Impact of Dehydroamino Acids on the Structure and Stability of Incipient 310-Helical Peptides

Article abstract of DOI:10.1021/acs.joc.9b02747

A comparative study of the impact of small, medium-sized, and bulky α,β-dehydroamino acids (AAs) on the structure and stability of Balaram's incipient 3₁₀-helical peptide (1) is reported. Replacement of the N-terminal Aib residue of 1 with a AA

Full text of DOI:10.1021/acs.joc.9b02747

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Article
Impact of Dehydroamino Acids on the Structure
and Stability of Incipient 310-Helical Peptides
Daniel Joaquin, Michael A. Lee, David W Kastner, Jatinder
Singh, Shardon T Morrill, Gracie Damstedt, and Steven L. Castle
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.9b02747 • Publication Date (Web): 15 Nov 2019
Downloaded from pubs.acs.org on November 15, 2019
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Impact of Dehydroamino Acids on the Structure and
Stability of Incipient 310-Helical Peptides
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Daniel Joaquin, Michael A. Lee, David W. Kastner, Jatinder Singh, Shardon T. Morrill, Gracie
Damstedt, and Steven L. Castle*
Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602
scastle@chem.byu.edu
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TOC Graphic.
ABSTRACT. A comparative study of the impact of small, medium-sized, and bulky ,-dehydroamino
acids (AAs) on the structure and stability of Balaram’s incipient 310-helical peptide (1) is reported.
Replacement of the N-terminal Aib residue of 1 with a AA afforded peptides 2a–c that maintained the
310-helical shape of 1. In contrast, installation of a AA in place of Aib-3 yielded peptides 3a–c that
preferred a -sheet-like conformation. The impact of the AA on peptide structure was independent of
size, with small (Ala), medium-sized (Z-Abu), and bulky (Val) AAs exerting similar effects. The
proteolytic stabilities of 1 and its analogs were determined by incubation with Pronase. Z-Abu and Val
increased the resistance of peptides to proteolysis when incorporated at the 3-position and had negligible
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impact on stability when placed at the 1-position, whereas Ala-containing peptides degraded rapidly
regardless of position. Exposure of peptides 2a–c and 3a–c to the reactive thiol cysteamine revealed that
Ala-containing peptides underwent conjugate addition at room temperature, while Z-Abu- and Val-
containing peptides were inert even at elevated temperatures. These results suggest that both bulky and
more accessible medium-sized AAs should be valuable tools for bestowing rigidity and proteolytic
stability on bioactive peptides.
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INTRODUCTION
The great potential of peptides as therapeutic agents1 continues to inspire the discovery of new methods
for increasing their short half-lives, which are caused by facile proteolytic degradation and rapid renal
clearance. A host of structural motifs including peptoids,2  and / peptides,3 stapled peptides,4 hydrogen
bond surrogates,5 AA-peptides,6 D-peptides,7 ,-disubstituted amino acids,8 N-amino peptides,9 and -
turn mimics10 can decrease their susceptibility to proteolysis. Additionally, techniques such as
PEGylation,11 conjugation to albumin or antibody fragments,12 and anchoring to plasma membranes13 are
effective at slowing renal clearance and/or proteolysis. In spite of these successes, only a modest number
of peptide drugs is currently reaching the market.14 Thus, more strategies for increasing the half-lives of
these potential therapeutics are urgently required.
In seminal work conducted 35–40 years ago, Stammer and co-workers established that ,-
dehydroamino acids (AAs) such as Ala, Z-Phe, and Z-Leu could improve the proteolytic stability
of the enkephalin peptide hormones.15 In regards to Phe and Leu, this phenomenon can be attributed
to the A1,3 strain caused by the alkene, which limits the conformational freedom of these residues and
increases the rigidity of peptides that contain them. This rigidity favors folded states instead of random
coil conformations and leads to greater stability since folded peptides undergo proteolysis more slowly
than unfolded peptides.16 Subsequent to Stammer’s pioneering investigations, Chauhan17,18 and others19
studied the impact of AAs containing trisubstituted alkenes (e.g., Phe, Leu) on peptide structure. In
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general, these residues promote the formation of -turns or 310 helices (Figure 1). In fact, Dong and co-
workers recently exploited the turn-inducing properties of Phe as an aid to cyclizing pentapeptides.20
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This AA and its analogs have been inserted into the backbones of bioactive peptides with promising
results.21 Phe and other AAs that incorporate trisubstituted alkenes are typically encountered as the
more stable Z-isomers due to challenges in constructing the E-isomers22 and E-to-Z isomerization that
often occurs during peptide coupling reactions.23
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Despite their presence in bioactive peptides including the yaku’amides,24 phomopsins,25 and
myxovalargin A,26 bulky dehydroamino acids such as Val and Ile that contain tetrasubstituted alkenes
have been less studied than their counterparts with trisubstituted Z-alkenes (Figure 1).27,28 We
hypothesized that the elevated A1,3 strain characteristic of bulky AAs would render them more effective
than smaller AAs at rigidifying peptides and increasing their proteolytic stability. However, this strain
also makes them more difficult to incorporate into peptides than their smaller relatives. A few years ago,
we developed a protocol for synthesizing peptides containing Val and its bis-homolog
dehydroethylnorvaline (Env) via solid-phase peptide synthesis.29 We applied this method to the
construction of -hairpins and demonstrated that inserting bulky AAs into these peptides leads to an
increased preference for the folded state as well as substantially higher proteolytic stability.30 The
synthetic challenges that we faced during this endeavor, which included construction of congested
azlactone dipeptides and sluggish couplings of these species, caused us to wonder how the effect of bulky
AAs on peptide structure and stability compared to that of medium-sized AAs with trisubstituted
alkenes. Specifically, we were unsure if the extra effort required to furnish peptides containing bulky
AAs was worthwhile. Comparisons of these two types of AAs in the literature have been limited to
studies of individual amino acids27f–h or dipeptides27a that are too small to exhibit secondary structure.
Accordingly, we resolved to examine the impact of small (i.e., Ala), medium-sized (i.e., dehydro-2-
aminobutyric acid or Abu), and bulky AAs (i.e., Val) on the structure and stability of incipient 310-
helical peptides.31 Herein, we report the results of this investigation.
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Figure 1. Two types of dehydroamino acids.
RESULTS AND DISCUSSION
In 1979, Balaram and co-workers synthesized tetrapeptide 1 (Figure 2) and studied its structure using
X-ray crystallography and NMR spectroscopy. They determined that 1 forms two overlapping -turns in
solution and in the solid state, representing an incipient 310 helix due to the presence of consecutive (i 
i + 3) hydrogen bonds.31 This compound is well-suited as a system for studying the impacts of different
AAs on peptide structure and stability, as the turn-inducing properties of these residues should cause
subtle yet measurable perturbations to its secondary structure. Moreover, the short and simple sequence
of 1 should render its AA-containing variants synthetically accessible and relatively straightforward to
study via NMR spectroscopy. Thus, we targeted peptides 2 (Figure 2) in which the Aib-1 residue is
replaced by Val (2a), Ala (2b), or Z-Abu (2c) in order to probe the impact of these AAs when they
are inserted at the (i + 1) position of one of the -turns contained in the incipient 310 helix. Similarly,
peptides 3 would permit evaluation of the effect of AAs when placed at the (i + 2) position.
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Figure 2. Balaram’s incipient 310 helical peptide (1) and proposed AA-containing variants (2 and 3).
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The synthesis of analog 2a incorporating a bulky AA in place of the Aib-1 residue in 1 commenced
with the construction of tripeptide 5 via coupling of Aib-Ala-OMe (4) and Cbz-Pro (Scheme 1). The
moderate yield is presumably due to the steric hindrance of the two coupling partners. Val derivative 7
was generated in good yield by using Martin sulfurane to dehydrate -OHVal derivative 6, which is
readily available via OsO4-catalyzed aminohydroxylation.29,32 Saponification of 7 was sluggish owing to
the A1,3 strain caused by the -methyl groups, with two days required to obtain complete conversion to
acid 8. Coupling of this acid with the amine derived from hydrogenolysis of tripeptide 5 furnished the
targeted tetrapeptide 2a in reasonable yield considering bulkiness of the reactants.
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Scheme 1. Synthesis of Val Analog 2a
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Preparation of Ala-containing analog 2b proceeded smoothly as outlined in Scheme 2. Dehydration
of the dipeptide Cbz-Ser-Pro-OMe (9) was accomplished by mesylation of the primary alcohol followed
by exposure to DBU and heat.33 No racemization of the Pro residue was detected under these conditions.
Saponification of the resulting Ala-containing dipeptide and coupling with dipeptide 4 delivered 2b in
an acceptable yield considering the hindered nature of the reactants.
