and
the organics were washed with NaHCO3 (30 mL, saturated, aqueous), 10% citric
acid (50 mL, aqueous), and brine (50 mL, saturated aqueous). The organics were
collected, dried over Na2SO4, filtered, and concentrated in vacuo to yield 3.26 g
of Boc-DPro-Val-Leu-OMe as a white solid (98% yield), the identity of which was
confirmed by UPLC-MS. MS (ESI-QToF) m/z: [M+Na]+ Calcd for
C22H39N3O6Na 464.27; Found 464.37.
Esterification: To a 250 mL round bottom flask containing Fmoc-DPro-Val-
Ser(tBu)-OH (287 mg, 0.495 mmol, 1.0 equiv.) was added EDC•HCl (190 mg,
0.990, 2.0 equiv.), HOBt•H2O (152 mg, 0.990 mmol, 2.0 equiv.), and 7.0 mL
MeOH. The reaction was stirred overnight. After 18 h, the reaction was concen-
trated in vacuo and redissolved in 30 mL CH2Cl2. The organics were washed with
NaHCO3 (30 mL, saturated, aqueous), 10% citric acid (30 mL, aqueous), and
brine (30 mL, saturated, aqueous). The organics were dried over Na2SO4, fil-
tered, and concentrated in vacuo. The crude solid was purified via automatic re-
verse phase flash chromatography, eluting with a gradient of 40% to 100%
MeCN/H2O to yield Fmoc-DPro-Val-Ser(tBu)-OMe as a white solid.
Deprotection #2: A 250 mL round bottom flask was charged with Boc-DPro-
Val-Leu-OMe (593 mg, 1.34 mmol, 1.1 equiv.) and a stirbar, and then capped
with a septum pierced with a Teflon cannula leading to a solution of NaHCO3
(saturated, aqueous). HCl (4.0 mL, 4N in 1,4-dioxane) was then added, and the
resulting solution was stirred vigorously for 1 h. After completion of the reaction,
Nitrogen gas was vigorously bubbled through the solution using a needle until all
Deprotection #2: To a flame dried 20 mL vial was added Fmoc-DPro-Val-
Ser(tBu)-OMe (388 mg, 0.823 mmol, 1.1 equiv.) and 3.6 mL 1:1 diethyla-
mine/CH2Cl2. The reaction for stirred for 30 min and then concentrated in vacuo.
The crude solid was dried in vacuo overnight.
solvent was removed, revealing HCl•H-DPro-Val-Leu-OMe as a crude white
solid. The solid was dried under vacuum overnight for 24 and was used in the next
step without further characterization or purification.
Peptide Coupling #3: To the 20 mL vial was added Fmoc-pThr(Bn)-OH (383
mg, 0.748 mmol, 1.0 equiv.). After cooling the solid mixture was to –10 °C using
a brine/ice bath, CH2Cl2 (4.8 mL) was added. To the mixture was added NMM
(470 μL, 4.27 mmol, 3.5 equiv.), followed by HATU (557 mg, 1.47 mmol, 1.2
equiv.). The vial was flushed with Ar, capped, and stirred for 18 h. Upon comple-
tion of the reaction, the solution was transferred to a separatory funnel, diluted
with CH2Cl2 (10 mL), and washed with 10% citric acid (25 mL, aqueous). The
organic layer was separated and the aqueous layer extracted with CH2Cl2 (25
mL). The combined organics were washed with brine (50 mL, saturated aque-
ous), dried over Na2SO4, filtered, and concentrated in vacuo to yield a pale yellow
solid. The crude product was purified via automatic reverse phase flash chroma-
tography with a gradient of 10% to 100% MeCN/H2O with a 0.1% formic acid
buffer solution, resulting in 440 mg P32 as a white solid (69% yield).
Variation of N-Terminal Protecting Group (General Procedure #6). See
Figure SI-6A. This procedure was used for peptides P23 (utilizing benzoic acid
as the carboxylic acid), P24 (p-methoxybenzoic acid), P22 (p-nitrobenzoic
acid), and P25 (2-furanoic acid). To an oven dried 8 mL scintillation vial was
added Fmoc-pThr(Bn)-DPro-Val-Leu-OMe (P12, 200 mg, 0.240 mmol, 1.0
equiv.), followed by 1.2 mL of a 1:1 solution of diethylamine and CH2Cl2. The
solution was stirred for 1 h and then concentrated in vacuo. The crude white solid
was dried under vacuum overnight for 24 h and was used in the next step without
Peptide Coupling #3: To the 25 mL round bottom flask containing HCl•H-
DPro-Val-Leu-OMe was added Fmoc-pThr(Bn)-OH (625 mg, 1.22 mmol, 1.0
equiv.). After cooling the solid mixture was to –10 °C using a brine/ice bath,
CH2Cl2 (7 mL) was added. To the mixture was added 4-methylmorpholine
(NMM, 470 μL, 4.27 mmol, 3.5 equiv.), followed by HATU (557 mg, 1.47 mmol,
1.2 equiv.). The round bottom was capped with a septum and placed under a bal-
loon of Ar and the reaction was stirred for 18 h. Upon completion of the reaction,
the solution was transferred to a separatory funnel, diluted with CH2Cl2 (10 mL),
and washed with 10% citric acid (25 mL, aqueous). The organic layer was sepa-
rated and the aqueous layer extracted with CH2Cl2 (25 mL). The combined or-
ganics were washed with brine (50 mL, saturated aqueous), dried over Na2SO4,
filtered, and concentrated in vacuo to yield a pale yellow solid. The crude product
was purified via automatic reverse phase flash chromatography with a gradient of
10% to 100% MeCN/H2O with a 0.1% formic acid buffer solution, resulting in
874 mg P12 as a white solid (86% yield).
Representative Synthesis of Peptide P26 (Procedure #2). See Figure SI-
2. The same procedure as shown in Peptide Coupling #1 was followed, instead
using methylamine hydrochloride (1.0 equiv.) as the amine source with Boc-Val-
OH as the carboxylic acid. These couplings were performed on 2.5 mmol scale
(wrt S2).
Synthesis of Peptide P30 (Procedure #3). See Figure SI-3. The same pro-
cedure as shown in Peptide Coupling #3 was followed, instead using dimethyla-
mine hydrochloride (1.1 equiv.) as the amine source and Fmoc-pThr(Bn)-OH
as the carboxylic acid. This coupling was performed on 1.00 mmol scale (wrt
S6).
further analysis or purification. To the
8 mL scintillation vial containing
Et2NH2•H-pThr(Bn)-DPro-Val-Leu-OMe was added relevant carboxylic acid
(0.288 mmol, 1.2 equiv.), followed by CH2Cl2 (2.0 mL). The solution was cooled
to –10 °C using a brine/ice bath. To the mixture was added NMM (66 μL, 0.600
mmol, 2.5 equiv.), followed by HATU (118 mg, 0.311 mmol, 1.3 equiv.). The vial
was flushed with Ar, capped with a teflon cap, and the reaction was stirred for 18
h. Upon completion of the reaction, the solution was transferred to a separatory
funnel, diluted with CH2Cl2 (5.0 mL), and washed with 10% citric acid (15 mL,
aqueous). The organic layer was separated and the aqueous layer extracted with
CH2Cl2 (15 mL). The combined organics were washed with brine (30 mL, satu-
rated aqueous), dried over Na2SO4, filtered, and concentrated in vacuo to yield a
pale yellow or white solid. The crude product was purified via automatic reverse
phase flash chromatography with a gradient of 10% to 100% MeCN/H2O with a
0.1% formic acid buffer solution, resulting in product.
Fmoc-allo-pThr(Bn)-OH (for P15) and Cbz-pThr(Bn)-OH (for P21)
were synthesized according to literature precedent.54
Representative Synthesis of Peptide P32 by Solid Phase Synthesis (for
P32–P34, General Procedure #5). Peptide Coupling #1: A 20 mL solid phase
synthesis tube was charged with HCl•Ser(tBu)-2-Cl-Trt-resin (1.1 meq/g, 1.00
g, 1.10 mmol, 1.0 equiv.) and 20 mL CH2Cl2. The vessel was rotated for 30 min
to swell the resin and drained upon completion. Simultaneously, a 20 mL vial was
charged with Fmoc-Val-OH (1.12 g, 3.30 mmol, 3.0 equiv.), HCTU (2.28 g, 5.50
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Acylation of N-deprotected Phosphopeptides (Procedure #7). See Fig-
1-(tert-butyl) 2-methyl (2R,4R)-4-(3-phenylureido)pyrrolidine-1,2-dicarbox-
ure SI-6B. To an oven dried 8 mL scintillation vial was added Fmoc-pThr(Bn)-
DPro-Val-Leu-OMe (P12, 200 mg, 0.240 mmol, 1.0 equiv.), followed by 1.2 mL
of a 1:1 solution of diethylamine and CH2Cl2. The solution was stirred for 1 h and
then concentrated in vacuo. The crude white solid was dried under vacuum over-
night for 24 h and was used in the next step without further analysis or purifica-
ylate (cis-12) was synthesized according to literature precedent.61 A flame dried
100 mL round bottom flask was charged with cis-11 (550 mg, 2.00 mmol, 1.00
equiv.), followed by 10 mL MeOH. To this solution was added Pd/C (60 mg).
