A strategy for dual inhibition of the proteasome and fatty acid synthase with belactosin C-orlistat hybrids

Article abstract of DOI:10.1016/j.bmc.2017.01.020

The proteasome, a validated cellular target for cancer, is central for maintaining cellular homeostasis, while fatty acid synthase (FAS), a novel target for numerous cancers, is responsible for palmitic acid biosynthesis. Perturbation of either enzymatic machine results in decreased proliferation and ultimately cellular apoptosis. Based on structural similarities, we hypothesized that hybrid molecules of belactosin C, a known proteasome inhibitor, and orlistat, a known inhibitor of the thioesterase domain of FAS, could inhibit both enzymes. Herein, we describe proof-of-principle studies leading to the design, synthesis and enzymatic activity of several novel, β-lactone-based, dual inhibitors of these two enzymes. Validation of dual enzyme targeting through activity-based proteome profiling with an alkyne probe modeled after the most potent inhibitor, and preliminary serum stability studies of selected derivatives are also described. These results provide proof of concept for dual targeting of the proteasome and fatty acid synthase-thioesterase (FAS-TE) enabling a new approach for the development of drug-candidates with potential to overcome resistance.

Full text of DOI:10.1016/j.bmc.2017.01.020

Accepted Manuscript
A Strategy for Dual Inhibition of the Proteasome and Fatty Acid Synthase with
Belactosin C-Orlistat Hybrids
Mingzhao Zhu, Wayne D. Harshbarger, Omar Robles, Joanna Krysiak, Kenneth
G. Hull, Sung Wook Cho, Robyn D. Richardson, Yanyan Yang, Andres Garcia,
Lindsey Spiegelman, Bianca Ramirez, Christopher T. Wilson, Ju Anne Yau,
James T. Moore, Caitlen B. Walker, James C. Sacchettini, Wenshe Liu, Stephan
A. Sieber, Jeffrey W. Smith, Daniel Romo
PII:
S0968-0896(16)31267-6
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.01.020
BMC 13499
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Accepted Date:
23 November 2016
13 January 2017
Please cite this article as: Zhu, M., Harshbarger, W.D., Robles, O., Krysiak, J., Hull, K.G., Cho, S.W., Richardson,
R.D., Yang, Y., Garcia, A., Spiegelman, L., Ramirez, B., Wilson, C.T., Anne Yau, J., Moore, J.T., Walker, C.B.,
Sacchettini, J.C., Liu, W., Sieber, S.A., Smith, J.W., Romo, D., A Strategy for Dual Inhibition of the Proteasome
and Fatty Acid Synthase with Belactosin C-Orlistat Hybrids, Bioorganic & Medicinal Chemistry (2017), doi: http://
dx.doi.org/10.1016/j.bmc.2017.01.020
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A Strategy for Dual Inhibition of the Proteasome and Fatty Acid
Synthase with Belactosin C-Orlistat Hybrids
Mingzhao Zhu,[a],[c] Wayne D. Harshbarger,[d] Omar Robles,[b] Joanna Krysiak, [e] Kenneth G. Hull,[a],[c]
Sung Wook Cho,[b] Robyn D. Richardson,[f] Yanyan Yang,[b] Andres Garcia,[b] Lindsey Spiegelman,[b]
Bianca Ramirez,[b] Christopher T. Wilson,[b] Ju Anne Yau,[b] James T. Moore,[b] Caitlen B. Walker,[b]
James C. Sacchettini,[d] Wenshe Liu,[b] Stephan A. Sieber [e] Jeffrey W. Smith,[f] and Daniel Romo*[a]
,
Abstract: The proteasome, a validated cellular target for cancer, is
central for maintaining cellular homeostasis, while fatty acid
synthase (FAS), a novel target for numerous cancers, is responsible
for palmitic acid biosynthesis. Perturbation of either enzymatic
machine results in decreased proliferation and ultimately cellular
apoptosis. Based on structural similarities, we hypothesized that
hybrid molecules of belactosin C, a known proteasome inhibitor, and
orlistat, a known inhibitor of the thioesterase domain of FAS, could
inhibit both enzymes. Herein, we describe proof-of-principle studies
leading to the design, synthesis and enzymatic activity of several
novel, -lactone-based, dual inhibitors of these two enzymes.
Validation of dual enzyme targeting through activity-based proteome
profiling with an alkyne probe modeled after the most potent
inhibitor, and preliminary serum stability studies of selected
derivatives are also described. These results provide proof of
concept for dual targeting of the proteasome and FAS-TE enabling a
new approach for the development of drug-candidates with potential
to overcome resistance.
kinase inhibitor lapatinib, which targets receptor tyrosine kinases
(EGFR) and ErbB-2 (HER2), and is currently used for the
[2]
treatment of metastatic breast cancer.
Drugs with single
targets may fall victim to drug resistance due to mutations or up-
regulation of the target protein, whereas polypharmacological
anticancer drugs are thought to evade drug resistance due to
the need for cells to become resistant across multiple pathways
through simultaneous protein/enzyme mutations. [3]
The human proteasome is responsible for the vast majority of
intracellular protein degradation, and thus plays a central role in
cellular homeostasis. [4] The 20S proteasome is the core particle
(CP) of the 26S proteasome, and is composed of 28 subunits
arranged as four stacked rings, in which each of the rings
contains either seven unique -chains or seven unique
chains. The organization of the CP is 1-7b1-7b1-71-7, and
possesses C2 symmetry [5]. Three of the 7 -subunits are
proteolytically active: 1, 2, and 5, and have been
characterized to have peptide cleavage preferences
corresponding to caspase-like (C-L, β1), trypsin-like (T-L, β2)
and chymotrypsin-like (ChT-L, β5) activity[6]. The proteasome is
a member of the N-terminal hydrolase super family, and all three
catalytic subunits act to degrade substrates utilizing an N-
terminal Thr1 residue. Due to accelerated cell cycles and
Introduction
Intentional and selective polypharmacology has emerged as
an exciting concept for rational drug design.[1] In cancer therapy,
prototypical chemotherapeutic agents are generally based on
the concept of “one-drug, one-target,” whereas selective
polypharmacology targets two or more enzyme targets with a
single drug. Selective polypharmacological drugs can be
efficacious, a notable example being the FDA approved multi-
metabolism in tumor cells, the CP is often upregulated to
[7]
increase turnover rates of proteins.
This dependence on CP
activity renders neoplastic cells highly susceptible to proteasome
inhibition. In addition, proteasome inhibitors increase the level of
tumor suppressors (e.g. cyclin kinase inhibitor p27) which induce
tumor apoptosis without significantly affecting quiescent cells.[8]
Thus, the proteasome CP has been extensively explored as a
drug target against various types of cancer, and many classes
of compounds have been found to inhibit proteasome function,
both by covalently binding to Thr1 and also non-covalent
inhibition [9]. The ChT-L site has been the main focus for the
design of antineoplastic therapeutics, however, the T-L and C-
L sites have also recently gained attention [10]. Two covalent
inhibitors with preference for the ChT-L site have received FDA
approval: the boronic acid, bortezomib (1, Velcade®), for the
treatment of multiple myeloma and relapsed or refractory
mantle cell lymphoma; and the epoxyketone carfilzomib (2,
Kyprolis®), for the treatment of refractory multiple myeloma. A
third promising class of proteasome inhibitors under
development possess a -lactone which covalently modifies
Thr1O to generate an acyl-enzyme adduct.[11] An example is
[a]
[b]
[c]
Dr. M. Zhu, Dr. K. Hull, Prof. Dr. Daniel Romo*
Department of Chemistry & Biochemistry and CPRIT Synthesis and
Drug-Lead Discovery Laboratory, Baylor University, Waco, TX
76706 (USA)
E-mail: Daniel_Romo@baylor.edu
Dr. O. Robles, Dr. S.-W. Cho, Dr. Y. Yang, A. Garcia, L.
Spiegelman, B. Ramirez, C. T. Wilson J. A. Yau, J. T. Moore, C. B.
Walker, Prof. Dr. Wenshe Liu
Department of Chemistry, Texas A&M University, College Station,
TX 77842 (USA)
Dr. J. Krysiak, Prof. Dr. S. A. Sieber
Department of Chemistry, Technische Universität München,
Lichtenbergstr. 4, 85747 Garching (Germany)
Dr. R. D. Richardson, Prof. Dr. J. W. Smith
Sanford Burnham Medical Research Institute, La Jolla, CA (USA)
Dr. W. Harshbarger, Prof. Dr. J. C. Sacchettini
Department of Biochemistry & Biophysics, Texas A&M University,
College Station, TX 77840 (USA)
[d]
[e]
salinosporamide
A (Marizomib, 3), which is a -lactone
containing proteasome inhibitor designated as an orphan drug
for multiple myeloma[12] and is in Phase I/II clinical studies for
the treatment of multiple myeloma, leukemia, and solid tumors
Supporting information for this article is given via a link at the end of
the document.

(Figure 1).[13] Despite these advances, drug resistance to
proteasome inhibitors through active site mutations and
upregulation of ChT-L subunits, continues to necessitate the
[22] which along with (–)-belactosin A (5) and congeners possess
inhibitory activity toward the 20S proteasome. X-ray studies of
the belactosin C analog, N-CBz-O-Bn homobelactosin C (6),
bound to the yeast 20S proteasome by Groll and coworkers[23]
revealed that the Thr1O is acylated by the β-lactone moiety, and
that the orientation of the inhibitor differs from other β-lactone-
containing proteasome inhibitors such as omuralide, which may
contribute to its selectivity for ChT-L subunits. A recent study
demonstrated the potential of belactosin derivatives bearing
minimal -lactone fragments to have selectivity for the
immunoproteasome.[24] Our concurrent studies of the belactosins
and orlistat derivatives led to a hypothesis that hybrid structures
of these -lactones might have the potential to inhibit both the
proteasome and FAS-TE. The common reactive acylating
moiety in these inhibitors is a (3S,4R)-trans-disubstituted -
lactone providing a starting point for the design of analogs with
dual inhibitory activity. Herein, we report our successful design
and synthesis of dual inhibitors of the proteasome and FAS-TE,
structure-activity studies (SAR) using fluorogenic assays for
each enzyme, and verification of the ability of these dual
inhibitors to target both FAS and the proteasome in HeLa cells
using activity-based protein profiling (ABPP).
[14]
development of novel inhibitors for cancer therapy.
Figure 1. Representative structures of human or yeast 20S proteasome
and fatty acid synthase inhibitors.
Fatty acid synthase (FAS) is a second vital enzyme which is
Results and Discussion
upregulated in rapidly proliferating cancers, including breast,
[15]
prostate, ovarian, and multiple myeloma.
FAS catalyzes the
Initial dual inhibitors. Given the presence of the common (3S,
4R)-trans-disubstituted--lactone (red) and similar -side chains
of orlistat and protected belactosins (e.g. N-Cbz-O-Bn-(–)-
homobelactosin C (7)), we reasoned that -lactones 8a-c that
were originally synthesized as belactosin derivatives to provide
SAR information regarding inhibition of the proteasome,[22a]
might also have inhibitory activity toward FAS (Figure 2).
de novo synthesis of fatty acids to provide lipids for membrane
formation and energy production via β-oxidation and lipid
modification of proteins. FAS inhibition suppresses cell
proliferation, adhesion, migration, and invasion. It also leads to
suppression of genes involved in production of arachidonic acid
and androgen hormones, both of which promote tumor
[16]
progression.
Studies have shown that FAS inhibition induces
apoptosis in various cancer cell lines,[17] synergizes with
common anti-cancer therapies (Herceptin® and Taxol®) and in
some cases reverses auto-resistance to those therapies, inhibits
tumor angiogenesis, and FAS inhibitors may even act as
-chain
-chain
-chain
-chain
Ph
NHCHO
O
O
O
O
O
H
O
H
H
O
O
O
H
N
N
O
chemopreventative agents for cancer making FAS an attractive
N
H
C10H21
2
H
18]
target for further development.[15a,
Tetrahydrolipstatin (4,
Me
O
Me
O
H
Orlistat®, Alli®, Xenical®) is a reduced form of the -lactone-
containing natural product lipstatin, and was approved by the
FDA in 1999 for the treatment of obesity due to its ability to
inhibit pancreatic lipase in the gut. Recently, however, it was
determined that orlistat also has antitumor activity, through
Orlistat (4)
N-Cbz-O-Bn-( )-Homobelactosin C (7)
fatty acid synthase-thioesterase
domain inhibitor
yeast 20S proteasome inhibitor
(IC50 low nM)
(IC50 4.63 ± 1.49 M)
O
O
O
O
O
H
inhibition of the thioesterase domain of FAS, FAS-TE (IC50
=
H
4
3
+
C3, C4 epimer
N
N
O
[19]
N
R
1.35 M).
X-ray analysis of the human FAS-TE bound with
H
H
H
Me
O
O
(dr 1:1)
orlistat by Kridel and Lowther revealed the mechanism of
8a ((S)-2-methylpropyl); 8b (R= n-Pr); 8c (R = n-Hex)
inhibition to be covalent bond formation between Ser2308 of
[20]
Initial Dual Inhibitors of FAS-TE and the Proteasome
FAS-TE and the -lactone of orlistat through acylation.
Poor
solubility and poor oral bioavailability limit the practicality of
orlistat as an antitumor drug, therefore it was crucial to
overcome these problems by modifying the structure of orlistat
Figure 2. Representative Initial dual inhibitors 8a-c displaying inhibitory
activity toward both FAS-TE and the proteasome and design process
based on structures of orlistat (4) and the belactosin C derivative 7.
[18e]
to develop the next generation of FAS-TE inhibitors.
Our group previously reported the synthesis of orlistat and a
series of analogs with FAS-TE inhibitory activity.[21] We also
described the synthesis of (–)-belactosin C (6) and derivatives,
These initial analogs 8a-c were synthesized as either a 1:1
(8a, 8b) or 1.3:1 (8c) mixture of C3, C4-diastereomers as a