Scheme 2. Synthesis of Ala Analog 2b
The synthesis of variant 2c possessing the medium-sized trisubstituted AA Z-Abu required coupling
of dipeptides 4 and 11, which was a challenging reaction (Scheme 3). Brief attempts at optimization
revealed that HBTU could promote the coupling in low but consistent yields. While it is possible that
either protecting the secondary alcohol of 11 or inverting the order of the dehydration and coupling steps
could have resulted in better yields, we elected not to explore these alternatives since we were able to
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generate the required amounts of 2c using the short sequence shown in Scheme 3. This peptide was
produced in modest yield by dehydrating tetrapeptide 12 with the Martin sulfurane.22a
Scheme 3. Synthesis of Z-Abu Analog 2c
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Analog 3a, which contains Val instead of the Aib-3 residue of Balaram’s peptide, was synthesized as
shown in Scheme 4. The dipeptide Cbz-Aib-Pro was coupled with racemic -OH-Val derivative 14 to
afford tripeptide 15 in good yield. Saponification of this peptide was followed by dehydration and
azlactone formation utilizing NaOAc and Ac2O in THF.30 Ring-opening and coupling of the hindered
azlactone intermediate was difficult. A brief survey of conditions revealed that serviceable yields of
tetrapeptide 3a could be obtained by first hydrolyzing the azlactone through treatment with NaOH and
then employing PyBOP to mediate coupling with Ala-OMe.34
Scheme 4. Synthesis of Val Analog 3a
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Construction of variant 3b wherein the Aib-3 residue of 1 is replaced by Ala is summarized in Scheme
5. Hydrogenolysis of the dipeptide Cbz-Ser-Ala-OMe (16) was followed by coupling with dipeptide 13,
furnishing tetrapeptide 17 in moderate yield. As in the coupling of 11, protection of the alcohol moiety in
16 was eschewed in favor of a short and direct synthetic route to 3b. Mesylation and elimination of the
primary alcohol in 17 was lower-yielding than the analogous transformation of dipeptide 9 (see Scheme
2), as 3b was obtained in 36% yield.
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Scheme 5. Synthesis of Ala Analog 3b
Preparation of analog 3c possessing Z-Abu in place of Aib-3 commenced with coupling of 13 to
dipeptide 18 (Scheme 6). We suspect that steric hindrance is the primary reason for the low yields of this
reaction. Exposure of the resulting tetrapeptide 19 to the Martin sulfurane afforded 3c. The poor yields
that we encountered while synthesizing some of the analogs of 1 are indicative of limits in current methods
for constructing hindered peptides containing AAs. While our concise routes enabled us to overcome
these problems and generate sufficient quantities of the small target peptides 2 and 3, we plan to address
these limitations in future studies.
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Scheme 6. Synthesis of Z-Abu Analog 3c
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The 1H NMR spectra of 2a–c and 3a–c in CDCl3 solutions provided evidence of a rigid and well-defined
major conformation for each peptide. The peptides in each series exhibit similar spectral characteristics,
suggesting that the position of the AA in the chain has a larger impact on peptide structure than the
nature of the AA (i.e., small, medium-sized, or bulky). Monitoring of the NH moieties of 2a–c in CDCl3
solutions doped with D2O revealed rapid H–D exchange of the AA carbamate NH and negligible H–D
exchange of the Aib and Ala amide NH groups. These observations are consistent with the 310-helical
pattern of intramolecular hydrogen bonds shown in Figure 3. In contrast, analogous experiments
conducted with 3a–c showed rapid H–D exchange of the AA amide NH and minimal H–D exchange of
the Aib carbamate NH and the Ala amide NH groups. These data fit the -sheet-like pattern of
intramolecular hydrogen bonds depicted in Figure 3. Interestingly, in CDCl3 solutions the AA residues
do not participate in intramolecular hydrogen bonding regardless of their position in the chain.
In an attempt to determine the impact of a polar environment on the hydrogen bonding patterns of 2 and
3, we conducted variable-temperature 1H NMR experiments with solutions of these peptides in DMSO-
d6. In this solvent, only the Ala amide NH groups of 2a–c, 3a, and 3c participate in intramolecular
hydrogen bonding. Apparently, the intramolecular hydrogen bonds involving the Aib amide or carbamate
NH groups that are present in the less polar CDCl3 solvent are weak enough to be disrupted by interactions
with the highly polar DMSO-d6 solvent molecules. Surprisingly, only the Ala amide NH of peptide 3b
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forms an intramolecular hydrogen bond in DMSO-d6 solution. Perhaps the absence of A1,3 strain in the
small Ala residue allows this peptide to adopt a conformation in a strongly polar solvent that is
unavailable to the other peptides with larger AA residues and intrinsic A1,3 strain.
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Figure 3. 310-Helical and -sheet-like conformations.
NOE-restrained structural calculations using the program CYANA 2.135 provided insights into the
solution structures of 1 and its analogs. First, ROESY spectra were acquired for each compound with a
mixing time of 200 ms. The resulting cross-peaks were then converted to upper-distance constraints. A
standard simulated annealing calculation was then performed for 100 conformers of each peptide with the
upper-limit file and experimentally determined hydrogen bonding pattern as input. The ten structures with
the lowest objective target function were then superimposed to create a final ensemble for each peptide,
which was then analyzed based on structure and RMSD values using the molecular visualization software
VMD. These ensembles are shown in Figure 4. The parent peptide 1 exhibits the highest RMSD value of
1.154 Å, suggesting that it has more conformational freedom than its analogs. As anticipated from the
intramolecular hydrogen bonding data, the peptides with a ΔAA in place of Aib-1 (i.e., 2a–c) form
incipient 310-helical structures and the peptides with a ΔAA in place of Aib-3 (i.e., 3a–c) resemble β-
turns. These structural data demonstrate that the position of the ΔAA plays an important role in
determining the conformation preferred by each analog.
Peptides 3a–c exhibit lower RMSD values than peptides 2a–c, indicating that placement of a AA at
the Aib-3 position leads to a greater increase in rigidity than does placement at the Aib-1 position.
Interestingly, the conformations of the Val and Z-Abu residues in peptides of the same series are quite
similar (i.e., 2a and 2c, 3a and 3c). However, the Z-Abu-containing analogs 2c and 3c exhibit higher
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RMSD values than the corresponding Val-containing analogs 2a and 3a. While both residues apparently
prefer similar low-energy conformations, it appears that the additional A1,3 strain inherent in a
tetrasubstituted alkene relative to a trisubstituted alkene destabilizes the high-energy conformations of
Val to a greater degree than those of Z-Abu. Thus, our data suggest that medium-sized and bulky AAs
exert similar effects on the structures of peptides, but that bulky AAs impart more rigidity.
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Figure 4. Solution-phase structures of 1, 2a–c, and 3a–c as calculated using NOE distance restraints.
The stability of 1 and its analogs to proteolytic degradation was determined by treating the peptides
with Pronase, a cocktail of proteases from Streptomyces griseus that aggressively cleaves peptide bonds
in a nonspecific fashion.36 Since 1 is relatively stable to proteolysis due to its two Aib residues, a high
concentration of Pronase was required for it to degrade on a reasonable timescale. As a result, the kinetics
of peptide degradation were affected by self-proteolysis of the enzymes. Although this prevented us from
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measuring half-lives, we were able to compare the amount of each peptide that remained after 30 minutes
of incubation with Pronase as recorded in Table 1. Peptides 2b and 3b containing Ala were significantly
more labile than Balaram’s peptide, as the relative abundance of the former peptides after 30 minutes of
exposure to Pronase was ca. 10–30% that of the latter. Thus, it is clear that Aib is more effective than
Ala at protecting peptides from proteolysis. Interestingly, the effects of Z-Abu and Val were
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dependent on their location in the peptide chain. Analogs 2a and 2c possessing a AA at the N-terminus
were equally or slightly less stable than 1, respectively. In contrast, variants 3a and 3c in which the AA
replaces Aib-3 were more stable than 1, as approximately twice as much of each remained after exposure
to Pronase for 30 minutes. While the Val-containing peptides 2a and 3a were slightly more stable than
the Z-Abu-containing peptides 2b and 3b, the differences in performance were not dramatic. Although
a more detailed and comprehensive study would be required to generate firm conclusions, these data
suggest that Z-Abu and Val might confer comparable increases in proteolytic stability upon peptides
that contain them. Importantly, our results show that in some cases the stabilizing impact of these two
AAs can exceed that of the ,-disubstituted residue Aib.
Table 1. Proteolysis of 1 and Analogs by Pronasea
amount remaining
peptide
after 30 min (%)
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a Solutions of peptides (0.5 mM, 500 L) in 1X PBS buffer (10 mM sodium phosphate, 137 mM NaCl,
2.7 mM KCl buffer, pH 7.4) were each treated with a solution of Pronase (0.88 mg/mL, 200 L).
Degradation of peptides was monitored using HPLC.