The vessel was fitted with a septum and placed under a balloon of H2. The reac-
1
2
3
4
5
6
7
8
tion was stirred at 40 °C until complete. The reaction was passed through a plug
of celite and concentrated in vacuo. The crude product was utilized in the next
step without further purification assuming quantitative yield. To a 100 mL round
bottom flask containing trans-12 (489 mg, 2.00 mmol, 1.0 equiv.) was added mL
CH2Cl2. The vessel was fit with a septum and placed under an atmosphere of Ar.
tion. To the 8 mL scintillation vial containing Et2NH2•H-pThr(Bn)-DPro-Val-
Leu-OMe was added CH2Cl2 (2.0 mL). The solution was cooled to –10 °C using
a brine/ice bath, followed by addition of acetic anhydride (340 µL, 0.360 mmol,
1.50 equiv.). The vial was flushed with Ar, capped with a teflon cap, and the reac-
tion was stirred for 18 h. Upon completion of the reaction, the solution was trans-
ferred to a separatory funnel, diluted with CH2Cl2 (5.0 mL), and washed with
10% citric acid (15 mL, aqueous). The organic layer was separated and the aque-
ous layer extracted with CH2Cl2 (15 mL). The combined organics were washed
with brine (30 mL, saturated aqueous), dried over Na2SO4, filtered, and concen-
trated in vacuo to yield a pale yellow or white solid. The crude product was puri-
fied via automatic reverse phase flash chromatography with a gradient of 10% to
100% MeCN/H2O with a 0.1% formic acid buffer solution, resulting in product.
Synthesis of 4-Amidoproline Substituted Peptides (Procedures #8 and
#9). See Figure SI-7. 1-(tert-butyl) 2-methyl (2R,4S)-4-azidopyrrolidine-1,2-di-
carboxylate (trans-11) and 1-(tert-butyl) 2-methyl (2R,4R)-4-azidopyrrolidine-
1,2-dicarboxylate (cis-11) were synthesized according to literature precedent.55,56
1-(tert-butyl) 2-methyl (2R,4S)-4-(3-phenylureido)pyrrolidine-1,2-dicarboxylate
(trans-12) was synthesized according to literature precedent.61 A 100 mL round
bottom flask was charged with trans-11 (550 mg, 2.00 mmol, 1.00 equiv.), fol-
lowed by 10 mL MeOH. To this solution was added Pd/C (60 mg). The vessel
was fitted with a septum and placed under a balloon of H2. The reaction was
Phenyl isocyanate (326 µL, 3.00 mmol, 1.50 equiv.) was then added slowly, and
the resulting solution was stirred overnight for 18 h. Upon completion of the re-
action, the solution was washed with 10% citric acid (60 mL, aqueous), followed
by brine (60 mL, saturated, aqueous). The organics were dried over Na2SO4, fil-
tered, and concentrated in vacuo. The resulting crude material was first purified
by automatic normal phase flash chromatography eluting with a gradient of 25%
to 100% EtOAc/Hex. Additional purification using automatic reverse phase flash
chromatography, eluting with a gradient of 25% to 100% MeCN/H2O was re-
quired, and yielded 430 mg white solid (59 % yield). 1H NMR (600 MHz,
CDCl3): δ 7.36–7.28 (comp, 4H), 7.05 (t, J = 6.7 Hz, 1H), 6.64 (d, J = 34.4 Hz,
1H), 5.82 (d, J = 69.1 Hz, 1H), 4.57 (s, 1H), 4.31 (ddd, J = 49.6, 9.7, 2.7 Hz, 1H),
3.80 3.57 (comp, 4H), 3.51 (dd, J = 46.8, 11.5 Hz, 1H), 2.49 (dddd, J = 24.8,
13.8, 9.8, 6.1 Hz, 1H), 1.99 (dd, J = 30.2, 13.8 Hz, 1H), 1.43 (s, 9H); 13C NMR
(151 MHz, CDCl3, mixture of rotamers): δ 175.4, 174.9, 155.0, 154.5, 153.8,
138.6, 129.3, 129.1, 123.7, 123.7, 120.6, 120.3, 80.8, 58.0, 57.8, 53.9, 53.1, 52.8,
52.5, 49.9, 48.9, 37.2, 36.0, 28.5, 28.4; HRMS (ESI-QToF) m/z: [M+H]+ Calcd
for C18H26N3O5 364.1872; Found 364.1867; IR (cm-1, neat): 2978, 1646, 1598,
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1468, 1393, 1247, 1158, 1129, 1001, 899, 854, 753, 693. [a]D20 14.6° (c = 0.010
g/mL, MeOH); TLC: Rƒ (1:2 Hexanes/EtOAc) 0.46, visualized with UV light.
(2R,4R)-1-(tert-butoxycarbonyl)-4-(3-phenylureido)pyrrolidine-2-carboxylic
acid (cis-13) was synthesized according to literature precedent.16 A flame dried
20 mL scintillation vial was charged with cis-12 (150 mg, 0.413 mmol, 1.0 equiv.),
4.0 mL THF, and 1.5 mL H2O. The vial was cooled to 0 °C and LiOH (35.0 mg,
0.826 mmol, 2.0 equiv.) was added. The reaction was warmed to RT and allowed
to stir for 2.5 h. Upon completion of the reaction, the solution was washed with
10% citric acid (20 mL, aqueous). The aqueous layer was extracted with CH2Cl2
(30 mL x 2). The combined organics were dried over Na2SO4, filtered, and con-
centrated in vacuo. The crude material was partially purified by automatic reverse
stirred at 40 °C for 12 h. Upon completion, the reaction was passed through a
plug of celite and concentrated in vacuo. The crude product was utilized in the
next step without further purification assuming quantitative yield. To a 100 mL
round bottom flask containing trans-12 (489 mg, 2.00 mmol, 1.0 equiv.) was
added 15 mL THF. The vessel was fitted with a septum and placed under an Ar
balloon. Phenyl isocyanate (240 μL, 2.20 mmol, 1.1 equiv.) was next added, and
the reaction was stirred at RT for 12 h. Upon completion, the solution was
washed with 10% citric acid (20 mL, aqueous) and brine (20 mL, saturated, aque-
ous). The organics were dried over Na2SO4, filtered, concentrated in vacuo to
yield crude product. The product was purified via automatic normal phase flash
chromatography, eluting with a gradient of 30% to 100% EtOAc/Hex, followed
by automatic normal phase flash chromatography, eluting with a gradient of 20%
to 100% MeCN/H2O to yield 253 mg pure, white solid (35% yield, 2 steps). 1H
NMR (500 MHz, CDCl3): δ 8.31 (d, J = 14.0 Hz, 1H), 7.33 (d, J = 7.4 Hz, 2H),
7.23–7.14 (m, 2H), 6.86 (t, J = 7.4 Hz, 1H), 6.46 (dd, J = 10.3, 6.9 Hz, 1H), 4.28–
4.13 (m, 2H), 3.64 (s, 3H), 3.60–3.50 (m, 1H), 3.22–3.10 (m, 1H), 2.49–2.44
(m, 2H), 2.23–2.00 (m, 2H), 1.34 (s, 9H); 13C NMR (151 MHz, d6-DMSO): δ
173.0, 172.5, 154.8, 153.5, 152.8, 140.1, 128.7, 121.3, 117.7, 79.2, 57.5, 57.2, 52.0,
51.6, 48.2, 47.66, 36.01, 34.97, 28.04, 27.87. HRMS (ESI-QToF) m/z [M+H]+
Calcd for C18H26N3O5 364.1872; Found364.1855. IR: (cm-1, neat): 3330, 2979,
1746, 1698, 1648, 1598, 1548, 1499, 1394, 1313, 1238, 1203, 1177, 1154, 1125,
phase flash chromatography, eluting with
a gradient of 25% to 100%
MeCN/H2O, to reveal 127 mg of mostly pure white solid that was taken onto the
next step without further purification (88% yield). 1H NMR (400 MHz,
CD3OD): δ 7.38–7.29 (m, 2H), 7.20 (t, J = 7.8 Hz, 2H), 6.93 (t, J = 7.3 Hz, 1H),
4.38–4.25 (m, 1H), 4.23–4.14 (m, 1H), 3.77–3.67 (m, 1H), 3.30–3.27 (m, 1H),
2.61–2.42 (m, 1H), 2.03–1.89 (m, 1H), 1.42 (s, 9H); HRMS (ESI-QToF) m/z:
[M+H]+ Calcd for C17H24N3O5 350.1716; Found 350.1706.