result of a non-diastereoselective, tandem Mukaiyama aldol-
lactonization with a glyoxamide dipeptide -side chain, as
previously described.[22a] Since these diastereomers were not
readily separable, they were assayed as diastereomeric
mixtures using fluorogenic substrates for both FAS-TE and the
proteasome as previously described. [18e],[25] All three compounds
exhibited inhibition of the proteasome with 8c (1.3:1 mixture of
N-CBz, O-Bn belactosin C[23b] and its C3,C4 diasteromer)
showing the greatest activity as expected. Importantly, all three
derivatives were more potent than orlistat toward FAS-TE with
IC50 values ranging from ~0.16 to 4.0 M.[26]
previously.[23a] On the other hand, less is known about the role
the small, hydrophobic sec-butyl -chain might play in binding to
the proteasome but the C3-n-hexyl group of orlistat binds to the
short-chain pocket in FAS-TE. As described above, replacement
of the -sec-butyl chain with an -n-hexyl group was tolerated
by the proteasome and given the structure of orlistat, we elected
to maintain this hydrophobic -chain in planned derivatives for
this study. Both the belactosins and orlistat, bear the same
relative (trans) and absolute configuration (3S, 4R) at C2 and C3
of the -lactone so this was maintained in most derivatives,
however the enantiomeric (3R, 4S)-trans--lactone was also
investigated.
To synthesize the targeted belactosin-orlistat hybrids, we
utilized our previously reported strategy toward (–)-belactosin
C.[22a] Synthesis of the disubstituted--lactones was enabled
through a chiral auxiliary-based, tandem Mukaiyama aldol-
lactonization (TMAL) process between glyoxylate ester 9 and
the silyl ketene acetal 10a to deliver the trans--lactone esters
11a and 11b’ (Scheme 1). The tartrate-derived chiral auxiliary,
while not significantly impacting the absolute configuration of the
newly created C2, C3 stereocenters (dr ~2:1) during the TMAL
process, served to facilitate separation of diastereomers
11a/11a’ produced. The two were separated and underwent
hydrogenolysis to cleave the chiral and auxiliary and enabled
exploration of the enantiomeric trans--lactone for SAR
purposes. In a similar manner, the -methyl -lactone esters
11b, 11b’, 11b’’, and 11b’’’ were synthesized and despite the
absence of diastereoselectivity, separation of the diastereomers
was again possible and subsequent deprotection of the two
trans-diastereomers allowed access to the -methyl -lactone
acids 11b and 11b’. A series of dipeptides, represented by
dipeptide amine 16a, were coupled to the -lactone carboxylic
acids, e.g. 11b, to deliver the dipeptide -lactone 17 (Scheme 2).
In this manner, a series of potential dual inhibitors 17-27 were
synthesized through variations of the dipeptide and -lactone
fragments.
The inhibitory activity of these initial -lactones verified our
hypothesis that simultaneous inhibition of the proteasome and
FAS was possible with a single compound by maintaining a
(3S,4R)-trans-disubstituted -lactone pharmacophore with
a
homobelactosin C dipeptide -sidechain. Encouraged by these
preliminary results, we set out to design and synthesize a series
of belactosin C-orlistat hybrids with the great challenge of
increasing potency toward both enzymes.
Design and synthesis of dual inhibitors of FAS and the
proteasome. A crystal structure of bis-benzylhomobelactosin C
bound to the yeast 20S proteasome suggests that the -
dipeptide side-chain plays a role in dictating which proteasomal
active site will be targeted via interaction with both the S1
specificity pocket and the primed substrate binding site.[23a]
Belactosins are the only class of proteasome inhibitors which
bind to the primed region, whereas all other proteasome
inhibitors bind to the non-primed sites.[23a] The cyclopropane ring
of belactosin A was proposed to adopt a position which blocks
the nucleophilic addition of water for enzyme turnover, and in
conjunction with cyclopropane stereochemistry, forces the
remainder of the compound into the primed site of the CP.
In the case of FAS, the co-crystal structure of FAS-TE with
bound orlistat shows the 11-carbon -chain of orlistat binding
predominately in a hydrophobic channel, termed the specificity
channel.[20] Given that orlistat possesses
a
relatively
hydrophobic -side chain also bearing an N-formyl amino ester
while N-Cbz, O-Bn-(–)-belactosin C has a protected dipeptide -
chain, we reasoned that this dipeptide might display a similar
binding mode and interact with both the specificity channel and
interface cavity of FAS-TE. As described previously, the -chain
dipeptide binds to the primed site of the proteasome and
optimization of binding interactions with primed sites has led to
success in improving potency and selectivity of proteasome
inhibitors.[27] Thus, it was reasonable to consider modifications of
the -dipeptide that might alter selectivity to the three
proteolytically active sites while at the same time improving
interactions with the specificity channel and interface cavity of
FAS-TE. We utilized L-(S)-lysine rather than L-(S)-ornithine as
the first amino acid of the -dipeptide side-chain resulting in a
four methylene unit spacer rather than three and protection of
the terminal nitrogen with a carbobenzyloxy group and the lysine
carboxylic acid as the benzyl ester was also maintained in all
derivatives given the high potency of N-Cbz, O-Bn
Scheme 1.
-lactone acids 12a and 12a’ and -
methyl--lactone acids 12b and 12b’ employing a chiral auxiliary-based,
tandem Mukaiyama aldol--lactonization. (a) ZnCl2, CH2Cl2, 23 oC, 30 min (R
= H) or 15 h (R = Me); (b) H2, Pd/C (10% w/w), THF.
homobelactosin
C
toward the
proteasome reported

unit by replacing L-(S)-lysine with L-(S)-ornithine, as found in the
belactosins, provided derivative 19 with similar activity to the
ChT-L site of the proteasome and reduced activity toward FAS-
TE, thus in all further derivatives (S)-lysine was retained in the
dipeptide (Table 1, entry 2 vs. 4). Replacing the terminal benzyl
carbamate of derivative 17 with a bromobenzoyl group provided
derivative 20, which maintains the hydrophobicity and hydrogen-
bonding capabilities, did not significantly alter inhibitory activity
(Table 1, entry 5). We also investigated reversed-amide variants
in the dipeptide wherein linkage is made through the carboxylic
acid rather than the amine of lysine leading to derivative 21
bearing a N-Cbz substituent and terminal benzyl ester. This
derivative exhibited the most potent activity toward FAS-TE
(1.50 ± 0.28 M) to this point, while also dramatically increasing
specificity toward the ChT-L sites of the proteasome (Table 1,
entry 6). A more rigid dipeptide was also explored through
synthesis of the cyclic imide urea 22 which also lacked a
hydrophobic benzyl substituent relative to other derivatives. The
restricted flexibility did not greatly alter ChT-L activity, but
reduced active site specificity with activity observed toward the
C-L site of the proteasome (IC50 7.93 ± 0.21 M) and reduced
FAS-TE activity (Table 1, entry 7).
Scheme 2. Representative coupling strategy for synthesis of dual inhibitors as
shown for dipeptide -lactone 17.
Enzymatic Assays: Inhibition of the 20S Proteasome and
FAS-TE. With the new analogs in hand, we performed an in vitro
enzyme assay to determine their potency as inhibitors of the
proteasome and FAS-TE. Both assays measure the rate of
hydrolysis of fluorogenic substrates in the presence and
absence of inhibitors (see SI for details). In the case of the
proteasome, the assay was run three times with a different
substrate specific for each of its catalytic sites; chymotrypsin-like
(ChT-L(b5)), caspase-like (C-L(b1)), or trypsin-like (T-L(b2)). As
summarized in Table 1, the 20S proteasome inhibition assay
reveals that all analogs have selectivity for the ChT-L
proteasomal subunit, with IC50 values ranging from 0.22 - 3.4
M. Inhibition is also seen against the C-L subunits with
generally higher IC50 values of 2.1-18 M, however, no inhibition
was seen against the TL subunit. Orlistat, used as a control for
the FAS-TE assay, showed no inhibition against any of the
proteasome catalytic sites at concentrations up to 100 µM (Table
1, entry 1). All of the analogs tested inhibited FAS-TE in the
range of ~1.2 – 9.6 M. The most potent dual inhibitor when
considering inhibitory activity toward both enzymes was
dipeptide -lactone 21, which exhibited an IC50 value of 1.50 ±
0.28 M against FAS-TE, which was 3X more potent compared
to orlistat ( IC50 4.63 ± 1.49 M), while also having an IC50 of
0.37 ± 0.01 against the proteasome with high selectivity for ChT-
L sites. A more detailed discussion of SAR established from
these derivatives is provided below with systematic and
sequential changes made in derivatives 18-33 highlighted (aqua)
in Table 1.
Effect of -lactone absolute configuration. To probe the effect
of the β-lactone relative and absolute configuration, analogs 23
and 24 were prepared from diastereomeric -lactones (cf. 12,
Scheme 1).
Derivative 23 maintained the reverse amide
variation in the dipeptide in analog 21, however the absolute
configuration was inverted at both the - and -positions of the
lactone to the 3R, 4S configuration. This alteration reduced
the proteasome ChT-L and FAS-TE activity ~8-fold of analog 23
compared to the diastereomeric -lactone 21 (Table 1, entry 6 vs
entry 8). Maintaining the same -lactone configuration but
returning to the previous amide connectivity with a Gly-Lys
dipeptide provided analog 24 (Table 1, entry 9) which regained
some activity toward both enzymes but did not rival activity
previously observed with the 3S, 4R configuration (cf. 18, Table
1, entry 3). The results validated the importance of the -lactone
configuration and is consistent with previous reports indicating
the importance of both relative and absolute stereochemistry for
-lactone inhibitors of both FAS-TE and the proteasome.
Overall, it is interesting to note that both the reversed amide
variation and alternate -lactone absolute configuration (Table 1,
entries 6, 8) led to inhibitors that had complete selectivity for the
ChT-L sites of the proteasome over the C-L site.
Variations of the dipeptide fragment. We first targeted
variations of the dipeptide side-chain to probe the effects these
changes would have on presumed binding to the S1 specificity
pocket of the proteasome and the specificity (hydrophobic)
channel of FAS-TE. Building on our preliminary data (vide infra),
our initial derivative 17 maintained the -n-hexyl side chain and
the N-Cbz-(S)-Ala-O-Bn(S)-Lys dipeptide and served as an
embarkation point for further structural modifications. Derivative
17 exhibited IC50 values of 0.50 ± 0.06 and 3.17 ± 0.17 M
toward the proteasome and FAS-TE, respectively (Table 1, entry
2). Removal of a methyl group in the dipeptide by replacement
of alanine with glycine in derivative 18 did not impact
proteasome inhibition, but led to an ~3-fold drop in potency
against FAS-TE (Table 1, entry 3). Removing one methylene
Effects of the C2-methyl group. Given the potential for hydrolysis
of these enzyme inhibitors prior to reaching their targets, we
explored introduction of an -methyl substituent on the -lactone
to slow the rate of hydrolysis. Maintaining the best dipeptide,
namely
N-Cbz-(S)-Ala-O-Bn-(S)-Lys,
we studied three
diastereomeric -lactones 25-27 prepared through a TMAL
process with an -methyl silylketene acetal (cf. 10b, Scheme 1;
see SI for further details). In general, and not unexpectedly, the
-quaternary carbon reduced the potency toward both
enzymatic targets (IC50 ~1-3 M, Table 1, entries 10-12) given
the expected slower rate of acylation of the active site serine
and threonine of the FAS-TE and the proteasome, respectively.

However, the stability of these -quaternary carbon-containing
-lactones was improved in culture media leading to greater
cytotoxicity in cellular assays (vida infra). Interestingly, -lactone
27 with an -quaternary carbon to the -lactone but inverted C3
stereochemistry relative to the most potent dual inhibitor 21,
rivaled the inhibitory activity against FAS-TE.
Table 1. Structure-activity relationships of novel orlistat-belactosin hybrids against the three proteolytic sites of the human 20S
proteasome, human FAS-TE, and HeLa cells.a
chymotrysin-
like (ChT-L)
IC50 (µM)
caspase-like
(C-L)
IC50 (µM)
trypsin-like
(T-L)
IC50 (µM)
FAS-TE IC50
(µM)
HeLa Cell
Growth
IC50 (µM)
entry
compound
1
>100
>100
>100
4.63 ± 1.49
ND[a]
Orlistat (4)
2
3
0.50 ± 0.06
0.57 ± 0.03
0.70 ± 0.05
0.51 ± 0.01
0.37 ± 0.01
0.51 ± 0.02
2.71 ± 0.17
1.38 ±0.03
2.43 ± 0.25
3.03 ± 0.05
3.51 ± 0.04
2.16 ± 0.05
2.81 ± 0.24
> 100
>100
>100
>100
>100
>100
>100
>100
>100
> 100
3.17 ± 0.17
9.56 ± 0.58
4.35 ± 1.11
4.81 ± 1.84
1.50 ± 0.28
5.55 ± 1.33
12.6 ± 0.6
62.5
51.1
ND[a]
ND[a]
45.8
ND[a]
ND[a]
ND[a]
10.0
17 [N-Cbz(S)-Ala-O-Bn(S)-Lys-(3S, 4R)]
O
O
H
H
N
N
CbzHN
n-C6H13
3
H
H
O
CO2Bn
O
18 [N-CbzGly-O-Bn(S)- Lys-(3S, 4R)]
O
Me
O
H
H
H
N
N
CbzHN
n-C6H13
4
2
H
O
CO2Bn
O
19 [N-Cbz(S)-Ala-N-Cbz(S)-Orn-(3S, 4R)]
5
20 [N-p-BrBz(S)-Ala-O-Bn(S)-Lys-(3S, 4R)]
6
21 [O-Bn(S)-Ala-N-Cbz(S)-Lys-(3S, 4R)]
7
7.93 ± 0.21
22 [cyclo-O-Bn(S)-Ala-O-Bn(S)-Lys-(3S, 4R)]
23 [O-Bn(S)-Ala-N-Cbz(S)-Lys-(3R, 4S)]
8
>100
9
8.72 ± 0.05
3.74 ± 0.04
8.37 ± 0.17
2.61 ± 0.94
24 [N-CbzGly-O-Bn(S)-Lys-(3R, 4S)]
25 [N-Cbz(S)-Ala-O-Bn(S)Lys-(3S, 4R)]
10

11
12
2.18 ± 0.08
3.39 ± 0.08
3.98 ± 0.63
3.62 ± 0.08
> 100
> 100
3.11 ±0.75
1.19 ± 0.42
37.5
26 [N-Cbz(S)-Ala-O-Bn(S)Lys-(3R, 4S)]
27 [N-Cbz(S)-Ala-O-Bn(S)Lys-(3R, 4R)]
ND[a]
13
0.26 ± 0.01
> 100
> 100
3.98 ± 0.74
ND[a]
28 [O-Bn(S)-Ala-N-Cbz(S)-Lys-(3S, 4R),
ABPP Probe]
[a]ND = not determined
Cell cytotoxicity toward HeLa cells. Several of the more
potent dual inhibitors, derivatives 17, 18, 21, 25, and 26,
identified through enzymatic assays, were selected to assess
their cytotoxicity toward HeLa cells using a colorimetric cell
viability assay (MTT). All analogs showed IC50 values in the 10-
60 µM range with the (3S, 4R)--disubstituted -lactone 25
demonstrating the highest cytotoxicity with an IC50 of 10.0 µM,
which was approximately 6X more potent than our initial lead
inhibitor 17 (IC50 62.5 µM). While cell permeability or efflux pump
effects could be responsible for the disparity observed between
cell cytoxicity and enzymatic inhibition, we reasoned that the
differential instability of these-lactones was a more likely
explanation. For example, the greatest cytotoxicity, which was
observed with derivative 25, did not correspond to the best IC50
values in enzymatic assays, but this could be a reflection of the
greater stability to hydrolysis of the -disubstituted -lactone.
This led us to study the relative rates of hydrolysis of selected -
lactone inhibitors in the buffer and serum used in both enzymatic
and cell-based activity studies.
run in DMEM buffer (pH 7.5) containing 5% fetal bovine serum
(FBS) and for comparison, stability studies were also run in
DMEM only. The half-lives of -lactone inhibitors in buffer with
and without added serum was studied through HPLC monitoring
over time. In the case of -monosubstituted -lactone 21, the
impact of added serum is dramatic leading to a change in half-
life from an already brief 20 min in buffer to < 3 min with added
serum (Table 2). However, the stability of the quaternary -
disubstituted--lactone 25 did not follow the same trend. The
half-life of -lactone 25 in DMEM buffer alone was nearly 3X that
of the -monosubstituted (t1/2 ~ 70 min). Futhermore, the
presence of serum led to a dramatically extended half-life (t1/2
~
120 min) for this -lactone and is consistent with improved
stability of labile molecules often observed due to interaction of
hydrophobic molecules with serum proteins protecting them from
the somewhat basic medium.[28] Furthermore, the hydrolysis
products of several -monosubstituted--lactones were
observed by LC-MS analysis despite the purity indicated by
NMR analysis and this was observed most often with
Stability of Selected Inhibitors in Buffer with and without
FBS. Given the disparity observed between enzymatic activity
and whole cell cytotoxicity and the potential for hydrolysis of
these -lactone containing dual inhibitors, we studied the relative
stability of two representative dual inhibitors in media employed
in cytotoxicity assays. We chose -lactone 21 which displayed
the greatest potency in the enzymatic assays (IC50 0.37 ± 0.01
and 1.50 ± 0.28 M vs ChT-L and FAS-TE, respectively) and -
lactone 25 which displayed the greatest cellular cytotoxicity
((IC50 10.0 µM) for these studies. The cell viability assay was
monosubstituted -lactones as expected.
Thus, it is
reasonable to deduce that -disubstituted -lactones are
generally better drug leads since their increased cytotoxicity may
be a reflection of their greater stability to hydrolysis and would
be expected to have a longer half-life in the blood stream.
These findings are consistent with observations made in recent
studies with belactosin A derivatives bearing -disbustituted -
lactones with the exception that increased stability was not
observed in the presence of human AB serum.[27a]

