Total synthesis of pseudacyclins A–E by an on-resin head-to-side chain concomitant cyclization-cleavage reaction

Article abstract of DOI:10.1016/j.tetlet.2018.10.025

Taking advantage of the nucleophile-sensitive ester link of oxime resin, a novel synthetic strategy was applied to the first synthesis of a type of cyclic peptides known as pseudacyclins A–E. The endocyclic ornithine side-chain part was incorporated by an on-resin acid-catalyzed concomitant cyclization-cleavage reaction after a selective deprotection of orthogonally protected ornithine. The synthetic methodology gives high macrocyclization yields and low oligomerization side-products. The combination used of solid-phase/solution-phase strategy was efficient to prepare pseudacyclins and could prove useful to prepare other natural cycle-tail peptides.

Full text of DOI:10.1016/j.tetlet.2018.10.025

Accepted Manuscript
Total synthesis of pseudacyclins A-E by an on-resin head-to-side chain con-
comitant cyclization-cleavage reaction
Christopher Bérubé, Alexandre Borgia, Normand Voyer
PII:
S0040-4039(18)31232-2
https://doi.org/10.1016/j.tetlet.2018.10.025
TETL 50335
DOI:
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
2 September 2018
10 October 2018
14 October 2018
Please cite this article as: Bérubé, C., Borgia, A., Voyer, N., Total synthesis of pseudacyclins A-E by an on-resin
head-to-side chain concomitant cyclization-cleavage reaction, Tetrahedron Letters (2018), doi: https://doi.org/
10.1016/j.tetlet.2018.10.025
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Christopher Bérubé1 Alexandre Borgia1 and Normand Voyer1
1Département de chimie and PROTEO, Université Laval, Québec, G1V 0A6, Canada
Graphical Abstract:
R2
O
O
O
O
O
O
H
N
NH
N
N
NH
N
O
N
N
O
H
NH
O
O
R3
O
Oxime resin
NH
O
ZHN
R1
1. Hydrogenolysis
2. Tail ligation
NH
NH
R1
R
1 = H or Me
O
H2N
On-resin head-to-side chain concomitant cyclization-cleaveage
Pseudacyclins A to E
Overall yields : 22 to 28%
Abstract:
Taking advantage of the nucleophile-sensitive ester link of oxime resin, a novel synthetic strategy
was applied to the first synthesis of a type of cyclic peptides known as pseudacyclins A-E. The
endocyclic ornithine side-chain part was incorporated by an on-resin acid-catalyzed concomitant
cyclization-cleavage reaction after a selective deprotection of orthogonally protected ornithine.
The synthetic methodology gives high macrocyclization yields and low oligomerization side-
products. The combination used of solid-phase/solution-phase strategy was efficient to prepare
pseudacyclins and could prove useful to prepare other natural cycle-tail peptides.
Key words: Oxime resin, cyclic peptides, pseudacyclins, cycle-tail peptides, peptide synthesis,
peptide cyclization
Total synthesis of pseudacyclins A-E by an on-resin head-to-side chain concomitant
cyclization-cleavage reaction
Introduction:
Cycle-tail peptides are one of the most versatile molecular scaffolds to be found among the rich
diversity of bioactive peptides found in nature,1,2. For example, cycle-tail peptides daptomycin3-5
and polymyxin6,7 are well-known and widely used antibiotics.8 Various synthetic methodologies
for cycle-tail peptides have been devised;7,9-11 generally, the final step is a solution
macrocyclization between an amine and the C-terminal carboxylic acid. However, this strategy
often requires high dilution to counterbalance entropic loss by cyclization. Also, activation of the
C-terminus by highly electrophilic ester species have to be used to cause cyclization, thus
increasing the probability of racemization. Although, agents can be used to minimize the
epimerization process, it is always a concerned in solution.12
Though any macrocyclization process is always challenging, considering that undesired
oligomerization and epimerization side reactions can occur, anchoring the linear peptide on a