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Conventional wisdom indicates that AAs are reactive electrophiles that undergo conjugate additions
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by biological nucleophiles such as thiols.37 In fact, enzyme-catalyzed conjugate additions of thiols to Ala
and Abu are involved in the biosynthesis of lantibiotics.38 However, a close look at the literature reveals
that uncatalyzed conjugate additions of thiols to AAs larger than Ala typically require large excesses
of thiol to proceed.39 Alternatively, placement of the AA at the C-terminus of a peptide instead of
embedded within the backbone can increase its electrophilicity.40 Based on these prior results, we
postulated that steric hindrance at the -carbon atom would significantly attenuate the reactivity of
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medium-sized and bulky AAs to thiols in the absence of enzymes. We probed this hypothesis by
exposing solutions of 2a–c and 3a–c in DMSO-d6 to ca. 2 equiv of cysteamine, a highly nucleophilic
thiol.41 As anticipated, Ala-containing peptides 2b and 3b were steadily consumed under these
conditions. The decomposition of cysteamine in solution complicated our efforts to calculate half-lives
for these reactions. Nonetheless, it was clear that 3b was consumed more rapidly than 2b, as 65% of the
former and 93% of the latter remained after 40 minutes of incubation at room temperature.
In contrast, Val-containing peptides 2a and 3a as well as Z-Abu-containing peptides 2c and 3c were
inert to cysteamine at temperatures up to 100 °C, at which point the thiol decomposed completely. Thus,
neither of these residues is reactive to thiols when embedded within a peptide (i.e., not located at the C-
terminus). While further investigation is warranted, these results suggest that both medium-sized and
bulky AAs can be incorporated into bioactive peptides without fear of in vivo degradation due to
conjugate addition. Interestingly, subjection of -methylene--butyrolactone to cysteamine caused
complete consumption of the electrophile within 5 minutes at room temperature. This result shows that
Ala-containing peptides 2b and 3b are only modestly reactive to cysteamine by comparison, implying
that peptides including this residue might also be useful for some biological applications.
CONCLUSIONS
In an effort to compare the impacts of small (i.e., Ala), medium-sized (i.e., Z-Abu), and bulky AAs
(i.e., Val) on peptide structure and stability, we synthesized AA-containing analogs 2a–c and 3a–c of
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Balaram’s incipient 310-helical peptide 1. The different types of AAs exerted similar effects on
conformation in CDCl3 solution according to NOE-restrained structural calculations. Variants 2a–c
possessing a AA in place of Aib-1 all preferred the 310-helical shape of the original peptide, whereas
analogs 3a–c with a AA replacing Aib-3 all adopted a -sheet-like orientation instead. Thus, the location
of the AA in the backbone played a larger role than the size of the residue in determining the structures
of the peptides. The proteolytic stabilities of 1 and its analogs were evaluated by exposure to Pronase.
Ala-containing analogs 2b and 3b were degraded rapidly, demonstrating that this small AA does not
protect peptides from proteolysis. Z-Abu and Val increased proteolytic stability to similar degrees,
with the most pronounced effect (ca. twice as stable as 1) occurring when the AA replaced Aib-3.
Intriguingly, peptides containing Z-Abu and Val were completely unreactive to conjugate addition by
the potent nucleophile cysteamine at temperatures up to 100 °C, whereas Ala-containing peptides
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reacted at room temperature. The comparable impacts of Z-Abu and Val on proteolytic stability
suggests that the former residue is likely to be preferred to the latter due to its relative ease of synthesis,
except in cases where the increased rigidity caused by the elevated A1,3-strain in Val is desired. The
inertness of both of these residues to thiols suggests that they can be incorporated into bioactive peptides
without fear of degradation by biological nucleophiles. While we hope that these conclusions will be valid
with peptides larger than 2 and 3, further experimentation is required to confirm this premise. When
viewed as a whole, our results provide indications of the promise of medium-sized and bulky AAs as
tools for imparting rigidity and proteolytic stability to peptides. Future studies will focus on the impact of
AAs on other secondary structures as well as the synthesis and study of additional AAs including
medium-sized AAs that are larger than Abu (e.g., Phe and Leu).42
EXPERIMENTAL SECTION
General Details. Dimethylformamide and tetrahydrofuran were dried by passage through
cylinders of activated alumina. Chloroform was dried over 4 Å molecular sieves and Na2SO4 under Ar
overnight. An oil bath was used to heat reactions that required elevated temperatures to proceed. Flash
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chromatography was conducted using 60–230 mesh silica gel. 1H NMR spectra were acquired on a 500
MHz spectrometer with chloroform (7.27 ppm) as internal reference. Signals are reported as follows: s
(singlet), d (doublet), t (triplet), q (quartet), quint (quintet), dd (doublet of doublets), br s (broad singlet),
m (multiplet). 13C NMR spectra were acquired on a spectrometer operating at 125 MHz with chloroform
(77.23 ppm) as internal reference. Infrared spectra were obtained on an FT-IR spectrometer. MS data
were obtained using ESI techniques.
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Methyl (2-amino-2-methylpropanoyl)-L-alaninate (4). A solution of Z-Aib-OH
(1.5034 g, 6.34 mmol) in anhydrous THF–DMF (5:1, 48 mL) at 0 °C under Ar was treated sequentially
with Ala-OMe (944.5 mg, 6.77 mmol, 1.1 equiv), HOBt (ca. 20% H2O content, 1.2988 g, 7.69 mmol, 1.2
equiv), EDC•HCl (1.7356 g, 9.05 mmol, 1.4 equiv), and NaHCO3 (1.2811 g, 15.25 mmol, 2.4 equiv). The
resulting mixture was slowly warmed to rt and stirred for 24 h. The reaction was quenched by the addition
of sat aq NaHCO3 (25 mL), diluted with H2O (30 mL), and extracted with EtOAc (5 × 40 mL). The
combined organic layers were dried (Na2SO4) and concentrated in vacuo. Flash chromatography (200 mL
of SiO2, 0–30% EtOAc in hexanes gradient elution) afforded Z-Aib-Ala-OMe (1.936 g, 6.01 mmol, 95%)
as a white solid: [α]25D –3.3 (c 1.2, CHCl3); 1H NMR (CDCl3, 500 MHz) δ 7.38–7.31 (m, 5H), 6.80 (br s,
1H), 5.29 (br s, 1H), 5.11 (s, 2H), 4.59–4.52 (m, 1H), 3.75 (s, 3H), 1.56 (s, 3H), 1.54 (s, 3H), 1.38 (br s,
3H); 13C{1H} NMR (CDCl3, 125 MHz) δ 173.8, 173.4, 155.0, 136.2, 128.6 (2C), 128.2, 128.1 (2C), 66.8,
56.9, 52.4, 48.2, 25.7, 25.2, 18.2; IR (film) υmax 3327, 2998, 1725, 1660, 1522, 1454, 1255, 1074 cm–1;
HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C16H22N2O5H 323.1607; Found 323.1600.
A solution of Z-Aib-Ala-OMe (1.001 g, 3.11 mmol) in CH3OH (10.0 mL) was treated with 10%
Pd/C (501.1 mg, 0.5 wt equiv) at rt under Ar. The resulting mixture was stirred at rt under H2 (500 psi)
for 72 h, then diluted with H2O (10 mL) and extracted with EtOAc (4 × 6 mL). The combined organic
layers were dried (Na2SO4) and concentrated in vacuo. The crude Aib-Ala-OMe (4, 559.6 mg, 2.97 mmol,
96%) was used directly in subsequent coupling reactions without further purification.
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Benzyl (S)-2-((1-(((S)-1-methoxy-1-oxopropan-2-yl)amino)-2-methyl-1-
oxopropan-2-yl)carbamoyl)pyrrolidine-1-carboxylate (5). A solution of Z-Pro-OH (44.8 mg, 0.180
mmol) in anhydrous THF–DMF (5:1, 6 mL) at 0 °C under Ar was treated sequentially with 4 (35.4 mg,
0.188 mmol, 1.0 equiv), HOBt (ca. 20% H2O content, 38.3 mg, 0.227 mmol, 1.3 equiv), EDC•HCl (50.5
mg, 0.263 mmol, 1.5 equiv), and NaHCO3 (38.3 mg, 0.456 mmol, 2.5 equiv). The resulting mixture was
slowly warmed to rt and stirred for 24 h. The reaction was quenched by the addition of sat aq NaHCO3 (3
mL), diluted with H2O (6 mL), and extracted with EtOAc (5 × 10 mL). The combined organic layers were
dried (Na2SO4) and concentrated in vacuo. Flash chromatography (100 mL of SiO2, 0–80% EtOAc in
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hexanes gradient elution) afforded 5 (39.3 mg, 0.0937 mmol, 52%) as a white film: [α]25 –55 (c 0.78,
D
CHCl3); 1H NMR (CDCl3, 500 MHz) δ 7.38–7.30 (m, 5H), 7.22 (br s, 1H), 6.67 (br s, 1H), 5.18 (d, J =
12.2 Hz, 1H), 5.12 (d, J = 12.4 Hz, 1H), 4.52 (quint, J = 7.2 Hz, 1H), 4.20–4.15 (m, 1H), 3.73 (s, 3H),
3.60–3.54 (m, 1H), 3.52–3.45 (m, 1H), 2.25–2.14 (m, 1H), 2.08–2.00 (m, 1H), 1.95–1.87 (m, 2H), 1.55
(s, 3H), 1.49 (s, 3H), 1.37 (d, J = 7.2 Hz, 3H); 13C{1H} NMR (CDCl3, 125 MHz) δ 173.6, 173.5, 171.4,
156.0, 136.3, 128.6 (2C), 128.2, 127.8 (2C), 67.4, 61.2, 57.3, 52.2, 48.3, 47.1, 28.9, 26.5, 24.7, 24.4, 17.8;
IR (film) υmax 3319, 2997, 1751, 1685, 1519, 1421, 1357, 1174, 1110 cm–1; HRMS (ESI-TOF) m/z: [M +
H]+ Calcd for C21H29N3O6H 420.2135; Found 420.2127.
