Cyclizing pentapeptides: Mechanism and application of dehydrophenylalanine as a traceless turn-inducer

Article abstract of DOI:10.1021/acs.orglett.6b03308

Dehydrophenylalanine is used as a traceless turn-inducer in the total synthesis of dichotomin E. Macrocyclization of the monomer is achieved in high yields and selectivity over cyclodimerization under conditions 100 times more concentrated than previously achieved. The enamide facilitates ring closing, and Rh-catalyzed hydrogenation of the unsaturated cyclic peptide results in selective formation of the natural product or its epimer, depending on our choice of phosphine ligand. NMR analysis and molecular modeling revealed that the linear peptide adopts a left-handed α-turn that preorganizes the N- and C-termini toward macrocyclization.

Full text of DOI:10.1021/acs.orglett.6b03308

Letter
pubs.acs.org/OrgLett
Cyclizing Pentapeptides: Mechanism and Application of
Dehydrophenylalanine as a Traceless Turn-Inducer
Diane N. Le,†,§ Jan Riedel,†,§ Natalia Kozlyuk,† Rachel W. Martin,†,‡ and Vy M. Dong*,†
†Department of Chemistry, University of California, Irvine, California 92697, United States
‡Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92697, United States
S
* Supporting Information
ABSTRACT: Dehydrophenylalanine is used as a traceless
turn-inducer in the total synthesis of dichotomin E. Macro-
cyclization of the monomer is achieved in high yields and
selectivity over cyclodimerization under conditions 100 times
more concentrated than previously achieved. The enamide
facilitates ring closing, and Rh-catalyzed hydrogenation of the
unsaturated cyclic peptide results in selective formation of the
natural product or its epimer, depending on our choice of
phosphine ligand. NMR analysis and molecular modeling
revealed that the linear peptide adopts a left-handed α-turn that preorganizes the N- and C-termini toward macrocyclization.
aturally occurring cyclic peptides have inspired the
1
Ninvention of strategies
for organic synthesis and
therapeutics for use as antibiotics2 and immunosuppressants.3
In comparison with their linear counterparts, these cyclic
structures show enhanced metabolic stability,4 conformational
rigidity,5 and potential to mimic protein−protein interactions.6
While significant progress has been made in the construction of
relatively large cyclic peptides, the construction of smaller
peptides (i.e., those containing five or fewer amino acids) remains
achallenge.7 Inaddition, cyclizingpeptidesathighconcentrations
on an industrial scale is important, and thus, a turn-inducer is
desirabletoensureanefficientandeconomicallyfeasibleprocess.8
Specific amino acids (e.g., proline, pseudoproline, D-amino acids,
and N-methylated amino acids) have been identified as turn-
inducers that can be incorporated into a linear precursor to
facilitate macrocyclizations.9 Ring closing of small peptides
without such turn-inducers is plagued by competitive dimeriza-
tion and epimerization.10 Toward a more general solution to this
challenge, we propose the use of dehydroamino acids as traceless
turn-inducers.
Dehydroamino acids modulate backbone conformations and
produce folded structures.11 The impact of dehydrophenylala-
nine on the conformation of small peptides has been studied
extensively over the past decade.12 For example, Singh has shown
Figure 1. (a) Crystal structure of a tetrapeptide containing
dehydrophenylalanine. (b) Proposed strategy using dehydrophenylala-
nine as a traceless turn-inducer.
that dehydrophenylalanines can induce β-turns in a linear
tetrapeptide on the basis of X-ray crystallography studies (Figure
1a).13 The ability of dehydroamino acids to impart folded
conformations has yet to be exploited to achieve efficient ring
closings in order to gain access to various cyclic peptides. We
envisioned that this unsaturated moiety could serve as a versatile
functional handle for further elaboration in the late-stage
preparation of natural product derivatives.14 Moreover, these
unsaturated derivatives could serve as analogues in structure−
activity relationship (SAR) studies or serve as potential epitope
mimetics.15,16 Here we report the first use of dehydrophenyla-
lanineasatracelessturn-inducerviaitsapplicationinthesynthesis
of dichotomin E (1).