solid support can reduce the rate of diffusion and decrease the probability of dimerization and
cyclodimerization (pseudodilution effect).13,14
Among different synthetic strategies to cyclize peptides, our research group focused on the use of
oxime resin. Discovered by Kaiser and DeGrado in the early 80s, the oxime resin has a unique
oxime ester link that can be cleaved by a nucleophilic displacement to release the peptide.15,16 As
the oxime ester linkage is stable to both acidic and non-nucleophilic basic conditions, anchoring
an amino acid on oxime resin allows peptide elongation using N-Boc strategy. After the desired
peptide synthesis and N-Boc removal, the susceptibility of oxime resin to nucleophiles allows an
easy release of the free or protected peptide via acid-catalyzed N-terminal macrocyclization in a
buffered AcOH/DIEA mixture. The macrocyclic peptide can then be recovered by simple
filtration and evaporation. This strategy was used for the preparation of Boc/Bzl/Cbz-protected
peptide segments and cyclic peptides.17-20 However, oxime resin was only exploited to afford
cyclic peptides in a head-to-tail fashion to yield cyclic dipeptides21-26 and larger peptides
macrocycles.27,28 Also, a dimerization-cyclization-cleavage sequence was used to prepare C2-
symmetrical cyclic peptides29 such as the well-known antibiotics tyrocidine A30 and gramicidin
S20. However, only two examples reported by Taylor described the use of head-to-side chain
cyclization on oxime resin to prepare amphiphilic -helical peptides with multiple lactam bridges
between Lysi + Glui+4 and Lysi + Aspi+4.31-33 Here, we report the first total synthesis of
pseudacyclins A-E (Figure 1) by a head-to-side chain concomitant cyclization-cleavage reaction
on oxime resin. Interestingly, pseudacyclin A exhibits cytotoxic and antiproliferative activities
against human lymphocytes. However, no bioactivity studies have been reported for
pseudacyclins B-E, probably due to the lack of materials required for such studies..34

Figure 1: Structures of pseudacyclins A-E (red: endocyclic ornithine)
R2
O
O
Exocyclic amidation
site
H
H
O
O
O
N
N
H
N
N
H2N
N
N
O
R2
O
R3
O
NH
NH
O
+
OH
HN
HN
N
Tail ligation
N
N
O
O
H
H
R3
O
O
O
R
1 = H or CH3
R1
R1
1. Macrocyclization
2. Hydrogenolysis
Pseudacyclins scaffold
Cyclization site
O
O
H
O
NHBoc
SPPS
N
N
N
+
N
O
N
HO
O
H
NH
O
ZHN
R1
Oxime resin
ZHN
COOH
Orthogonally protected ornithine
R
1 = H or Me
H2N
On-resin head-to-side chain concomitant cyclization-cleaveage
Figure 2: Retrosynthetic approach towards pseudacyclin cycle-tail peptide scaffold
Results and discussion
Pseudacyclins A-E (1-5) are characterized by 18-membered cyclic peptides that incorporate an
endocyclic L-ornithine side-chain. The exocyclic -amino group is linked to a tail: an acetylated
valine or isoleucine. Our retrosynthetic approach is described in Figure 2. We envisioned that

