Solid-phase synthesis of peptides containing bulky dehydroamino acids

Article abstract of DOI:10.1016/j.tetlet.2014.12.097

A method of incorporating bulky α,β-dehydroamino acids such as dehydrovaline and dehydroethylnorvaline into peptides via solid-phase peptide synthesis is reported. The key step involves ring opening of an azlactone (i.e., oxazolone) containing the dehydro

Full text of DOI:10.1016/j.tetlet.2014.12.097

Accepted Manuscript
Solid-phase synthesis of peptides containing bulky dehydroamino acids
Jintao Jiang, Shi Luo, Steven L. Castle
PII:
S0040-4039(14)02174-1
DOI:
http://dx.doi.org/10.1016/j.tetlet.2014.12.097
TETL 45621
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Accepted Date:
1 December 2014
17 December 2014
Please cite this article as: Jiang, J., Luo, S., Castle, S.L., Solid-phase synthesis of peptides containing bulky
dehydroamino acids, Tetrahedron Letters (2014), doi: http://dx.doi.org/10.1016/j.tetlet.2014.12.097
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Solid-phase synthesis of peptides containing
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Jintao Jiang, Shi Luo, Steven L. Castle∗

1
Tetrahedron Letters
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Solid-phase synthesis of peptides containing bulky dehydroamino acids
Jintao Jiang, Shi Luo, Steven L. Castle∗
Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT 84602, USA
ARTICLE INFO
ABSTRACT
Article history:
Received
A method of incorporating bulky α,β-dehydroamino acids such as dehydrovaline and
dehydroethylnorvaline into peptides via solid-phase peptide synthesis is reported. The key step
involves ring opening of an azlactone (i.e., oxazolone) containing the dehydroamino acid by the
amino group of a resin-bound peptide. The use of Alloc-protected azlactones was key to the
success of the process.
Received in revised form
Accepted
Available online
2009 Elsevier Ltd. All rights reserved.
Keywords:
Peptide synthesis
Dehydroamino acids
Oxazolones
Dehydrations
Ring opening reactions
α,β-Dehydroamino acids (∆AAs) are characterized by planar
geometry and restricted rotational freedom relative to standard
amino acids. They can enhance the proteolytic stability of
peptides that contain them, presumably by altering the shape of
the backbone and by stabilizing folded states through their
rigidifying effect.1 The conformational preferences of ∆AAs that
contain at least one hydrogen atom at the β carbon (e.g., ∆Ala,
∆Abu, ∆Phe) have been well-studied, and design rules governing
their inclusion in secondary structures have been established.2,3
However, much less is known about bulky ∆AAs that are fully
substituted at the β carbon (e.g., ∆Val), which should exhibit a
more pronounced rigidifying effect on peptides due to their high
levels of A1,3 strain (Figure 1).4 While the structures of simple
∆Val derivatives5 and ∆Val-containing di-, tri-, and tetrapeptides6
have been examined, we are unaware of any studies of larger
peptides that include this residue. This gap in knowledge can be
attributed to a lack of methods for incorporating bulky ∆AAs into
peptides via solid-phase synthesis techniques.
In the course of our efforts to synthesize natural products that
contain tetrasubstituted ∆AAs,7 we became interested in
examining the effects of bulky residues such as ∆Val and its
homologue dehydroethylnorvaline (∆Env, Figure 1) on the
structures of peptides. As a first step in this direction, we have
developed protocols for the efficient solid-phase synthesis of
simple peptides that include these bulky ∆AAs. This work sets
the stage for the preparation and study of more complex ∆AA-
containing peptides including therapeutically relevant
compounds.
Our synthetic strategy is summarized in Scheme 1 and was
inspired by the fact that C-terminal dehydroamino acids are
rapidly transformed into azlactones (i.e., oxazolones) upon
activation of their carboxylate group.8 Although this process
causes the stereochemical scrambling of unsymmetrical ∆AAs
such as E- or Z-∆Ile,9 it can be used to facilitate couplings to
symmetrical ∆AAs such as ∆Val and ∆Env. Thus, we envisioned
that dipeptides 1 bearing either ∆Val or ∆Env at the C terminus
would undergo dehydration and cyclization in a single pot to
generate azlactones 2. Then, heating 2 in the presence of a resin-
bound peptide should trigger a coupling reaction, furnishing
elongated resin-bound peptides 3. Next, subjection of 3 to
standard solid-phase peptide synthesis (SPPS) protocols followed
by cleavage from the resin and purification would provide the
targeted peptides with bulky ∆AAs located at the desired
positions. Prior reports of thermally promoted ring openings of
azlactones by amines in solution10 provided some support for our
strategy. However, the lack of examples involving resin-bound
amines was concerning.
Figure 1. Normal and bulky dehydroamino acids.
———
∗ Corresponding author. Tel.: +1-801-422-1780; fax: +1-801-422-0153; e-mail: scastle@chem.byu.edu.

