The effect of sulfur stereochemistry of l-β,β-dimethylmethionine S-oxide on the physicochemical properties of truncated polytheonamides

Article abstract of DOI:10.1016/j.tetlet.2010.06.126

L-β,β-Dimethylmethionine S-oxide is a unique amino acid found in polytheonamides. Four designed hexapeptides containing the sulfide, (S _R)-sulfoxide, (S_S)-sulfoxide, and sulfone derivatives of l-dimethylmethionine were synthesized and functionally analyzed. Our results indicate that the sulfoxide stereochemistry of the peptides controls their overall physicochemical properties.

Full text of DOI:10.1016/j.tetlet.2010.06.126

Tetrahedron Letters 51 (2010) 4644–4647
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
The effect of sulfur stereochemistry of L-b,b-dimethylmethionine S-oxide on
the physicochemical properties of truncated polytheonamides
*
Shigeru Matsuoka, Yuki Mizoguchi, Hiroaki Itoh, Ken Okura, Naoki Shinohara, Masayuki Inoue
Graduate School of Pharmaceutical Sciences, The University of Tokyo, and PRESTO, Japan Science and Technology Agency, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113 0033, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
L
-b,b-Dimethylmethionine S-oxide is a unique amino acid found in polytheonamides. Four designed hexa-
Received 20 May 2010
Revised 19 June 2010
Accepted 28 June 2010
Available online 1 July 2010
peptides containing the sulfide, (SR)-sulfoxide, (SS)-sulfoxide, and sulfone derivatives of -dimethylmethi-
L
onine were synthesized and functionally analyzed. Our results indicate that the sulfoxide
stereochemistry of the peptides controls their overall physicochemical properties.
Ó 2010 Elsevier Ltd. All rights reserved.
Polytheonamides A (1) and B (2), isolated from the marine
sponge Theonella swinhoei, exhibit antitumor activities against
P388 murine leukemia cells at concentrations of less than
100 pg/mL.1,2 Compounds 1 and 2 are the largest non-ribosomal
peptides identified to date (Fig. 1).3 Their linear 48 residues with
amino acid only found in polytheonamides. Sulfoxides are a
stereogenic functionality, and compounds 1 and 2 were in fact
shown to be a pair of sulfoxide stereoisomers of the 44th resi-
dues.3 Most recently, the absolute configuration of the sulfoxide
of polytheonamide B (2) was determined to be R by our total
synthesis.6 Interestingly, distinct retention times for these iso-
mers (1: Tr = 24.75 min; 2: Tr = 29.03 min) were observed upon
reversed-phase HPLC analyses (Inertsil C8 column with n-PrOH/
H2O solvent system),6 suggesting that a single stereochemical
change in the side chains affects the hydrophobicity of the entire
molecule.
alternating
D- and L-amino acids include 13 non-proteinogenic
amino acids and an N-terminal cap. The sequences are also rich
in b-branched amino acids, which are generally known to contrib-
ute to low conformational flexibility of the peptide backbone.4,5
Among the non-proteinogenic amino acid residues, the 44th
L-b,b-dimethylmethionine S-oxide
[L
-Me2Met(O)] is
a
unique
OH
16
O
O
O
O
O
H
O
O
O
O
H
N
H
N
H
N
H
N
H
N
H
N
H
N
N
O
N
H
N
H
N
H
N
H
N
H
N
H
N
H
N
H
O
O
O
O
O
O
O
O
O
NH
1
MeHN
O
O
HN
O
H2N
NH
O
O
O
O
O
O
H
N
H
N
H
N
H
N
H
N
H
N
H
N
MeHN
N
H
N
H
N
H
N
H
N
H
N
H
N
H
O
O
O
O
O
O
O
O
O
O
O
NH
OH
NHMe
O
O
O
OH
OH
O
NHMe
NHMe
NHMe
O
HN
O
H2N
S
O
43
45
OH
OH
O
O
O
O
O
O
NH
H
H
N
H
N
H
N
H
N
H
N
N
N
H
N
H
N
H
N
H
N
H
N
H
O
O
O
O
O
O
O
O
O
O
O
OH
OH
OH
polytheonamide A (1): SS
polytheonamide B (2): SR
44
48
NHMe
NHMe
NH2
NH2
Figure 1. Structures of polytheonamides A (1) and B (2), which are sulfoxide epimers at the 44th residue. The SR stereochemistry of 2 was determined by our total synthesis.
* Corresponding author. Tel.: +81 3 5841 1354; fax: +81 3 5841 0568.
E-mail address: inoue@mol.f.u-tokyo.ac.jp (M. Inoue).
0040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.06.126