Synthesis of pThr-Embedded Peptides. Fmoc-pThr(Bn)-DPro-Acpc-Met-
OMe (P1). Synthesized using General Procedure #1 on 0.600 mmol scale wrt
Fmoc-pThr(Bn)-OH. Yield: 502 mg white solid (63% yield); 31P NMR (162
MHz, CDCl3)
41H50N4O11PS 837.2934; Found 837.2916.
Fmoc-pThr(Bn)-DPro-Tle-Leu-OMe (P2). Synthesized using General Proce-
δ
–0.20; MS (ESI-QToF) m/z: [M+H]+ Calcd for
20
890, 854, 750; [a]D 23.1° (c = 0.0060 g/mL, MeOH); TLC: Rƒ (1:2 Hexa-
nes/EtOAc) 0.42, visualized with UV light.
C
(2R,4S)-1-(tert-butoxycarbonyl)-4-(3-phenylureido)pyrrolidine-2-carboxylic
acid (trans-13) was synthesized according to literature precedent.16 A flame dried
20 mL scintillation vial was charged with trans-12 (140 mg, 0.385 mmol, 1.0
dure #1 on 0.606 mmol scale wrt Fmoc-pThr(Bn)-OH. Yield: 221 mg white solid
(43% yield); 31P NMR (162 MHz, CDCl3): δ –1.75; HRMS (ESI-QToF) m/z:
[M+H]+ Calcd for C44H58N4O11P 849.3840; Found 849.3821.
equiv.), 4.0 mL THF, and 1.5 mL H2O. The vial was cooled to 0 °C and LiOH
(35.0 mg, 0.826 mmol, 2.0 equiv.) was added. The reaction was warmed to RT
and allowed to stir for 2h. Upon completion of the reaction, the solution was
washed with 10% citric acid (20 mL, aqueous). The aqueous layer was extracted
with CH2Cl2 (30 mL x 2). The combined organics were dried over Na2SO4, fil-
tered, and concentrated in vacuo to reveal 135 mg pure, white solid without need
for further purification (<99% yield). 1H NMR (400 MHz, CD3OD): δ 7.31 (d, J
= 7.8 Hz, 2H), 7.22 (t, J = 7.8 Hz, 2H), 6.95 (t, J = 7.3 Hz, 1H), 4.42–4.24 (comp,
2H), 3.77–3.68 (m, 1H), 3.36–3.31 (m, 1H), 2.31–2.18 (m, 2H), 1.44 (s, 9H);
13C NMR (151 MHz, d6-DMSO, mixture of rotamers): δ 174.0, 173.5, 171.3,
154.9, 154.8, 154.7, 153.5, 153.1, 140.2, 140.1, 128.7, 121.3, 117.7, 117.6, 79.1,
57.6, 57.3, 52.0, 51.6, 48.2, 48.1, 47.6, 47.6, 47.6, 47.5, 36.2, 36.1, 35.2, 35.1, 28.1,
28.0; HRMS (ESI-QToF) m/z: [M+H]+ Calcd for C17H24N3O5 350.1716;
Found 350.1703; IR (cm-1, neat): 2970, 1733, 1649, 1622, 1472, 1427, 1366,
Fmoc-pThr(Bn)-DPro-Trp-Leu-OMe (P3). Synthesized using General Proce-
dure #1 on 0.780 mmol scale wrt Fmoc-pThr(Bn)-OH. Yield: 296 mg white solid
(51% yield); 31P NMR (162 MHz, CDCl3): δ –1.43; HRMS (ESI-QToF) m/z:
[M+H]+ Calcd for C49H57N5O11P 922.3792; Found 922.3770.
Fmoc-pThr(Bn)-DPro-Thr(Me)-Leu-OMe (P4). Synthesized using General
Procedure #1 on 0.648 mmol scale wrt Fmoc-pThr(Bn)-OH. Yield: 228 mg
white solid (38% yield); 31P NMR (202 MHz, CDCl3): δ –1.13; HRMS (ESI-
QToF) m/z: [M+H]+ Calcd for C42H54N4O12P 837.3476; Found 837.3466.
Fmoc-pThr(Bn)-DPro-Chg-Leu-OMe (P5). Synthesized using General Proce-
dure #1 on 0.648 mmol scale wrt Fmoc-pThr(Bn)-OH. Yield: 407 mg white solid
(71% yield); 31P NMR (162 MHz, CDCl3): δ –1.32; HRMS (ESI-QToF) m/z:
[M+H]+ Calcd for C46H60N4O11P 875.3996; Found 875.3979.
Fmoc-pThr(Bn)-DPro-Cha-Leu-OMe (P6). Synthesized using General Proce-
dure #1 on 0.648 mmol scale wrt Fmoc-pThr(Bn)-OH. Yield: 379 mg white solid
(66% yield); 31P NMR (162 MHz, CDCl3): δ –0.70; HRMS (ESI-QToF) m/z:
[M+H]+ Calcd for C47H62N4O11P 889.4153; Found 889.4127.
1313, 1203, 1160, 1142, 1002, 909, 894, 854, 801, 764, 740, 696. [a]D20 13.8° (c
= 0.0098 g/mL, MeOH); TLC: Rƒ (9:1 CH2Cl2/MeOH) 0.13, visualized with
UV light.
Fmoc-pThr(Bn)-DPro-Phg-Leu-OMe (P7). Synthesized using General Proce-
dure #1 on 0.648 mmol scale wrt Fmoc-pThr(Bn)-OH. Yield: 347 mg white solid
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The Journal of Organic Chemistry
(62% yield); 31P NMR (202 MHz, CDCl3): δ –0.40; HRMS (ESI-QToF) m/z:
(84% yield); 31P NMR (162 MHz, CDCl3): δ –1.05; HRMS (ESI-QToF) m/z:
[M+H]+ Calcd for C36H52N4O11P 747.3370; Found 747.3365.
[M+H]+ Calcd for C46H54N4O11P 869.3527; Found 869.3510.
1
2
3
4
5
6
7
8
Fmoc-pThr(Bn)-DPro-Phe-Leu-OMe (P8). Synthesized using General Proce-
dure #1 on 0.648 mmol scale wrt Fmoc-pThr(Bn)-OH. Yield: 210 mg white solid
(37% yield); 31P NMR (162 MHz, CDCl3): δ –1.30; HRMS (ESI-QToF) m/z:
[M+H]+ Calcd for C47H56N4O11P 883.3683; Found 883.3661.
Furanoyl-pThr(Bn)-DPro-Val-Leu-OMe (P25). Synthesized using General
Procedures #1 and #6 on 0.400 mmol scale wrt P11. Yield: 136 mg white solid
(48% yield); 1H NMR (600 MHz, Benzene-d6): δ 8.70 (bs, 1H), 7.82 (bd, J = 8.6
Hz, 1H), 7.49 (s, 1H), 7.31–7.27 (m, 2H), 7.19 (d, J = 3.5 Hz, 1H), 7.11–7.06
(m, 2H), 7.06–7.00 (m, 2H), 5.93 (dd, J = 3.5, 1.7 Hz, 1H), 5.15–5.05 (comp,
3H), 4.90 (td, J = 8.6, 5.8 Hz, 1H), 4.82 (t, J = 6.1 Hz, 1H), 4.68 (d, J = 8.3 Hz,
1H), 4.53 (t, J = 8.5 Hz, 1H), 3.89 (bt, J = 7.7 Hz, 1H), 3.30 (s, 3H), 3.20–3.12
(m, 1H), 2.94 (bs, 1H), 2.44–2.37 (m, 1H), 2.17 (dt, J = 13.8, 4.8 Hz, 1H), 1.99
(q, J = 10.5, 9.7 Hz, 1H), 1.87–1.80 (m, 1H), 1.80–1.73 (m, 0H), 1.73–1.64 (m,
2H), 1.50 (ddt, J = 12.3, 9.2, 5.2 Hz, 1H), 1.33 (d, J = 6.2 Hz, 3H), 1.06 (d, J = 6.7
Hz, 3H), 0.96 (d, J = 6.7 Hz, 3H), 0.93 (d, J = 6.6 Hz, 3H), 0.89 (d, J = 6.6 Hz,
3H). 31P NMR (162 MHz, CDCl3): δ –0.58; HRMS (ESI-QToF) m/z: [M+H]+
Calcd for C33H48N4O11P 707.3057; Found 707.3046.
Fmoc-pThr(Bn)-DPro-Ala-Leu-OMe (P9). Synthesized using General Proce-
dure #1 on 0.648 mmol scale wrt Fmoc-pThr(Bn)-OH. Yield: 309 mg white solid
(59% yield); 31P NMR (162 MHz, CDCl3): δ –1.25; HRMS (ESI-QToF) m/z:
[M+H]+ Calcd for C41H52N4O11P 807.3370; Found 807.3362.
