Synthesis of a 6,6-spiroketal amino acid and its incorporation into a peptide turn sequence using solid-phase peptide synthesis

Article abstract of DOI:10.1002/chem.201204546

Spiropins for SPPS: The rigid structure of an anomerically stabilised spiroketal motif enables the appendage of substituents in a fixed conformation. To assess the ability of a spiroketal motif to induce a turn structure and participate in solid-phase peptide synthesis (SPPS), an Fmoc-spiroketal amino acid was synthesised and incorporated into a spiroketal-containing cyclic peptide (see figure). Copyright

Full text of DOI:10.1002/chem.201204546

COMMUNICATION
DOI: 10.1002/chem.201204546
Synthesis of a 6,6-Spiroketal Amino Acid and Its Incorporation into a Peptide
Turn Sequence Using Solid-Phase Peptide Synthesis
Jui Thiang Brian Kueh,[a] Ka Wai Choi,[a] Geoffrey M. Williams,[a] Kerstin Moehle,[b]
Bernadett Bacsa,[b] John A. Robinson,[b] and Margaret A. Brimble*[a]
Reverse turns are a secondary structure element found in
proteins[1] and are known to be involved in the molecular
recognition of various biological processes including pro-
tein–protein[2] and protein–DNA interactions[3] and protein
folding.[4] A variety of turn structures exist, which can be
broadly categorised into several classes.[5] Due to the biolog-
ical significance of this secondary structure, various confor-
mationally constrained structures have been developed to
mimic turn motifs. For example, benzodiazepinones 1,[6] var-
ious spirocyclic lactams, such as 2,[7] (S)-aminobicyclo-
lised spiroketal is able to project substituents in a well-de-
fined orientation. Notably, Ley et al.[13] replaced the N-ace-
tylglucosamine-galactose disaccharide core of sialyl Lewis X
with a spiroketal to generate a sialyl Lewis X mimetic,
which exhibited anti-inflammatory activity by preventing
binding of sialyl Lewis X to E-selectin. Another example
was reported by Smith et al.[14] who identified several con-
formers of the AB spiroketal rings in the natural macrolide
(+)-spongistatin that were locked in a turn-like structure.
The synthesis of spiroketals on solid support has been re-
ported in natural product synthesis[15] and for the prepara-
tion of natural-product-inspired spiroketal libraries.[16] How-
ever, very few examples have been reported of spiroketals
being incorporated into peptides by solid-phase peptide syn-
thesis (SPPS). A recent example by Wipf et al.[17] entailed
ACHTUNGTRENNUNG
[2.2.2]octane-2-carboxylic acid (ABOC; 3),[8] azetidine 4,[9]
triazole 5,[10] and azabicycloalkane amino acids, such as 6[11]
have all been reported to induce turn structures (Figure 1).
Spiroketals are cyclic motifs found in many natural prod-
ucts that exhibit a wide range of biological activities.[12] The
conformationally locked structure of an anomerically stabi-
the use of Fmoc-SPPS to construct spiroACTHNUTRGNEkNUG etal–steroid–RGD
hybrids that exhibited antagonistic activity for the CD11b/
CD18 integrin.
Inspired by these studies, we decided to assess the ability
of a spiroketal scaffold to induce turn-like structures by its
incorporation into a suitable peptide sequence. For example,
spiroketal 7 is a relatively rigid scaffold that contains anchor
groups to which the N and C termini of a peptide chain can
be attached. Initial modelling studies suggested that the
spiroACTHNURGTNEkNUG etal might mimic the i+1 and i+2 positions of a b-
turn. This building block might in particular influence the
conformations available to cyclic peptides that are already
constrained by backbone cyclisation. We set out to test this
idea by incorporating this spiroketal template into the cyclic
peptide 8 (Figure 2). The related b-hairpin-shaped peptide 9
is a known inhibitor of the p53–HDM2 protein–protein in-
teraction, and has been well-studied structurally, both in so-
Figure 1. Synthetic reverse turn mimics or turn inducers.
AHCTUNGTREGUNlNN ution and when bound to the human double minute 2
(HDM2) protein.[18]
The key building block 7 was prepared as described in
Scheme 1. (R)-Glycidol (10) was initially converted to lac-
tone 11 and then to alkene 12 in eight steps.[19] Sharpless di-
hydroxylation[20] of alkene 12 gave an inseparable mixture of
diols 13a and 13b in 74% yield (13a/13b, 6:1 d.r.).[21] Treat-
ment of the diastereomerically enriched mixture of diols
13a and 13b with CSA afforded a mixture of spiroketals
containing the major spiroketal 14 that was isolated in 69%
yield, and an inseparable mixture of minor isomers.[22] The
primary alcohol was converted to azido-spiroketal 15 in
good yield (81% over two steps). A nOe interaction be-
[a] J. T. B. Kueh, Dr. K. W. Choi, Dr. G. M. Williams,
Prof. M. A. Brimble
School of Chemical Sciences, The University of Auckland
23 Symonds Street, Auckland 1010 (New Zealand)
Fax : (+64)93737422
E-mail: m.brimble@auckland.ac.nz
[b] Dr. K. Moehle, Dr. B. Bacsa, Prof. J. A. Robinson
Institute of Organic Chemistry, University of Zꢀrich
Winterthurerstrasse 190, 8057 Zꢀrich (Switzerland)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201204546.
Chem. Eur. J. 2013, 19, 3807 – 3811
ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3807