S. Matsuoka et al. / Tetrahedron Letters 51 (2010) 4644–4647
4645
Oxidation of methionine to methionine S-oxide in proteins has
S
been correlated with the changes in peptide conformation,7 associ-
ation properties8–12 and biological functions,13,14 presumably due
to the marked difference in polarity. However, precise structural
role of methionine S-oxide in such functional changes is still un-
clear. In this context, we are interested in the effects of the stereo-
chemistry and oxidation state of the unique dimethylmethionine
on the overall properties of the compounds. As a preliminary study,
Ot-Bu
FmocHN
O
3
S-4 (0.02 eq)
H2NCONH2·H2O2
MeOH, 0 ºC
R-4 (0.02 eq)
the physicochemical and biological properties of the
derivatives were evaluated at both amino acid and peptide levels.
The two stereoisomers of -Me2Met(O) could be generated
by non-stereoselective oxidation of -b,b-dimethylmethionine
-Me2Met). Further oxidation of -Me2Met(O) gives the sulfone
derivative [ -Me2Met(O2)]. Therefore, the 44th residue of polythe-
onamide is capable of generating four analogues in terms of stereo-
chemistry and oxidation state. Accordingly, we designed,
synthesized, and functionally analyzed the four protected amino
acids and the four model hexapeptides, which contain the dimeth-
L-Me2Met
O
S
O
S
L
L
Fmoc
OR
N
Fmoc
N
OR
(L
L
H
L
H
O
O
5: R = t-Bu
97% (85% de)
6: R = t-Bu
92% (97% de)
TFA/CH2Cl2, 0 ºC to rt
7: R = H
85% (96% de)
8: R = H
95% (99% de)
ylmethionine derivatives,
L-Me2Met,
L
-(SR)-Me2Met(O),
L-(SS)-
Me2Met(O), and -Me2Met(O2), in their structures.
L
1. Et2NH, CH2Cl2
For experiments at the amino acid level, we selected an N-ben-
zoyl, C-methylamide-capped structure to evaluate the properties
of the single residual side chain. Scheme 1 shows syntheses of
2. BzCl, PhMe, carbonate buffer (pH = 10)
3. IBCF, NMM, THF, -36ºC
; then aq. MeNH2
the four analogues of N- and C-protected
cific sulfur stereochemistries and oxidation states. Sulfide 3, which
was previously synthesized from
-aspartic acid,6 was stereo- and
chemoselectively oxidized to (SR)-sulfoxide 5 and (SS)-sulfoxide 6
by using Katsuki catalysts S-4 and R-4, respectively.15 For the
purpose of further derivatization, 5 and 6 were treated with TFA/
CH2Cl2 for removal of the t-Bu group to afford diastereomeric car-
boxylic acids 7 and 8, respectively.
Next, Fmoc-protected acids 7 and 8 were derivatized into the
corresponding model amides 9 and 10, respectively, through
base-mediated Fmoc removal, Bz protection, and condensation
with methyl amine. To prepare the analogues at different oxidation
states, compound 9 was reduced by SO3ꢀpyr-NaI to generate sulfide
1116 or oxidized by Oxone to generate sulfone 12.17
The hydrophobicities of the synthesized four protected amino
acids were evaluated next. Namely, log kw, a hydrophobic parame-
ter,18,19 was deduced from the retention times on reversed-phase
HPLC for the four protected peptides 9–12. First, each amino acid
was eluted with a solvent system of CH3CN/H2O at three different
mixing ratios. The resulting three retention times for each amino
acid were then analyzed according to the method reported by
Braumann to calculate its log kw (Table 1).19 It was found that sul-
fide 11 had the largest value of log kw, and thus possessed the high-
est hydrophobicity among the four protected amino acids, while
sulfone 12 had the lowest hydrophobicity. The log kw difference
between 9 and 10, the sulfoxide stereoisomers, was negligible.
Thus, only the oxidation states of sulfur affected the hydrophobic-
ities of the protected mono-amino acids [sulfide > (SR)-sulfox-
ide = (SS)-sulfoxide > sulfone].
L-Me2Met with the spe-
O
O
S
S
L
O
O
H
N
H
N
Ph
N
H
Me
Ph
N
H
Me
O
O
10: 67% (3 steps)
9: 53% (3 steps)
SO3·pyr, NaI
CH3CN
Oxone, aq.n-PrOH
O
O
S
S
O
O
H
N
H
N
Ph
N
H
Me
Ph
N
H
Me
O
O
11: 91%
12: 90%
O
O
N
N
O
O
Ti
O
N
N
N
N
OH HO
Ph Ph
N
Ti
O
R-4 =
;
=
N
OH HO
Scheme 1. Chemical structures and reduction/oxidation of N- and C-protected L-
Me2Met(O), (SR)-sulfoxide 9, and (SS)-sulfoxide 10. Sulfide 11 and sulfone 12 were
obtained by single-step reactions from 9. IBCF = isobutyl chloroformate; NMM = N-
methylmorpholine.
To investigate the physicochemical influence of the sulfur state
on the peptide backbone, we designed four truncated hexapeptide
models of polytheonamides containing the L-Me2Met analogues
Table 1
Hydrophobic parameters (log kw) of protected amino acids 9–12 and hexapeptides
(14–17, Scheme 2). The amino acid sequences of the peptides are
composed of the seven units, which are characteristic structures
in the polytheonamide sequence, that is, the N-terminal cap, gly-
14–17
a
a
Oxidation states
Compound
Log kw
Compound
Log kw
(SR)-Sulfoxide
(SS)-Sulfoxide
Sulfide
9
10
11
12
2.29
2.30
2.93
2.03
14
15
16
17
1.91
3.88
NDb
1.36
cine-1,
L
-b-hydroxyvaline-16,
D-asparagine-43, an analogue of L-
Me2Met(O)-44,
D
-asparagine-45, and glycine as the C-terminal car-
boxylic acid (Fig. 1).
Sulfone
The hexapeptide sequences of 14 and 15, bearing L-(SR)-Me2M-
et(O) and L-(SS)-Me2Met(O), respectively, were synthesized from
a
Values were determined from retention times in reversed-phase HPLC. Analyses
were performed at 30 °C for 9–12 and at 40 °C for 14–17. Detailed experimental
commercially available Fmoc-Gly-Wang resin (13) by Fmoc chem-
istry on an automatic solid-phase peptide synthesizer.20 After elon-
gation of the peptide using the corresponding N-Fmoc-amino acids,
conditions are described in the Supplementary data.
Compound 16 failed to give clear peaks to calculate a log kw value due to its
high hydrophobicity (ND = not determined).
b