Fmoc-pThr(Bn)-DPro-Ile-Leu-OMe (P10). Synthesized using General Proce-
dure #1 on 0.648 mmol scale wrt Fmoc-pThr(Bn)-OH. Yield: 424 mg white solid
(77% yield); 31P NMR (162 MHz, CDCl3): δ –0.33; HRMS (ESI-QToF) m/z:
[M+H]+ Calcd for C44H58N4O11P 849.3840; Found 849.3827.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Fmoc-pThr(Bn)-DPro-Leu-Leu-OMe (P11). Synthesized using General Proce-
dure #1 on 0.648 mmol scale wrt Fmoc-pThr(Bn)-OH. Yield: 406 mg white solid
(56% yield); 31P NMR (162 MHz, CDCl3): δ –0.68; HRMS (ESI-QToF) m/z:
[M+H]+ Calcd for C44H58N4O11P 849.3840; Found 849.3829.
Fmoc-pThr(Bn)-DPro-Val-NHMe (P26). Synthesized using General Proce-
dures #1 and #2 on 1.47 mmol scale wrt Fmoc-pThr(Bn)-OH. Yield: 997 mg
white solid (94% yield); 31P NMR (162 MHz, CDCl3): δ –1.16; HRMS (ESI-
QToF) m/z: [M+H]+ Calcd for C37H46N4O9P 721.3002; Found 721.2991.
Fmoc-pThr(Bn)-DPro-Val-OMe (P27). Synthesized using General Procedure
#1 on 2.77 mmol scale wrt Fmoc-pThr(Bn)-OH. Yield: 1.63 g white solid (82%
yield); 31P NMR (162 MHz, CDCl3): δ –0.86; HRMS (ESI-QToF) m/z:
[M+H]+ Calcd for C37H45N3O10P 722.2843; Found 722.2836.
Fmoc-pThr(Bn)-DPro-Val-Leu-OMe (P12). Synthesized using General Proce-
dure #1 on 1.22 mmol scale wrt Fmoc-pThr(Bn)-OH. Yield: 874 mg white solid
(86% yield); 31P NMR (162 MHz, CDCl3): δ –0.79; HRMS (ESI-QToF) m/z:
[M+H]+ Calcd for C43H56N4O11P 835.3683; Found 835.3671.
Fmoc-pThr(Bn)-DPro-DTle-Leu-OMe (P13). Synthesized using General Proce-
dure #1 on 0.648 mmol scale wrt Fmoc-pThr(Bn)-OH. Yield: 223 mg white solid
(41% yield); 31P NMR (162 MHz, CDCl3): δ –1.44; HRMS (ESI-QToF) m/z:
[M+H]+ Calcd for C44H58N4O11P 849.3840; Found 849.3833.
Fmoc-pThr(Bn)-DPro-NHMe (P28). Synthesized using General Procedures
#1 and #2 on 2.16 mmol scale wrt Fmoc-pThr(Bn)-OH. Yield: 1.07 g white solid
(80% yield); 31P NMR (162 MHz, CDCl3): δ –0.41; HRMS (ESI-QToF) m/z:
[M+H]+ Calcd for C32H37N3O8P 622.2318; Found 622.2316.
Fmoc-pThr(Bn)-Pro-DTle-Leu-OMe (P14). Synthesized using General Proce-
dure #1 on 0.648 mmol scale wrt Fmoc-pThr(Bn)-OH. Yield: 378 mg white solid
(69% yield); 31P NMR (162 MHz, CDCl3): δ –2.66; HRMS (ESI-QToF) m/z:
[M+H]+ Calcd for C44H58N4O11P 849.3840; Found 849.3826.
Fmoc-pThr(Bn)-DPro-OMe (P29). Synthesized using General Procedure #1
on 0.250 mmol scale wrt Fmoc-pThr(Bn)-OH (32% yield); 31P NMR (202 MHz,
CDCl3): δ –0.06; HRMS (ESI-QToF) m/z: [M+H]+ Calcd for C32H36N2O9P
623.2158; Found 623.2150.
Fmoc-allo-pThr(Bn)-DPro-Val-Leu-OMe (P15). Synthesized using General
Procedures #1 and #4 on 0.293 mmol scale wrt Fmoc-allo-pThr(Bn)-OH. Yield:
210 mg white solid (86% yield); 31P NMR (162 MHz, CDCl3): δ –0.74; HRMS
Fmoc-pThr(Bn)-NHMe (P30). Synthesized using General Procedures #3 on
1.00 mmol scale wrt Fmoc-pThr(Bn)-OH. Yield: 353 mg white solid (66%
yield); 31P NMR (162 MHz, CDCl3): δ –1.51; HRMS (ESI-QToF) m/z:
[M+H]+ Calcd for C28H32N2O7P 539.1947; Found 539.1945.
(ESI-QToF) m/z: [M+H]+ Calcd for C43H56N4O11
P 835.3683; Found
835.3671.
Fmoc-DpThr(Bn)-DPro-Val-Leu-OMe (P16). Synthesized using General Pro-
cedure #1 on 0.196 mmol scale wrt Fmoc-DpThr(Bn)-OH. Yield: 108 mg white
solid (66% yield); 31P NMR (162 MHz, CDCl3): δ –2.01; HRMS (ESI-QToF)
m/z: [M+H]+ Calcd for C43H56N4O11P 835.3683; Found 835.3678.
Fmoc-pSer(Bn)-DPro-Val-Leu-OMe (P17). Synthesized using General Proce-
dure #1 on 0.606 mmol scale wrt Fmoc-pSer(Bn)-OH. Yield: not reported; 31P
NMR (162 MHz, CDCl3): δ –1.59; HRMS (ESI-QToF) m/z: [M+H]+ Calcd for
C42H54N4O11P 821.3527; Found 821.3520.
p-MeO-Bz-pThr(Bn)-DPro-Val-OMe (P31). Synthesized using General Proce-
dures #1 and #6 on 0.240 mmol scale wrt P25. Yield: 62.6 mg white solid (41%
yield); 31P NMR (162 MHz, CDCl3): δ –0.37; HRMS (ESI-QToF) m/z:
[M+H]+ Calcd for C30H41N3O10P 634.2530; Found 634.2526.
p-MeO-Bz-pThr(Bn)-DPro-Val-Ser(tBu)-OMe (P32). Synthesized using Gen-
eral Procedures #1 and #6 on 0.173 mmol scale wrt Fmoc-pThr(Bn)-DPro-Val-
Ser(tBu)-OMe. Yield: 440 mg white solid (69% yield); 31P NMR (162 MHz,
CDCl3): δ –0.27; HRMS (ESI-QToF) m/z: [M+H]+ Calcd for C37H54N4O12
P
Fmoc-pThr(Bn)-DPro(4-trans-Ph-urea)-Val-Leu-OMe (P18). Synthesized us-
ing General Procedures #1 and #8 on 0.614 mmol scale wrt Fmoc-pThr(Bn)-OH.
Yield: 440 mg white solid (73% yield); 31P NMR (162 MHz, CDCl3): δ –1.81;
HRMS (ESI-QToF) m/z: [M+H]+ Calcd for C51H64N6O12P 983.4320; Found
983.4301.
777.3476; Found 777.3488.
Fmoc-pThr(Bn)-Pro-DLys(Boc)-DPro-Tyr(tBu)-OMe (P33). Synthesized using
General Procedure #5 on 0.433 mmol scale wrt Fmoc-pThr(Bn)-OH. Yield: 339
mg white solid (67% yield); 31P NMR (162 MHz, CDCl3): δ –2.26; HRMS (ESI-
QToF) m/z: [M+H]+ Calcd for C61H80N6O15P 1167.5419; Found 1167.5402.
Fmoc-pThr(Bn)-Pro-DLys(Boc)-DPro-Phe-OMe (P34). Synthesized using Gen-
eral Procedure #5 on 0.621 mmol scale wrt Fmoc-pThr(Bn)-OH. Yield: 429 mg
white solid (57% yield); 31P NMR (162 MHz, CDCl3): δ –2.13; HRMS (ESI-
QToF) m/z: [M+H]+ Calcd for C57H72N6O14P 1095.4844; Found 1095.4833.
Synthesis of 3-Amidocyclohexanone Substrates (4). See Figure SI-9. Tert-
butyl (3-oxocyclohexyl)carbamate (14) was synthesized according to literature
precedent.56 A flame dried 100 mL round bottom flask was charged with tert-bu-
tyl carbamate (11.7 g, 100 mmol, 1.0 equiv.) and bismuth nitrate pentahydrate
(9.70 g, 20.0 mmol, 0.20 equiv.), followed by addition of cyclohexenone (9.68
mL, 100 mmol, 1.0 equiv.). The neat solution was stirred vigorously until for-
mation of a pale yellow solid inhibited stirring. The crude solid was suspended in
CH2Cl2 and partially purified by flash chromatography, eluting with a gradient of
2:1 to 1:4 Hex/EtOAc, revealing 14.5 g of pale yellow, mostly pure solid (68%
yield), which was used in the next step without further purification. 1H NMR
(400 MHz, CDCl3): δ 4.68–4.31 (m, 1H), 3.86 (s, 1H), 2.63 (ddt, J = 14.1, 4.6,
1.6 Hz, 1H), 2.32 (dddd, J = 13.6, 6.7, 3.0, 1.4 Hz, 1H), 2.25–2.15 (m, 2H), 2.07–
1.87 (m, 2H), 1.73–1.47 (m, 2H), 1.39 (m, 9H); TLC: Rƒ (1:1 Hex/EtOAc)
0.47, visualized using KMNO4 stain.