Received: November 14, 2016
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.6b03308
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Organic Letters
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Isolated from the chickweed plant, Stellaria dichotoma, 1 is a
cyclic peptide containing five amino acids with cell growth
inhibitory activity against leukemia cells.17 Our retrosynthetic
analysisforconstructionofthissmallcyclicpeptideissummarized
in Figure 1b. First, we imagined that the natural product could be
obtained from cyclic peptide 2, containing a (Z)-dehydropheny-
lalanine,18 by catalytic hydrogenation. In contrast to the
incorporation of other turn-inducers, the dehydrophenylalanine
can be easily unveiled to the L- or D-amino acid. Next, we chose to
disconnect the glycine−alanine peptide bond to reveal the linear
and unsaturated peptide 3. We chose this disconnection to help
favor an effective macrocyclization by placing the dehydrophe-
nylalanineatthei+2position, whereitwaspreviously reportedto
induce a β-turn.19 A similar disconnection was used in the
previous synthesis of 1 by Tam.20 In general, macrocyclizations
are more favorable using glycine because it is a relatively
unhindered nucleophile.21
Scheme 2. Effect of Dehydroamino Acid on Macrocyclization
(n = Number of Dehydroamino Acid Residues)
a b
,
With this retrosynthetic analysis in mind, we prepared
unsaturated pentapeptide 3 (Scheme 1). Boc-L-alanine (6) was
Scheme 1. Synthesis of Unsaturated Pentapeptide 3
a
b
Isolated yields are shown. Selectivity was determined by HPLC.
tration afforded the macrolactam in 87% yield, our approach
circumvents the need for high dilution by using 100 times less
solvent in the macrocyclization.
Next, we prepared pentapeptide 12 bearing two dehydroamino
acids (see the SI) and subjected this linear precursor to ring
closing. Under the same cyclization conditions at 0.1 M
concentration, cyclic pentapeptide 13 was isolated in 84% yield
with improved 26:1 selectivity for the monomer over the
cyclodimer (cf. Scheme 2b,c).23 Together, these results
demonstrate that dehydrophenylalanines act as turn-inducers
that greatly favor macrocyclization even at high concentrations.
With unsaturated cyclic peptides 2 and 13 in hand, we applied
hydrogenation to install the final stereocenters. Hydrogenation of
cyclic peptide 2 using Rh(cod)2BF4 and the achiral dppp ligand
resulted in the formation of an 8:1 mixture favoring epimer 1′ of
dichotomin E (eq1). To overcome theinherent substrate bias, we
coupled to DL-(β-OH)-Phe-OMe (7) toafford dipeptide 8 in 76%
yield. Treatment of 8 with Boc anhydride and tetramethylgua-
nidineaffordedunsaturateddipeptide9in91%yield.22 Subjecting
9 to hydrolysis, peptide coupling, and deprotection gave
tripeptide 4 in 63% yield. 4 was then coupled to dipeptide 5 in
61% yield to afford pentapeptide 10. After hydrolysis and
deprotection of 10, unsaturated pentapeptide 3 was obtained in
97% yield. For comparison, we also prepared saturated linear
peptide 11 in 64% yield using solid-phase peptide synthesis
(SPPS) (see the Supporting Information (SI)).
When saturated linear pentapeptide 11 was subjected to
macrocyclization at 0.1 M, only a 15% yield of 1 was obtained,
with 1.5:1 selectivity for the desired monomer over the
cyclodimer (Scheme 2a). In stark contrast, treatment of
unsaturated pentapeptide 3 under the same conditions resulted
in the formation of cyclic pentapeptide 2 in 74% yield, and the
selectivity improved to 20:1 for the monomer versus the
cyclodimer. Subsequently, cyclic pentapeptide 2 was isolated in
81% yield with 39:1 selectivity for the desired monomer over
cyclodimer at 0.05 M. In comparison to Tam’s method, where a
silver-ion-assisted orthogonal cyclization at 0.001 M concen-
turned to asymmetric hydrogenation, which is commonly used in
the synthesis of medicines in industry.24 Liu and Zhang25
previously reported the asymmetric hydrogenation of enamides
using Rh(I) with Duanphos as the ligand to afford the
corresponding amide with 99% ee. By using 5mol %Rh(cod)2BF4
B
DOI: 10.1021/acs.orglett.6b03308
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Organic Letters
Letter
Scheme 3. Asymmetric Hydrogenation Affords Dichotomin E
Figure 3. Minimized-energy conformation 15 of unsaturated peptide 3
consistent with NMR analysis.
