Total synthesis, structure, and oral absorption of a thiazole cyclic peptide, sanguinamide A

Article abstract of DOI:10.1021/ol3027347

The first total synthesis and three-dimensional solution structure are reported for sanguinamide A, a thiazole-containing cyclic peptide from the sea slug H. sanguineus. Solution phase fragment synthesis, solid phase fragment assembly, and solution macroc

Full text of DOI:10.1021/ol3027347

ORGANIC
LETTERS
2
012
Vol. 14, No. 22
720–5723
Total Synthesis, Structure, and Oral
Absorption of a Thiazole Cyclic Peptide,
Sanguinamide A
5
,†
Daniel S. Nielsen, Huy N. Hoang, Rink-Jan Lohman, Frederik Diness,* and
David P. Fairlie*
Division of Chemistry and Structural Biology, Institute for Molecular Bioscience,
The University of Queensland, Brisbane, Qld 4072, Australia
d.fairlie@imb.uq.edu.au; fdi@farma.ku.dk
Received October 3, 2012
ABSTRACT
The first total synthesis and three-dimensional solution structure are reported for sanguinamide A, a thiazole-containing cyclic peptide from the
sea slug H. sanguineus. Solution phase fragment synthesis, solid phase fragment assembly, and solution macrocyclization were combined to give
(1) in 10% yield. Spectral properties were identical for the natural product, requiring revision of its structure from (2) to (1). Intramolecular
transannular hydrogen bonds help to bury polar atoms, which enables oral absorption from the gut.
Hexabranchus sanguineus (Spanish dancer) is one of the
1
largest and most colorful nudibranchs (sea slugs), marine
invertebrates conspicuous throughout Indian and Pacific
ocean coral reefs for their spectacular red or yellow colors
and undulating shapes. Many bioactive peptides with
oxazole/oxazoline or thiazole/thiazoline heterocycles
incorporated in the peptide backbone have been isolated
2ꢀ4
from H. sanguineus.
peptides featuring such azoles
We have suggested that the many
6
be named azotides, and
2
ꢀ7
our interest has been on how these kinds of heterocycles
can act as conformational constraints to influence molec-
†
Present address: Department of Drug Design and Pharmacology, University
of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark.
7
8
(
(
1) Coleman, N. Nudibranchs of the South Pacific 1989, 7.
2) (a) Pawlik, J. R.; Kernan, M. R.; Molinski, T. F.; Harper, M. K.;
ular shape and biological activities of cyclic peptides
more generally. Limited quantities of azotides are acces-
sible through isolation from organisms, prompting a need
for synthetic routes in order to permit studies of their
chemical structures and properties.
Sanguinamide A is a novel thiazole-containing cyclic
peptide isolated as a minor component in extracts from
3
H. sanguineus. Unlike the cyclic octapeptide analogue
Faulkner, D. J. J. Exp. Marine Biol. Ecol. 1988, 119, 99. (b) Chattopadhyay,
S. K.; Pattenden, G. J. Chem. Soc., Perkin Trans. 1 2000, 2429.
(
3) Dalisay, D. S.; Rogers, E. W.; Edison, A. S.; Molinski, T. F.
J. Nat. Prod. 2009, 72, 732.
4) Singh, E. K.; Ramsey, D. M.; McAlpine, S. R. Org. Lett. 2012,
4, 1198.
5) (a) Jin, Z. Nat. Prod. Rep. 2011, 28, 1143. (b) Houssen, W. E.;
Jaspars, M. ChemBioChem 2010, 11, 1803.
6) Diness, F.; Nielsen, D. S.; Fairlie, D. P. J. Org. Chem. 2011, 76,
845.
7) (a) Abbenante, G.; Fairlie, D. P.; Gahan, L. R.; Hanson, G. R.;
(
1
(
(
9
sanguinamide B, cyclo-[Val-Ala(Thz)-Leu-Pro(Thz)-
Ox-Pro], that features all L-amino acids, two thiazoles,
(
Pierens, G.; van den Brenk, A. L. J. Am. Chem. Soc. 1996, 118, 10384. (b)
Singh, Y.; Sokolenko, N.; Kelso, M. J.; Gahan, L. R.; Abbenante, G.;
Fairlie, D. P. J. Am. Chem. Soc. 2001, 123, 333. (c) Singh, Y.; Stoermer,
M. J.; Lucke, A.; Glenn, M. P.; Fairlie, D. P. Org. Lett. 2002, 4, 3367. (d)
Singh, Y.; Stoermer, M. J.; Lucke, A.; Guthrie, T.; Fairlie, D. P. J. Am.
Chem. Soc. 2005, 127, 6563.
(8) (a) Fairlie, D. P.; Abbenante, G.; March, D. Curr. Med. Chem.
1995, 2, 672. (b) McGeary, R. P.; Fairlie, D. P. Curr. Opin. Drug Discuss.
Dev. 1998, 1, 208.
1
0.1021/ol3027347 r 2012 American Chemical Society
Published on Web 11/06/2012