Ethyl 2-(((Benzyloxy)carbonyl)amino)-3-methylbut-2-enoate (7). A solution of 632 (172
mg, 0.582 mmol) in anhydrous CHCl3 (3 mL) at 0 °C was treated with Martin sulfurane (783.2 mg, 1.16
mmol, 2.0 equiv) and stirred at 0 °C to rt for 1 h. The mixture was then dried (Na2SO4) and concentrated
in vacuo. Flash chromatography (150 mL of SiO2, 0–1.5% MeOH in CH2Cl2 gradient elution) afforded 7
(139.4 mg, 0.503 mmol, 86%) as a yellow oil: 1H NMR (CDCl3, 500 MHz) δ 7.41–7.30 (m, 5H), 5.99 (br
13
s, 1H), 5.16 (s, 2H), 4.26–4.19 (m, 2H), 2.19 (s, 3H), 1.91 (s, 3H), 1.31–1.25 (m, 3H); C{1H} NMR
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(CDCl3, 125 MHz) δ 164.9, 159.9, 145.6, 136.3, 128.5 (2C), 128.2, 128.1 (2C), 126.2, 67.1, 60.8, 22.5,
21.3, 14.2; IR (film) υmax 3324, 2924, 1715, 1499, 1454, 1373, 1308, 1249, 1090 cm–1; HRMS (ESI-TOF)
m/z: [M + H]+ Calcd for C15H19NO4H 278.1392; Found 278.1383.
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2-(((Benzyloxy)carbonyl)amino)-3-methylbut-2-enoic acid (8). A solution of 7 (22.0 mg,
0.0793 mmol) in THF–EtOH–H2O (3:2:1, 3 mL) at rt was treated with LiOH•H2O (15.2 mg, 0.362 mmol,
4.6 equiv). The resulting mixture was stirred at 50 °C for 48 h. The reaction was acidified using 1N HCl
until pH ~2, and the mixture was then extracted with EtOAc (4 × 10 mL). The combined organic layers
were washed with brine (8 mL), dried (Na2SO4), and concentrated in vacuo. The crude acid 8 (20 mg,
0.079 mmol, quant.), was obtained as a yellow oil and used in subsequent reactions without further
purification.
Methyl
(2-((S)-1-(2-(((Benzyloxy)carbonyl)amino)-3-methylbut-2-
enoyl)pyrrolidine-2-carboxamido)-2-methylpropanoyl)-L-alaninate (2a). A solution of 5 (34.3 g,
0.0818 mmol) in MeOH (4 mL) at rt under Ar was treated with 10% Pd/C (21.5 mg, 0.63 wt equiv). The
resulting mixture was stirred at rt under H2 (500 psi) for 72 h, then filtered through Celite, dried (Na2SO4),
and concentrated in vacuo. The crude Pro-Aib-Ala-OMe product (18.5 mg, 0.0648 mmol, 79%) was used
directly in subsequent coupling reactions without further purification.
A solution of acid 8 (20.0 mg, 0.0802 mmol, 1.2 equiv) in anhydrous THF–DMF (6:1, 3.5 mL) at
0 °C under Ar was treated sequentially with Pro-Aib-Ala-OMe (18.5 mg, 0.0648 mmol, 1.0 equiv), HOBt
(ca. 20% H2O content, 17.0 mg, 0.101 mmol, 1.6 equiv), EDC•HCl (21.7 mg, 0.113 mmol, 1.7 equiv),
and NaHCO3 (14.6 mg, 0.174 mmol, 2.7 equiv). The resulting mixture was stirred at 0 °C to rt for 48 h.
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The reaction was quenched by the addition of sat aq NaHCO3 (3 mL) and diluted with H2O (6 mL), and
the mixture was then extracted with EtOAc (5 × 10 mL). The combined organic layers were dried
(Na2SO4) and concentrated in vacuo. Flash chromatography (100 mL of SiO2, 0–80% EtOAc in hexanes
gradient elution) afforded 2a (15.8 mg, 0.0306 mmol, 47%) as a white solid: [α]25D –16 (c 0.9, CHCl3);
1H NMR (CDCl3, 500 MHz) δ 7.41 (br s, 1H), 7.40–7.34 (m, 5H), 7.33 (br s, 1H), 6.35 (s, 1H), 5.14 (d,
J = 12.2 Hz, 1H), 5.06 (d, J = 12.2 Hz, 1H), 4.53–4.48 (m, 2H), 3.69 (s, 3H), 3.66–3.61 (m, 1H), 3.55–
3.49 (m, 1H), 2.35–2.28 (m, 1H), 2.18–2.09 (m, 1H), 2.04–1.91 (m, 2H), 1.76 (s, 3H), 1.73 (s, 3H), 1.55
(s, 3H), 1.51 (s, 3H), 1.37 (d, J = 7.2 Hz, 3H); 13C{1H} NMR (CDCl3, 125 MHz) δ 174.4, 173.4, 170.6,
166.7, 153.9, 135.4, 128.7 (2C), 128.6, 128.1 (2C), 124.6, 122.7, 67.6, 61.0, 57.3, 52.1, 48.4, 48.3, 29.0,
25.6, 25.4, 24.5, 19.8, 18.3, 17.6; IR (film) υmax 3322, 2924, 1640, 1518, 1445, 1240, 1261 cm–1; HRMS
(ESI-TOF) m/z: [M + H]+ Calcd for C26H36N4O7H 517.2662; Found 517.2677.
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Methyl ((Benzyloxy)carbonyl)-L-serylprolinate (9). A solution of Z-Ser-OH (500.5
mg, 2.09 mmol) in anhydrous THF–DMF (5:1, 18 mL) under Ar at 0 °C was treated sequentially with
Pro-OMe•HCl (363.4 mg, 2.19 mmol, 1.05 equiv), HOBt (ca. 20% H2O content, 458.8 mg, 2.72 mmol,
1.3 equiv), EDC•HCl (680.9 mg, 3.55 mmol, 1.7 equiv), and NaHCO3 (403.7 mg, 4.81 mmol, 2.3 equiv).
The resulting mixture was slowly warmed to rt and stirred for 24 h. The reaction was quenched by the
addition of sat aq NaHCO3 (15 mL), diluted with H2O (25 mL), and extracted with EtOAc (4 × 25 mL).
The combined organic layers were dried (Na2SO4) and concentrated in vacuo. Flash chromatography (185
mL of SiO2, 0–5% MeOH in CH2Cl2 gradient elution) afforded 9 (597.3 mg, 1.70 mmol, 81%) as a white
solid: [α]25D –25 (c 0.6, CHCl3); 1H NMR (CDCl3, 500 MHz) δ 7.36–7.29 (m, 5H), 5.95–5.89 (m, 1H),
5.09 (s, 2H), 5.07 (br s, 1H), 4.70–4.64 (m, 1H), 4.59–4.55 (m, 1H), 3.92–3.88 (m, 1H), 3.86–3.79 (m,
1H), 3.76–3.70 (m, 1H), 3.73 (s, 3H), 3.41–3.39 (m, J = 7.2 Hz, 1H), 2.27–2.15 (m, 2H), 2.03–1.94 (m,
2H); 13C{1H} NMR (CDCl3, 125 MHz) δ 172.9, 169.9, 156.2, 136.2, 128.5 (2C), 128.1, 128.0 (2C), 67.0,
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63.8, 58.9, 53.8, 52.7, 47.3, 28.9, 24.8; IR (film) υmax 3315, 2953, 2348, 1720, 1639, 1529, 1449, 1216,
1061 cm–1; HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C17H22N2O6H 351.1556; Found 351.1549.