Fmoc-pThr(Bn)-DPro(4-cis-Ph-urea)-Val-Leu-OMe (P19). Synthesized using
General Procedures #1 and #9 on 0.244 mmol scale wrt Fmoc-pThr(Bn)-OH.
Yield: 188 mg white solid (78% yield); 31P NMR (202 MHz, CDCl3): δ –0.60;
HRMS (ESI-QToF) m/z: [M+H]+ Calcd for C51H64N6O12P 983.4320; Found
983.4320.
Ac-pThr(Bn)-DPro-Val-Leu-OMe (P20). Synthesized using General Proce-
dures #1 and #7 on 0.240 mmol scale wrt P11. Yield: 56.5 mg white solid (36%
yield); 31P NMR (202 MHz, CDCl3): δ –1.68; HRMS (ESI-QToF) m/z:
[M+H]+ Calcd for C30H48N4O10P 655.3108; Found 655.3109.
Cbz-pThr(Bn)-DPro-Val-Leu-OMe (P21). Synthesized using General Proce-
dures #1 and #6 on 0.228 mmol scale wrt P11. Yield not reported; 31P NMR (202
MHz, CDCl3): δ –1.84; HRMS (ESI-QToF) m/z: [M+H]+ Calcd for
C36H52N4O11P 747.3370; Found 747.3364.
p-O2N-Bz-pThr(Bn)-DPro-Val-Leu-OMe (P22). Synthesized using General
Procedures #1 and #6 on 0.400 mmol scale wrt P11. Yield: 72.2 mg white solid
(39% yield); 31P NMR (162 MHz, CDCl3) δ –0.54; HRMS (ESI-QToF) m/z:
[M+H]+ Calcd for C35H49N5O12P 762.3115; Found 762.3118.
Bz-pThr(Bn)-DPro-Val-Leu-OMe (P23). Synthesized using General Proce-
dures #1 and #6 on 0.400 mmol scale wrt P11. Yield: 143 mg white solid (50%
yield); 31P NMR (162 MHz, CDCl3): δ 0.01; HRMS (ESI-QToF) m/z: [M+H]+
Calcd for C35H50N4O10P 717.3265; Found 717.3254.
Variation of N-Protecting Group of 3-Amidocylohexanones (General
Procedure #10). See Figure SI-10. To a 250 mL round bottom flask containing
14 (1.0 equiv.) was added CH2Cl2 (1.0 mL/mmol 14), followed by trifluoroace-
tic acid (1.0 mL/mmol 14). The flask was fitted with a septum pierced with a
cannula leading to a solution of NaHCO3 (saturated, aqueous). The solution was
allowed to stir for 1 h. Upon completion of the deprotection, N2 was blown into
p-MeO-Bz-pThr(Bn)-DPro-Val-Leu-OMe (P24). Synthesized using General
Procedures #1 and #6 on 0.240 mmol scale wrt P11. Yield: 150 mg white solid
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the solution via a needle inlet. After 1 h, the remaining solution was concentrated
in vacuo, using a base trap connected to the round bottom filled with KOH to
neutralize any volatile TFA. After drying under reduced pressure for a few hours,
mmol, 1.0 equiv.), p-anisidine (62.0 mg, 0.500 mmol, 1.25 equiv.), Hantzsch es-
ter (152 mg, 0.600 mmol, 1.5 equiv.), diphenyl phosphate (50.0 mg, 0.200 mmol,
0.50 equiv.), and calcium sulfate (4.0 g, 100 g/mmol 2), followed by 10.0 mL sol-
vent (see below). The vial’s headspace was flushed with Ar, capped with a Teflon
1
2
3
4
5
6
7
8
9
the vessel was charged with CH2Cl2 (6.0 mL/mmol 14) and cooled to 0 °C with
an ice bath. Electrophile (1.2 equiv.) was then added and the flask was fitted with
a septum and pierced with an argon balloon. After this, Hünig’s base (2.4 equiv.)
was added, and the reaction was allowed to stir overnight, slowly warming to rt.
Upon completion of the reaction, the solution was transferred to a separatory fun-
nel and washed with NaHCO3 (6.0 mL/mmol 14, saturated, aqueous) and 10%
citric acid (6.0 mL/mmol 14, aqueous), dried over Na2SO4, filtered, and concen-
trated in vacuo. The crude material was purified via flash chromatography.
N-(3-oxocyclohexyl)-2-phenylacetamide (4a). General Procedure #8 was used
on 7.00 mmol scale (wrt 14). Phenylacetyl chloride (1.12 mL, 8.44 mmol, 1.2
equiv.) was used as the electrophile. The crude material was purified via flash
chromatography, using a gradient eluent of 2:1 to 1:1 Hex/EtOAc, yielding 625
mg 4a as a white solid (38% yield). 1H NMR (400 MHz, CDCl3): δ 7.36–7.19
(comp, 5H), 5.37 (bd, J = 7.5 Hz, 1H), 4.19 (dddd, J = 13.1, 8.8, 6.9, 4.0 Hz, 1H),
3.53 (s, 3H), 2.65–2.56 (m, 1H), 2.37–2.28 (m, 1H), 2.23 – 2.09 (m, 2H), 2.04–
1.95 (m, 1H), 1.88–1.77 (m, 1H), 1.68 (dddd, J = 15.2, 13.7, 6.8, 3.5 Hz, 1H),
1.54 (dtd, J = 13.2, 9.5, 3.7 Hz, 1H); 13C NMR (151 MHz, CDCl3): δ 208.5,
170.2, 134.6, 129.3, 129.1, 127.5, 48.5, 47.4, 43.8, 40.8, 30.6, 22.0; HRMS (ESI-
QToF) m/z: [M+H]+ Calcd for C14H18NO2 232.1338; Found 232.1335; IR:
(cm-1, neat) 3255, 3080, 2934, 1714, 1635, 1557, 1495, 1454, 1421, 1345, 1267,
1221, 1174, 1032.4, 978, 903, 717; TLC: Rƒ (1:4 Hexanes/EtOAc) 0.32, visual-
ized with a KMnO4 stain; HPLC: Chiralpak IC column, 1.0 mL/min, 15%
EtOH/Hex, 254 nm and 210 nm, peaks observed at 16.2 and 22.8 min.
N-(3-oxocyclohexyl)benzamide (4b). General Procedure #8 was used on 14.0
mmol scale (wrt 14). Benzoyl chloride (2.20 mL, 16.8 mmol, 1.2 equiv.) was used
as the electrophile. The crude material was purified via flash chromatography, us-
ing a gradient eluent of 4:1 to 0:1 Hex/EtOAc, yielding 1.27 g of mostly pure 4b
as a beige solid. The mixture was further purified using automatic normal phase
flash chromatography, eluting with a gradient of 0 to 100% EtOAc/Hex to yield
992 mg 4b as a beige solid (33% yield). 1H NMR (400 MHz, CDCl3): δ 7.73–
7.68 (m, 2H), 7.52–7.42 (m, 1H), 7.38 (t, J = 7.5 Hz, 1H), 6.31 (d, J = 8.4 Hz,
1H), 4.46–4.34 (m, 1H), 2.75 (ddt, J = 14.1, 4.8, 1.6 Hz, 1H), 2.43–2.32 (m, 2H),
2.26 (dddd, J = 16.3, 8.9, 5.6, 1.4 Hz, 1H), 2.19–2.10 (m, 1H), 2.04–1.94 (m,
1H), 1.83–1.69 (m, 2H); 13C NMR (151 MHz, CDCl3): δ 208.8, 166.8, 134.2,
131.6, 128.6, 126.9, 49.0, 47.6, 40.9, 30.8, 22.2; HRMS (ESI-QToF) m/z:
[M+H]+ Calcd for C13H16NO2 218.1181; Found 218.1182; IR: (cm-1, neat):
3323, 2960, 2937, 1709, 1634, 1580, 1525, 1490, 1452, 1325, 1311, 1290, 1270,
1221, 1151, 1070, 1051, 1029, 802, 721; TLC: Rƒ (1:1 Hexanes/EtOAc) 0.2, vis-
ualized with a KMnO4 stain.