aStability studies were run in triplicate. DMEM = Dulbecco’s
modified eagle medium; FBS = fetal bovine serum.
several protein targets could be observed with dominant bands
at molecular weights of ca. 250, 50, and 20 kDa. To validate the
identity of these proteins, we applied western blot analysis with
anti-FAS and anti-CT-L (PSB-5) antibodies. The fluorescent
bands at 250 kDa and 20 kDa overlapped with the
corresponding antibody signals suggesting that these two
protein bands represent the expected FAS and CT-L targets
(Figure 3B). Finally, we first added the dual inhibitors 21 and 25
in various concentrations (0  100 μM) to MCF-7 cells prior to
probe addition (Figure 3C). Subsequent labeling with probe 28
revealed a clear concentration dependent reduction in signal
intensity only for 21 emphasizing that this inhibitor competes
with the ABPP probe 28 for the same active sites. FAS was less
sensitive to this competition (10-fold excess resulted in signal
reduction) compared to ChT-L (2.5-fold excess resulted in signal
reduction) which is in line with the lower IC50 of 21 for FAS. On
the other hand, inhibitor 25 was not able to compete effectively
with labeling by probe 28 even at 10-fold excess of the
compound. This is likely due to slower reaction rates of this
inhibitor with actives site nucleophiles (i.e. serine and threonine
hydroxyl groups) due to the presence of the more hindered, -
disubstituted -lactone and the short (30 min) pre-incubation
Dual target validation via activity-based protein profiling. To
determine if cytotoxicity observed in cancer cells might be due to
dual inhibition of both FAS-TE and the proteasome, we
conducted activity-based protein profiling (ABPP) experiments
with cell lysates.[29] Previous whole cell ABPP studies led to
orlistat as an inhibitor of FAS.[18h] We sought to verify that both
FAS-TE and the proteasome were indeed labeled in a sensitive
cancer cell line with the designed activity-based probe 28, which
is based on the most potent dual inhibitor 21 in terms of
enzymatic inhibition. Activity-based probe 28 includes an alkyne
group in the para-position of the benzyl ester moiety for
subsequent
Sharpless-Hüisgen
cycloaddition
following
incubation with a proteome. Given their potency in enzymatic
and cell-based assays, dual inhibitors 21 and 25 were selected
for competition experiments to validate selectivity if any.[30]
Satisfyingly, probe 28 retained good activity compared to the
parent inhibitor 21 with slightly improved activity to the ChT-L
site of the proteasome and ~2.5X drop in activity toward FAS-TE
suggesting no major adverse effects of the alkyne tag (Table 2).
Both MCF-7 (breast adenocarcinoma) and HeLa (cervix
adenocarcinoma) cells were incubated in situ with varying
concentrations (0  50 μM) of probe 28 for 1 h at 37 °C. The
labeling was followed by cell lysis and attachment of the TAMRA
fluorophore under copper (I) catalyzed Sharpless-Hüisgen
time utilized prior to adding the ABPP probe 28.
This
observation is consistent with findings made during enzymatic
assays with inhibitor 25 which required longer incubation times
to achieve comparable IC50 values to other -monosubstituted -
Table 2. Serum stability studies (t1/2
)
with an -
monosubstituted 21 versus an -disubstituted 25 -lactone
in DMEM with and without serum (FBS).a.
-lactone
DMEM
(pH = 7.5)
DMEM
inhibitor [a]
+ 5% FBS[b]
21
25
20
70
<3
120
cycloaddition conditions.[31] SDS-PAGE and in-gel fluorescence
scanning were employed to separate and detect labeled
proteins, respectively (Figure 3A). Importantly, labeling of
lactones and also the greater stability to buffer/serum (120 vs <
3 min) leading to greater whole cell cytotoxicity for inhibitor 25
compared to 21 (10.0 vs 45.8 M, respectively).
Figure 3. A) In situ ABPP labeling of MCF-7 and HeLa cells with increasing concentrations of alkyne ABPP probe 28. B) Identification
of FAS and proteasome beta-5 by Western Blotting using specific anti-FAS and anti-PSB-5 antibodies. C) Competitive labeling with
inhibitors 21, 25 and ABPP probe 28. (PSB-5: proteasome subunit beta type, FAS: Fatty acid synthase)

thioester (S3) required for the preparation of ketene acetal 10b are
provided in the SI. Aldehyde 9 was prepared according to our previously
described procedure.[22a] Dipeptide 15c was prepared according to the
previously published procedure and data matched that previously
reported.[22a] Commercially available protected amino acids, 13a, 13b,
13c, 13d, 13e, 14b were purchased and used as received. Protected
amino acids, 14a[23b] and 4-ethynylbenzyl (tert-butoxycarbonyl)-L-
alaninate[32] were prepared based on known procedures.
Conclusions
Several dual inhibitors of the proteasome and fatty acid
synthase were designed and synthesized based on the original
structures of orlistat and belactosin C. Exploiting the trans-
disubstituted -lactone pharmacophore which covalently binds to
these two enzyme active sites and modifying the dipeptide,
which was optimized based on analysis of the X-ray structures
of
proteasome-homobelactosin
C
and
FAS-TE-orlistat
(E/Z)-2-((1-((Triethylsilyl)oxy)oct-1-en-1-yl)thio)pyridine (10a). The
synthesis was performed as previously described[26] and related to a
published procedure.[22a] To a solution of S-(pyridin-2-yl) octanethioate[33]
(5.30 g, 22.7 mmol, 1 equiv), DMF (2.11 mL, 27.2 mmol, 1.2 equiv) and
Et3N (3.78 mL, 27.2, 1.2 equiv) in 100 mL of DCM was added LiHMDS
(1M in THF, 52.3 mL, 52.3 mmol, 2.3 equiv) at -78 oC under N2. After
stirring for 30 min, TESCl (7.69 mL, 45.4 mmol, 2 equiv) was added. The
mixture was continued to stir at -78 oC for another 2 h. The reaction was
quenched with pH 7 buffer (40 mL) and the organic layer was separated,
dried over MgSO4 and concentrated. The residue was purified by flash
chromatography (SiO2, hexanes:MTBE = 30:1) to give ketene acetal 10a
as a yellow oil (5.1 g, 64%, ~13:1 ratio of E/Z geometrical isomers). Data
provided for major E-olefin isomer: 1H NMR (500 MHz, CDCl3) δ 8.42-
8.41 (m, 1H), 7.56-7.52 (m, 1H), 7.34 (d, J = 8.0 Hz, 1H), 7.01-6.98 (m,
1H), 5.38 (t, J = 7.3 Hz, 1H), 2.18 (q, J = 7.3 Hz, 2H), 1.44-1.23 (m, 11H),
0.90-0.84 (m, 9H), 0.67-0.62 (m, 6H). 13C NMR (75 MHz, CDCl3) δ 160.6,
149.6, 139.4, 136.7, 124.5, 121.6, 119.8, 31.8, 29.2, 27.0, 22.8, 14.3, 6.7
(2C), 6.0, 5.3 (2C).
complexes and structure-activity relationships, we successfully
identified a dual inhibitor 21 with greater enzymatic inhibition to
both targets (ChT-L: IC50 = 0.37 µM; FAS IC50 = 1.5 µM; HeLa
cells IC50 = 45.8 µM) compared to our original lead compound
17. In addition, an -disubstituted -lactone dual inhibitor 25
displayed the highest potency in a cell-based cytotoxicity assay
(HeLa cells IC50 = 10.0 µM) which along with serum stability
studies suggest greater hydrolytic stability of this -lactone with
attendant improved cell-based activity. We confirmed targeting
of the proteasome and FAS-TE in HeLa cells through activity
based-protein profiling with an alkyne probe modeled after our
best enzymatic inhibitor 21. These studies provide proof of
concept for the potential of dual inhibition of FAS-TE and the
proteasome with orlistat-belactosin hybrid molecules.
In
addition, it raises the possibility that previously described
belactosin derivatives should be analyzed for cross-reactivity
with FAS-TE. The ability to target an enzyme that is a validated
target for chemotherapy, namely the proteasome, and an
enzyme that has attracted great interest for cancer
chemotherapy, namely FAS-TE, by a single molecule provides
proof-of-principle for targeting both these enzymes with -
lactone-based dual inhibitors.
Ketene acetal 10b. To a solution of LiHMDS in THF (1 M, 8.76 mL, 2.0
equiv) was added DMF (406 L, 5.26 mmol, 1.2 equiv) at -78 oC followed
by TESCl (742 L, 4.38 mmol, 1 equiv). After stirring for 10 min, a
solution of thioester S3 (1.10 g, 4.38 mmol, 1.0 equiv) in 40 mL of THF
was added and the solution was stirred for another 5 h at the same
temperature. The reaction was quenched by aq. satd. NH4Cl solution,
and was extracted with MTBE (3 x 50 mL). The combined organic layers
were washed with brine, dried over anhydrous MgSO4 and concentrated
in vacuo. The residue was purified by flash chromatography
(SiO2,hexanes:MTBE = 100:150:1) to give 10b as an ~1:1 mixture of
E- and Z-isomers (1.01 g, 63%). 1H NMR (300 MHz, CDCl3)8.43 (m,
1H), 7.54 (td, J = 7.9, 2.0 Hz, 1H), 7.26 (t, J = 7.9 Hz, 1H), 7.00 (m, 1H),
2.29 (m, 2H), 1.89/1.86 (s, 3H), 1.36-1.24 (m, 8H), 1.03-0.86 (m, 12H),
0.69-0.60 (m, 6H). 13C NMR (75 MHz, CDCl3) δ 200.9 181.7, 151.8,
150.3, 137.4, 130.4, 123.6, 105.2, 34.2, 31.8, 29.4, 27.3, 22.8, 17.7,
14.3, 7.0 (3C), 6.6 (3C).
Experimental Section
General Information. All reactions were carried out under a nitrogen
atmosphere in oven-dried glassware. Acetonitrile, dichloromethane,
tetrahydrofuran, and methanol were purified by passage through
activated molecular sieves or alumina (solvent drying system). DMF was
dried over molecular sieves before use. All commercial reagents were
used as received. 1H and 13C NMR spectra were recorded on INOVA-
500. 1H NMR chemical shifts are reported as δ values in ppm relative to
CDCl3 (7.26 ppm,), coupling constants (J) are reported in Hertz (Hz), and
multipilicity
follows
convention.
Unless
indicated
otherwise,
deuterochloroform (CDCl3) served as an internal standard (77.2 ppm) for
all 13C spectra. Flash column chromatography was performed using 60Å
Tandem Mukaiyama Aldol-Lactonization: Synthesis of -lactone
esters 11 and 11a’. ZnCl2 (1.47 g, 10.82 mmol, 2 equiv) was heated to
fuse under vacuum and was cooled to ambient temperature, to which a
solution of aldehyde 9 (2.08 g, 5.41 mmol, 1.0 equiv) in 50 mL of CH2Cl2
was added. After stirring at ambient temperature (23 oC) for 10 min, a
solution of ketene acetal 10a (2.09 g, 5.95 mmol, 1.1 equiv) in 10 mL of
CH2Cl2 was added. After 30 minutes the dark suspension was diluted
with MTBE (100 mL), washed with H2O (5 mL) and brine (5 mL), and
dried over MgSO4. After concentration the residue was purified by flash
chromatography (SiO2, hexanes:MTBE = 5:1) to obtain a diastereomeric
mixture of 11a and 11a’. The diastereomeric mixture was further
silica gel as
a stationary phase using a gradient solvent system
(EtOAc/n-hexane as eluent unless specified otherwise). Mass spectra
were obtained at the Center for Chemical Characterization and Analysis
(Texas A&M University). Thin layer chromatography (TLC) was
performed using glass-backed silica gel 60F254. LC/MS was carried out
using an ion trap HPLC/MS instrument with an Agilent Poroshell EC-C-18
2.7 micron column (50
x 3.0 mm), eluting with a gradient of 5%
acetonitrile/95% water  95% acetonitrile/5% water over 12 min or 20
min. Signals were detected with UV 254 nm and MS ion trap (ionization
modes: negative or positive APCI or positive or negative ESI, scan range
separated
by
a
second
flash
chromatography
((SiO2,
100-1000 a.m.u.).
Synthetic details and characterization data for