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Methyl (2-(((Benzyloxy)carbonyl)amino)acryloyl)-L-prolinate (10). A solution of
9 (380.7 mg, 1.09 mmol) in 1,2-dichloroethane (15 mL) at 0 oC was treated with Et3N (380 L, 276 mg,
2.73 mmol, 2.5 equiv), then stirred at 0 oC for 10 min. The resulting mixture was treated dropwise with
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o
MsCl (170 L, 252 mg, 2.20 mmol, 2.0 equiv) and stirred at 0 C to rt for 1 h. It was then treated with
DBU (810 L, 825 mg, 5.42 mmol, 5.0 equiv) and stirred at 90 oC for 2 h. The mixture was concentrated
in vacuo, and the residue was dissolved in EtOAc (15 mL) and washed with 10% aq citric acid (10 mL),
sat aq NaHCO3 (2 × 8 mL), and brine (10 mL). The combined organic layers were dried (Na2SO4) and
concentrated in vacuo. Flash chromatography (110 mL of SiO2, 0–13% MeOH in CH2Cl2 gradient elution)
afforded 10 (223.2 mg, 0.672 mmol, 62%) as a white solid: [α]25D –43 (c 0.6, CHCl3); 1H NMR (CDCl3,
500 MHz) δ 7.38–7.30 (m, 5H), 7.23 (br s, 1H), 6.13 (s, 1H), 5.14 (s, 3H), 4.55–4.48 (m, 1H), 3.83–3.77
(m, 2H), 3.74 (s, 3H), 2.30–2.21 (m, 1H), 2.10–2.01 (m, 1H), 2.00–1.84 (m, 2H); 13C{1H} NMR (CDCl3,
125 MHz) δ 172.3, 165.0, 153.3, 136.0, 134.8, 128.5 (2C), 128.3, 128.1 (2C), 102.6, 66.8, 59.8, 52.3,
50.4, 28.9, 25.4; IR (film) υmax 3303, 2953, 2881, 1738, 1622, 1511, 1438, 1219, 1088 cm–1; HRMS (ESI-
TOF) m/z: [M + H]+ Calcd for C17H20N2O5H 333.1445; Found 333.1438.
Methyl
(2-(1-(2-(((Benzyloxy)carbonyl)amino)acryloyl)pyrrolidine-2-
carboxamido)-2-methylpropanoyl)-L-alaninate (2b). A solution of 10 (150.6 mg, 0.453 mmol) in t-
BuOH–H2O (3:1, 10 mL) at 0 °C was treated with LiOH•H2O (95.1 mg, 2.27 mmol, 5.0 equiv) and stirred
at rt for 4 h. The reaction was quenched by the addition of 1N HCl until pH ~4, and the mixture was
extracted with EtOAc (4 × 10 mL). The combined organic layers were washed with brine (10 mL), dried
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(Na2SO4), and concentrated in vacuo. Crude acid Z-ΔAla-Pro-OH (105.2 mg, 0.330 mmol, 73%) was
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obtained as a colorless oil and was used in subsequent reactions without further purification.
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A solution of Z-ΔAla-Pro-OH (105 mg, 0.330 mmol) in anhydrous THF–DMF (5:1, 18 mL) at 0
°C under Ar was treated sequentially with Aib-Ala-OMe (4, 65.2 mg, 0.346 mmol, 1.05 equiv), HOBt
(ca. 20% H2O content, 71.7 mg, 0.425 mmol, 1.3 equiv), EDC•HCl (88.5 mg, 0.462 mmol, 1.4 equiv),
and NaHCO3 (66.5 mg, 0.792 mmol, 2.4 equiv). The resulting mixture was slowly warmed to rt and stirred
for 24 h. The reaction was quenched by the addition of sat aq NaHCO3 (5 mL), diluted with H2O (5 mL),
and extracted with EtOAc (4 × 8 mL). The combined organic layers were dried (Na2SO4) and concentrated
in vacuo. Flash chromatography (60 mL of SiO2, 0–3% MeOH in CH2Cl2 gradient elution) afforded 2b
(93.7 mg, 0.192 mmol, 58%, 42% from 10) as a white solid: [α]25D –14 (c 0.5, CHCl3); 1H NMR (CDCl3,
500 MHz) δ 7.40–7.31 (m, 5H), 7.27 (br s, 1H), 7.17 (br s, 1H), 6.96 (br s, 1H), 5.60 (s, 1H), 5.15 (d, J =
12.3 Hz, 1H), 5.12 (d, J = 12.4 Hz, 1H), 5.01 (s, 1H), 4.51 (quint, J = 7.2 Hz, 1H), 4.42–4.36 (m, 1H),
3.75–3.67 (m, 2H), 3.66 (s, 3H), 2.22–2.12 (m, 2H), 2.11–2.03 (m, 1H), 1.93–1.86 (m, 1H), 1.56 (s, 3H),
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1.52 (s, 3H), 1.33 (d, J = 7.2 Hz, 3H); C{1H} NMR (CDCl3, 125 MHz) δ 174.0, 173.6, 170.7, 165.7,
153.4, 135.8, 135.6, 128.6 (2C), 128.5, 128.2 (2C), 102.6, 67.4, 61.7, 57.3, 52.2, 50.2, 48.3, 28.7, 26.3,
25.3, 24.6, 17.7; IR (film) υmax 3324, 2951, 1715, 1649, 1523, 1450, 1243, 1090 cm–1; HRMS (ESI-TOF)
m/z: [M + H]+ Calcd for C24H32N4O7H 489.2349; Found 489.2357.
((Benzyloxy)carbonyl)-L-threonyl-L-proline (11). A solution of Z-Thr-OH (1.004 g,
3.96 mmol) in anhydrous THF–DMF (5:1, 24 mL) under Ar at 0 °C was treated sequentially with Pro-
OMe•HCl (707 mg, 4.27 mmol, 1.1 equiv), HOBt (ca. 20% H2O content, 811.5 mg, 4.80 mmol, 1.2 equiv),
EDC•HCl (1.085 g, 5.66 mmol, 1.4 equiv), and NaHCO3 (798.1 mg, 9.50 mmol, 2.4 equiv). The resulting
mixture was allowed to warm to rt and was stirred for 24 h. The reaction was quenched by the addition of
sat aq NaHCO3 (25 mL), diluted with H2O (30 mL), and extracted with EtOAc (5 × 40 mL). The combined
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organic layers were dried (Na2SO4) and concentrated in vacuo. Flash chromatography (200 mL of SiO2,
0–30% EtOAc in hexanes gradient elution) afforded Z-Thr-Pro-OMe (1.3584 g, 3.73 mmol, 94%) as a
white solid: [α]25D –88 (c 0.49, CHCl3); 1H NMR (CDCl3, 500 MHz) δ 7.40–7.31 (m, 5H), 5.70 (d, J =
9.0 Hz, 1H), 5.14 (d, J = 12.2 Hz, 1H), 5.10 (d, J = 12.4 Hz, 1H), 4.57–4.53 (m, 1H), 4.48 (dd, J = 9.2,
2.0 Hz, 1H), 4.24–4.17 (m, 1H), 3.86–3.80 (m, 1H), 3.78–3.73 (m, 1H), 3.75 (s, 3H), 3.38 (br s, 1H),
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2.30–2.21 (m, 1H), 2.10–1.97 (m, 3H), 1.24 (d, J = 6.4 Hz, 3H); C{1H} NMR (CDCl3, 125 MHz) δ
172.5, 170.6, 156.6, 136.2, 128.5 (2C), 128.2, 128.0 (2C), 67.3, 67.1, 58.8, 56.0, 52.5, 47.3, 28.9, 24.9,
18.5; IR (film) υmax 3399, 2977, 1744, 1720, 1634, 1524, 1439, 1218, 1176, 1027 cm–1; HRMS (ESI-
TOF) m/z: [M + H]+ Calcd for C18H24N2O6H 365.17126; Found 365.17005.
A solution of Z-Thr-Pro-OMe (800.0 mg, 2.20 mmol) in t-BuOH–H2O (3:1, 12 mL) at 0 oC was
treated with LiOH•H2O (480.5 mg, 11.45 mmol, 5.2 equiv) and stirred for 4 h. The reaction was quenched
by the addition of 1 N HCl until pH ~2, and the mixture was extracted with EtOAc (4 × 15 mL). The
combined organic layers were washed with brine (10 mL), dried (Na2SO4), and concentrated in vacuo.
Crude acid 11 (616.7 mg, 1.76 mmol, 80%) was obtained as a colorless oil and was used in subsequent
reactions without further purification.