Scheme 1. Total synthesis of cis,trans-Sanguinamide A (1)
4
oxazole, and all trans amide bonds, sanguinamide A is a
cleaved from resin with TFA/DCM (1:4). The crude
peptide was purified on an RP-18 column and obtained
as the TFA-salt 6. Cyclization was achieved in solution
with HBTU, HATU, or BOP at 1 mM concentrations in
DMF. LC-MS confirmed rapid, exclusive formation of
cyclic product 1, the highest yield (94%) obtained with use
of HBTU. Sufficient quantities of pure synthetic sangui-
namide A (1) enabled acquisition of multiple 1D and 2D
heptapeptide derivative cyclo-[Ile(Thz)-Ala-Phe-Pro-Ile-Pro]
with all L-amino acids, two prolines, phenylalanine,
alanine, isoleucine and an isoleucine-thiazole dipeptide
surrogate. Two cis-amide bonds were reported in this
3
natural product, as shown in 2. We wished to synthesize
sanguinamide A to enable determination of its structure
and to identify how the heterocycles might constrain the
cyclic peptide shape. Here, we report a facile total synthe-
sis of cis,trans-sanguinamide A (1) in 10% overall yield
and show extensive NMR data that support this as also
being the identity of the reported natural product sangui-
namide A, ratherthanthe cis,cis-sanguinamideA structure
1
13
NMR spectra (1D H, COSY, TOCSY, NOESY, 1D C,
HSQC, HMBC) in CDCl and d -DMSO (Supporting
3
6
Information (SI)).
NMR spectral data for synthetic sanguinamide A (1)
3
matched well with published data for the slightly impure
1
3
2
attributed previously.
The synthesis was accomplished by combining solution
and solid phase synthesis (Scheme 1). Boc-L-isoleucine 3
natural product, except for the β-carbon C chemical
shift of Pro6 previously assigned as 30.5 ppm. A detailed
3
1
13
study of the Hꢀ C HSQC spectrum (Figure 1) showed a
clear signal at 25.88 ppm for synthetic 1, with unambig-
uous single-bond correlations to the β-protons of Pro6
7
was converted to thioamide 4, followed by a modified
7
,9
Hantzsch procedure, to give the thiazole-containing
dipeptide surrogate 5a (overall yield 54%). The linear
peptide 6 was assembled via solid phase synthesis on a
polystyrene resin using a 2-chloro-trityl linker. Macrocy-
clization was effected between Pro6 and the isoleucine-
thiazole, since 5b was Boc-protected and required acid for
N-deprotection with simultaneous cleavage of the linker.
Fmoc-L-proline was therefore linked to the solid support
for subsequent peptide couplings, including coupling of
building block 5b, mediated by HBTU/DIPEA. This gave
polymer-bound Boc-protected linear precursor (Boc-Ile-
3
1
13
(Figure 1A). Examination of the published Hꢀ C
HSQC spectrum (Figure 1B) for the natural product
assigned as 2 revealed a signal at 30.5 ppm previously
thought to couple with the two β-protons of Pro6 at 1.83
and 2.71 ppm. Instead, the published spectrum (Figure 1B)
1
13
displayed the same correlative Hꢀ C resonances as the
synthetic compound 1 (Figure 1A).
The above HSQC data for both synthetic and isolated
sanguinamide A are thus consistent with 1 rather than 2.
The availability of more compound here enabled reinves-
tigation of the macrocycle structure, leading to this
(
Thz)-Ala-Phe-Pro-Ile-Pro), which was deprotected and
(
9) (a) Brendenkamp, M. W.; Holzapfel, C. W.; van Zyl, W. Synth.
(10) (a) Zimmerman, S. S.; Scheraga, H. A. Macromolecules 1976, 9,
408. (b) Dorman, D. E.; Bovey, F. A. J. Org. Chem. 1973, 38, 2379. (c)
W u€ thrich, K.; Billeter, M.; Braun, W. J. Mol. Biol. 1984, 180, 715.
Commun. 1990, 20, 2235. (b) Bredenkamp, M. W.; Holzapfel, C. W.;
Snyman, R. M.; van Zyl, W. J. Synth. Commun. 1992, 22, 3029.
Org. Lett., Vol. 14, No. 22, 2012
5721