pseudacyclins A-E (1-5) could be prepared at the final step by the ligation of the N-acetylated
amino acid to the free amine macrocycle, and that the latter could be obtained by on-resin acid-
catalyzed head-to-side chain concomitant cyclization-cleavage of the required peptide using
suitably protected ornithine as a nucleophile.
The peptide chain elongation on the resin involves the use of peptide coupling strategies with N-
Boc protected amino acids to assemble the four first amino acids. The key step involves the
incorporation of Z-Orn(Boc)-OH at the fifth position. After removal of the N-Boc group under
acidic conditions, head-to-side chain concomitant cyclization-cleavage could be performed, while
keeping the Orn N-terminal protected by a carboxybenzyl group, thus avoiding undesired head-
to-tail cyclization products. Then, hydrogenolysis liberates the -amino group to achieve the
exocyclic ligation with proper acetylated amino acids.
All peptides were synthesized on oxime resin with a low substitution level (0.3 mmol/g), using
the protocols described in Figure 3 (see ESI for details). Low oxime functionalization on
polystyrene was used, since higher resin loadings increased the occurrence of the oligomerization
process.29
Briefly, the first amino acid was coupled for three hours using diisopropylcarbodiimide (DIC) as
a coupling reagent. The N-Boc protecting group was removed using a mixture of 1:1
trifluoroacetic acid (TFA)/dichloromethane, while the second amino acid was activated with 6-
chloro-1-hydroxybenzotriazole (6-Cl-HOBt) and 1-[Bis(dimethylamino)methylene]-1H-1,2,3-
triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HCTU). The Kaiser test showed that each
coupling step on the resin was quantitative. After appropriate coupling/deprotection steps, the
linear peptides were simultaneously cyclized and cleaved from the resin in the presence of
diisopropylethylamine (DIEA; 2.5 equiv) and acetic acid (AcOH; 5 equiv) in dichloromethane at
a precursor concentration of 10-2 M on the basis of the starting loading resin, leading to high
macrocyclization yield (70–72%) of Z-protected cyclic peptides 6 and 7.
As observed in the literature, incorporating an amino acid with a flexible aliphatic side-chain-
bearing amine, such as ornithine in the present case, facilitates the macrocyclization process.35 An
HPLC-TOF-MS investigation was performed on the crude cyclization products. In both cases,
results revealed only two peaks; the first was assigned to the cyclic monomer and the second to
the dimer, the latter being obtained at 5% and 8% respectively in the preparation of 6 and 7 (see
ESI for details). No other side products that might be expected to arise from higher
oligomerization were identified in the cyclization-cleavage process.
Due to solubility problems, the desired cyclic monomers were purified from the cyclic dimers
after the N-carboxybenzyl deprotection step. This deprotection of peptides 6 and 7 was performed

by a catalytic palladium hydrogenation using mild conditions in EtOH with 1 atm of hydrogen to
afford the desired macrocyclic peptides 8 and 9.
Usually, HPLC preparative purification was used to separate the desired cyclic monomers from
the cyclic oligomers formed during the cyclization process. This required a long purification time
and yielded small quantities of the desired compounds. Therefore, we developed a powerful
normal-phase chromatography method for rapid separation of 8 and 9 from their cyclic dimers.
Purification was performed using medium-pressure liquid chromatography equipped with a 25
µm silica flash cartridge as the stationary phase and a gradient of dichloromethane and methanol
as eluent (see ESI for details). The pure desired peptides 8 and 9 were obtained with satisfying
yields of 56% and 52% respectively.
We finally added the required tails to peptides 8 and 9 by a direct amidation between the
exocyclic amine and the carboxylic acid from Ac-Val, Ac-Ile or Ac-NMe-Ile, leading to
pseudacyclins A-E (1-5) using 6-Cl-HOBt, EDC and Et3N. An HPLC semi-preparative
purification afforded pure peptides 1-5 with purities over 95% and structures confirmed by high-
resolution ESI-mass spectrometry with (M+H+) ions as the major species. Spectroscopic data of
synthetic samples of 1-5 entirely matched those reported for the isolated natural pseudacyclins A-
E (see ESI for details).34

Figure 3: Procedure for the total synthesis of pseudacyclins A-E (1-5).
Conclusion
We achieved the first total synthesis of the cycle-tail peptides known as pseudacyclins A-E with
high overall yields (22% to 28%). The cyclization process of the linear precursors is highly
selective leading to over 92% of the desired cyclic monomer products, with less of 8% of the
cyclic dimers. The strategy is based on the use of orthogonally protected ornithine to yield
protected-cyclic peptides. Hydrogenolysis then gave the free exocyclic amine and side-chain
ligation yielded pseudacyclins A-E. To the best of our knowledge, this constitutes the first report
of an on-resin head-to-side chain concomitant cyclization-cleavage reaction using oxime resin to
prepare naturally occurring peptide macrocycles. The procedure described could be exploited to
prepare other cycle-tail peptides efficiently. Work is currently underway to prepare larger
quantities of pseudocyclins A-E for bioactivity measurements and to demonstrate the generality
of the synthetic strategy for the preparation of various ring-size cyclic peptides.
Acknowledgments