hexanes:DCM:MTBE = 30:10:1) to give 11a (1.13 g, 41%) and 11a’ (530
mg, 19%) as colorless oils. Data for 11a: Rf = 0.33 (hexanes:MTBE =
5:1), []D -22 (c = 1.0, CHCl3). 1H NMR (500 MHz, CDCl3) 7.34-7.32
(R)-((4S,5S)-5-((R)-methoxy(phenyl)methyl)-2,2-dimethyl-1,3-
dioxolan-4-yl)(phenyl)methyl (3R, 4S)-3-hexyl-3-methyl-4-
23
oxooxetane-2-carboxylate (11b’). Obtained as a colorless oil (72 mg,
9%). []D25 + 43 (c = 1.0, CHCl3). 1H NMR (500 MHz, CDCl3) 7.34-7.25
(m, 6H), 7.12-7.10 (m, 4H), 5.25 (d, J = 5.8 Hz, 1H), 4.70 (s, 1H), 4.22
(dd, J = 6.6, 5.8 Hz, 1H), 4.02 (t, J = 6.2 Hz, 1H), 3.71 (d, J = 6.2 Hz, 1H),
3.15 (s, 3H), 1.76-1.72 (m, 2H), 1.47 (s, 6H), 1.35-1.25 (m, 8H), 1.01 (s,
3H), 0.90 (t, J = 7.0 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 172.61,
166.68, 137.28, 136.03, 129.15, 128.85 (2C), 128.73, 128.68 (2C),
127.93 (2C), 127.76 (2C), 111.14, 84.27, 80.98, 79.20, 77.08, 75.31,
62.26, 56.97, 35.22, 31.66, 29.30, 27.87, 27.59, 24.45, 22.63, 15.46,
14.18. LRMS (ESI+): Calcd. For C31H40O7Na ([M+Na]+), 547.3. Found:
547.4.
(m, 3H), 7.30-7.27 (m, 3H), 7.14-7.12 (m, 2H), 7.09-7.07 (m, 2H), 5.16 (d,
J = 5.3 Hz, 1H), 4.60 (d, J = 4.4 Hz, 1H), 4.17 (dd, J = 6.8, 5.4 Hz, 1H),
4.04 (t, J = 6.7 Hz, 1H), 3.73 (d, J = 6.2 Hz, 1H), 3.63-3.59 (m, 1H), 3.16
(s, 3H), 1.93-1.78 (m, 2H), 1.46 (s, 3H), 1.45 (s, 3H), 1.45-1.41 (m, 2H),
1.35-1.26 (m, 6H), 0.90 (t, J = 6.9 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ
169.1, 167.2, 137.3, 136.0, 129.1, 128.89 (2C), 128.83 (2C), 128.79,
127.8 (2C), 127.5 (2C), 111.1, 84.4, 80.8, 79.4, 77.1, 71.5, 57.7, 57.0,
31.6, 28.8, 27.84, 27.78, 27.5, 26.8, 22.7, 14.2. LRMS (ESI+): Calcd. For
C30H38O7Na ([M+Na]+), 533.3. Found: 533.9. Data for 11a’: Rf = 0.33
23
(hexanes:MTBE = 5:1), []D = -0.50 (c = 1.0, CHCl3). 1H NMR (500
MHz, CDCl3) 7.34-7.32 (m, 3H), 7.31-7.27 (m, 3H), 7.15-7.11 (m, 4H),
5.19 (d, J = 5.4 Hz, 1H), 4.59 (d, J = 4.3 Hz, 1H), 4.18 (dd, J = 7.0, 5.4
Hz, 1H), 4.03 (dd, J = 7.0, 6.2 Hz, 1H), 3.71 (d, J = 6.2 Hz, 1H), 3.68
(ddd, J = 8.7, 6.6, 4.3 Hz, 1H), 3.15 (s, 3H), 1.94-1.79 (m, 2H), 1.47 (s,
3H), 1.45 (s, 3H), 1.34-1.26 (m, 8H), 0.89 (t, J = 6.9 Hz, 3H); 13C NMR
(75 MHz, CDCl3) δ 169.2, 167.2, 137.3, 136.0, 129.1, 128.83 (2C),
128.80 (2C), 128.75, 127.7 (2C), 127.5 (2C), 111.0, 84.2, 80.7, 79.4,
77.1, 71.5, 57.6, 57.0, 31.5, 28.8, 27.8, 27.7, 27.4, 26.7, 22.6, 14.2.
LRMS (ESI+): Calcd. For C30H38O7Na ([M+Na]+), 533.3. Found: 533.9.
(R)-((4S,5S)-5-((R)-methoxy(phenyl)methyl)-2,2-dimethyl-1,3-
dioxolan-4-yl)(phenyl)methyl
(3S,
4S)-3-hexyl-3-methyl-4-
oxooxetane-2-carboxylate (11b’’). Obtained as a colorless oil (125 mg,
25
14%). []D + 66 (c = 1.0, CHCl3). 1H NMR (500 MHz, CDCl3)7.35-
7.26 (m, 6H), 7.15-7.11 (m, 4H), 5.19 (d, J = 5.5 Hz, 1H), 4.60 (s, 1H),
4.22 (dd, J = 6.9, 5.5 Hz, 1H), 4.01 (t, J = 6.4 Hz, 1H), 3.70 (d, J = 6.1
Hz, 1H), 3.16 (s, 3H), 1.52 (s, 3H), 1.49 (s, 3H), 1.48 (s, 3H), 1.40-1.30
(m, 2H), 1.27-1.20 (m, 4H), 1.16-1.03 (m, 4H), 0.85 (t, J = 7.2 Hz, 3H).
13C NMR (125 MHz, CDCl3) δ 172.51, 166.43, 137.36, 136.04, 129.10,
128.82 (2C), 128.72 (2C), 128.66, 127.90 (2C), 127.71 (2C), 111.12,
84.17, 80.90, 79.19, 77.63, 77.17, 61.83, 56.93, 31.61, 31.44, 29.42,
27.87, 27.51, 23.88, 22.63, 19.59, 14.10. LRMS (ESI+): Calcd. For
C31H40O7Na ([M+Na]+), 547.3. Found: 547.4.
Tandem Mukaiyama Aldol-Lactonization: Synthesis of -lactone
esters 11b, 11b’, 11b’’and 11b’’’. ZnCl2 (559 mg, 4.10 mmol, 2.5 equiv)
was fused by heating under vacuum and then cooled to ambient
temperature and then 0 oC. A solution of aldehyde 9 (630 mg, 1.64
mmol, 1.0 equiv) in 5 mL of CH2Cl2 was added. After stirring for 10 min, a
solution of ketene acetal 10b (599 mg, 1.64 mmol, 1 equiv) in 5 mL of
CH2Cl2 was added. The mixture was stirred at ambient temperature (23
oC) for 15 h and then the reaction was quenched by adding water (5 mL).
The mixture was poured into 125 mL of MTBE and the organic layer was
washed with brine. After drying over MgSO4, the organic solution was
concentrated. The residue was purified by a flash chromatography (SiO2,
hexanes:MTBE = 5:1) to isolate the trans-diastereomers (containing 11b
and 11b’) and the cis-diastereomers (containing 11b’’ and 11b’’’). The
trans-diastereomers were submitted to a 2nd flash chromatographic
separation ((SiO2, hexanes:CH2Cl2:MTBE = 5:4:0.08) to isolate 11b and
11b’. A 2nd flash chromatographic separation enabled separation of the
(R)-((4S,5S)-5-((R)-methoxy(phenyl)methyl)-2,2-dimethyl-1,3-
dioxolan-4-yl)(phenyl)methyl
(3R,
4R)-3-hexyl-3-methyl-4-
oxooxetane-2-carboxylate (11b’’’). Obtained as a colorless oil (125 mg,
25
14%). []D
+ 48 (c = 1.0, CHCl3). 1H NMR (500 MHz, CDCl3) 7.34-
7.24 (m, 6H), 7.15-7.09 (m, 4H), 5.31 (d, J = 5.9 Hz, 1H), 4.62 (s, 1H),
4.26 (t, J = 6.3 Hz, 1H), 4.001 (t, J = 6.3 Hz, 1H), 3.67 (d, J = 6.0 Hz, 1H),
3.14 (s, 3H), 1.494 (s, 3H), 1.486 (s, 3H), 1.47 (s, 3H), 1.30-1.27 (m, 2H),
1.20-1.12 (m, 4H), 1.04-0.97 (m, 4H), 0.82 (t, J = 7.3 Hz, 3H). 13C NMR
(125 MHz, CDCl3) δ 172.40, 166.46, 137.35, 135.83, 129.16, 128.80
(2C), 128.71 (2C), 128.64, 128.08 (2C), 127.74 (2C), 111.16, 84.07,
81.06, 79.10, 77.45, 77.32, 61.79, 56.97, 31.60, 31.34, 29.37, 27.90,
27.61, 23.88, 22.62, 19.67, 14.10. LRMS (ESI+): Calcd. For C31H40O7Na
([M+Na]+), 547.3. Found: 547.4.
syn-diastereomers 11b’’ and 11b’’’ (SiO2, hexanes:CH2Cl2:MTBE
=
20:20:1).
(R)-((4S,5S)-5-((R)-methoxy(phenyl)methyl)-2,2-dimethyl-1,3-
Representative procedure for hydrogenolytic cleavage of the chiral
auxiliary: Synthesis of (3S, 4R)-3-hexyl-4-oxooxetane-2-carboxylic
acid (12a). In a round-bottomed flask was added Pd/C (80 mg, 10%
w/w, 0.076 mmol, 0.09 equiv) and the flask was evacuated by vacuum for
~30 minutes, and then refilled with N2. A THF solution of 11a (429 mg,
0.840 mmol, 1 equiv, 0.05M) was added. The flask was charged with a
hydrogen balloon and the reaction was kept at ambient temperature (23
oC) for 15 h with stirring. The mixture was filtered through a short celite
pad and washed with EtOAc (5 mL). After concentration the crude
product was purified by silica gel flash chromatography (hexanes:MTBE
dioxolan-4-yl)(phenyl)methyl
(3S,
4R)-3-hexanoyl-3-methyl-4-
oxooxetane-2-carboxylate (11b). Obtained as a colorless oil (72 mg,
9%). []D25 + 59 (c = 1.0, CHCl3). 1H NMR (500 MHz, CDCl3)7.35-7.27
(m, 6H), 7.15-7.11 (m, 4H), 5.21 (d, J = 5.8 Hz, 1H), 4.69 (s, 1H), 4.22
(dd, J = 6.8, 5.8 Hz, 1H), 4.00 (t, J = 6.3 Hz, 1H), 3.68 (d, J = 6.0 Hz, 1H),
3.16 (s, 3H), 1.80-1.77 (m, 2H), 1.48 (s, 3H), 1.47 (s, 3H), 1.39-1.30 (m,
8H), 1.22 (s, 3H), 0.91 (t, J = 6.6 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ
172.7, 166.64, 137.34, 136.06, 129.11, 128.81 (2C), 128.74 (2C),
128.72, 127.79 (2C), 127.70 (2C), 110.99, 84.13, 80.91, 79.15, 76.94,
75.48, 62.26, 56.95, 35.31, 31.63, 29.30, 27.76, 27.55, 24.43, 22.63,
15.73, 14.17. LRMS (ESI+): Calcd. For C31H40O7Na ([M+Na]+), 547.3.
Found: 547.4.
23
= 2:1) to give 12a as a colorless oil (116 mg, 69%). []D -20 (c = 5.8,
CHCl3). 1H NMR (300 MHz, CDCl3) 10.19 (brs, 1H), 4.65 (d, J = 4.4 Hz,
1H), 3.82 (ddd, J = 8.7, 6.7, 4.3 Hz, 1H), 2.01-1.80 (m, 2H), 1.54-1.23 (m,
8H), 0.88 (t, J = 6.8 Hz, 3H). 13C NMR (75 MHz, CDCl3) 173.8, 168.9,

71.0, 58.2, 31.6, 28.8, 27.9, 26.7, 22.6, 14.2. HRMS (ESI-): Calcd. for
C10H15O4 ([M-H]-), 199.0976. Found: 199.0982.
14a (1.51 g, 4.47 mmol) to give the known dipeptide 15a (2.12 g, 87%
yield) as a white solid. The characterization data are identical to the
literature.[23b]
(3S, 4R)-3-Hexyl-4-oxooxetane-2-carboxylic acid (12a’). Based on the
representative hydrogenolysis procedure, the enantiomeric -lactone
Cbz-Gly-BnO-Lys(Boc) dipeptide (15b). N-Cbz-Gly (13b) (1.03 g, 4.92
mmol) was dissolved in dry CH2Cl2 (20 mL) in a dry, round-bottomed
flask under a positive atmosphere of nitrogen at 22 ºC and then the
solution was cooled to 0 ºC. The EDAC (1.03 g, 5.37 mmol), HOBt (0.726
g, 5.37 mmol) and i-Pr2NEt (2.34 mL, 13.43 mmol) were then added
sequentially and the resulting solution was stirred at 0 ºC for 30 min.
BnO-Lys(Boc) (14a) (1.51 g, 4.47 mmol) was then added via cannula
dissolved in dry CH2Cl2 (10 mL). After the addition was complete, the
reaction mixture was allowed to warm to 22 ºC and stirred at this
temperature for 4 h. The reaction was quenched by addition of water (30
mL), the layers were separated, and the organic fraction was dried over
Na2SO4, filtered and concentrated under reduced pressure. The crude
mixture was purified by MPLC (SiO2, gradient: 100% hexane 100%
EtOAc) to afford the desired dipeptide (2.01 g, 85% yield) as a viscous
colorless oil. 1H NMR (500 MHz, CDCl3) 7.36-7.30 (m, 10H), 6.89 (bs,
1H), 5.75 (bs, 1H), 5.18 (d, J = 12.0 Hz, 1H), 5.12 (d, J = 12.0 Hz, 1H),
5.11 (s, 2H), 4.72 (bs, 1H), 4.64-4.60 (m, 1H), 3.91 (d, J = 5.0 Hz, 2H),
3.05-2.96 (m, 2H), 1.87-1.78 (m, 1H), 1.70-1.62 (m, 1H), 1.46-1.38 (m,
2H), 1.42 (s, 9H), 1.29-1.22 (m, 2H). 13C NMR (125 MHz, CDCl3)
172.16, 169.19, 156.79, 156.26, 136.33, 135.37, 128.78, 128.66 (2C),
128.45 (2C), 128.31, 128.19, 79.27, 67.34, 67.26, 52.23, 44.54, 40.11,
31.81, 29.55, 28.57 (3C), 22.39. FT-IR (neat, cm-1): 3326, 2934, 1682,
1520. FT-IR (neat, cm-1): 3326, 2934, 1682, 1520. HRMS (ESI+): Calcd.
for C28H38N3O7 ([M+H]+), 528.2710. Found: 528.2681.
25
carboxylic acid 12a’ was obtained as a colorless oil (135 mg, 66%). []D
+18 (c = 6.8, CHCl3). 1H NMR and 13C NMR spectra were identical to that
of 12a. HRMS (ESI+): Calcd. For C10H15O4 ([M-H]-), 199.0976. Found:
199.0963.
(3S, 4R)-3-hexyl-3-methyl-4-oxooxetane-2-carboxylic acid (12b).
According to the representative hydrogenolysis procedure, anti--lactone
acid 12b was prepared from ester 11b as a colorless oil (20.5 mg, 75%).
[]D25 +11.6 (c = 2.1, CHCl3). 1H NMR (500 MHz, CDCl3) 8.49 (brs, 1H),
4.75 (s, 1H), 1.83-1.80 (m, 2H), 1.53-1.46 (m, 1H), 1.36 (s, 3H), 1.33-
1.24 (m, 7H), 0.89 (t, J = 7.0 Hz, 3H). 13C NMR (125 MHz, CDCl3)
172.52, 172.36, 75.2, 62.45, 35.24, 31.66, 29.32, 24.47, 22.67, 15.81,
14.19. HRMS (ESI-): Calcd. For C11H17O4 ([M-H]-), 213.1132. Found:
213.1131.
(3R, 4S)-3-hexyl-3-methyl-4-oxooxetane-2-carboxylic acid (12b’).
According to the general hydrogenolysis procedure, the enantiomeric
anti--lactone 12b’ was prepared from ester 11b’ as a colorless oil (12.8
mg, 68%). []D25 -10.5 (c = 1.3, CHCl3). The 1H NMR and 13C NMR were
identical to the enantiomeric anti--lactone acid 12b. HRMS (ESI-):
Calcd.
(3R, 4R)-3-hexyl-3-methyl-4-oxooxetane-2-carboxylic acid (12b’’).
According to the general hydrogenolysis procedure, the diastereomeric
syn--lactone 12b’’ was prepared as a colorless oil (16.7 mg, 64%).
4-BrBzAla-BnO-Lys(Boc) dipeptide (15d). 4-BrBz-Ala-OH (13c) (0.536
g, 1.97 mmol) was dissolved in dry CH2Cl2 (10 mL) in a round-bottomed
flask under positive atmosphere of nitrogen at 22 °C then the solution
was cooled to 0 °C. EDAC (0.412 g, 2.15 mmol), HOBt (0.290 g, 2.15
mmol), and i-Pr2-NEt (0.935 mL, 5.37 mmol) were then added
sequentially and the resulting solution was stirred at 0 °C for 30 minutes.
BnO-Lys(Boc) (14a) (0.602 g, 1.79 mmol) was then added via cannula
dissolved in dry CH2Cl2 (5 mL). After the addition was completed, the
reaction mixture was allowed to warm to 22 °C and stirred at this
temperature for 12 h. To quench the reaction, the solution was diluted
with deionized water (15 mL), the organic fraction was dried over MgSO4,
filtered and concentrated under reduced pressure. The crude was
purified by MPLC (SiO2, gradient: 100% hexane 100% EtOAc) to
afford 15d (0.326 g, 31% yield) as a white solid and as a 2:1 mixture of
rotamers. Data for the major rotamer: 1H NMR (500 MHz, CDCl3) δ 7.65
(d, J = 8.5 Hz, 2H), 7.54 (d, J = 8.5 Hz, 2H), 7.35-7.29 (m, 5H), 7.18 (d, J
= 8.0 Hz, 1H), 7.10 (d, J = 4.5 Hz, 1H), 5.16 (d, J = 12.0 Hz, 1H), 5.08 (d,
J = 12.0 Hz, 1H), 4.81-4.74 (m, 1H), 4.66-4.59 (m, 2H), 3.09-2.97 (m,
2H), 1.90-1.84 (m, 1H), 1.76-1.69 (m, 1H), 1.49 (d, J = 7.0, 3H) 1.47-1.40
(m, 2H), 1.44 (s, 9H), 1.36-1.26 (m, 2H). 13C NMR (125 MHz, CDCl3) δ
172.49, 172.04, 166.40, 156.29, 135.37, 132.77, 131.96 (2C), 128.94
(2C), 128.79 (2C), 128.69 (2C), 128.46, 126.67, 79.38, 67.37, 52.38,
49.37, 40.10, 31.79, 29.60, 28.62 (3C), 22.52, 18.89. FT-IR (neat, cm-1):
3417, 1642, 1535, 1171. HRMS (ESI+): Calcd. For C28H37BrN3O6
([M+H]+), 590.1866. Found: 590.1895.
25
[]D -3.7 (c = 1.7, CHCl3). 1H NMR (500 MHz, CDCl3) 8.49 (brs, 1H),
4.75 (s, 1H), 1.83-1.80 (m, 2H), 1.53-1.46 (m, 1H), 1.36 (s, 3H), 1.33-
1.24 (m, 7H), 0.89 (t, J = 7.0 Hz, 3H). 13C NMR (125 MHz, CDCl3)
172.52, 172.36, 75.2, 62.45, 35.24, 31.66, 29.32, 24.47, 22.67, 15.81,
14.19. HRMS (ESI-): Calcd. For C11H17O4 ([M-H]-), 213.1132. Found:
213.1131.
The relative configuration of -lactone acids 12a, 12a’, 11b, 11b’, 11b’’,
11b’’’was assigned based on coupling constant analysis or nOe
correlations (via ROESY experiments). The absolute configuration was
assigned by comparison of optical rotations to known, related -
lactones.[27e, 34] See Supporting Information for details.
4-Ethynylbenzyl-L-ala (14c). To the solution of 4-ethynylbenzyl (tert-
butoxycarbonyl)-L-alaninate[32] (1.00 g, 3.30 mmol) in 12 mL of DCM was
slowly added TFA (1.5 mL) at 0 oC. The solution was kept in a 4 oC
refrigerator for 40 h. After concentration the crude product was purified
on silica gel chromatography (DCM:MeOH = 20:1) to give the product as
a yellow oil (880 mg, 89%) in the form of a TFA salt. 1H NMR (500 MHz,
CDCl3) 8.43 (brs, 3H), 7.44 (d, J = 8.1 Hz, 2H), 7.23 (d, J = 8.1 Hz, 2H),
5.14 (d, J = 12.5 Hz, 1H), 5.08 (d, J = 12.5 Hz, 1H), 4.08 (q, J = 7.2 Hz,
1H), 3.09 (s, 1H), 1.55 (d, J = 7.2 Hz, 3H). 13C NMR (125 MHz, CDCl3)
170.2, 135.1, 132.6, 128.2, 122.8, 83.1, 78.2, 67.9, 49.3, 15.9. HRMS
(ESI+): Calcd. for C12H14N2O+ ([M]+), 204.1019. Found: 204.1023.
Cbz-Ala-BnO-Lys(Boc) dipeptide (15a). Based on the literature
procedure,[23b] N-Cbz-Ala (1.1 g, 4.92 mmol) (13a) coupled with amine
BnO-Ala-Cbz-Lys(Boc) dipeptide (15e). Cbz-Lys(Boc)-OH (13d) (2.0 g,
5.26 mmol) was dissolved in dry CH2Cl2 (20 mL) in a dry round-bottomed