Benzyl (3-oxocyclohexyl)carbamate (4c). General Procedure #8 was used on
17.8 mmol scale (wrt 14). Benzyl chloroformate (3.0 mL, 21.4 mmol, 1.2 equiv.)
was used as the electrophile. The crude material was purified via flash chromatog-
raphy, using a gradient eluent of 4:1 to 1:1 Hex/EtOAc, yielding 3.33 g 4c as a
white solid (35% yield). 1H NMR (600 MHz, CDCl3): δ 7.32–7.23 (comp, 5H),
5.44 (bd, J = 8.5 Hz, 1H), 5.10 (s, 2.5H, major conformer), 5.02 (d, J = 2.3 Hz,
0.5H, minor conformer), 3.89 (s, 1H), 2.65–2.55 (m, 1H), 2.33–2.09 (comp,
3H), 2.05–1.95 (m, 1H), 1.94–1.85 (m, 1H), 1.65–1.47 (comp, 2H); 13C NMR
(151 MHz, CDCl3): δ 209.1, 157.1, 155.4, 136.4, 128.5, 128.5, 128.1, 128.1,
128.0, 66.7, 50.1, 47.8, 40.7, 30.9, 21.8; HRMS (ESI-QToF) m/z: [M+H]+
Calcd for C14H18NO3 248.1287; Found 248.1282; IR: (cm-1, neat): 3332, 2949,
1715, 1686, 1528, 1453, 1284, 1266, 1246, 1215, 1142, 1083, 1034, 1024, 971,
844, 764, 726; TLC: Rƒ (1:1 Hexanes/EtOAc) 0.52, visualized with a KMnO4
stain.
4-methyl-N-(3-oxocyclohexyl)benzenesulfonamide (4d). General Procedure #8
was used on 8.90 mmol scale (wrt 14). p-Toluenesulfonyl chloride (2.04 g, 10.7
mmol, 1.2 equiv.) was used as the electrophile. The crude material was purified
via flash chromatography, using a gradient eluent of 2:1 to 1:1 Hex/EtOAc, yield-
ing 821 mg 4d as a white solid (35% yield). 1H NMR (400 MHz, CDCl3): δ 7.75
(d, J = 8.3 Hz, 2H), 7.31 (d, J = 7.8 Hz, 2H), 4.90 (bd, J = 7.4 Hz, 1H), 3.59–3.49
(m, 1H), 2.50 (ddt, J = 14.2, 4.8, 1.7 Hz, 1H), 2.45 (s, 3H), 2.36–2.26 (m, 1H),
2.26–2.14 (comp, 2H), 2.06–1.90 (m, 2H), 1.70–1.54 (comp, 2H); 13C NMR
(151 MHz, CDCl3): δ 208.0, 143.7, 137.5, 129.9, 126.9, 52.4, 48.5, 40.6, 31.9,
21.7, 21.5; HRMS (ESI-QToF) m/z: [M+H]+ Calcd for C13H18NO3S 268.1007;
Found 268.0999; IR: (cm-1, neat): 3232, 2957, 1701, 1596, 1446, 1327, 1230,
1150, 1112, 1086, 1022, 1010, 906, 818, 694;; TLC: Rƒ (1:1 Hexanes/EtOAc)
0.42, visualized with a KMnO4 stain.
cap, and stirred for a designated amount of time at either RT or 40 °C. The crude
mixtures were next filtered through a fine fritted funnel to remove the CaSO4,
washing with additional CH2Cl2 and EtOAc. The solution was concentrated in
vacuo, redissolved in a minimal amount of CH2Cl2 and purified via automatic nor-
mal phase flash chromatography (specific conditions listed below). Further puri-
fication was required via preparative HPLC, using a Waters SymmetricPrep C8 7
μm (19 x 300 mm) column. Gradient conditions with H2O (0.1% formic acid)
and MeCN (0.1% formic acid) were utilized as follows: Held at 15% MeCN for
5 min, ramped to 20% MeCN for 5 min, ramped to 22% MeCN for 30 min,
ramped to 35% MeCN for 10 min, ramped to 95% MeCN for 5 min, ramped to
5% MeCN for 1 min, held at 5% MeCN for 5 min. The relevant fractions were
collected and concentrated in vacuo. The crude material was then redissolved in
20 mL CH2Cl2 and washed with NaHCO3 (20 mL, saturated, aqueous). The
aqueous layer was extracted with 30 mL CH2Cl2. The combined organics were
dried over Na2SO4, filtered, and concentrated in vacuo.
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Trans-3-((4-methoxyphenyl)amino)cyclohexyl)-2-phenylacetamide (trans-7a).
See Figure SI-11. General Procedure #10 was used, with CHCl3 as the solvent,
40 °C as the temperature, and for 96 h. Five individual reactions were performed
in tandem and combined prior to purification. The crude material was purified by
automatic normal phase flash chromatography, eluting with a gradient of 40% to
100% EtOAc/Hex, yielding 524 mg of a mixture of trans-7a, cis-7a, and 4a. The
three compounds were further separated by preparative HPLC to yield 79.9 mg
light brown solid (11% combined yield). 1H NMR (600 MHz, CDCl3): δ 7.33–
7.30 (comp, 2H), 7.29–7.23 (m, 1H), 7.21 (d, J = 7.1 Hz, 2H), 6.74 (d, J = 8.8
Hz, 2H), 6.52 (d, J = 8.8 Hz, 2H), 5.38 (bd, J = 8.3 Hz, 1H), 3.87 (tdt, J = 11.7,
8.1, 4.0 Hz, 1H), 3.72 (s, 3H), 3.51 (s, 2H), 3.22 (tt, J = 10.8, 3.8 Hz, 1H), 2.94
(s, 1H), 2.26 (bd, J = 12.5 Hz, 1H), 2.01 (d, J = 13.0 Hz, 1H), 1.89 (d, J = 11.2
Hz, 1H), 1.75 (dp, J = 14.7, 3.7 Hz, 1H), 1.44–1.34 (m, 1H), 1.01–0.92 (m, 2H),
0.83 (q, J = 11.4 Hz, 1H); 13C NMR: (151 MHz, CDCl3): δ 170.0, 152.2, 140.9,
134.9, 129.4, 129.0, 127.3, 115.0, 114.9, 55.8, 51.9, 47.3, 43.9, 39.7, 32.8, 32.4,
22.6; HRMS (ESI-QToF) m/z: [M+H]+ Calcd for C21H27N2O2 339.2073;
Found 339.2063; IR: (cm-1, neat): 3365, 3321, 2934, 2865, 1646, 1528, 1511,
1461, 1353, 1240, 1182, 1035, 964, 893, 819, 762, 709; Mp: 124–125 °C; TLC:
Rƒ (1:4 Hexanes/EtOAc) 0.60, visualized with UV light. HPLC: Chiralpak IC,
1.0 mL/min, 15% EtOH/Hex, 254 nm and 210 nm, peaks observed at 10.5 and
11.8 min.
Cis-3-((4-methoxyphenyl)amino)cyclohexyl)-2-phenylacetamide (cis-7a). See
Figure SI-11. General Procedure #10 was used, with CHCl3 as the solvent, 40 °C
as the temperature, and for 96 h. Five individual reactions were performed in tan-
dem and combined prior to purification. The crude material was purified by au-
tomatic normal phase flash chromatography, eluting with a gradient of 40% to
100% EtOAc/Hex, yielding 524 mg of a mixture of trans-7a, cis-7a, and 4a. The
three compounds were further separated by preparative HPLC to yield 49.8 mg
light brown solid (7% combined yield). 1H NMR (600 MHz, CDCl3): δ 7.36 (t,
J = 7.5 Hz, 2H), 7.30 (t, J = 7.4 Hz, 1H), 7.25 (d, J = 6.8 Hz, 2H), 6.73 (d, J = 8.8
Hz, 2H), 6.44 (d, J = 8.7 Hz, 2H), 5.50 (d, J = 7.8 Hz, 1H), 4.16–4.08 (m, 1H),
3.72 (s, 3H), 3.55 (s, 2H), 3.28 (s, 1H), 3.17 (s, 1H), 1.74–1.53 (m, 5H), 1.43–
1.29 (m, 3H); 13C NMR (151 MHz, CDCl3): δ 170.2, 152.0, 141.1, 135.1, 129.3,
129.1, 127.4, 114.9, 114.6, 55.8, 48.7, 44.8, 44.0, 37.0, 31.1, 31.0, 19.9; HRMS
(ESI-QToF) m/z: [M+H]+ Calcd for C21H27N2O2 339.2073; Found 339.2068;
IR: (cm-1, neat): 3212, 2931, 2856, 1635, 1541, 1513, 1456, 1352, 1230, 1175,
1033, 821, 726; Mp: 116–118 °C; TLC: Rƒ (1:4 Hexanes/EtOAc) 0.60, visual-
ized with UV light; HPLC: Chiralpak IC, 1.0 mL/min, 15% EtOH/Hex, 254 nm
and 210 nm, peaks observed at 14.5 and 19.9 min.