2
Tetrahedron
CbzHN–OMs
OsO4 (10 mol %)
1. H2 (650 psi)
10% Pd/C, MeOH
CO2Et
CbzHN
CO2Et
CH3CN–H2O 8:1
45 °C
2. Alloc-Gly or Phe
OH
COMU,
-collidine
sym
6
7
75%
DMF
R
R
H
H
N
N
CO2Et
CO2H
LiOH•H2O
AllocHN
AllocHN
O
O
-BuOH–H O
t
2
3:1
OH
OH
1c
(93%)
5c
(R = H, 77%)
5d (R = Bn, 63%)
1d (84%)
COMU:
O
N
NaOAc
Ac2O
N+
O
Scheme 1. Strategy for synthesis of ∆AA-containing peptides via SPPS.
N
O
O
Our initial attempts to execute this plan employed Fmoc-
AllocHN
–
PF6
50 °C
protected dipeptides of type 1. Although formation of the
azlactones was facile, heating solutions of these species in the
presence of resin-bound peptides yielded only trace amounts of
the coupled products according to MS analysis. Further
investigation suggested that the Fmoc group was not stable under
the reaction conditions. Accordingly, we decided to examine
Alloc-protected substrates, since this group would presumably be
more stable than the Fmoc group under the weakly basic
coupling conditions. Moreover, cleavage of the Alloc moiety
from solid-supported peptides has been accomplished under
conditions that are compatible with the acid-labile side-chain
protecting groups that are typically employed in Fmoc-based
SPPS.11
N
R
2c
(85%)
2d (64%)
NC
CO2Et
Scheme 3. Synthesis of ∆Env-based azlactones.
With the requisite azlactones 2a–d in hand, we explored their
incorporation into resin-bound peptides. We wished to use a
therapeutically relevant peptide as a system for determining the
viability of our synthetic strategy. Accordingly, we decided to
synthesize analogues of the C-terminal region of enfuvirtide, an
FDA-approved inhibitor of HIV membrane fusion.16 Thus,
attachment of Fmoc-Phe-OH to Rink amide resin and elaboration
using standard Fmoc-based SPPS techniques afforded resin-
bound pentapeptide 9 (Scheme 4). Different solvents (DMF
versus NMP), reaction temperatures (50 °C versus 60 °C), and
additives (DMAP versus no additive) were then evaluated in the
coupling of 9 with azlactone 2a. Analysis of the reactions by
LC/MS revealed that heating the mixture at 60 °C in NMP for 24
h in the presence of DMAP yielded the best results, although the
coupling also proceeded in the absence of the additive. Therefore,
these conditions were employed to mediate the ring openings of
azlactones 2b–2d by 9. Each reaction afforded the desired
product as evidenced by MS, but the yields decreased somewhat
as the size of the azlactones increased (vide infra).
Based on this logic, we constructed ∆Val-containing
dipeptides 1 and azlactones 2 as outlined in Scheme 2.
Hydrogenolysis of racemic Cbz-protected β-OHVal derivative
47a and coupling of the resulting amine with Alloc-Gly or Alloc-
Phe afforded dipeptides 5a and 5b, respectively, in good yields.
Saponification furnished the free acids 1a and 1b, which
underwent facile dehydration and cyclization upon exposure to
Ac2O and NaOAc.12 The azlactones 2 were typically used in
subsequent reactions without purification due to their sensitivity
to SiO2.
The Alloc groups of peptides 10a–d were then removed using
conditions (Pd(PPh3)4, PhSiH3) that were previously developed
for the deprotection of resin-bound peptides.11 This process was
repeated to ensure complete conversion. Coupling of the
resulting resin-bound amines to Fmoc-Trp(Boc)-OH was then
accomplished under standard conditions. Finally, TFA-mediated
deprotection and cleavage of the octapeptides from the solid
support furnished the targeted compounds 11a–11d, which were
purified via reverse-phase HPLC.
The nature of SPPS renders it impossible to precisely
determine the yield of a single step in a synthetic sequence.
Notwithstanding, we were interested in comparing the yields of
the couplings of azlactones 2a–2d with 9. The HPLC traces of
the crude final products 11a–11d provided us with a qualitative
means of making this comparison. The trace for crude 11a, which
was derived from the smallest azlactone 2a, was very clean with
only a handful of minor impurities present.17 The traces for crude
11b and 11c, which are derived from medium-sized azlactones
2b and 2c, were similar to each other and showed a few more
impurities than the trace for 11a. Still more impurities were
visible in the trace for crude 11d, which is obtained from the
largest azlactone 2d. Although it is possible that the Alloc
deprotection, Trp coupling, and final cleavage/deprotection steps
could have proceeded with varying yields, it is likely that the
differences in the amounts of 11a–11d are primarily due to the
ring opening reaction proceeding more smoothly with less-
Scheme 2. Synthesis of ∆Val-based azlactones.
In preparation for synthesizing the corresponding ∆Env-
containing azlactones, we prepared racemic β-hydroxy
ethylnorvaline derivative
7
in one-step via base-free
aminohydroxylation7,13 of known enoate 614 (Scheme 3). This
compound was transformed into azlactones 2c and 2d via the
same sequence employed to convert 4 into 2a and 2b. COMU15
was found to be superior to EDC•HCl and HOBt for mediating
peptide couplings of the hindered amine derived from 7. The
slightly lower yields of the couplings and dehydration–
cyclization reactions in the ∆Env series are presumably a
consequence of the bulkier nature of this amino acid relative to
∆Val.