flask under a positive atmosphere of nitrogen at 22 ºC and then the
solution was cooled to 0 ºC. The EDAC (1.21 g, 6.31 mmol), HOBt (0.852
g, 6.31 mmol) and i-Pr2NEt (2.75 mL, 15.77 mmol) were then added
sequentially and the resulting solution was stirred at 0 ºC for 30 min. The
H-Ala-OBn p-tosylate (14b) (2.03 g, 5.78 mmol) was then added via
cannula as a solution in dry CH2Cl2 (10 mL). After the addition was
completed, the reaction mixture was allowed to warm to 22 ºC and stirred
at this temperature for 4 h. To quench the reaction, the solutions was
diluted with water (30 mL), the organic fraction was dried over Na2SO4,
filtered and concentrated under reduced pressure. The crude was
purified by MPLC (SiO2, gradient: 100% hexane 100% EtOAc) to
afford the desired dipeptide 15e (2.52 g, 89% yield) as a white solid. 1H
NMR (500 MHz, CDCl3) δ 7.37-7.32 (m, 10H), 6.93 (bs, 1H), 5.68 (bs,
1H), 5.20 (d, J = 12.0 Hz, 1H), 5.12 (d, J = 12.0 Hz, 1H), 5.08 (s, 2H),
4.80 (bs, 1H), 4.60 (p, J = 7.0 Hz, 1H), 4.24 (t, J = 7.0 Hz, 1H), 3.09-3.03
(m, 2H), 1.84-1.78 (m, 1H), 1.67-1.60 (m, 1H), 1.48-1.35 (m, 4H), 1.41 (s,
9H), 1.37 (d, J = 7.0 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 172.67,
171.72, 156.39, 156.29, 136.33, 135.42, 128.72 (2C), 128.60 (2C),
128.54, 128.27 (2C) , 128.24, 128.15 (2C), 79.17, 67.26, 67.08, 54.64,
48.25, 39.88, 32.37, 29.50, 28.55 (3C), 22.36, 18.01. FT-IR (neat, cm-1):
3306, 2937, 1694, 1538. FT-IR (neat, cm-1): 3306, 2937, 1694, 1538.
HRMS (ESI+) Calcd. for C29H40N3O7 ([M+H]+): 542.2866. Found:
542.2850.
and the crude residue was purified by MPLC (SiO2, gradient: 100%
hexanes 100 % EtOAc) to afford the desired hydantoin 15ff (0.141 g,
41% yield) as a colorless viscous oil. 1H NMR (500 MHz, CDCl3) 7.37-
7.31 (m, 5H), 6.43 (bs, 1H), 5.20 (d, J = 12.5 Hz, 1H), 5.13 (d, J = 12.5
Hz, 1H), 4.79 (q, J = 7.5 Hz, 1H), 4.62 (bs, 1H), 4.01 (t, J = 6.0 Hz, 1H),
3.12-3.03 (m, 2H), 1.89-1.82 (m, 1H), 1.70-1.61 (m, 1H), 1.66 (d, J = 7.5
Hz, 3H), 1.48-1.32 (m, 4H), 1.45 (s, 9H). 13C NMR (125 MHz, CDCl3)
173.55, 169.56, 156.78, 156.42, 135.40, 128.78 (2C), 128.61, 128.48
(2C), 79.58, 67.77, 57.05, 48.38, 40.04, 31.15, 29.72, 28.67 (3C), 21.63,
15.00. FT-IR (neat, cm-1): 3333, 2931, 1717, 1510. HRMS (ESI+) Calcd.
for C22H32N3O6 ([M+H]+): 434.2291. Found: 434.2276.
4-Ethynylbenzyl N-Cbz-N-Boc-L-lysyl-L-alaninate (15g). A solution of
N-Cbz-N-Boc-L-lysine (13d) (253 mg, 0.666 mmol, 1.0 equiv), TFA salt of
4-ethynylbenzyl-L-ala (14c) (200 mg, 0.666 mmol, 1 equiv), EDCI (255
mg, 1.33 mmol, 2 equiv), HOBt (90 mg, 0.666 mmol, 1 equiv) in 3 mL of
DMF and 12 mL of DCM was cooled to 0 oC and TMP (87 mL, 0.666
mmol, 1 equiv) was added. The solution was kelp in a 4 oC refrigerator
for 15 h, diluted with 50 mL of EtOAc and washed with H2O (5 mL) and
brine (5mL). The organic layer was dried over MgSO4 and concentrated.
The residue was purified on a silica gel chromatography (hexanes:EtOAc
= 4:1) to give the product as a yellow oil (250 mg, 66%). 1H NMR (500
MHz, CDCl3) 7.41 (d, J = 8.1 Hz, 2H), 7.28-7.23 (m, 5H), 7.22 (d, J =
8.1 Hz, 2H), 5.76 (d, J = 8.0 Hz, 1H), 5.12 (d, J = 12.6 Hz, 1H), 5.04 (d, J
= 12.6 Hz, 1H), 5.02 (s, 2H), 4.82 (brs, 1H), 4.58-4.48 (m, 1H), 4.21-4.14
(m, 1H), 3.06 (s, 1H), 30.3-2.95 (m, 2H), 1.80-1.69 (m, 1H), 1.63-1.51 (m,
1H), 1.42-1.30 (m, 4H), 1.36 (s, 9H), 1.33 (d, J = 7.3 Hz, 3H). ). 13C NMR
(125 MHz, CDCl3) 172.6, 171.9, 156.5, 136.1, 132.5 (2C), 128.7 (2C),
128.2 (2C), 128.1 (2C), 127.2, 122.4, 116.5, 111.8, 83.3, 79.5, 78.0,
67.2, 66.7, 54.8, 48.4, 39.9, 32.2, 29.5, 28.5, 22.4, 18.0. HRMS (ESI+):
Calcd. for C31H40N3O7+ ([M+H]+), 566.2861. Found: 566.2884.
BnO-Ala-Fmoc-Lys(Boc) dipeptide (15f). Fmoc-Lys(Boc)-OH (13e)
(2.0 g, 4.27 mmol) was dissolved in dry CH2Cl2 (20 mL) in a dry round-
bottomed flask under a positive atmosphere of nitrogen at 22 ºC and then
the solution was cooled to 0 ºC. The EDAC (0.982 g, 5.12 mmol), HOBt
(0.692 g, 5.12 mmol) and i-Pr2NEt (2.23 mL, 12.81 mmol) were then
added sequentially and the resulting solution was stirred at 0 ºC for 30
min. The H-Ala-OBn p-tosylate (14b) (1.65 g, 4.70 mmol) was then
added via cannula as a solution in dry CH2Cl2 (10 mL). After the addition
was complete, the reaction was allowed to warm to 22 ºC and stirred at
this temperature for 1 h. To quench the reaction, the solution was diluted
with water (30 mL), the organic fraction was dried over Na2SO4, filtered
and concentrated under reduced pressure. The crude was purified by
MPLC (SiO2, gradient: 100% hexane 100% EtOAc) to afford the
desired dipeptide 15f (2.28 g, 85% yield) as a white solid. 1H NMR (500
MHz, CDCl3) 7.77 (d, J = 7.5 Hz, 2H), 7.60 (d, J = 7.0 Hz, 2H), 7.40 (t, J
= 7.5 Hz, 2H), 7.36-7.29 (m, 7H), 6.73 (bs, 1H), 5.60 (bs, 1H), 5.20 (d, J
= 12.0 Hz, 1H), 5.13 (d, J = 12.0 Hz, 1H), 4.73 (bs, 1H), 4.62 (p, J = 7.0
Hz, 1H), 4.38 (d, J = 7.0 Hz, 2H), 4.21 (t, J = 7.0 Hz, 2H), 3.15-3.03 (m,
2H), 1.89-1.82 (m, 1H), 1.70-1.63 (m, 1H), 1.48-1.39 (m, 4H), 1.44 (s,
9H), 1.42 (d, J = 7.0 Hz, 3H). 13C NMR (125 MHz, CDCl3) 172.69,
171.57, 156.36, 144.02, 141.4, 135.43, 128.80 (2C), 128.64 (2C), 128.38
(2C), 128.33 (2C), 127.90 (2C), 127.26 (2C), 125.29 (2C), 120.16 (2C),
79.34, 77.43, 67.42, 54.72, 48.39, 47.30, 39.93, 32.45, 29.62, 28.63
(3C), 22.45, 18.24. FT-IR (neat, cm-1): 3311, 2934, 1684, 1532. HRMS
(ESI+) Calcd. for C36H44N3O7 ([M+H]+): 630.3179. Found: 630.3203.
Cbz-Ala-BnO-Lys dipeptide (16a). Cbz-Ala-BnO-Lys(Boc) dipeptide
(15a) (1.0 g, 1.85 mmol) was dissolved in dry CH2Cl2 (10 mL) in a dry,
round-bottomed flask under a positive atmosphere of nitrogen at 22 ºC
and then TFA was added (1.41 mL, 18.46 mmol). The reaction mixture
was stirred for 8 h at 22 ºC and then concentrated under reduced
pressure and diluted with toluene (2 x 10 mL) and concentrated to
provide the desired Cbz-Ala-BnO-Lys dipeptide (1.03 g, 98% yield) in the
form a TFA salt as a viscous colorless oil. 1H NMR (500 MHz, CDCl3)
7.97 (bs, 3H), 7.44 (d, J = 7.5 Hz, 1H), 7.35-7.27 (m, 10H), 6.01 (d, J =
7.5 Hz, 1H), 5.16 (d, J = 12.0 Hz, 1H), 5.08 (d, J = 12.0 Hz, 1H), 5.06 (d,
J = 12.0 Hz, 1H), 4.99 (d, J = 12.0 Hz, 1H), 4.56-4.51 (m, 1H), 4.32-4.29
(m, 1H), 2.82-2.74 (m, 2H), 1.82-1.75 (m, 1H), 1.67-1.56 (m, 2H), 1.55-
1.46 (m, 1H), 1.30 (d, J = 7.0 Hz, 3H), 1.37-1.25 (m, 2H). 13C NMR (125
MHz, CDCl3) 173.41, 171.84, 162.67 (q, J = 26.75 Hz, CF3CO2–),
156.64, 136.28, 135.38, 128.77 (2C), 128.72, 128.67 (2C), 128.46,
128.35 (2C), 127.95 (2C), 116.75 (q, J = 288.75 Hz, CF3CO2–), 67.42,
67.18, 52.19, 50.50, 39.28, 30.88, 26.63, 21.93, 18.61. FT-IR (neat, cm-
1): 3300, 2939, 1673, 1531. HRMS (ESI+): Calcd. for C24H32N3O5+ ([M]+),
442.2336. Found: 442.2365.
Boc-hydantoin (15ff). BnO-Ala-Fmoc-Lys(Boc) dipeptide (15f) (0.5 g,
0.793 mmol) was dissolved in dry CH2Cl2 (5 mL) in a dry round-bottomed
flask under a positive atmosphere of nitrogen at 22 ºC and then the
triphosgene was added (94.2 mg, 0.318 mmol). The solution was then
cooled to 0 ºC and then the DBU (0.475 mL, 3.18 mmol) was added
dropwise. The reaction mixture was stirred at 0 ºC for 3 h and then at 50
ºC for 8 h. The mixture was then concentrated under reduced pressure
Cbz-Gly-BnO-Lys dipeptide (16b). Cbz-Gly-BnO-Lys(Boc) dipeptide
(15b, 1.0 g, 1.90 mmol) was dissolved in dry CH2Cl2 (10 mL) in a dry,
round-bottomed flask under a positive atmosphere of nitrogen at 22 ºC
and then TFA was added (1.41 mL, 18.46 mmol). The reaction mixture
was stirred for 8 h at 22 ºC and then concentrated under reduced