Trans-N-(3-((4-methoxyphenyl)amino)cyclohexyl)benzamide (trans-7b). See
Figure SI-12. General Procedure #10 was used, with PhMe as the solvent, at
room temperature, and for 144 h. The crude material was purified by automatic
normal phase flash chromatography, eluting with a gradient of 5% to 100%
EtOAc/Hex, yielding a mixture of trans-7b, cis-7b and 4b. The three compounds
were further separated by preparative HPLC to yield 25.0 mg beige solid (19%
yield). 1H NMR (400 MHz, CDCl3): δ 7.75 (d, J = 6.9 Hz, 2H), 7.53–7.46 (m,
1H), 7.43 (t, J = 7.5 Hz, 2H), 6.79–6.75 (m, 2H), 6.63–6.56 (m, 2H), 6.11 (bd, J
= 7.7 Hz, 1H), 4.41–4.31 (m, 1H), 3.73 (s, 3H), 3.67– 3.58 (m, 1H), 1.99–1.81
(comp, 3H), 1.75 (ddt, J = 13.6, 8.8, 3.9 Hz, 2H), 1.66–1.55 (m, 2H), 1.54–1.43
(m, 1H); 13C NMR (151 MHz, CDCl3): δ 167.0, 152.2, 141.2, 135.0, 131.5,
128.7, 127.0, 115.1, 114.9, 55.9, 49.2, 45.3, 37.3, 31.9, 30.7, 20.1; HRMS (ESI-
QToF) m/z: [M+H]+ Calcd for C20H25N2O2 325.1916; Found 325.1908; Mp:
Synthesis and Isolation of Racemic 1,3-Diamine Products 7 (General
Procedure #10). To a flame dried 20 mL scintillation vial was added 4 (0.400
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The Journal of Organic Chemistry
Cis-N-(3-((4-methoxyphenyl)amino)cyclohexyl)-4-methylbenzenesulfonamide
118–124 °C; TLC: Rƒ (1:2 Hexanes/EtOAc) 0.60, visualized with UV light;
HPLC: Chiralpak IC, 1.0 mL/min, 20% iPrOH/Hex, 254 nm, peaks observed at
20.4 and 32.7 min.
(cis-7d) with CH2Cl2 as the solvent, at room temperature, and for 96 h. See Fig-
ure SI-14. General Procedure #10 was used. The crude material was purified by
automatic normal phase flash chromatography, eluting with a gradient of 10% to
100% EtOAc/Hex, yielding a peak with trans-7d, and a second peak with cis-7d
and 4d. Both mixtures were further purified with preparative HPLC to yield 19.0
mg beige solid (13% yield). 1H NMR (600 MHz, CDCl3): δ 7.71 (d, J = 8.2 Hz,
2H), 7.26–7.23 (m, 2H), 6.73 (d, J = 8.9 Hz, 2H), 6.48 (d, J = 8.8 Hz, 2H), 4.82
(s, 1H), 3.73 (s, 3H), 3.22 (dtt, J = 11.1, 7.4, 3.4 Hz, 1H), 3.13–3.05 (m, 1H),
2.40 (s, 3H), 2.09 (d, J = 12.3 Hz, 1H), 1.96 (d, J = 13.3 Hz, 1H), 1.86–1.79 (m,
1H), 1.75 (dt, J = 14.0, 3.9 Hz, 1H), 1.33–1.20 (m, 1H), 1.17–1.07 (m, 1H), 0.98
(td, J = 12.4, 11.8, 8.7 Hz, 2H); 13C NMR (151 MHz, CDCl3): δ 152.7, 143.4,
140.5, 138.4, 129.8, 127.0, 115.5, 115.0, 55.9, 52.2, 51.8, 40.3, 33.7, 32.4, 22.4,
21.7; HRMS (ESI-QToF) m/z: [M+H]+ Calcd for C20H27N2O3S 375.1742;
Found 375.1736; IR: (cm-1, neat): 3372, 3292, 2962, 1511, 1423, 1319, 1290,
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Cis-N-(3-((4-methoxyphenyl)amino)cyclohexyl)benzamide (cis-7b). See Fig-
ure SI-12. General Procedure #10 was used, with PhMe as the solvent, at room
temperature, and for 144 h. The crude material was purified by automatic normal
phase flash chromatography, eluting with a gradient of 5% to 100% EtOAc/Hex,
yielding a mixture of trans-7b, cis-7b and 4b. The three compounds were further
separated by preparative HPLC to yield 14.8 mg beige solid (11% yield). 1H
NMR (600 MHz, Chloroform-d): δ 7.74–7.69 (m, 2H), 7.50–7.45 (m, 1H),
7.43–7.39 (m, 2H), 6.80–6.76 (m, 2H), 6.65–6.60 (m, 2H), 6.25 (s, 1H), 4.12
(dddt, J = 14.9, 11.3, 8.1, 3.8 Hz, 1H), 3.74 (s, 3H), 3.36 (ddt, J = 10.6, 7.7, 3.8
Hz, 1H), 2.45 (d, J = 11.8 Hz, 1H), 2.15–2.04 (m, 2H), 1.87 (dt, J = 13.9, 4.0 Hz,
1H), 1.51 (dddd, J = 15.9, 14.2, 7.9, 3.6 Hz, 1H), 1.30–1.21 (m, 1H), 1.21–1.10
(comp, 2H); 13C NMR (151 MHz, CDCl): δ 166.7, 152.7, 140.8, 134.9, 131.5,
128.7, 127.0, 115.6, 115.1, 55.9, 52.3, 47.8, 39.6, 33.0, 32.7, 22.6; HRMS (ESI-
QToF) m/z: [M+H]+ Calcd for C20H25N2O2 325.1916; Found 325.1913; Mp:
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1233, 1155, 109, 1038, 904, 812, 669; Mp: 130–134 °C; TLC: Rƒ (2:1 Hex-
anes/EtOAc) 0.16, visualized with UV light; HPLC: Chiralpak AD-H column,
1.0 mL/min, 20% EtOH/Hex, 254 nm, peaks observed at 51.0 and 84.7 min. See
144–147 °C; TLC: Rƒ (1:2 Hexanes/EtOAc) 0.70, visualized with UV light;
HPLC: Chiralpak OD-H column, 1.0 mL/min, 20% iPrOH/Hex, 254 nm, peaks
observed at 20.9 and 55.2 min.
supporting information for crystal structures of (1R,3S)-cis-7d and (±)-cis-7d.
Trans -Benzyl (3-((4-methoxyphenyl)amino)cyclohexyl)carbamate (trans-7c).
See Figure SI-13. General Procedure #10 was used, with CH2Cl2 as the solvent,
at room temperature, and for 120 h. The crude material was purified by automatic
normal phase flash chromatography, eluting with a gradient of 15% to 100%
EtOAc/Hex, yielding a mixture of trans-7c, cis-7c and 4c. The three compounds
were further separated by preparative HPLC to yield 20.9 mg yellow oil (14%
yield). 1H NMR (400 MHz, CDCl3): δ 7.42–7.28 (comp, 5H), 6.77 (d, J = 8.8
Hz, 2H), 6.57 (d, J = 8.6 Hz, 2H), 5.10 (s, 2H), 4.79 (bd, J = 7.9 Hz, 1H), 3.94 (s,
1H), 3.74 (s, 3H), 3.51 (s, 1H), 3.32 (bs, 1H), 1.88–1.60 (comp, 5H), 1.60–1.36
(comp, 3H); 13C NMR (151 MHz, CDCl3): δ 155.7, 152.3, 141.1, 136.6, 128.7,
128.3, 128.3, 115.1, 115.0, 66.8, 55.9, 49.0, 46.6, 37.5, 31.8, 31.1, 10.0; HRMS
(ESI-QToF) m/z: [M+H]+ Calcd for C21H27N2O3 355.2022; Found 355.2018;
TLC: Rƒ (2:1 Hexanes/EtOAc) 0.30, visualized with UV light; HPLC: Chiralpak
IC column, 1.0 mL/min, 10% EtOH/Hex, 254 nm, peaks observed at 14.1 and
17.0 min.
Cis-Benzyl (3-((4-methoxyphenyl)amino)cyclohexyl)carbamate (cis-7c). See
Figure SI-13. General Procedure #10 was used, with CH2Cl2 as the solvent, at
room temperature, and for 120 h. The crude material was purified by automatic
normal phase flash chromatography, eluting with a gradient of 15% to 100%
EtOAc/Hex, yielding a mixture of trans-7c, cis-7c and 7c. The three compounds
were further separated by preparative HPLC to yield 3.5 mg yellow oil (2% yield).
1H NMR (400 MHz, CDCl3): δ 7.39–7.29 (comp, 5H), 6.78 (d, J = 8.9 Hz, 2H),
6.68 (d, J = 8.4 Hz, 2H), 5.08 (s, 2H), 4.67 (d, J = 8.1 Hz, 1H), 4.01 (s, 1H), 3.75
(s, 3H), 3.58 (d, J = 12.3 Hz, 1H), 3.24 (s, 1H), 2.40 (d, J = 12.1 Hz, 1H), 2.12–
1.96 (comp, 2H), 1.82 (dt, J = 14.1, 3.6 Hz, 1H), 1.48–1.34 (m, 1H), 1.14–0.94
(comp, 3H); 13C NMR (151 MHz, CDCl3): δ 163.5, 155.6, 153.4, 136.6, 128.7,
128.3, 128.3, 116.7, 115.1, 66.8, 55.9, 53.5, 49.3, 40.0, 33.0, 32.4, 22.9; HRMS
(ESI-QToF) m/z: [M+H]+ Calcd for C21H27N2O3 355.2022; Found 355.2012;
TLC: Rƒ (2:1 Hexanes/EtOAc) 0.23, visualized with UV light; HPLC: Chiralpak
IC column, 1.0 mL/min, 10% EtOH/Hex, 254 nm, peaks observed at 20.1 and
26.6 min.