We are grateful to NSERC of Canada and to FRQNT of Québec for financial support. CB also
thanks FRQNT for a graduate scholarship and PROTEO for a travel award. The authors wish to
thank François Otis for his technical assistance with HPLC, Claudia Carpentier for MPLC
purification, and Pierre Audet for mass spectrometry analyses.
References
1. Harvey, A. L.; Edrada-Ebel, R.; Quinn, R. J. Nat. Rev. Drug Discovery 2015, 14, 111-129.
2. Newman, D. J.; Cragg, G. M. J. Nat. Prod. 2016, 79, 629-661.
3. Fowler, V. G.; Boucher, H. W.; Corey, G. R.; Abrutyn, E.; Karchmer, A. W.; Rupp, M. E.; Levine,
D. P.; Chambers, H. F.; Tally, F. P.; Vigliani, G. A.; Cabell, C. H.; Link, A. S.; DeMeyer, I.; Filler, S. G.;
Zervos, M.; Cook, P.; Parsonnet, J.; Bernstein, J. M.; Price, C. S.; Forrest, G. N.; Faetkenheuer, G.;
Gareca, M.; Rehm, S. J.; Brodt, H. R.; Tice, A.; Cosgrove, S. E.; Akins, R.; Albrecht, H.; Barron, M.;
Bessesen, M.; Carson, P.; Fierer, D.; Foltzer, M.; Goodenberger, D.; High, K.; Hirsch, B.; Hoen, B.;
Hsiao, C.; Lampiris, H.; Levison, M.; Suh, B.; Le, T. P.; Peetermans, W.; Pitrak, D.; Quinn, J. P.;
Segreti, J.; Ramirez, J.; Rice, P. A.; Salzberger, B.; Stone, J.; Tan, J.; Temesgen, Z.; Freeman, K.;
John, J., Jr.; Longworth, D.; Alder, J.; Anastasiou, D.; Arbeit, R.; Aziz, J.; Blackerby, K.; Campanaro,
E.; Campion, M.; Eisenstein, B.; Lufkin, J.; Luperchio, S.; Otradovec, C.; Rundlett, C.;
Stoutenburgh, U.; Thorne, G.; Tipirneni, P. N. Engl. J. Med. 2006, 355, 653-665.
4. Silverman, J. A.; Perlmutter, N. G.; Shapiro, H. M. Antimicrob. Agents Chemother. 2003, 47,
2538-2544.
5. Arbeit, R. D.; Maki, D.; Tally, F. P.; Campanaro, E.; Eisenstein, B. I. Clin. Infect. Dis. 2004, 38,
1673-1681.
6. Falagas, M. E.; Kasiakou, S. K. Clin. Infect. Dis. 2005, 40, 1333-1341.
7. Ramesh, S.; Govender, T.; Kruger, H. G.; Albericio, F.; de la Torre, B. G. Tetrahedron. Lett. 2016,
57, 1885-1888.
8. Van, B. T. P.; Caudron, Q.; Gandra, S.; Ashok, A.; Grenfell, B. T.; Levin, S. A.; Laxminarayan, R.
Lancet Infect Dis. 2014, 14, 742-750.
9. Barnawi, G.; Noden, M.; Taylor, R.; Lohani, C.; Beriashvili, D.; Palmer, M.; Taylor, S. D.
Biopolymers 2018. DOI : 10.1002/bip.23094
10. Low, K. E.; Ler, S.; Chen, K. J.; Campbell, R. L.; Hickey, J. L.; Tan, J.; Scully, C. C. G.; Davies, P. L.;
Yudin, A. K.; Zaretsky, S. J. Med. Chem. 2016, 59, 5403-5415.
11. Rotstein, B. H.; Winternheimer, D. J.; Yin, L. M.; Deber, C. M.; Yudin, A. K. Chem. Commun.
2012, 48, 3775-3777.
12. El-Faham, A.; Albericio, F. Chem. Rev. 2011, 111, 6557-6602.
13. Scott, L. T.; Rebek, J.; Ovsyanko, L.; Sims, C. L. J. Am. Chem. Soc. 1977, 99, 625-626.
14. Mazur, S.; Jayalekshmy, P. J. Am. Chem. Soc. 1979, 101, 677-683.
15. DeGrado, W. F.; Kaiser, E. T. J. Org. Chem. 1980, 45, 1295-1300.
16. DeGrado, W. F.; Kaiser, E. T. J. Org. Chem. 1982, 47, 3258-3261.
17. Arai, T.; Imachi, T.; Kato, T.; Ogawa, H. I.; Fujimoto, T.; Nishino, N. Bull. Chem. Soc. Jpn. 1996,
69, 1383-1389.
18. Jarrett, J. T.; Lansbury, P. T. Tetrahedron Lett. 1990, 31, 4561-4564.
19. Kaiser, E. T. Acc. Chem. Res. 1989, 22, 47-54.
20. Tamaki, M.; Honda, K.; Kikuchi, S.; Ishii, R. Tetrahedron. Lett. 2006, 47, 8475-8478.
21. Smith, R. A.; Bobko, M. A.; Lee, W. Bioorg. Med. Chem. Lett. 1998, 8, 2369-2374.
22. Bérubé, C.; Cardinal, S.; Boudreault, P. L.; Barbeau, X.; Delcey, N.; Giguère, M.; Gleeton, D.;
Voyer, N. Tetrahedron 2015, 71, 8077-8084.
23. Bérubé, C.; Voyer, N. Synth. Commun. 2016, 46, 395-403.