Figure 2. Sanguinamide A with residue numbers (R-, β-, γ-,
δ-positions labeled for Pro4, Pro6). Double headed arrows
indicate NOE correlations between R- and δ-protons that define
cis- versus trans- amides. Two intramolecular hydrogen bonds
between Ala2 and Ile5 are shown by dashed lines.
Ile5 exchanged more slowly than Phe3 (SI Figure S10B).
Together, these data support the presence of two intramo-
lecular hydrogen bonds (Figure 2), which direct Ala2 and
Ile5 polar atoms to the interior of the cycle while restricting
adjacent side chain locations.
Figure 1. HSQC spectrum of aliphatic region for (A) synthetic cis,
trans-sanguinamide A (1) and(B) isolated“cis,cis-sanguinamide A”
The solution structure for 1 was determined in d -DMSO
6
1
at 298 K using NOESY 2D H NMR spectra, calculated
(
2) reported in reference 3. Cross-peaks for differently assigned
β-protons and carbon of Pro1 circled red.
from 36 NOE distance restraints, 4 backbone φ-dihedral
, one cis-amide
3
angle restraints derived from J
NHꢀCHR
between Phe3-Pro4, and without any hydrogen bond
1
1a
reassignment. Second, peptide bondstothe proline tertiary
restraints. Structures were calculated in XPLOR-NIH
using a dynamic simulated annealing protocol in a geometric
force field and energy minimized using the CHARMm force
1
0a
nitrogen can adopt a cis- or trans-conformation, with
trans-amide bonds generally preferred over a smaller
percentage of cis-amide. These geometric isomers can be
11b
field. The 19 lowest energy structures (Figure 3A) for 1
˚
13
distinguished by C NMR chemical shifts, which differ
between C-β and C-γ (Δδβγ = δ ꢀ δ ), a cis configuration
had no distance (g0.2 A) or dihedral angle (g2°) violations
and were quite rigid, convergent structures (ave. pairwise
β
γ
1
0b
having a larger Δδ compared to a trans configuration.
˚
backbone RMSD 0.01 A). The structure for 1 supported
βγ
1
Third, NOE H NMR correlations also reflect a shorter
distance in the cis isomer between R(Xaa)-R(Pro) protons
giving a strong NOE, whereas the trans configuration
has a shorter distance between R(Xaa)-δ(Pro) protons
observations made in the VT NMR and HꢀD exchange
experiments, with reciprocal Ala2 NH...OC Ile5 and Ile5
NH...OC Ala2 amide hydrogen bonds forming an antipar-
allel β-sheet connected by a hairpin turn centered at Phe3-
Pro4 (Figure 3A). Pro6 and Ile1(Thz) form an R-turn at the
other end of 1. Side chains from Pro4-Ile5-Pro6-Ile1 create a
contiguous hydrophobic surface along one side of the mole-
cule (Figure 3B), which shields polar atoms in Ile5 and Ile1
from solvent. The opposite side of the molecule exposes
amide protons from Phe3 and, to some extent, Ala2 making
them more accessible to solvent.
1
0c
(
Figure 2). The Δδβγ for Pro4 and Pro6 (Figure 2) in the
natural product were measured at 8.54 and 0.86 ppm,
respectively. These values clearly indicated a cis amide
for Phe3-Pro4 and a trans amide for Ile5-Pro6 (Figure 2).
NOEs (SI Figure S9) also strongly supported cis- and
trans-conformations for Phe3-Pro4 and Ile5-Pro6 amides,
respectively. All these NMR spectral parameters for syn-
thetic compound 1 are thus identical to those for the
natural product and preclude two cis-amide bonds as
required for structure 2. Thus, the geometry can be reas-
signed to cis,trans-, rather than cis,cis-, sanguinamide A,
requiringrevision of the naturalproduct structure from the
reported structure 2 to the structure 1 found here.
Since the cyclic peptide structure forces amides to the
interior of the molecule through two hydrogen bonds, and
shields others from water through hydrophobic side
chains, we were interested in whether 1 would be absorbed
into the bloodstream after oral administration to rats.
1
2
Although Lipinski’s rule-of-five is violated by three
parameters (MW 721, HBA 13, CLogP 5.4) with high
1
Variable temperature H NMR experiments revealed
that two amide-NH protons had low temperature coeffi-
cients in d -DMSO (Δ∂/T =þ0.5 ppb/K, Ala2; ꢀ1.5 ppb/K,
6
(
11) (a) Brunger, A. T. X-PLOR Manual, version 3.1; Yale University
Ile5), consistent with being protected from solvent likely
due to intramolecular hydrogen bonds (SI Figure S10A).
This was supported by HꢀD exchange experiments in
d -DMSO containing D O, which showed that Ala2 and
Press: New Haven, CT, 1992. (b) Brooks, B. R.; Bruccoleri, R. E.; Olafson,
B. D.; States, D. J.; Swaminathan, S.; Karplus, M. J. Comput. Chem. 1983,
4
, 18.
12) Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J.
Adv. Drug Delivery Rev. 2001, 46, 3.
(
6
2
5
722
Org. Lett., Vol. 14, No. 22, 2012