4646
S. Matsuoka et al. / Tetrahedron Letters 51 (2010) 4644–4647
O
O
FmocHN
O
Fmoc-Gly-Wang resin (13)
1. solid phase peptide synthesis
(i) Nα-Fmoc-amino acid (or Ncap-OH)
HATU, HOAt, i-Pr2NEt, NMP
(ii) 22% piperidine/NMP
2. TFA/H2O=95/5, rt
O
O
S
S
OH
O
OH
O
43
45
O
O
O
O
O
O
O
O
H
N
H
N
H
N
H
N
H
N
H
N
OH
OH
N
H
N
H
N
H
N
H
N
H
N
H
O
O
O
O
O
O
O
O
44
O
O
1
16
44
NH2
NH2
NH2
NH2
15: 22% (13 steps)
14: 30% (13 steps)
SO3·pyr, NaI, CH3CN
S
Oxone, aq. n-PrOH
O
O
O
S
OH
O
OH
O
O
O
O
O
O
O
H
N
H
H
N
H
N
H
N
H
N
OH
N
OH
N
H
N
H
N
H
N
H
N
H
N
H
O
O
O
O
O
O
O
O
O
O
O
44
44
NH2
NH2
NH2
17: 56%
NH2
16: 66%
Scheme 2. Structures of truncated model peptides of polytheonamides. N-capped hexapeptides with a specific stereochemistry at sulfoxide, (SR)-sulfoxide 14 and (SS)-
sulfoxide 15, were obtained by SPPS. Sulfide 16 and sulfone 17 were derived from 14 according to the same methods used for the preparation of 11 and 12, respectively.
HATU = O-(7-azabenzotriazole-1-yl)-N,N,N0,N0-tetramethyluronium hexafluorophosphate; HOAt = 1-hydroxy-7-azabenzotriazole; NMP = N-methylpyrrolidone.
the N-terminal cap was manually introduced, because the acti-
vated carboxylate intermediate of the dicarbonyl unit was chemi-
cally sensitive to the automated coupling conditions. Finally, the
peptides were cleaved from the Wang resin under acidic condi-
tions, and subjected to HPLC purification to generate 14 and 15.
Similar to the syntheses of the protected amino acid derivatives,
14 was then reduced to produce sulfide 16 or oxidized to produce
sulfone 17.
Table 2
Hemolytic activities of hexapeptides 14, 15, and 17
a
Oxidation states
Compound
EC50 (mM)
(SR)-Sulfoxide
(SS)-Sulfoxide
Sulfone
14
15
17
0.38
>1.0
>1.0
a
Values are evaluated using 107 cells/mL of rabbit red blood cells in PBS at 37 °C
for 15 h.
The four model peptides synthesized with the different stereo-
chemistries and sulfur oxidation states (14–17) were subjected to
reversed-phase HPLC analysis to estimate the hydrophobic param-
eter, log kw. The analysis conditions were the same as described
above for protected amino acids, except for column temperature.
The resulting log kw values are shown in Table 1. Although sulfide
16 was too hydrophobic to give a precise log kw value in the
CH3CN/H2O solvent system, the order of hydrophobicities among
the three oxidation states in model peptides (sulfide > sulfox-
ide > sulfone) coincided with that of the protected amino acids.
The significant difference in log kw for 14 and 15, the pair of sulfox-
ide stereoisomers, was remarkable. Log kw of (SS)-sulfoxide 15 was
about twice as large as that of (SR)-sulfoxide 14 [(SS)-sulfox-
ide > (SR)-sulfoxide]. The effect of the stereochemistry of the sulf-
oxide on the overall hydrophobicity was clearly enhanced in
hexapeptides 14 and 15 in comparison to mono-amino acids 9
and 10. These results should reflect the large structural difference
between 14 and 15 induced by the single stereochemistry of the
sulfoxide.
water to the hydrophobic membrane.21,22 Hemolytic activities of
the three truncated model peptides, 14, 15, and 17, were evaluated
against rabbit erythrocytes at 37 °C for 15 h (Table 2). Both (SS)-
sulfoxide 15 and sulfone 17 showed no activities at concentrations
up to 1 mM. On the other hand, (SR)-sulfoxide 14 displayed hemo-
lytic activity with an EC50 of 0.38 mM. Curiously, the activity order
[14 > 15, 17 (Table 2)] did not reflect the hydrophobic order
[15 > 14 > 17 (Table 1)]. These findings indicated that 14 and 15
differ in molecular properties in addition to hydrophobicity.
To investigate whether the sulfoxide moiety controlled the pep-
tide structures, NMR and CD studies of hexapeptides 14 and 15
were carried out. 1H NMR spectra were recorded at 2 mM in
CD3CN/H2O (10:1) at 40 °C. A large shift in the amide proton signal
of the L-Me2Met(O) residue was observed between the model pep-
tides 14 and 15 of ꢁ0.73 ppm (dSR–dSS). In addition, an obvious shift
was observed for the amide proton of the adjacent residue, aspar-
agine-5, of +0.34 ppm.23
Drastically different behavior between 14 and 15 was also ob-
served by CD spectra. At 0.1 mM in CH3CN/H2O (10:1), the peak
at 213 nm was increased in the spectrum of 14 compared to that
of 15 (Fig. 2A). Interestingly, while the spectrum of 14 remained
The profound effects of the sulfoxide stereochemistries were
also observed in the hemolytic activities of the model peptides.
The potency of hemolytic activity has a positive correlation with
the hydrophobic parameter, because membrane disruption of
erythrocytes requires partitioning of amphiphilic compounds from