Methyl (2-((S)-1-(((Benzyloxy)carbonyl)-L-threonyl)pyrrolidine-2-carboxamido)-
2-methylpropanoyl)-L-alaninate (12). A solution of acid 11 (240.4 mg, 0.686 mmol) in anhydrous DMF
(10 mL) at 0 °C under Ar was treated sequentially with HBTU (262.3 mg, 0.692 mmol, 1.0 equiv), i-
Pr2NEt (360 L, 267 mg, 2.07 mmol, 3.0 equiv), and amine 4 (159.9 mg, 0.850 mmol, 1.2 equiv). The
resulting mixture was warmed to rt and stirred for 2 h, then diluted with EtOAc (15 mL) and washed with
sat aq NH4Cl (3 × 15 mL), sat aq NaHCO3 (2 × 15 mL), and brine (15 mL). The organic layer was dried
(Na2SO4) and concentrated in vacuo. Flash chromatography (100 mL of SiO2, 2–4.5% MeOH in CH2Cl2
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Page 22 of 41
gradient elution) afforded 12 (89.5 mg, 0.172 mmol, 25%) as a white solid: [α]25D –34 (c 0.34, CHCl3);
1H NMR (CDCl3, 500 MHz) δ 7.37–7.32 (m, 6H), 7.00 (br s, 1H), 6.94 (d, J = 7.0 Hz, 1H), 5.75 (d, J =
8.7 Hz, 1H), 5.14 (d, J = 12.2 Hz, 1H), 5.10 (d, J = 12.2 Hz, 1H), 4.56–4.52 (m, 2H), 4.50–4.48 (m, 1H),
4.26–4.21 (m, 1H), 3.74 (s, 3H), 3.73–3.70 (m, 2H), 2.27–2.18 (m, 1H), 2.13–2.07 (m, 1H), 2.06–1.96
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(m, 2H), 1.59 (s, 3H), 1.46 (s, 3H), 1.37 (d, J = 7.2 Hz, 3H), 1.26 (d, J = 4.0 Hz, 3H); C{1H} NMR
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(CDCl3, 125 MHz) δ 173.9, 173.8, 171.1, 170.7, 156.6, 136.2, 128.5 (2C), 128.2, 128.0 (2C), 67.2, 67.1,
60.5, 57.1, 56.5, 52.5, 48.3, 47.7, 27.3, 26.8, 25.2, 23.9, 19.0, 18.1; IR (film) υmax 3312, 2923, 1735, 1633,
1529, 1454, 1256 cm–1; HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C25H36N4O8H 521.2611; Found
521.2602.
Methyl (2-((S)-1-((Z)-2-(((Benzyloxy)carbonyl)amino)but-2-enoyl)pyrrolidine-2-
carboxamido)-2-methylpropanoyl)-L-alaninate (2c). A solution of tetrapeptide 12 (55.0 mg, 0.106
mmol) in anhydrous CHCl3 (3 mL) at 0 °C was treated with Martin sulfurane (152 mg, 0.226 mmol, 2.1
equiv). The resulting mixture was stirred at 0 °C to rt for 1 h, dried (Na2SO4), and concentrated in vacuo.
Flash chromatography (150 mL of SiO2, 2–4.5% MeOH in CH2Cl2 gradient elution) afforded 2c (23.1
mg, 0.0460 mmol, 44%) as a white film: [α]25D –32 (c 0.85, CHCl3); 1H NMR (CDCl3, 500 MHz) δ 7.40–
7.31 (m, 7H), 6.49 (br s, 1H), 5.52 (q, J = 6.8 Hz, 1H), 5.16 (d, J = 12.1 Hz, 1H), 5.10 (d, J = 12.1 Hz,
1H), 4.54–4.45 (m, 2H), 3.70 (s, 3H), 3.63–3.50 (m, 2H), 2.24–2.12 (m, 2H), 1.98–1.85 (m, 2H), 1.73 (d,
J = 7.0 Hz, 3H), 1.55 (s, 3H), 1.53 (s, 3H), 1.37 (d, J = 7.2 Hz, 3H);13C{1H} NMR (CDCl3, 125 MHz) δ
174.4, 173.5, 170.6, 166.6, 153.7, 135.4, 130.7, 128.7 (2C), 128.6, 128.3 (2C), 117.1, 67.8, 61.4, 57.3,
52.1, 49.6, 48.3, 29.0, 25.6, 25.4, 24.9, 17.6, 11.7; IR (film) υmax 3320, 2924, 2359, 1669, 1521, 1456,
1255 cm–1; HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C25H34N4O7H 503.2506; Found 503.2513.
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(2-(((Benzyloxy)carbonyl)amino)-2-methylpropanoyl)-L-proline (13). A solution of
Z-Aib-OH (750.6 mg, 3.16 mmol) in anhydrous THF–DMF (5:1, 24 mL) at 0 °C under Ar was treated
sequentially with Pro-OMe•HCl (557.6 mg, 3.37 mmol, 1.1 equiv), HOBt (ca. 20% H2O content, 645.1
mg, 3.82 mmol, 1.2 equiv), EDC•HCl (861.2 mg, 4.49 mmol, 1.4 equiv), and NaHCO3 (641.7 mg, 7.64
mmol, 2.4 equiv). The resulting mixture was allowed to warm to rt and was stirred for 24 h. The reaction
was quenched by the addition of sat aq NaHCO3 (15 mL), diluted with H2O (30 mL), and extracted with
EtOAc (5 × 30 mL). The combined organic layers were dried (Na2SO4) and concentrated in vacuo. Flash
chromatography (150 mL of SiO2, 0–30% EtOAc in hexanes gradient elution) afforded Z-Aib-Pro-OMe
(750.7 mg, 2.15 mmol, 68%) as a white solid: [α]25D –52 (c 2.64, CHCl3); 1H NMR (CDCl3, 500 MHz) δ
7.36–7.29 (m, 5H), 5.65 (br s, 1H), 5.11 (d, J = 11.8 Hz, 1H), 5.03 (d, J = 11.6 Hz, 1H), 4.58–4.52 (m,
1H), 3.70 (s, 3H), 3.50–3.42 (m, 1H), 3.38–3.29 (m, 1H), 2.08–1.94 (m, 2H), 1.91–1.80 (m, 2H), 1.59 (s,
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3H), 1.54 (s, 3H); C{1H} NMR (CDCl3, 125 MHz) δ 173.0, 172.1, 154.2, 136.6, 128.4 (2C), 128.2,
128.1 (2C), 66.4, 60.8, 56.7, 52.1, 47.9, 27.7, 25.8, 24.7, 24.3; IR (film) υmax 3303, 2986, 1720, 1623,
1525, 1411, 1364, 1258, 1168, 1074 cm–1; HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C18H24N2O5H
349.1763; Found 349.1757.
A solution of Z-Aib-Pro-OMe (56.2 mg, 0.161 mmol) in t-BuOH (1.0 mL) and H2O (0.4 mL) was
treated with LiOH•H2O (34.1 mg, 0.813 mmol, 5.0 equiv), then stirred at rt for 4 h. The resulting mixture
was acidified to pH 4~5 by the addition of 1 N HCl, diluted with H2O (2 mL), and extracted with EtOAc
(3 × 5 mL). The combined organic layers were dried (Na2SO4) and concentrated in vacuo. The crude
carboxylic acid 13 was used directly in subsequent coupling reactions without further purification.
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Ethyl
(S)-2-((S)-1-(2-(((Benzyloxy)carbonyl)amino)-2-
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methylpropanoyl)pyrrolidine-2-carboxamido)-3-hydroxy-3-methylbutanoate (15). A solution of 13
(506.8 mg, 1.52 mmol) in anhydrous THF–DMF (5:1, 12 mL) at 0 °C under Ar was treated with 1432
(248.4 mg, 1.54 mmol, 1.0 equiv), HOBt (ca. 20% H2O content, 322.3 mg, 1.91 mmol, 1.3 equiv),
EDC•HCl (413.5 mg, 2.16 mmol, 1.4 equiv), and NaHCO3 (314.0 mg, 3.74 mmol, 2.5 equiv). The
resulting mixture was warmed to rt and stirred for 24 h. The reaction was quenched by the addition of sat
aq NaHCO3 (15 mL), diluted with H2O (30 mL), and extracted with EtOAc (5 × 40 mL). The combined
organic layers were dried (Na2SO4) and concentrated in vacuo. Flash chromatography (150 mL of SiO2,
0–60% EtOAc in hexanes gradient elution) afforded 15 (558.4 mg, 1.17 mmol, 77%) as a colorless oil
that was a 1:1 mixture of diastereomers: 1H NMR (CDCl3, 500 MHz) δ 7.68–7.60 (m, 1H), 7.42–7.32 (m,
5H), 5.22–5.02 (m, 3H), 4.70–4.55 (m, 1H), 4.40–4.15 (m, 3H), 4.06 and 3.85 (2 br s, 1H), 3.72–3.60 (m,
1H), 3.467–3.35 (m, 1H), 2.21–2.12 (m, 1H), 2.02–1.80 (m, 3H), 1.61 (s, 6H), 1.48 and 1.45 (2s, 3H),
1.44 and 1.36 (2s, 3H), 1.33–1.29 (m, 3H); 13C{1H} NMR (CDCl3, 125 MHz) δ 172.4, 171.6 and 171.5,
170.7, 155.7 and 154.9, 135.8 and 135.4, 128.9 and 128.7 (2C), 128.6, 128.5 (2C), 72.2 and 71.9, 68.2,
67.3, 63.0 and 62.2, 61.0 and 60.6, 57.2, 48.2 and 48.0, 28.8, 27.6, 27.1 and 26.6, 26.3 and 26.2, 25.7,
24.7, 14.2; IR (film) υmax 3294, 2980, 1725, 1651, 1536, 1456, 1400, 1274, 1196, 1089 cm–1; HRMS (ESI-
TOF) m/z: [M + H]+ Calcd for C24H35N3O7H 478.2553; Found 478.2546.