24. Bérubé, C.; Carpentier, C.; Voyer, N. Tetrahedron Lett. 2017, 58, 2334-2336.
25. Bérubé, C.; Barbeau, X.; Cardinal, S.; Boudreault, P.-L.; Bouchard, C.; Delcey, N.; Lagüe, P.;
Voyer, N. Supramol. Chem. 2017, 29, 330-349.
26. Bérubé, C.; Voyer, N. Supramol. Chem. 2018, 30, 184-195.
27. Blanchette, J.-P.; Ferland, P.; Voyer, N. Tetrahedron. Lett. 2007, 48, 4929-4933.
28. Bérubé, C.; Borgia, A.; Simon, G.; Grenier, D.; Voyer, N. Phytochem. Lett. 2018, 26, 101-105.
29. Nishino, N.; Hayashida, J.; Arai, T.; Mihara, H.; Ueno, Y.; Kumagai, H. J. Chem. Soc., Perkin
Trans. 1 1996, 939-946.
30. Ösapay, G.; Profit, A.; Taylor, J. W. Tetrahedron. Lett. 1990, 31, 6121-6124.
31. Osapay, G.; Goodman, M. J. Chem. Soc., Chem. Commun. 1993, 1599-1600.
32. Osapay, G.; Taylor, J. W. J. Am. Chem. Soc. 1992, 114, 6966-6973.
33. Osapay, G.; Taylor, J. W. J. Am. Chem. Soc. 1990, 112, 6046-6051.
34. Pavlaskova, K.; Nedved, J.; Kuzma, M.; Zabka, M.; Sulc, M.; Sklenar, J.; Novak, P.; Benada, O.;
Kofronova, O.; Hajduch, M.; Derrick, P. J.; Lemr, K.; Jegorov, A.; Havlicek, V. J. Nat. Prod. 2010,
73, 1027-1032.
35. Ahmed, M. I.; Harper, J. B.; Hunter, L. Org. Biomol. Chem. 2014, 12, 4598-4601.
Highlights



Convergent approach exploiting oxime resin towards pseudacyclin scaffold
Novel on-resin head-to-side chain concomitant cyclization-cleavage reaction
Devised approach to facilitate pseudacyclin analogs preparation