3
hindered azlactones. Nonetheless, it is important to note that all
four targeted octapeptides were abundant and were easily
purified to homogeneity.
References and notes
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O
NHTr
O
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H
N
H
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SPPS
H2N
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H
N
H
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N
H
N
H
H
N
N
AllocN
H
N
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N
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H
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O
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10a (R1 = Me, R2 = H)
10b R1
R2
= Bn)
(
(
= Me,
10c R1
R2
= H)
= Et,
= Et,
10d R1
R2
= Bn)
(
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1. Pd(PPh3)4, PhSiH3, CH2Cl2
10 min
2. Fmoc-Trp(Boc)-OH, iPr2NEt
HBTU, HOBt, NMP
2
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HSCH2CH2SH, iPr3SiH
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O
NH2
R2
O
O
O
O
H
H
H
FmocHN
N
N
N
NH2
N
N
N
N
H
H
H
H
O
O
O
O
R1
R1
NH
NH
NH
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R2
R2
= H)
(
= Me,
= Me,
= Et,
= H)
= Bn)
11b R1
(
11c R1
R2
(
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Scheme 4. Solid-phase synthesis of ∆AA-containing peptides.
In summary, we have devised a protocol for incorporating
bulky tetrasubstituted α,β-dehydroamino acids into peptides via
solid-phase peptide synthesis. Our method capitalizes on the
facile generation of azlactones (i.e., oxazolones) from dipeptides
that possess C-terminal β-hydroxy amino acids via a one-pot
dehydration–cyclization process. The azlactone rings are readily
opened by the amino groups of resin-bound peptides. Efforts to
improve the couplings of hindered azlactones and to synthesize
full-length therapeutic peptides by this method are in progress
and will be reported in due course.
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17. The HPLC traces can be found in the Supplementary Data section.
Supplementary Data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.tetlet.xxxx.xx.
xxx. These data include experimental procedures, spectral data,
and HPLC traces.
Acknowledgments
We thank Brigham Young University (Undergraduate
Research Award to S.L.) for support of this work. We are
grateful to Prof. Joshua L. Price for providing access to
equipment used in SPPS and peptide purification. We also thank
Paul Lawrence and Ankur Jalan for technical assistance.