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Methyl
(2-((S)-1-(2-(((Benzyloxy)carbonyl)amino)-2-
methylpropanoyl)pyrrolidine-2-carboxamido)-3-methylbut-2-enoyl)-L-alaninate (3a). A solution of
tripeptide 15 (75.8 mg, 0.159 mmol) in t-BuOH–H2O (3:1, 8 mL), was treated with LiOH•H2O (36.0 mg,
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The Journal of Organic Chemistry
0.858 mmol, 5.4 equiv). The mixture was stirred at rt for 4 h, and the reaction was quenched by the
addition of 1N HCl until pH ~2. The mixture was then extracted with EtOAc (4 × 15 mL), and the
combined organic layers were washed with brine (10 mL), dried (Na2SO4), and concentrated in vacuo.
The crude acid was then dissolved in anhydrous THF (8 mL) and treated with NaOAc (19.3 mg, 0.235
mmol, 1.5 equiv) and Ac2O (75 L, 81.2 mg, 0.795 mmol, 5.0 equiv). The resulting mixture was stirred
at rt under Ar for 12 h, then diluted with MeOH (10 mL), stirred for an additional 30 min, and diluted
with H2O (10 mL). The mixture was extracted with EtOAc (4 × 15 mL), and the combined organic layers
were washed with brine (10 mL), dried (Na2SO4), and concentrated in vacuo.
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The crude azlactone (40.2 mg, 0.0972 mmol) was then dissolved in MeOH (2 mL) and treated
dropwise with 1 N NaOH (970 L, 0.97 mmol, 10 equiv). The resulting mixture was stirred at rt for 24 h,
then washed with sat aq NaHCO3 (2 × 5 mL) and acidified by the addition of 1 N HCl to pH ~2. The
mixture was extracted with EtOAc (3 × 5 mL), and the combined organic layers were dried (Na2SO4) and
concentrated in vacuo. The crude C-terminal dehydroamino acid was dissolved in anhydrous THF–DMF
(5:1, 3 mL) and treated sequentially with Ala-OMe (42.6 mg, 0.305 mmol, 3.1 equiv), PyBOP (152.4 mg,
0.293 mmol, 3.0 equiv), and i-Pr2NEt (100 L, 74.2 mg, 0.574 mmol, 3.6 equiv). The resulting mixture
was stirred at rt under Ar for 24 h, then extracted with EtOAc (5 × 5 mL). The combined organic layers
were dried (Na2SO4) and concentrated in vacuo. Flash chromatography (25 mL of SiO2, 2–4.5% MeOH
in CH2Cl2 gradient elution) afforded 3a (17.0 mg, 0.0329 mmol, 34% from azlactone and 21% from 15)
as a white film: [α]25D +12.7 (c 0.41, CHCl3); 1H NMR (CDCl3, 500 MHz) δ 8.14 (s, 1H), 7.39–7.30 (m,
6H), 5.30 (s, 1H), 5.16 (d, J = 12.1 Hz, 1H), 4.96 (d, J = 12.1 Hz, 1H), 4.59–4.51 (m, 2H), 3.71 (s, 3H),
3.70–3.65 (m, 1H), 3.26–3.19 (m, 1H), 2.30–2.23 (m, 1H), 2.23 (s, 3H), 1.90–1.78 (m, 3H), 1.78 (s, 3H),
13
1.55 (s, 3H) 1.46 (d, J = 7.2 Hz, 3H), 1.44 (s, 3H); C{1H} NMR (CDCl3, 125 MHz) δ 173.5, 172.4,
171.0, 165.4, 155.3, 144.6, 136.0, 128.6 (2C), 128.5, 128.4 (2C), 122.3, 67.3, 63.2, 57.0, 52.1, 48.5, 48.3,
28.7, 26.5, 26.0, 24.6, 22.1, 21.1, 17.6; IR (film) υmax 3306, 2987, 1666, 1625, 1535, 1454, 1273, 1077
cm–1; HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C26H36N4O7H 517.2662; Found 517.2670.
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Methyl ((Benzyloxy)carbonyl)-L-seryl-L-alaninate (16). A solution of Z-Ser-OH
(500.2 mg, 2.09 mmol) in anhydrous THF–DMF (5:1, 18 mL) under Ar at 0 °C was treated sequentially
with Ala-OMe•HCl (291.7 mg, 2.09 mmol, 1.0 equiv), HOBt (ca. 20% H2O content, 423.6 mg, 2.51
mmol, 1.2 equiv), EDC•HCl (520.8 mg, 2.72 mmol, 1.3 equiv), and NaHCO3 (403.8 mg, 4.81 mmol, 2.3
equiv). The resulting mixture was slowly warmed to rt and stirred for 24 h. The reaction was quenched
by the addition of sat aq NaHCO3 (20 mL), diluted with H2O (25 mL), and extracted with EtOAc (4 × 25
mL). The combined organic layers were dried (Na2SO4) and concentrated in vacuo. Flash chromatography
(170 mL of SiO2, 0–5% MeOH in DCM gradient elution) afforded 16 (489.3 mg, 1.51 mmol, 72%) as a
white solid: [α]25D –11 (c 0.6, CHCl3); 1H NMR (CDCl3, 500 MHz) δ 7.39–7.31 (m, 5H), 7.13–7.01 (m,
1H), 5.92–5.83 (m, 1H), 5.13 (s, 2H), 5.10 (br s, 1H), 4.57 (quint, J = 7.3 Hz, 1H), 4.33–4.28 (m, 1H),
4.08–4.00 (m, 1H), 3.76 (s, 3H), 3.68 (dd, J = 11.2, 6.2 Hz, 1H), 1.41 (d, J = 7.1 Hz, 3H); 13C{1H} NMR
(CDCl3, 125 MHz) δ 173.4, 170.6, 156.5, 136.0, 128.6 (2C), 128.3, 128.1 (2C), 67.3, 63.0, 55.4, 52.7,
48.3, 17.7; IR (film) υmax 3314, 1736, 1665, 1562, 1547, 1460, 1217, 1058 cm–1; HRMS (ESI-TOF) m/z:
[M + H]+ Calcd for C15H20N2O6H 325.1400; Found 325.1408.
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Methyl (2-(((Benzyloxy)carbonyl)amino)-2-methylpropanoyl)-L-prolyl-L-seryl-
L-alaninate (17). A solution of 16 (106.7 mg, 0.329 mmol) in MeOH (6.0 mL) was treated with 10%
Pd/C (70.2 mg, 0.66 wt equiv) at rt under Ar. The resulting mixture was stirred at rt under H2 (500 psi)
for 48 h, then diluted with H2O (10 mL) and extracted with EtOAc (4 × 6 mL). The combined organic
layers were dried (Na2SO4) and concentrated in vacuo. The crude Ser-Ala-OMe (54.4 mg, 0.286 mmol,
87%) was used directly in subsequent couplings without further purification.
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A solution of crude carboxylic acid 13 (94.6 mg, 0.283 mmol) in anhydrous DMF (3.0 mL) at 0
1
2
°C under Ar was treated with HBTU (107.4 mg, 0.282 mmol, 1.0 equiv) and i-Pr2NEt (51.0 L, 37.8 mg,
0.293 mmol, 1.0 equiv). The resulting mixture was treated dropwise with a solution of Ser-Ala-OMe (54.4
mg, 0.286 mmol, 1.0 equiv) in anhydrous DMF (1 mL) and stirred at 0 °C under Ar for 2 h. It was then
diluted with H2O (8 mL) and extracted with EtOAc (3 × 10 mL). The combined organic layers were
washed with sat aq NH4Cl (3 × 5 mL), sat aq NaHCO3 (2 × 4 mL), and brine (6 mL), dried (Na2SO4), and
concentrated in vacuo. Flash chromatography (110 mL of SiO2, 0–10% MeOH in CH2Cl2 gradient elution)
afforded 17 (61.6 mg, 0.122 mmol, 37% from 16) as a white solid: [α]25D + 22 (c 0.6, CHCl3); 1H NMR
(CDCl3, 500 MHz, ca. 1.2:1 mixture of rotamers) δ 7.63 and 7.57 (2d, J = 8.8 Hz and J = 7.4 Hz, 1H),
7.38–7.32 (m, 5H), 7.21 (br s, 1H), 5.75 (br s, 1H), 5.34 (br s, 1H), 5.23–5.19 and 5.17 (m and d, J = 11.8
Hz, 1H), 5.00 and 4.99 (2d, J = 12.2 and 12.1 Hz, 1H), 4.60–4.50 (m, 2H), 4.06–4.00 (m, 1H), 3.83 (dd,
J = 11.4, 4.7 Hz, 1H), 3.71 (s, 3H), 3.67–3.60 (m, 1H), 3.18–3.09 (m, 2H), 2.33–2.27 (m, 1H), 2.17–2.10
and 1.92–1.85 (2m, 1H), 1.80–1.70 (m, 2H), 1.59 and 1.54 (2s, 3H), 1.46 (d, J = 7.4 Hz, 3H) 1.45 and
1.42 (2s, 3H); 13C{1H} NMR (CDCl3, 125 MHz) δ 174.9, 172.9 and 172.8, 171.4, 170.9, 155.9 and 155.3,
135.9 and 135.6, 128.7 (2C), 128.6, 128.3 (2C), 67.7 and 67.3, 63.3 and 63.0, 62.4, 57.2, 54.7, 52.3, 48.7,
48.3 and 48.1, 28.6 and 28.5, 26.7 and 26.5, 26.0 and 25.7, 24.7 and 24.3, 17.4; IR (film) υmax 3306, 2987,
1666, 1625, 1535, 1454, 1273, 1077 cm–1; HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C24H34N4O8H
507.2455; Found 507.2461.