pressure and diluted with toluene (2 x 10 mL) and concentrated to
provide the desired Cbz-Gly-BnO-Lys dipeptide (16b, 1.04 g, 99% yield)
as a viscous colorless oil. 1H NMR (500 MHz, CDCl3) 7.67 (bs, 3H),
7.44 (d, J = 7.5 Hz, 1H), 7.35-7.26 (m, 10H), 6.12 (bs, 1H), 5.14 (d, J =
12.5 Hz, 1H), 5.09 (d, J = 12.5 Hz, 1H), 5.04 (s, 2H), 4.55-4.50 (m, 1H),
3.83 (d, J = 5.0 Hz, 2H), 2.86-2.77 (m, 2H), 1.83-1.75 (m, 1H), 1.65-1.49
(m, 3H), 1.34-1.23 (m, 2H). 13C NMR (125 MHz, CDCl3) 172.05,
170.66, 162.67 (q, J = 37.50 Hz, CF3CO2–), 157.46, 136.16, 135.20,
128.84 (2C), 128.79 (2C), 128.75 (2C), 128.43 (2C), 127.96, 116.05 (q, J
= 290.62 Hz, CF3CO2–), 67.67, 67.49, 52.24, 44.21, 39.68, 30.96, 26.52,
21.80. FT-IR (neat, cm-1): 3385, 2987, 1781, 1650, 1586. HRMS (ESI+)
Calcd. for C23H30N3O5+ ([M]+): 428.2180. Found: 428.2205.
5.13 (d, J = 12.5 Hz, 1H), 5.05 (d, J = 12.0 Hz, 1H), 5.03 (d, J = 12.0 Hz,
1H), 5.01 (d, J = 12.0 Hz, 1H), 4.51 (q, J = 7.0 Hz, 1H), 4.23 (t, J = 6.0
Hz, 1H), 2.82 (bs, 2H), 1.75-1.67 (m, 1H), 1.63-1.54 (m, 3H), 1.42-1.29
(m, 2H), 1.35 (d, J = 7.0 Hz, 3H). 13C NMR (125 MHz, CDCl3) 172.85,
172.01, 162.04 (q, J = 35.75 Hz, CF3CO2–), 156.65, 136.24, 135.44,
128.75 (2C), 128.67, 128.56 (2C), 128.33 (2C), 128.21(2C), 127.99,
116.56 (q, J = 289.5 Hz, CF3CO2–), 67.30, 67.21, 54.10, 48.39, 39.30,
32.20, 26.49, 21.95, 17.31. FT-IR (neat, cm-1): 3280, 2939, 1681, 1538.
HRMS (ESI+) Calcd. for C24H32N3O5+ ([M]+): 442.2336. Found: 442.2341.
Hydantoin (16f). Boc-hydantoin dipeptide (15ff) (100 mg, 0.230 mmol)
was dissolved in dry CH2Cl2 (3 mL) in a dry, round-bottomed flask under
a positive atmosphere of nitrogen at 22 ºC and then TFA was added
(0.176 mL, 2.31 mmol). The reaction mixture was stirred for 8 h at 22 ºC
and then concentrated under reduced pressure and diluted with toluene
(2 x 3 mL) and concentrated to provide the desired hydantoin dipeptide
(103 mg, 99% yield) as a viscous colorless oil. 1H NMR (500 MHz,
CDCl3) 7.70 (bs, 3H), 7.40 (bs, 1H), 7.36-7.29 (m, 5H), 5.18 (d, J = 12.5
Hz, 1H), 5.12 (d, J = 12.5 Hz, 1H), 4.76 (q, J = 7.5 Hz, 1H), 4.04 (dd, J =
7.0, 4.5 Hz, 1H), 2.93 (bs, 2H), 1.87-1.80 (m, 1H), 1.63-1.56 (m, 3H),
1.62 (d, J = 7.5 Hz, 3H), 1.47-1.38 (m, 2H). 13C NMR (125 MHz, CDCl3)
N-CBz-Ala-O-Bn-Orn dipeptide (16c). N-CBz-Ala-O-Bn-Orn(Boc)
(15c)32 (300 mg, 569 µmol) was dissolved in dry CH2Cl2 (5 mL) in a
round bottomed flask under positive atmosphere of nitrogen at 22 °C.
TFA was added (0.4 mL, 5.54 mmol) to the solution. The reaction mixture
was stirred for 8 h at 22 °C and then concentrated under reduced
pressure and diluted with toluene (2 x 4 mL) and concentrated to provide
16c (315 mg, 99% yield) as viscous yellow oil in the form of a TFA salt.
1H NMR (500 MHz, CD3OD) 7.37-7.30 (m,10 H), 5.18 (d, J = 12.2 Hz,
1H), 5.16 (d, J = 12.2 Hz, 1H), 5.10 (d, J = 12.4 Hz, 1H), 5.04 (d, J =
12.4Hz, 1H), 4.56-4.52 (m, 1H), 4.11 (q, J = 7.3 Hz, 1H), 2.92 (t, J = 7.5
Hz, 2H), 2.04-1.97 (m, 1H), 1.78-1.73 (m, 2H), 1.30 (d, J = 7.2 Hz, 3H).
13C NMR (125 MHz, CD3OD) 176.0, 172.6, 163.0 (q, J = 34.5 Hz,
CF3CO2–), 158.3, 138.1, 137.0, 129.6 (2C), 129.5 (2C), 129.42, 129.37
(2C), 129.0, 128.7, 118.2 (q, J = 289.4 Hz, CF3CO2–), 68.2, 67.6, 52.8,
51.9, 40.1, 29.2, 24.9, 18.1. HRMS (ESI+) Calcd. for C23H30N3O5+ ([M]+):
428.2180. Found: 428.2289.
-
173.64, 169.72, 161.61 (q, J = 38.50 Hz, CF3CO2 ), 157.57, 135.25,
-
128.77 (2C), 128.67, 128.30 (2C), 116.05 (q, J = 286.12 Hz, CF3CCO2 ),
67.84, 56.84, 48.43, 39.78, 30.57, 26.60, 21.07, 14.76. FT-IR (neat, cm-
1): 3179, 2948, 1776, 1713, 1673, 1530. HRMS (ESI+): Calcd. for
C17H24N3O4+ ([M]+), 334.1761. Found: 334.1750.
4-Ethynylbenzyl N-Cbz-L-lysyl-L-alaninate (16g). To the solution of
15g (280 mg, 0.495 mmol) in 10 mL of DCM was slowly added TFA (1.2
mL) at 0 oC. The solution was kept in a 4 oC refrigerator for 15 h. Toluene
(10 mL) was added and most of the DCM and TFA were evaporated in
vacuo. The residue was purified on silica gel chromatography
(DCM:MeOH = 20:1) to give the product 16g as a yellow oil (130 mg,
62%) together with unseparable methyl ketone (~ 40%) derived from
hydration of the alkyne. 1H NMR (500 MHz, CDCl3) 7.83 (brs, 3H), 7.43
(d, J = 8.1 Hz, 2H), 7.29-7.22 (m, 5H), 7.22 (d, J = 8.1 Hz, 2H), 6.02 (brs,
1H), 5.40 (brs, 1H), 5.11 (d, J = 12.5 Hz, 1H), 5.03-4.99 (m, 3H), 4.57-
4.51 (m, 1H), 4.28-4.23 (m, 1H), 3.08 (s, 1H), 2.85 (brs, 2H), 1.71-1.52
(m, 4H), 1.38-1.32 (m, 5H). HRMS (ESI+) Calcd. for C29H32N3O5+ ([M]+):
466.2336. Found: 466.2338.
4-BrBzAla-BnO-Lys dipeptide (16d). 4-BrBz-Ala-BnO-Lys(Boc) (15d)
(326 mg, 552.06 µmol) was dissolved in dry CH2Cl2 (5 mL) in a round-
bottomed flask under a positive atmosphere of nitrogen at 22 °C. TFA
was added (0.4 mL, 5.54 mmol) to the solution. The reaction mixture was
stirred for 8 h at 22 °C and then concentrated under reduced pressure
and diluted with toluene (2 x 4 mL). Concentration in vacuo provided the
desired 4-BrBz-Ala-BnO-Lys dipeptide (330 mg, 99% yield) as viscous
yellow oil and and as a 2:1 mixture of rotamers. Data for the major
rotamer: 1H NMR (500 MHz, CDCl3) 7.98 (bs, 3H), 7.67 (bs, 1H), 7.59
(d, J = 8.5 Hz, 2H), 7.50 (d, J = 8.5, 2H), 7.34-7.24 (m, J = 7.8, 5H), 5.43
(bs, 1H), 5.17-5.09 (m, 2H), 4.69-4.46 (m, 2H), 3.05-2.85 (m, 2H), 1.85-
1.57 (m, 4H), 1.4-1.35 (m, 2H), 1.41 (d, J = 6.0, 3H). 13C NMR (125 MHz,
CDCl3) 172.65, 171.76, 166.79, 135.22, 131.90, 129.08 (2C), 128.76
(2C), 128.66 (2C), 128.36 (2C), 128.32, 126.79, 67.51, 52.60, 49.83,
39.57, 30.67, 26.83, 22.24, 18.11. FT-IR (neat, cm-1): 3275, 3068, 1740,
Representative procedure for coupling of dipeptides and carboxylic
acids: synthesis of -lactone dipeptide 17. To a round-bottomed flask
was added dipeptide 16a (13.0 mg, 0.023 mmol, 1 equiv), -lactone acid
12a’ (4.7 mg, 0.023 mmol, 1 equiv), EDCI (8.9 mg, 0.047 mmol, 2.0
equiv), HOBt (3.1 mg, 0.023 mmol, 1 equiv), DMF (0.1 mL), and DCM
(0.4 mL), followed by TMP (3.0 L, 0.023 mmol, 1.0 equiv). The reaction
mixture was stirred at 0 oC under N2 for 4 h, transferred to a separatory
funnel, and diluted with EtOAc (10 mL) and MTBE (10 mL). The organic
layer was washed with aq. NaHSO4 soln. (1 M), water and brine. The
solution was dried over MgSO4 and concentrated in vacuo. The residue
was purified by a flash chromatography (hexanes/acetone) to afford 17
as a viscous colorless oil (11.3 mg, 77%). []D20 -7.0 (c = 2.4, CHCl3). 1H
NMR (500 MHz, CDCl3) 7.39-7.30 (m, 10 H), 6.70 (d, J = 6.3 Hz, 1H),
6.58 (bs, 1H), 5.49 (d, J = 6.3 Hz, 1H), 5.20 (d, J = 12.4 Hz, 1H), 5.13 (d,
J = 12.4 Hz, 1H), 5.09 (s, 2H), 4.59-4.52 (m, 2H), 4.30-4.23 (m, 1H),
3.64-3.61 (m, 1H), 3.36-3.29 (m, 1H), 3.16-3.11 (m, 1H), 1.90-1.79 (m,
+
1675, 1541, 1204. HRMS (ESI+) Calcd. For C23H29BrN3O4 ([M]+):
490.1336. Found: 490.1320.
BnO-Ala-Cbz-Lys dipeptide (16e). BnO-Ala-Cbz-Lys(Boc) dipeptide
(15e) (1.0 g, 1.85 mmol) was dissolved in dry CH2Cl2 (10 mL) in a dry
round-bottomed flask under a positive atmosphere of nitrogen at 22 ºC
and then TFA was added (1.41 mL, 18.46 mmol). The reaction mixture
was stirred for 8 h at 22 ºC and then concentrated under reduced
pressure and diluted with toluene (2 x 10 mL) and concentrated to
provide the desired BnO-Ala-Cbz-Lys dipeptide 16e (1.015 g, 99% yield)
as a viscous colorless oil. 1H NMR (500 MHz, CDCl3) 8.04 (bs, 3H),
7.61 (d, J = 6.5 Hz, 1H), 7.32-7.27 (m, 10H), 6.08 (d, J = 8.0 Hz, 1H),