a
b
For 2, MeOH was used. For 13, THF was used. Pd/C (20 mol %),
H2, 28 h, 30°, 99%.
left-handed α-turn, which is preorganized toward macrocycliza-
tion (Figure 3). Intramolecular H-bonding was also investigated
using the temperature coefficients of the N−H chemical shifts
(Δδ/ΔT), which can be used as an indicator of intramolecular H-
bonding as opposed to H-bonds to solvent.30,31 A value of
−0.0039 ppm/K was obtained for the internal alanine N−H in
dehydropeptide 3, in contrast to the value of −0.0052 ppm/K
observed for saturated pentapeptide 11 (see the SI). This
difference suggests that there is dynamic intramolecular H-
bonding in dehydropeptide 3 but not in saturated pentapeptide
11, consistent with the ensemble of structures predicted by the
molecular modeling. Together, these results demonstrate that a
single dehydrophenylalanine residue can induce a left-handed α-
turn.
and 5 mol % (S,S′,R,R′)-Duanphos in THF under 30 atm
hydrogen, we were able to hydrogenate peptide 2 and obtain
dichotomin E (1) in 96% yield with >95:5 dr (Scheme 3). It is
worthyofnotethatreductionofcyclicpeptide2using(R,R′,S,S′)-
Duanphosaffordstheepimer1′in82%yieldwith>95:5dr. Cyclic
peptide 13 bearing two enamides can also be transformed to
dichotomin E by tandem asymmetric reduction followed by
debenzylation (Scheme 3).
To better understand the mechanism of macrocyclization, we
performed CD spectroscopy experiments on pentapeptides 11, 3,
and 2 in MeOH (298 K) to investigate the presence of secondary
structure (Figure 2).26 Uncyclized dehydropeptide 3 showed
When we replaced the phenyl substituent with a cyclohexyl
substituent in 3, cyclic monomer formation was observed in a
promisingyetlessefficient54%yieldby1HNMRanalysis(seethe
SI). This result suggests that the steric impact of the substituent
influences the cyclization. In view of the higher-yielding
macrocyclizations weobservedinScheme2, conjugationbetween
the phenyl substituent and the alkene helps promote ring closing
by increasing the steric impact of the phenyl group. Weiss,
Lawrence, and co-workers used dehydrophenylalanine as a β-
breaker to study insulin and showed that extended conjugation of
the aromatic π electrons with the neighboring CC and CO
electrons enforces near-planarity.32 The near-planar conforma-
tion ofdehydrophenylalanine results inagreatersteric interaction
between the phenyl group and the adjacent amide group, as
shown in 15, which ultimately restricts the ϕ angle of the
dehydroamino acid.33 This restriction, through the increased A1,3
strain, biases the N- and C-termini toward cyclization.
Interestingly, a peptide containing three consecutive dehydroa-
lanine units has been shown to adopt an extended conformation
in which all of the amide groups show near-planarity.34 This
example suggests that the steric interactions of the group at the β-
carbon of the dehydroamino acid are correlated to its ability to
induce a turn.
In conclusion, we have demonstrated dehydrophenylalanine as
an effective and traceless turn-inducer in the synthesis of
dichotomin E. NMR analysis revealed that unsaturated
pentapeptide 3 adopts a cyclic, preorganized structure. The
enamide serves as a turn-inducer to facilitate ring closing without
the need for high dilution. Moreover, it is a convenient functional
handle for the late-stage construction of natural products and
their derivatives. In SPPS, the overall yield is typically exceptional
because each step in this linear approach is driven by exploiting
excess reagents.35 Combined with the need for dilute solvent
conditions, the amount of waste generated in this traditional
approach to cyclic peptide construction is significant. Our
approach aims for a more efficient synthesis of cyclic peptides,
especially on a large scale, while SPPS enables the rapid synthesis
Figure 2. CD spectra of pentapeptides 11, 3, and 2.
absorption patterns consistent with a cyclized structure, similar to
cyclic dehydropeptide 2. In contrast, uncyclized, saturated
pentapeptide 11 showed no absorption patterns indicative of
any secondary structural motif. The CD spectrum supports the
pronounced effect of the presence of dehydrophenylalanine on
the secondary structure of uncyclized dehydropeptide 3, which
helps to facilitate macrocyclization.