tion. Staudinger reduction of the azide group proceeded in
high yield to afford aminospiroketal 16.
Fmoc protection followed by acidic cleavage of the silyl
protecting group using BF3·OEt2 and 4-methoxysalicylalde-
hyde gave Fmoc-alcohol 18.[23] Finally, TEMPO oxidation in
an acetonitrile-aqueous phosphate buffer furnished the de-
sired spiroketal amino acid 7.[24]
With the key spiroketal amino acid 7 in hand, the synthe-
sis of cyclic spiroketal peptide 8 was undertaken using
Fmoc-SPPS (Scheme 2). Employing standard conditions, the
Figure 2. a) Proposed synthesis of cyclic spiroketal peptide 8 by SPPS.
b) Cyclic b-hairpin peptide 9 synthesised by Fasan et al.[18]
Scheme 1. Synthesis of spiroketal amino acid 7. a) (DHQ)2AQN, OsO4,
K3Fe(CN)6, K2CO3, NaHCO3, CH3SO2NH2, tBuOH, H2O, 0 8C, 74%
(13a/13b, 6:1 d.r.); b) CSA, aq. EtOH, RT, 69%; c) TsCl, NEt3, DMAP,
CH2Cl2, 08C to RT; d) NaN3, DMF, 808C, 81% over two steps; e) PPh3,
aq. THF, 89%; f) Fmoc-OSu, DIPEA, CH2Cl2/1,4-dioxane (1:1), 08C to
RT, 90 %; g) BF3·OEt2, 4-methoxysalicylaldehyde, CH2Cl2, RT, 63%;
Scheme 2. Synthesis of cyclic spiroketal peptide 8. a) i. Fmoc-amino acid,
HBTU, NMM, DMF; ii. DMF wash; iii. 20% piperidine/DMF; iv. DMF
wash; b) i. spiroketal amino acid 7, HOAt, HATU, DIPEA, DMF, RT,
two cycles; ii. DMF wash; iii. 20% piperidine/DMF; iv. DMF wash;
c) i. Fmoc-Phe-OH, HOAt, PyBoP, DIPEA, DMF, RT; ii. DMF wash;
iii. 20% piperidine/DMF; iv. DMF wash; d) 20% AcOH/CH2Cl2, then
80% AcOH/CH2Cl2; e) HOAt, HATU, DIPEA, RT, CH2Cl2/DMF (9:1);
f) 95% TFA/H2O.
h) TEMPO, PhIACHTUNGTRENNUNG(OAc)2, CH3CN/aq. NaH2PO4·2H2O (3:1), 64%.
tween 2’-H and 8’-H established that azido-spiroketal 15
adopts the expected bis-anomerically stabilised conforma-
3808
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Chem. Eur. J. 2013, 19, 3807 – 3811