3H), 1.69-1.64 (m, 1H), 1.57-1.43 (m, 4H), 1.37 (d, J = 7.0 Hz, 3H), 1.34-
1.25 (m, 8H), 0.88 (t, J = 6.7 Hz, 3H). 13C NMR (125 MHz, CDCl3)
172.69, 172.07, 169.83, 168.42, 156.23, 136.49, 135.45, 128.90 (2C),
128.80, 128.78 (2C), 128.60 (2C), 128.42, 128.25 (2C), 73.10, 67.51,
67.20, 57.99, 52.14, 50.72, 45.20, 38.70, 31.76/31.67 (rotamers), 28.98,
28.67, 28.32, 26.71, 22.74, 22.31, 18.63, 14.25. HRMS (ESI+): Calcd. For
C34H45N3O8Li ([M+Li]+), 630.3367. Found: 630.3334.
amine 16e (13.9 mg, 0.025 mmol) and obtained as a viscous, colorless
oil (7.0 mg, 47%). []D20 -4.0 (c = 0.8, CHCl3). 1H NMR (500 MHz, CDCl3)
7.39-7.30 (m, 10H), 6.54-6.50 (m, 2H), 5.46 (d, J = 7.7 Hz, 1H), 5.22 (d,
J = 12.2 Hz, 1H), 5.15 (d, J = 12.2 Hz, 1H), 5.11 (s, 2H), 4.62 (pent, J =
7.2Hz, 1H), 4.49 (d, J = 4.1 Hz, 1H), 4.21-4.15 (m, 1H), 3.65 (ddd, J =
8.3Hz, J = 7.1 Hz, J = 4.7 Hz, 1H), 3.36 (dq, J = 13.6 Hz, J = 7.0 Hz, 1H),
3.28-3.21 (m, 1H), 1.90-1.80 (m, 3H), 1.71-1.63 (m, 1H), 1.58-1.45 (m,
4H), 1.42 (d, J = 7.0 Hz, 3H), 1.39-1.26 (m, 8H), 0.89 (t, J = 6.8 Hz, 3H).
13C NMR (125 MHz, CDCl3) 172.76, 171.34, 169.74, 168.27, 156.41,
136.37, 135.48, 128.85 (2C), 128.77 (2C), 128.68, 128.45, 128.37 (2C),
128.32 (2C), 73.03, 67.48, 67.31, 57.98, 54.64, 48.40, 38.65, 32.18,
31.63, 29.92, 28.95/28.85 (rotamers), 28.32, 26.66, 22.71, 22.26, 18.25,
14.21. HRMS (ESI+) Calcd. For C34H46N3O8 ([M+H]+): 624.3285. Found:
624.3303.
-lactone dipeptide 18. Prepared according to the representative
procedure from -lactone acid 12a’ (5.0 mg, 0.025 mmol) and dipeptide
amine 16b (13.5 mg, 0.025 mmol) and obtained as a viscous colorless oil
(9.0 mg, 59%). 1H NMR (500 MHz, CDCl3) 7.39-7.31 (m, 10H), 6.66 (d,
J = 7.1 Hz, 1H), 6.53 (bs, 1H), 5.55 (bs, 1H), 5.20 (d, J = 12.1 Hz, 1H),
5.15 (d, J = 12.1 Hz, 1H), 5.14 (s, 2H), 4.66-4.62 (m, 1H), 4.54 (d, J = 4.5
Hz, 1H), 3.91 (d, J = 5.7 Hz, 2H), 3.64 (td, J = 7.6 Hz, J = 4.4 Hz, 1H),
3.29-3.18 (m, 2H), 1.93-1.80 (m, 3H), 1.72-1.64 (m, 1H), 1.58-1.45 (m,
4H), 1.37-1.26 (m, 8H), 0.89 (t, J = 6.5 Hz, 3H). 13C NMR (125 MHz,
CDCl3) 172.09, 169.89, 169.27, 168.27, 156.87, 136.36, 135.33, 128.89
(2C), 128.82, 128.77 (2C), 128.56 (2C), 128.44, 128.23 (2C), 73.07,
67.56, 67.43, 57.98, 52.02, 44.70, 38.83, 31.97, 31.63, 28.95, 28.61,
28.29, 26.66, 22.70, 22.31, 14.23. HRMS (ESI+) Calcd. For C33H44N3O8
([M+H]+): 610.3128. Found: 610.3114. (~ 85% purity by HPLC).
-lactone dipeptide 22. Prepared according to the representative
procedure from -lactone acid 12a’ (5.0 mg, 0.025 mmol) and dipeptide
amine 16f (11.2 mg, 0.025 mmol) and obtained as a viscous, colorless oil
20
(7.4 mg, 57%). []D -25 (c = 0.8, CHCl3). 1H NMR (500 MHz, CDCl3)
7.38-7.31 (m, 5H), 6.49 (t, J = 6.3 Hz, 1H), 5.97 (bs, 1H), 5.21 (d, J =
12.4 Hz, 1H), 5.15 (d, J = 12.4 Hz, 1H), 4.79 (q, J = 7.2 Hz, 1H), 4.55 (d,
J = 4.3 Hz, 1H), 4.04 (t, J = 5.4 Hz, 1H), 3.68 (ddd, J = 8.1 Hz, J = 6.8
Hz, J = 4.4 Hz, 1H), 3.33-3.23 (m, 2H), 1.96-1.83 (m, 3H), 1.75-1.69 (m,
1H), 1.66 (d, J = 7.2 Hz, 3H), 1.56-1.46 (m, 4H), 1.40-1.26 (m, 8H), 0.89
(t, J = 7.2 Hz, 3H). 13C NMR (125 MHz, CDCl3) 173.29, 169.69, 169.49,
168.42, 156.53, 135.45, 128.79 (2C), 128.61, 128.45 (2C), 73.01, 67.82,
58.13, 56.78, 48.49, 38.55, 31.65, 30.61, 29.92, 29.00/28.95 (rotamers),
28.40, 26.63, 22.72, 21.02, 14.94, 14.23. HRMS (ESI+) Calcd. For
C27H36N3O7 ([M-H]-): 514.2553. Found: 514.2576. (~85% purity by
HPLC).
-lactone dipeptide 19. Prepared according to the representative
procedure from -lactone acid 12a’ (5.0 mg, 0.025 mmol) and dipeptide
amine 16c (13.5 mg, 0.025 mmol) and obtained as a viscous colorless oil
(9.5 mg, 62%). 1H NMR (500 MHz, CDCl3) 7.40-7.30 (m, 10 H), 6.78 (d,
J = 7.7 Hz, 1H), 6.62 (bs, 1H), 5.429 (d, J = 7.2 Hz, 1H), 5.21 (d, J = 12.3
Hz, 1H), 5.14 (d, J = 12.3 Hz, 1H), 5.11 (s, 2H), 4.62 (td, J = 7.6 Hz, J =
5.1 Hz, 1H), 4.49 (d, J = 4.4 Hz, 1H), 4.27 (pent, J = 7.0 Hz, 1H), 3.62
(ddd, J = 8.3 Hz, J = 6.9 Hz, J = 4.4 Hz, 1H), 3.32-3.25 (m, 1H), 3.24-
3.17 (m, 1H), 1.92-1.78 (m, 3H), 1.71-1.61 (m, 2H), 1.54-1.42 (m, 3H),
1.38 (d, J = 7.2 Hz, 3H), 1.35-1.27 (m, 6H), 0.89 (t, J = 6.8 Hz, 3H). 13C
NMR (125 MHz, CDCl3)  172.51, 171.80, 169.74, 168.21, 156.31,
136.39, 135.28, 128.91 (2C), 128.86, 128.76 (2C), 128.62 (2C), 128.43,
128.25 (2C), 73.03, 67.63, 67.27, 57.98, 51.98, 50.73, 38.58, 31.63,
29.91/29.50 (rotamers), 28.96, 28.93, 26.63, 25.33, 22.70, 18.50, 14.23.
HRMS (ESI+) Calcd. For C33H44N3O8 ([M+H]+): 610.3128. Found:
610.3105.
-lactone dipeptide 23. Prepared according to the representative
procedure from -lactone acid 12a (10.0 mg, 0.050 mmol) and dipeptide
amine 16e (27.8 mg, 0.050 mmol) and obtained as a viscous colorless oil
20
(9.3 mg, 60%). []D -13 (c = 3.1, CHCl3). 1H NMR (500 MHz, CDCl3)
7.39-7.30 (m, 10H), 6.65 (d, J = 6.7 Hz, 1H), 6.60 (t, J = 5.6 Hz, 1H),
5.50 (d, J = 8.1 Hz, 1H), 5.21 (d, J = 12.3 Hz, 1H), 5.15 (d, J = 12.3 Hz,
1H), 5.10 (s, 2H), 4.60 (pent, J = 7.4Hz, 1H), 4.53 (d, J = 4.4 Hz, 1H),
4.20 (q, J = 7.3 Hz, 1H), 3.65 (ddd, J = 8.1Hz, J = 6.9 Hz, J = 4.3Hz, 1H),
3.33 (dq, J = 13.3 Hz, J = 6.6 Hz, 1H), 3.24 (dq, J = 13.3 Hz, J = 6.6 Hz,
1H), 1.93-1.79 (m, 3H), 1.69-1.62 (m, 1H), 1.58-1.44 (m, 4H), 1.41 (d, J =
7.4 Hz, 3H), 1.37-1.28 (m, 8H), 0.88 (t, J = 6.8 Hz, 3H). 13C NMR (125
MHz, CDCl3) 172.74, 171.42, 169.86, 168.12, 156.38, 136.37, 135.50,
128.82 (2C), 128.73 (2C), 128.64, 128.39, 128.31 (2C), 128.23 (2C),
73.04, 67.45, 67.27, 57.90, 54.55, 48.39, 38.91, 32.44, 31.61, 29.89,
28.95/28.83 (rotamers), 28.29, 26.63, 22.60, 22.43, 18.14, 14.20. HRMS
(ESI+) Calcd. For C34H46N3O8 ([M+H]+): 624.3285. Found: 624.3304.
-lactone dipeptide 20. Prepared according to the representative
procedure from -lactone acid 12a’ (5.0 mg, 0.025 mmol) and dipeptide
amine 16b (15.1 mg, 0.025 mmol) and obtained as a viscous colorless oil
(5.6 mg, 51%). 1H NMR (500 MHz, CDCl3) 7.69-7.65 (m, 2H), 7.58-7.56
(m, 2H), 7.38-7.20 (m, 5H), 6.86 (d, J = 7.5 Hz, 1H), 6.73-6.72 (m, 1H),
6.58-6.55 (m, 1H), 5.22-5.16 (m, 1H), 5.12 (d, J = 12.3 Hz, 1H), 4.76-4.67
(m, 1H), 4.54 (d, J = 4.5 Hz, 1H), 3.70-3.62 (m, 1H), 3.34-3.26 (m, 2H),
1.92-1.83 (m, 4H), 1.76-1.71 (m, 1H), 1.66-1.60 (m, 4H), 1.50 (d, J = 7.0
Hz, 3H), 1.37-1.27 (m, 8H), 0.88 (t, J = 6.7 Hz, 3H). 13C NMR (125 MHz,
CDCl3) 172.4, 171.9, 170.0, 168.2, 166.6, 135.3, 132.7, 132.0 (2C),
129.0, 128.9 (2C), 128.84 (2C), 128.79, 128.5 (2C), 73.2, 67.6, 57.9,
52.1, 49.5, 39.0, 32.2, 31.6, 29.9, 28.6, 28.3, 26.6, 22.6, 22.5, 18.6, 14.2.
HRMS (ESI-) Calcd. For C33H41BrN3O7 ([M-H]-): 670.2128. Found:
670.2113.
-Lactone dipeptide 24. Prepared according to the representative
procedure from -lactone acid 12a (10.0 mg, 0.050 mmol) and dipeptide
amine 16b (27.0 mg, 0.050 mmol) and obtained as a viscous, colorless
oil (16.0 mg, 52%). 1H NMR (500 MHz, CDCl3) 7.40-7.31 (m, 10H), 6.67
(d, J = 6.8 Hz, 1H), 6.53 (bs, 1H), 5.56 (bs, 1H), 5.20 (d, J = 12.1 Hz,
1H), 5.15 (d, J = 12.1 Hz, 1H), 5.14 (s, 2H), 4.68-4.64 (m, 1H), 4.54 (d, J
= 4.2 Hz, 1H), 3.91 (d, J = 5.9 Hz, 2H), 3.64 (td, J = 7.6 Hz, J = 4.2 Hz,
1H), 3.31-3.24 (m, 1H), 3.22-3.16 (m, 1H), 1.95-1.82 (m, 3H), 1.73-1.65
(m, 1H), 1.59-1.44 (m, 4H), 1.38-1.24 (m, 8H), 0.90 (t, J = 6.7 Hz, 3H).
-lactone dipeptide 21. Prepared according to the representative
procedure from -lactone acid 12a’ (5.0 mg, 0.025 mmol) and dipeptide

13C NMR (125 MHz, CDCl3) 172.05, 169.94, 169.33, 168.18, 156.88,
136.36, 135.34, 128.87 (2C), 128.79, 128.75 (2C), 128.53 (2C), 128.42
(2C), 128.20, 73.06, 67.53, 67.43, 57.86, 52.00, 44.73, 38.93, 31.98,
31.61, 28.93, 28.59, 28.27, 26.65, 22.68, 22.27, 14.20. HRMS (ESI+)
Calcd. For C33H44N3O8 ([M+H]+): 610.3128. Found: 610.3114.
1H), 5.49 (d, J = 7.8 Hz, 1H), 5.19 (d, J = 12.4 Hz, 1H), 5.12 (d, J = 12.4
Hz, 1H), 5.11 (d, J = 12.5 Hz, 1H), 5.08 (d, J = 12.5 Hz, 1H), 4.59 (pent, J
= 7.2 Hz, 1H), 4.48 (d, J = 4.2 Hz, 1H), 4.17 (q, J = 6.1 Hz, 1H), 3.64 (td,
J = 7.7 Hz, J = 4.6 Hz, 1H), 3.37-3.31 (m, 1H), 3.26-3.20 (m, 1H), 3.10 (s,
1H), 1.90-1.25 (m, 16H), 1.41 (d, J = 7.2 Hz, 3H), 0.88 (t, J = 6.7 Hz, 3H).
13C NMR (125 MHz, CDCl3) 172.6, 171.3, 169.6, 168.2, 156.1, 136.3,
136.2, 132.5 (2C), 128.7 (2C), 128.4, 128.2 (2C), 128.1 (2C), 122.4,
83.3, 78.0, 73.0, 67.3, 66.8, 58.0, 54.7, 48.4, 38.7, 32.2, 316., 29.0, 28.9,
28.3, 26.6, 22.7, 22.3, 18.1, 14.2. HRMS (ESI+) Calcd. For C36H46N3O8
([M+H]+): 648.3285. Found: 648.3278. (~ 80% purity by HPLC)
-Methyl--lactone dipeptide 25. Prepared according to the
representative procedure from -lactone acid 12b (10.0 mg, 0.045 mmol)
and dipeptide amine 16a (24 mg, 0.045 mmol) and obtained as a viscous
colorless oil (15 mg, 55%). 1H NMR (500 MHz, CDCl3) 7.38-7.29 (m,
10H), 6.76-6.70 (m, 1H), 6.58 (brs, 1H), 5.51/5.38 (d, J = 6.4 Hz, 1H),
5.19 (d, J = 12.2 Hz, 1H), 5.13 (d, J = 12.2 Hz, 1H), 5.10 (s, 2H), 4.62-
4.56 (m, 1H), 4.59 (s, 1H), 4.30-4.23 (m, 1H), 3.26-3.16 (m, 2H), 1.90-
1.82 (m, 1H), 1.76-1.73 (m, 2H), 1.72-1.63 (m, 1H), 1.54-1.42 (m, 3H),
1.37 (d, J = 7.1 Hz, 3H), 1.36-1.25 (m, 9H), 1.23 (s, 3H), 0.88 (t, J = 6.8
Hz, 3H). 13C NMR (125 MHz, CDCl3) 173.5, 172.4, 171.9, 167.2, 156.2,
136.4, 135.3, 128.8 (2C), 128.74 (2C), 128.70 (2C), 128.6 (2C), 128.4,
128.2, 76.8, 67.4, 67.2, 61.4, 52.1, 50.7, 38.7, 35.4, 31.8, 31.7, 29.4,
28.9, 24.4, 22.7, 22.3, 18.4, 15.9, 14.2. HRMS (ESI-) Calcd. For
C35H47ClN3O8 ([M+Cl]-): 672.3057. Found: 672.3026.
Fluorogenic Assay for Detection of 20S Human Proteasome
Inhibition. For determination of IC50 values, 50 ng of 20S proteasome,
purified from human red blood cells [5], was incubated in assay buffer (20
mM Tris, pH 7.5) at 37 oC with varying concentrations of each inhibitor.
After twenty minutes, the reaction was initiated by the addition of
fluorogenic substrate for the chymotrypsin-like (Suc-Leu-Leu-Val-Tyr-
AMC), trypsin-like (Boc-Leu-Arg-Arg-AMC), or caspase-like (Z-Leu-Leu-
Glu-AMC) activity of the proteasome, with final substrate concentrations
of 10 M, 20 M, and 20 M respectively. The rates of hydrolysis of the
substrates were monitored using
a POLARstar Omega microplate
-Methyl--lactone dipeptide 26. Prepared according to the
representative procedure from -lactone acid 12b’ (8.0 mg, 0.035 mmol)
and dipeptide amine 16a (19.5 mg, 0.035 mmol) and obtained as a
viscous colorless oil (11.5 mg, 51%). 1H NMR (500 MHz, CDCl3) 7.38-
7.30 (m, 10H), 6.67 (d, J = 7.2 Hz, 1H), 6.53 (brs, 1H), 5.47 (d, J = 7.3
Hz, 1H), 5.19 (d, J = 12.2 Hz, 1H), 5.13 (d, J = 12.2 Hz, 1H), 5.10 (s, 2H),
4.64 (s, 1H), 4.60-4.56 (m, 1H), 4.29-4.23 (m, 1H), 3.33-3.27 (m, 1H),
3.21-3.15 (m, 1H), 1.91-1.84 (m, 1H), 1.76-1.68 (m, 3H), 1.56-1.44 (m,
3H), 1.37 (d, J = 7.1 Hz, 3H), 1.34-1.26 (m, 9H), 1.24 (s, 3H), 0.88 (t, J =
6.8 Hz, 3H). 13C NMR (125 MHz, CDCl3) 173.3, 172.5, 172.0, 167.4,
156.1, 136.5, 135.4, 128.8 (2C), 128.74, 128.70 (2C), 128.6 (2C), 128.4
(2C), 128.2, 76.9, 67.5, 67.2, 61.4, 52.1, 50.7, 38.4, 35.4, 31.7, 29.9,
29.4, 28.8, 24.4, 22.7, 22.2, 18.5, 15.9, 14.2. HRMS (ESI+) Calcd. For
C35H48N3O8 ([M+H]+): 638.3441. Found: 638.3455.
reader, with excitation/emission wavelengths of 360 nm/460 nm. The
rates of hydrolysis can be directly correlated to the enzymatic activity of
the proteasome. The reaction was monitored for 600 seconds, and the
linear portion for each curve was used in calculating the IC50 values
(inhibitor concentration to cause 50% inhibition of the enzyme active
site).This was accomplished by plotting the residual proteasomal activity
against the applied inhibitor concentration using GraphPad Prism version
6
for Windows, and fitting the experimental data to the equation:
Y=Bottom + (Top-Bottom)/(1+10^((LogIC50-X)*HillSlope)) where X is the
logarithm of inhibitor concentration and Y is the residual activity. Results
are average of triplicate measurements. IC50 values were deduced from
the fitted data. They depend on enzyme concentration and are
comparable only within the same experimental settings.
Fluorogenic Assay for Detection of Human FAS-TE Inhibition. The
synthetic fluorogenic substrate, 4-methylumbelliferyl heptanoate (4-
MUH), was purchased from Sigma (St. Louis, MO). The reaction mixture
consisted of 500 nM human FAS-TE in buffer (100mM Tris-HCl, 50 mM
NaCl at pH 7.4) which was pre-incubated with 2.5 μL test compounds
dissolved in DMSO at final concentrations of 0.32-100 μM and/or 0.08-10
µM at 37 ºC for 30 minutes. The reaction was initiated by addition of 5 μL
of 1.25 mM 4-MUH in 1:1 DMSO:buffer A. The resulting fluorescence
from liberated 4-methylumbelliferone was measured every five minutes at
350/450nm for 60 minutes. 4-MUH incubated without enzyme served as
a background control. Results are the average of triplicate time points.
IC50 values were determined in similar fashion as described for the
proteasome using GraphPad Prism 6 for Windows. IC50 values were
deduced from the fitted data. They depend on enzyme concentration and
are comparable only within the same experimental settings.
-Methyl--lactone dipeptide 27. Prepared according to the
representative procedure from -lactone acid 12b” (10 mg, 0.045 mmol)
and dipeptide amine 16a (24 mg, 0.045 mmol) and obtained as a viscous
colorless oil (18 mg, 63%). 1H NMR (500 MHz, CDCl3) 7.39-7.30 (m,
10H), 6.67 (d, J = 7.3 Hz, 1H), 6.56 (brs, 1H), 5.51 (d, J = 6.9 Hz, 1H),
5.20 (d, J = 12.2 Hz, 1H), 5.13 (d, J = 12.2 Hz, 1H), 5.09 (s, 2H), 4.60-
4.56 (m, 1H), 4.56 (s, 1H), 4.26 (pent, J = 7.1 Hz, 1H), 3.38-3.31(m, 1H),
3.17-3.11 (m, 1H), 1.90-1.83 (m, 1H), 1.73-1.66 (m, 1H), 1.62-1.59 (m,
2H), 1.51-1.45 (m, 1H), 1.49 (s, 3H), 1.40-1.36 (m, 2H), 1.37 (d, J = 7.1
Hz, 3H), 1.28-1.23 (m, 9H), 0.85 (t, J = 6.8 Hz, 3H). 13C NMR (125 MHz,
CDCl3) 173.1, 172.5, 172.0, 167.1, 156.1, 136.4, 135.3, 128.8 (2C),
128.74, 128.71 (2C), 128.5 (2C), 128.4, 128.2 (2C), 79.0, 67.5, 67.2,
61.0, 52.0, 50.7, 38.4, 31.8, 31.6, 29.9, 29.7, 28.7, 24.1, 22.7, 22.2, 20.0,
18.7, 14.2. HRMS (ESI-) Calcd. For C35H47ClN3O8 ([M+Cl]-): 672.3057.
Found: 672.3058.
Cell Cytotoxicity Assays. HeLa cells were grown in DMEM containing
10% fetal bovine serum (FBS). Cells were seeded at the density of 5000
cells per well in a 96-well plate (Thermo) and were incubated overnight at
37ºC with 5% CO2. The medium was removed and the cells were treated
with 100 µL of fresh culture medium with 17, 18, 21, 25 and 26 (1-100
M) or vehicle in duplicate. After 24 h, a stock solution of 3-(4,5-
ABPP probe 28. Prepared according to the representative procedure
from -lactone acid 12a’ (6.8 mg, 0.034 mmol) and dipeptide amine 16g
(19 mg, 0.034 mmol) and obtained as a viscous colorless oil (11 mg,
50%). 1H NMR (500 MHz, CDCl3) 7.48 (d, J = 8.2 Hz, 2H), 7.36-7.32
(m, 5H), 7.30 (d, J = 8.2 Hz, 2H), 6.60 (d, J = 6.8 Hz, 1H), 6.65-6.53 (m,

dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 10 L,
5
concentration assay (Carl Roth) and adjusted to the same concentration
in PBS (typically 1.0 mg/mL).
mM) was added to each well. As a negative control 10 µL of the MTT
stock solution was added to 100 µL of medium alone. The plate was
incubated at 37 °C for 4 h. All the medium was removed from the wells
and 50 µL of DMSO was added to each well and the solution was mixed
thoroughly with a pipette. Absorbance was read at 570 nm with a
microplate reader (BioTek, Winooski, VT). The data are shown as mean
± SEM based on three independently repeated experiments. IC50 values
were derived from dose-response curves. All statistical analyses were
performed using GraphPad Prism version 5.01 for Windows (GraphPad
Software, San Diego, CA). . IC50 values were deduced from the fitted
data. They depend on enzyme concentration and are comparable only
within the same experimental settings.
Activity-Based In situ competitive labeling. For labeling experiments,
cells were grown to ca. 80-90% confluency in a complete medium in a 6-
well plate. Then the medium was aspirated, cells were washed with pre-
warmed PBS (1.0 mL), followed by removal of PBS by suction. Then, 1.0
mL serum-free fresh medium containing either DMSO (as blank control),
10 µM competitive dual inhibitor 21 or 25 (for competitive labeling) at the
appropriate concentration (0  100 M) were added. DMSO content in
the medium did not exceed 0.1% (1000x dilution from DMSO stock).
Cells were incubated for 30 min with varying concentration of the
competitive inhibitor (10  100 µM corresponding to 1, 2.5, 5, 10-fold
excess compared to the alkyne probe 28) at 37 °C and 5% CO2. Then, 1
µL 100 mM alkyne probe 28 was added directly to the medium to achieve
a 10 µM final concentration. Cells were incubated for another 30 min at
37 °C and 5% CO2. Subsequently, the medium was carefully aspirated,
cells were washed with cold PBS (2 x 1.0 mL) to remove the excess of
the probe and then harvested in 1.0 mL fresh cold PBS by scraping. Cell
pellets were isolated by centrifugation (800 g, 3 min, 4 °C), resuspended
in 100 µL lysis buffer (1% NP-40, 1% sodium deoxycholate in PBS,
supplemented with protease inhibitor cocktail tablets (Roche)) and lysed
for 15 min on ice. Soluble and insoluble fractions were separated by
centrifugation at 14800 rpm for 45 min at 4 °C. Protein concentration
(soluble fraction) was determined using BCA protein concentration assay
(Carl Roth) and adjusted to the same concentration in PBS (typically 1.0
mg/mL).
Stability studies of -lactones 21 and 25
In DMEM without FBS: A stock solution of 21 or 25 in DMSO (1 mM, 5
L) was added to 995 L of DMEM (pH = 7.5) to reach the final
concentration of 5 M. The vial was loosely capped and placed in an
incubator at 37 oC with circulating 5% CO2. At regular time intervals 25
L of the solution was transferred to another vial and mixed with 50 L of
phosphate buffer (pH = 2.0, 1M). The solution was submitted to HPLC
analysis (Phenomenex® Gemini-NX 3 C18, 110A. Size: 150 x 4.60 mm;
Mobile Phase A: water; mobile phase B: acetonitrile) with a constant
injection volume (20 L). The % -lactone remaining was based on
integration in comparison to the initial pure -lactone. All compounds
were assayed in triplicate.
In DMEM with 5% FBS: A stock solution of 21 or 25 in DMSO (1 mM, 5
L) was added to the pre-mixed 5% fetal bovine serum (FBS) in DMEM
(995 L) to reach the final concentration of 5 M. The vial was loosely
capped and placed in an incubator at 37 oC with circulating 5% CO2. At
regular time intervals 100 L of the solution was transferred to another
vial and mixed with 100 L of phosphate buffer (pH = 2.0, 1M) and 150
L of CH3CN. The vial was vortexed for 1 minute to precipitate out the
serum and 80 L of the clear solution was transferred to another vial for
HPLC analysis with a constant injection volume (20 L) and analyzed by
HPLC in a manner identical to that described above. The ‘time vs % -
lactone remaining’ curves are provided in the SI.
Sharpless-Hüisgen (Click) Reactions, Protein Electrophoresis (SDS-
PAGE) and In-Gel Fluorescence Scanning (IG-FS). To 88 µL probe
28-bound cell lysate, 10 µL of a freshly prepared master mix containing 2
µL TAMRA-N3 fluorescent tag (5 mM stock in DMSO), 2 µL TCEP (50
mM stock in water) and 6 µL TBTA ligand (1.7 mM stock in DMSO:tert-
BuOH 1:4 (v/v)) were added. The samples were gently vortexed and 2 µL
CuSO4 (50 mM stock in water) was added to initiate the Sharpless-
Hüisgen cycloaddition reaction, giving a total reaction volume of 100 µL.
The final concentrations were as follows: 100 µM TAMRA-N3, 1 mM
TCEP, 100 µM TBTA and 1 mM CuSO4. Samples were incubated at RT
(25 °C) for 1-1.5 h in the dark. After click chemistry reaction, 400 µL pre-
chilled acetone were added, the samples were gently vortexed and left
overnight at -20 °C. Then, samples were centrifuged at 14800 rpm for 30
min at 4 °C. Supernatant was discarded and the protein pellets were
washed with 200 µL pre-chilled MeOH, resuspended by sonication,
centrifuged at 14800 rpm for 30 min at 4 °C and supernatant removed
(repeated twice if necessary). After removal of MeOH, the samples were
left to warm up to RT and dried in air for ca. 5 min. Then, samples were
redissolved in 0.2% SDS in PBS, an equal volume of 2 x SDS-PAGE
loading buffer (reducing) was added, samples were vortexed and
denaturated at 95 °C for 5 min. Proteins were separated by 1D SDS-
PAGE on 10% polyacrylamide gels (ca. 25 µg of protein/gel lane)
applying 300 V and then visualized by in-gel fluorescence scanning.
Fluorescence was recorded using Fujifilm LAS-4000 Luminescent Image
Analyzer with a Fujinon VRF43LMD3 Lens and a 575DF20 filter. Gels
were then subjected to Coomassie Brilliant Blue staining to verify
equivalent protein loading.
Activity-Based In situ proteome labeling. For labeling experiments,
cells were grown to ca. 80-90% confluency in a complete medium in a 6-
well plate. Then the medium was aspirated, cells were washed with pre-
warmed PBS (1.0 mL), followed by removal of PBS by suction. Then, 1.0
mL serum-free fresh medium containing either DMSO (as blank control)
or probe at the appropriate concentration (10-50 µM) were added. DMSO
content in the medium did not exceed 0.1% (1000x dilution from DMSO
stock). Cells were incubated for 1 h with varying concentrations of alkyne
probe 28 at 37 °C and 5% CO2. Subsequently, the medium was carefully
aspirated, cells were washed with cold PBS (2 x 1.0 mL) to remove the
excess of the probe and then harvested in 1.0 mL fresh cold PBS by
scraping. Cell pellets were isolated by centrifugation (800 g, 3 min, 4 °C),
resuspended in 100 µL lysis buffer (1% NP-40, 1% sodium deoxycholate
in PBS, supplemented with protease inhibitor cocktail tablets (Roche))
and lysed for 15 min on ice. Soluble and insoluble fractions were
separated by centrifugation at 14800 rpm for 45 min at 4 °C. Protein
concentration (soluble fraction) was determined using a BCA protein

Western Blot. Proteins were separated by 1D SDS-PAGE on 10%
-lactones 21 and 25. 1H and 13C NMR spectra for all new compounds,
and LC-MS data for -lactone inhibitors 17-28.
polyacrylamide gels and visualized by in-gel fluorescence scanning as
described above. Then, the proteins were transferred to
a PVDF
Keywords:
beta-lactones
•inhibitor•activity-based
protein
membrane (VWR) with a semi-dry blotter for 70 min at 14 kV. Prior to the
transfer, gel was incubated for ca. 5 min in the transfer buffer (48 mM
Tris, 39 mM glycine, 0.38 % w/v SDS, 20% v/v MeOH) while the
membrane in 100% MeOH. After the transfer, the membrane was
saturated with 5% BSA in 0.1% TBS-T pH 8.0 for 1 h at RT. Then, the
blot was cut into three parts for probing with three different antibodies.
The upper part was incubated with rabbit anti-FAS primary antibody
(1:1000 in 5% BSA in 0.1% TBS-T pH 8.0 (Cell Signaling C20G5))
overnight at 4 °C. Middle part was probed with goat anti-actin primary
antibody (1: 1000 in 5% BSA in 0.1% TBS-T pH 8.0 (SantaCruz
Biotechnology sc-1616)) overnight at 4 °C. Lower part was incubated
with rabbit anti-proteasome beta-5 (anti-PSB-5) primary antibody (1:
1000 in 5% BSA in 0.1% TBS-T pH 8.0 (Enzo BML-PW8895)) overnight
at 4 °C. The blots were then washed three times 15 min with TBS-T
followed by the probing with the secondary antibody. Goat anti-rabbit IgG
HRP-conjugated antibody (1:10,000 dilution in 5% BSA in TBS-T pH 8.0
(Pierce 32260)) and donkey anti-goat IgG HRP-conjugated antibody
(1:10,000 dilution in 5% BSA in TBS-T pH 8.0 (SantaCruz Biotechnology
sc-2020) were incubated for 1 h at RT (25 °C) with anti-FAS, anti-PSB-5
and anti-actin probed blots, respectively. Signals were detected using
Amersham™ ECL™ Prime Western Blotting Detection Reagent (GE
HealthCare RPN2232). Chemiluminescence was recorded for 10-60 sec
using CCD camera of Fujifilm LAS-4000 Luminescent Image Analyzer.
profiling•structure-activity relationship studies•serum stability
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Corresponding Author
Daniel Romo, Daniel_Romo@baylor.edu
Acknowledgements
We thank the NIH (R37 GM052964 to D.R.; R01 CA188694 to
J.W.S; R01 CA161158 to W.S.L.), the NSF (CHE 1546973 to
D.R.; for support of the Texas A&M University Undergraduate
MiniPharma), and the Robert A. Welch Foundation (AA-1280 to
D.R.; A-1715 to W.S.L.; A-0015 to J.C.S.) for generous support.
We thank the Office of the Vice President for Research and the
Dept. of Chemistry at Texas A&M University for support of the
Natural Products LINCHPIN Laboratory at Texas A&M and the
Undergraduate Research Scholars at Texas A&M for support of
TAMU MiniPharma Team Leaders during Summers of 2012,
2013, and 2014. We thank Dr. Lisa M. Perez of the Texas A&M
Laboratory for Molecular Simulation and members of the TAMU
MiniPharma Molecular Modeling Team, in particular Evelyn
Hoover, Deren Koseoglu, Robert Blando, Prithvi Vangal, Clifton
Molak, Emily Brackhahn, and Asuka Orr for assistance in
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Supporting Information. Synthetic procedures and characterization
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