We used solution-state NMR spectroscopy and molecular
modeling to elucidate the structure of unsaturated pentapeptide
3. The 3J couplings for the Tyr residue and the two Ala residues
27
1
were obtained from 2D J-resolved H NMR spectra. These
couplings were used to calculate HNHα ϕ dihedral angles via the
Karplus relation.28 Using these dihedral angle and 2D NOESY
restraints, we performed molecular modeling studies with
Maestro29 to obtain 20 low-energy conformations that were
consistent with our experimental observations (see the SI). These
calculations support the lowest-energy structure 15 containing a
C
DOI: 10.1021/acs.orglett.6b03308
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
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ASSOCIATED CONTENT
* Supporting Information
■
S
TheSupportingInformationisavailablefreeofchargeontheACS
Publications website at DOI: 10.1021/acs.orglett.6b03308.
Procedures and additional data (PDF)
V. S. Biopolymers 1993, 33, 209. (d) Tang, W.; Jimen
́ ́
ez-Oses, G.; Houk,
K. N.; van der Donk, W. A. Nat. Chem. 2015, 7, 57.
AUTHOR INFORMATION
■
(12) For selected examples, see: (a) Jiang, J.; Ma, Z.; Castle, S. L.
Tetrahedron 2015, 71, 5431. (b) Gupta, M.; Chauhan, V. S. Biopolymers
2011, 95, 161. (c) Mathur, P.; Ramakumar, S.; Chauhan, V. S. Pept. Sci.
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Crystallogr. 2005, 35, 761.
Corresponding Author
*dongv@uci.edu
ORCID
Rachel W. Martin: 0000-0001-9996-7411
Vy M. Dong: 0000-0002-8099-1048
Author Contributions
§D.N.L. and J.R. contributed equally.
(14) Bonauer, C.; Walenzyk, T.; Konig, B. Synthesis 2006, 2006, 1.
̈
(15) Hill, T. A.; Shepherd, N. E.; Diness, F.; Fairlie, D. P. Angew. Chem.,
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(17) Morita, H.; Kayashita, T.; Shishido, A.; Takeya, K.; Itokawa, H.;
Shiro, M. Tetrahedron 1996, 52, 1165.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
(18) Studying the effect of (E)-dehydrophenylalanine was of interest
but was not pursued because of its acid/base sensitivity and propensity to
isomerize tothe Z olefin. See:Nitz, T. J.; Holt, E. M.;Rubin, B.; Stammer,
C. H. J. Org. Chem. 1981, 46, 2667.
(19) Patel, H. C.; Singh, T. P.; Chauhan, V. S.; Kaur, P. Biopolymers
1990, 29, 509.
■
Funding was provided by the National Science Foundation
(NSF) (CHE-1465263 for V.M.D. and CHE-1308231 for
R.W.M.). D.N.L. is grateful for an NSF Graduate Fellowship.
We thank Dr. Nathan Bennett (AbbVie), Dr. I-Hon Chen
(Chugai), and Dr. Kritika Mohan (Stanford) for helpful
(20) Zhang, L.; Tam, J. P. J. Am. Chem. Soc. 1999, 121, 3311.
(21) Kopple, K. D. J. Pharm. Sci. 1972, 61, 1345.
́ ́
discussions. Weacknowledge SrideviRamanoudjame (Universite
de Strasbourg) for help with the synthesis of 11 and Daniel
Droege (UCSF) for help with various dipeptides. We appreciate
the Nowick lab for use of HPLC instrumentation.
(22)Monteiro, L. S.;Andrade, J. J.;Suarez, A.C. Eur. J. Org. Chem. 2011,
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formation (see the SI).
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