Synthesis of a 6,6-Spiroketal Amino Acid
COMMUNICATION
À
linear peptide chain was constructed on 2-chlorotrityl resin
preloaded with leucine using automated Fmoc-SPPS condi-
tions. However, the valuable Fmoc-spiroketal amino acid 7
was coupled using a manual procedure employing HATU,
HOAt and DIPEA. The second Phe residue was also incor-
porated using a manual procedure employing the phospho-
nium coupling reagent, PyBoP, as initial attempts to couple
the second Phe residue using HBTU resulted in formation
of an N-terminal tetramethyl guanidine by-product.
full list). Notably, however, an important strong Ha Ha rOe
was observed between Trp3 and Phe8.
Based on rOe-derived distance restraints, average solution
structures were calculated for peptide 8 by restrained molec-
ular dynamics in torsion angle space using the simulated an-
nealing protocol in the program DYANA,[26] with the spiro-
ketal in the bis-anomerically stabilised conformation. The
resulting calculated average structures superimpose with a
backbone rmsd of approximately 1.2 ꢂ (Figure 4 and
Table 1), and clearly reflect a significant backbone flexibility,
Upon completion of the peptide sequence, the resin was
carefully treated with 20% AcOH/CH2Cl2, followed by
80% AcOH/CH2Cl2 to release the linear protected peptide
22 (Scheme 2). A dilute solution (1 mgmLÀ1) of this materi-
al was then treated with HATU to induce the macrocyclisa-
tion, the progress of which was monitored by observing the
consumption of the linear peptide 22 by analytical RP-
HPLC.[25] Upon completion of the reaction, the solvent was
removed in vacuo and the crude protected cyclic peptide 23
was immediately treated with 95% TFA in H2O to cleave
the side chain protecting groups (Scheme 2). Subsequent pu-
rification by preparative RP-HPLC afforded cyclic spiro-
ACHTUNGTRENNUNG
ketal peptide 8 with no epimerisation of Leu4 Ca observed
by NMR spectroscopy or HPLC analysis (>96% purity,
[M+H]+ =1364.4, Figure 3).
Figure 4. Left: backbone representation and superimposition of the 20
DYANA NMR structures for 8. All backbone heavy atoms were used for
the superimposition. Right: one typical structure is shown. MOLMOL[27]
and PyMOL[28] were used for structure analysis and visualisation.
with large torsion angle fluctuations (Table 2). Nevertheless,
the backbone conformations show similarities and adopt dis-
torted hairpin-like structures. Interestingly, the spiroketal
motif itself does not mimic the central i+1/i+2 region of a
b-turn. Rather, the calculated structures all contain the
spiroACTHUNRTGNEUNGketal in a b-turn-like structure, with the spiroketal oc-
cupying positions i and i+1, and Phe1 and Glu2 at positions
i+2 and i+3, respectively. Thus, the spiroketal does not
induce a full 1808 turn in the peptide chain, but rather indu-
ces an approximate 908 turn in the backbone conformation.
This is illustrated in Figure 5, showing the turn-like region
Figure 3. HPLC and MS spectra of purified cyclic spiroketal peptide 8
using a Gemini C18 column (5 mm, 4.6ꢃ150 mm), 1–81% CH3CN/H2O
over 20 min, 1.5 mLminÀ1
.
Table 1. Summary of experimental distance restraints used and resulting
statistics for the final 20 NMR structures calculated for peptide 8, as su-
perimposed and shown in Figure 4.
The solution structure of spiroketal peptide 8 was investi-
gated by 1H NMR spectroscopy in [D6]DMSO, due to its
limited solubility in water. At room temperature, the spec-
trum showed broadening of the NH resonances although
sharp signals were observed in the aromatic region (dH =
6.7–6.8 ppm). Reducing the peptide concentration had little
effect on the chemical shifts or shape of the signals, howev-
er, raising the temperature to 355 K in [D6]DMSO caused
sharpening of the NH resonances. These findings are consis-
tent with relatively slow exchange process(es) on the NMR
time scale at room temperature. In 2D ROESY plots, with a
mixing time of 250 ms, most of the observed rOes were
Experimental distance restraints
Calcd
number of NOE upper-distance limits
intraresidue
65
21
sequential
35
medium- and long-range
9
residual target function value [ꢂ2]
0.32Æ0.08
mean rmsd values [ꢂ]
all backbone atoms
all heavy atoms
1.17Æ0.40
2.61Æ0.80
residual NOE violations
number >0.2 ꢂ
5
intra
ACHTUNGTRENNUNGresidue or sequential, with only a few weak long range
maximum [ꢂ]
0.25
rOes being observed (see the Supporting Information for a
Chem. Eur. J. 2013, 19, 3807 – 3811
ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
3809