Methods for Measurement of Conversion by LC/MS. The following meth-
ods utilized a Waters CORTECS UPLC C18 (1.6 µm, 3.0 x 50 mm) column at
40 °C with a 0.8 mL/min flow rate.
4a/7a: Gradient conditions with H2O (0.01% formic acid) and MeCN
(0.01% formic acid) were utilized as follows: Held at 10% MeCN for 2 min,
ramped to 90% MeCN for 12.5 min, ramped to 10% MeCN for 2.5 min, held at
10% MeCN for 2 min. Peaks observed: 4a = 3.53 min; cis-7a = 3.90 min; trans-7a
= 3.98 min.
4b/7b: Gradient conditions with H2O (0.01% formic acid) and MeCN
(0.01% formic acid) were utilized as follows: Held at 10% MeCN for 2 min,
ramped to 90% MeCN for 12.5 min, ramped to 10% MeCN for 2.5 min, held at
10% MeCN for 2 min. Peaks observed: 4b = 3.18 min; cis-7b = 3.77 min; trans-
7b = 3.93 min.
4c/7c: Gradient conditions with H2O (0.01% formic acid) and MeCN (0.01%
formic acid) were utilized as follows: Held at 10% MeCN for 0.2 min, ramped to
98% MeCN for 3.8 min, held at 98% MeCN for 0.25 min, ramped to 10% MeCN
for 0.45 min, held at 10% MeCN for 3.3 min. Peaks observed: 4c = 1.88 min; cis-
7c = 1.62 min; trans-7c = 1.65 min.
4d/7d: Gradient conditions with H2O (0.01% formic acid) and MeCN
(0.01% formic acid) were utilized as follows: Held at 10% MeCN for 2 min,
ramped to 90% MeCN for 12.5 min, ramped to 10% MeCN for 2.5 min, held at
10% MeCN for 2 min. Peaks observed: 4d = 4.80 min; 7d = 4.48 min.
Calculation of Relative Response Factors (RRF) of 7 to 4. To correct for
different response factors between ketone 4 and diamine 7 on the LC/MS in con-
version measurements, external standard calibration on the compounds’ relative
response factors were calculated. The LC/MS was set to monitor from 220 nm
to 400 nm, the HPLC was set to monitor 254 nm.
Example Method for LC/MS: To a vial containing 4 or 7 was 10 mL MeCN.
From this solution was taken 0.75 mL, which was transferred into a separate
LC/MS vial and diluted with 0.25 mL 0.1% aqueous formic acid. The sample was
subjected to LC/MS analysis, with the response factor measured.
RRF for cis-7a/4a: 2.48
RRF for trans-7a/4a: 2.60
Average RRF for 7a/4a (used for some screening): 2.54
RRF for cis-7b/4b: 1.25
RRF for trans-7b/4b: 1.08
RRF for cis-7c/4c: 2.90
RRF for trans-7c/4c: 3.87
Trans-N-(3-((4-methoxyphenyl)amino)cyclohexyl)-4-methylbenzenesulfona-
mide (trans-7d) with CH2Cl2 as the solvent, at room temperature, and for 96 h.
See Figure SI-14. General Procedure #10 was used. The crude material was pu-
rified by automatic normal phase flash chromatography, eluting with a gradient
of 10% to 100% EtOAc/Hex, yielding a peak with trans-7d, and a second peak
with cis-7d and 4d. Both mixtures were further purified with preparative HPLC
to yield 47.7 mg colorless oil (33% yield). 1H NMR (600 MHz, CDCl3): δ 7.74
(d, J = 7.9 Hz, 2H), 7.20 (d, J = 7.9 Hz, 2H), 6.71 (d, J = 8.3 Hz, 2H), 6.47 (d, J =
8.3 Hz, 2H), 5.32 (d, J = 7.7 Hz, 1H), 3.74 (s, 3H), 3.51 (s, 1H), 3.46 (tt, J = 7.8,
3.7 Hz, 1H), 3.02 (s, 1H), 2.38 (s, 3H), 1.82–1.66 (comp, 2H), 1.64–1.51 (comp,
3H), 1.51–1.35 (comp, 2H), 1.35–1.23 (comp, 1H); 13C NMR (151 MHz,
CDCl3): δ 152.1, 143.3, 141.1, 137.9, 129.8, 126.9, 114.9, 114.9, 55.9, 49.4, 48.3,
37.7, 32.2, 31.4, 21.6, 19.8; HRMS (ESI-QToF) m/z: [M+H]+ Calcd for
C20H27N2O3S 375.1742; Found 375.1732; IR: (cm-1, neat): 3393, 3266, 2940,
1511, 1439, 1312, 1234, 1155, 1073, 1046, 936, 886, 812, 660; TLC: Rƒ (2:1 Hex-
anes/EtOAc) 0.32, visualized with UV light; HPLC: Chiralpak AD-H column,
1.0 mL/min, 20% EtOH/Hex, 254 nm, peaks observed at 33.2 and 39.1 min.
RRF for cis-7d/4d: 1.66
RRF for trans-7d/4d: 1.69
Average RRF for 7d/4d: 1.67
Example Method HPLC: (This was required for 4d/7d, as cis-7d and trans-7d
co-eluted on LC/MS) To a vial containing 7d was added 3.0 mL EtOH. From
this solution was taken 0.40 mL, which was transferred into a separate LC/MS
vial and diluted with 0.20 mL Hex. The sample was subjected to HPLC analysis,
with the response factor measured.
RRF for cis-7d/trans-7d: 1.59
20
[a]D –12.8° (c = 0.0058 g/mL). See supporting information for crystal struc-
ture of (±)-trans-7d.
ASSOCIATED CONTENT
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Supporting Information
The Supporting Information is available free of charge on the ACS Pub-
lications website.
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Schemes for peptide and substrate synthesis (Schemes S1–S13); H
and 13C NMR spectra for all compounds; 31P NMR spectra for all pep-
tide catalysts; 1H–1H COSY, 1H–13C HSQC, and 1H–1H NOESY NMR
spectra for peptide P25; HPLC traces for compounds 4 and 7; DFT and
M06 calculations for 4a, 8a, and 10.
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X-ray crystallographic data for compound (±)-trans-7d.
X-ray crystallographic data for compound (1R,3S)-cis-7d.
X-ray crystallographic data for compound (±)-cis-7d.
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AUTHOR INFORMATION
(34) Garrett, R. H.; Grisham, C. M. Biochemistry, 5th ed; Cengage: United States
of America, 2013.
Corresponding Author
*E-mail: scott.miller@yale.edu
(35) Pollak, N.; Dolle, C.; Ziëgler, M. Biochem. J. 2007, 402, 205–218.
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(43) See supporting information for synthetic details.
(44) Additional peptides in Table 1 were screened and followed the same pattern
as P32 and P34, with minute changes to er with marginal or no increase in dr
favoring cis-4a.
(45) Vedejs, E.; Chen, X. J. Am. Chem. Soc. 1997, 119, 2584–2585.
(46) Dehli, J. R.; Gotor, V. Chem. Soc. Rev. 2002, 31, 365–370.
(47) See Supporting Information for computational details.
(48) Zhao, Y. & Truhlar, D. G. Theor. Chem. Acc. 2008, 120, 215–241.
(49) For recent energy difference calculations performed on chair conformations
using M06-2X functional, see: Jahn, M. K.; Dewald, D.; Vallejo-Lopez, M.; Co-
cinero, E. J.; Lesarri, A.; Grabow, J.-U. J. Phys. Chem. A 2013, 117, 13673–13679.
(50) For a review on solvation models, see: Tomasi, J.; Mennucci, B.; Cammi, R.
Chem. Rev. 2005, 105, 2999–3093.
(51) For recent combinations of the M06-2x functional with the IEF-PCM solv-
ation model, see: a. Simon, A.; Lam, Y.- h.; Houk, K. N. J. Am. Chem. Soc. 2016,
138, 503– 506.
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Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
This work was supported by the National Institutes of Health (NIH
R01-GM096403). We graciously thank Dr. Brandon Q. Mercado for ac-
quiring the X-ray crystallographic data. C.R.S. thanks the National Sci-
ence Foundation Graduate Research Fellowship Program for funding
(DGE1122492). A.L. would like to acknowledge funding from the Yale
College Dean’s Office in support of the Science, Technology, and Re-
search Scholars (STARS) summer research program.
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