contiguous hydrophobic surface is important for mem-
brane permeation, with water-exposed polar groups mostly
on one surface of the molecule.
Insummary, a total synthesishas beendescribedhere for
cis,trans-sanguinamide A (1), featuring a cis-amide at
1
13
Phe3-Pro4. H, C, HSQC, and NOESY spectra establish
unambiguously that the synthetic product is identical to a
3
previously reported natural product, assigned at that time
to cis,cis-sanguinamide A (2) with two cis-amide bonds,
one at Phe3-Pro4 and one at Ile5-Pro6. The structure of
the natural product must now be revised to cis,trans-
1
sanguinamide A (1). H NMR studies support two intra-
molecular hydrogen bonds that, together with hydropho-
bic side chains, protect residues Ile1, Ala2, and Ile5 from
water solvation. These structural features were predicted
to favor a degree of membrane permeability, which was
verified by oral absorption from the gut in rats. This
finding adds to our knowledge of how cyclic peptides can
promote oral bioavailability of peptides that normally do
not survive oral delivery in vivo. It may help in the design of
orally bioavailable compounds that defy rule-of-five para-
meters normally used to guide medicinal chemists in the
development of orally bioavailable drugs.
Figure 3. (A) Backbone superimposition of the 19 lowest energy
structures for 1 in d -DMSO. Nonpolar hydrogens omitted for
6
clarity; hydrogen bonds indicated by dashed lines. (B) Surface of
1
showing hydrophobic surface (gray) impregnated by polar
groups (nitrogen, blue; oxygen, red). Ile1 and Ile5 amides are
shielded from solvent, while Thz-S (nonpolar sulfur, yellow),
Thz-N, Ile1-CO, Ala2-NH (slightly), Phe3-NH, Phe3-CO,
Pro4-CO, and Pro6-CO are solvent exposed. Ala2 and Ile5
polar atoms are all hydrogen bonded.
Acknowledgment. We acknowledge the Carlsberg
Foundation (Denmark) for a postdoctoral fellowship
2
polar surface area (tPSA 169 A ), compound 1 was found
in sera after oral administration at 10 mg/kg to Wistar rats
(F 7 ( 4%, Cmax 40 nM, Tmax 60 min), despite rapid
clearance (70 mL/min) and a short half-life (t1/2 23 min).
˚
(to F.D.); the Australian Research Council for a Federation
Fellowship (to D.F.) and the National Health and Medical
Research Council for a Senior Principal Research Fellow-
ship (to D.F); and both agencies for Grants (FF0668733,
DP1096290).
This oral availability is higher than that for most peptides
1
3
of this size and is attributed to (a) replacement of three
amide NH protons by heterocycles, (b) shielding of some
polar groups from water by hydrophobic side chains, and
Supporting Information Available. Detailed experimen-
tal procedures, NMR, HPLC, and characterization data
of synthetic compounds, and solution structure data for
1. This material is available free of charge via the Internet
at http://pubs.acs.org.
(c) two intramolecular hydrogen bonds that force polar
atoms to the interior of the macrocycle. It is likely that a
(
13) (a) Paulettia, G. M.; Gangwara, S; Siahaana, T. J.; Aub ꢀe d, J;
Borchardta, R. T. Adv. Drug Delivery Rev. 1997, 27, 235. (b) Hamman,
J. H.; Enslin, G. M.; Kotz ꢀe , A. F. BioDrugs 2005, 19, 165.
The authors declare no competing financial interest.
Org. Lett., Vol. 14, No. 22, 2012
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