M. A. Brimble et al.
Table 2. Average backbone torsion angles and their standard deviations
found in the final 20 NMR structures shown in Figure 4.[a]
In conclusion, the synthesis and incorporation of the
novel spiroketal amino acid 7 into the cyclic spiroketal pep-
tide 8 has been achieved using Fmoc-SPPS. The data ob-
tained from NMR structure calculations indicate that the
spiroketal template 7 has an unexpected and unprecedented
influence on the backbone conformation of peptide 8. The
results provide synthetic access to this interesting new tem-
plate 7, which may find many applications in the design of
other novel turn-containing peptidomimetics.
Residue
Phe1
Dihedral
Average
S.D.[a]
f
y
f
y
f
y
f
y
f
y
f
y
f
y
f
y
f
y
À54.9
À26.0
À106.7
165.4
Æ99.2
Æ63.1
Æ64.0
Æ54.3
Æ40.1
Æ48.3
Æ64.6
Æ92.6
Æ81.9
Æ23.4
Æ57.8
Æ67.5
Æ92.5
Æ18.2
Æ34.4
Æ33.1
Æ34.6
Æ40.5
Glu2
Trp3
À135.8
136.6
Leu4
Asp5
Trp6
À175.8
À66.3
À73.7
157.6
À2.6
Acknowledgements
76.1
Glu7
Phe8
spiro9[b]
44.9
170.0
The University of Auckland is acknowledged for the award of a Doctoral
Scholarship (J.T.B.K.). The authors thank Dr. Chris Squire for his assis-
tance in generating the visual models of the peptide structures.
À74.7
130.4
À105.1
À153.7
Keywords: amino acids · peptidomimetics · reverse turns ·
solid-phase peptide synthesis · spiroketals
[a] Standard deviations. [b] For the spiroketal, f/y correspond to the di-
hedrals following N and preceding the CO termini, respectively.
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including the spiroketal, found in the NMR structures. As a
result, the backbone as well as the side chain orientation of
the residues are influenced in a novel and unusual manner
by the spiroketal template (Figure 4). In the NMR struc-
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point to the same side of the macrocycle, whereas Trp6 and
Phe1 are significantly separated and located in opposite turn
regions.
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compared to that seen earlier with the cyclic peptide 9, con-
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conformation in both aqueous and mixed water/methanol
solution. A related analogue of 9 containing 6-chlorotrypto-
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Synthesis of a 6,6-Spiroketal Amino Acid
COMMUNICATION
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Received: December 20, 2012
Published online: February 19, 2013
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