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Methyl
(2-((S)-1-(2-(((Benzyloxy)carbonyl)amino)-2-
methylpropanoyl)pyrrolidine-2-carboxamido)acryloyl)-L-alaninate (3b). A solution of 17 (40.4 mg,
0.0798 mmol) in 1,2-dichloroethane (6 mL) at 0 oC was treated with Et3N (28 L, 20.3 mg, 0.20 mmol,
2.5 equiv), then stirred at 0 oC for 10 min. The resulting mixture was treated dropwise with MsCl (12 L,
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17.8 mg, 0.155 mmol, 1.9 equiv), warmed to rt, and stirred for 1 h. It was then treated with DBU (60 L,
61.1 mg, 0.401 mmol, 5.0 equiv) and stirred at 90 oC for 16 h. The mixture was concentrated in vacuo,
and the residue was dissolved in EtOAc (10 mL) and washed with 10% aq citric acid (6 mL), sat aq
NaHCO3 (2 × 5 mL), and brine (10 mL). The combined organic layers were dried (Na2SO4) and
concentrated in vacuo. Flash chromatography (50 mL of SiO2, 0–10% MeOH in CH2Cl2 gradient elution)
afforded 3b (14.2 mg, 0.0291 mmol, 36%) as a white solid: [α]25D +6.1 (c 0.6, CHCl3); 1H NMR (CDCl3,
500 MHz) δ 8.45 (s, 1H), 7.42–7.30 (m, 5H), 7.10 (br s, 1H), 5.90 (s, 1H), 5.88 (s, 1H), 5.29 (s, 1H), 5.13
(d, J = 11.6 Hz, 1H), 5.00 (d, J = 12.0 Hz, 1H), 4.63 (quint, J = 7.1 Hz, 1H), 4.59–4.53 (m, 1H), 3.76 (s,
3H), 3.62–3.55 (m, 1H), 3.30–3.22 (m, 1H), 2.17–2.09 (m, 1H), 1.96–1.90 (m, 1H), 1.88–1.75 (m, 2H),
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1.57 (s, 3H), 1.50 (s, 3H), 1.46 (d, J = 7.2 Hz, 3H); C{1H} NMR (CDCl3, 125 MHz) δ 176.7, 173.0,
172.6, 171.0, 156.0, 136.1, 128.7 (2C), 128.6 (2C), 128.5, 128.1, 111.9, 67.2, 66.9, 63.2, 57.0, 52.5, 48.6,
29.4, 26.7, 26.0, 24.6, 18.0; IR (film) υmax 3301, 2950, 1668, 1532, 1455, 1263, 1170, 1075 cm–1; HRMS
(ESI-TOF) m/z: [M + H]+ Calcd for C24H32N4O7H 489.2349; Found 489.2357.
Methyl L-Threonyl-L-alaninate (18). A solution of Z-Thr-OH (1.0021 g, 3.96 mmol)
in anhydrous THF–DMF (5:1, 24 mL) under Ar at 0 °C was treated sequentially with Ala-OMe•HCl
(594.0 mg, 4.26 mmol, 1.1 equiv), HOBt (ca. 20% H2O content, 800.5 mg, 4.74 mmol, 1.2 equiv),
EDC•HCl (1.0621 g, 5.54 mmol, 1.4 equiv), and NaHCO3 (801.4 mg, 9.54 mmol, 2.4 equiv). The resulting
mixture was allowed to warm to rt and was stirred for 24 h. The reaction was quenched by the addition of
sat aq NaHCO3 (25 mL), diluted with H2O (30 mL), and extracted with EtOAc (5 × 40 mL). The combined
organic layers were dried (Na2SO4) and concentrated in vacuo. Flash chromatography (200 mL of SiO2,
0–30% EtOAc in hexanes gradient elution) afforded Z-Thr-Ala-OMe (1.2778 g, 3.78 mmol, 95%) as a
white solid: [α]25D –23 (c 0.50, CHCl3); 1H NMR (CDCl3, 500 MHz) δ 7.39–7.32 (m, 5H), 6.96 (d, J =
6.7 Hz, 1H), 5.77 (d, J = 7.6 Hz, 1H), 5.17 (d, J = 12.2 Hz, 1H), 5.12 (d, J = 12.2 Hz, 1H), 4.56 (quint, J
= 7.3 Hz, 1H), 4.38–4.32 (m, 1H), 4.19 (dd, J = 7.8, 2.0 Hz, 1H), 3.76 (s, 3H), 3.17 (br s, 1H), 1.40 (d, J
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The Journal of Organic Chemistry
= 7.2 Hz, 3H), 1.20 (d, J = 6.5 Hz, 3H); 13C{1H} NMR (CDCl3, 125 MHz) δ 173.1, 170.5, 156.8, 136.0,
128.6 (2C), 128.3, 128.0 (2C), 67.3, 66.9, 58.2, 52.6, 48.1, 18.0, 17.9; IR (film) υmax 3324, 3066, 3035,
2956, 1742, 1661, 1538, 1455, 1384, 1293, 1219, 1150, 1051 cm–1; HRMS (ESI-TOF) m/z: [M + H]+
Calcd for C16H22N2O6H 339.15561; Found 339.15492.
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A solution of Z-Thr-Ala-OMe (1.200 g, 3.55 mmol) in MeOH (10.0 mL) was treated with 10%
Pd/C (602.5 mg, 0.5 wt equiv) at rt under Ar. The suspension was then pressurized under hydrogen gas
(500 psig) and stirred for 72 h. The reaction was filtered through Celite, dried (Na2SO4), and concentrated
in vacuo. Amine 18 (534.1 mg, 2.62 mmol, 74%) was obtained as a white solid and was used in subsequent
reactions without further purification.
Methyl
(2-(((Benzyloxy)carbonyl)amino)-2-methylpropanoyl)-L-prolyl-L-
threonyl-L-alaninate (19). A solution of acid 13 (500.0 mg, 1.50 mmol, 1.0 equiv) in anhydrous THF–
DMF (5:1, 12 mL) at 0 °C under Ar was treated with amine 18 (301.1 mg, 1.48 mmol), HOBt (ca. 20%
H2O content, 310.6 mg, 1.84 mmol, 1.2 equiv), EDC•HCl (396.0 mg, 2.07 mmol, 1.4 equiv), and NaHCO3
(304.1 mg, 3.62 mmol, 2.5 equiv). The resulting mixture was warmed to rt, then stirred for 48 h. The
reaction was quenched by the addition of sat aq NaHCO3 (15 mL), diluted with H2O (30 mL), and
extracted with EtOAc (5 × 40 mL). The combined organic layers were dried (Na2SO4) and concentrated
in vacuo. Flash chromatography (150 mL of SiO2, 2–4.5% MeOH in CH2Cl2 gradient elution) afforded
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19 (167.6 mg, 0.322 mmol, 22%) as a white solid: [α]25 –8.1 (c 0.23, CHCl3); H NMR (CDCl3, 500
D
MHz) δ 7.44 (d, J = 9.2 Hz, 1H), 7.40–7.34 (m, 7H), 5.17 (s, 1H), 5.15 (d, J = 12.4 Hz, 1H), 5.05 (d, J =
12.1 Hz, 1H), 4.61–4.53 (m, 4H), 3.78–3.73 (m, 1H), 3.73 (s, 3H), 3.32–3.27 (m, 1H), 2.36–2.31 (m, 1H),
1.89–1.83 (m, 3H), 1.55 (s, 3H), 1.47–1.44 (m, 3H), 1.46 (s, 3H), 1.21 (d, J = 6.4 Hz, 3H); 13C{1H} NMR
(CDCl3, 125 MHz) δ 173.0, 172.5, 171.8, 170.6, 155.5, 135.4, 128.7 (2C), 128.7, 128.4 (2C), 67.8, 67.1,
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