S. Matsuoka et al. / Tetrahedron Letters 51 (2010) 4644–4647
4647
yses of the peptides and the parent polytheonamides are currently
underway in our laboratory.
Acknowledgments
This work was supported in part by Grant-in-Aids for Young
Scientists (S) to M.I., and Grant-in-Aids for Young Scientists (start
up) to S.M.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2010.06.126.
References and notes
Figure 2. CD spectra of 14 and 15 recorded at room temperature in CH3CN/H2O
(10:1) at concentrations of 0.1 mM (A) and 1 mM (B).
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unchanged at higher concentrations (1 mM, Fig. 2B), the peak at
213 nm in 15 was diminished. It clearly suggests that 15 undergoes
conformational change in
a concentration-dependent fashion,
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5. Pal, D.; Chakrabarti, P. Acta Crystallogr., D 2000, 56, 589.
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15 is more hydrophobic than 14. Therefore, starkly different behav-
iors of 14 and 15 observed by 1H NMR, CD spectra, and the hemo-
lysis assay appear to originate in the different aggregation states of
14 and 15.
Sulfoxides are known as strong hydrogen bond acceptors due to
their substantial dipole moments.24–26 The sulfoxide stereochemis-
try defines the orientation of its S+–Oꢁ bond and confers the geom-
etry of the hydrogen bond. Based on our results, it is highly likely
that the chirality of sulfoxides in 14 and 15 serves as a controlling
structural element at least at two levels; it controls the overall
molecular shape by introducing intramolecular hydrogen bonds,
thereby controlling the hydrophobicities, and it controls aggrega-
tion of the peptides. Peptide 15 with (SS)-sulfoxide promotes
aggregation through increased hydrophobicity,27 whereas peptide
14 with (SR)-sulfoxide disfavors aggregation, which is apparently
critical in exerting its hemolytic activity.
In conclusion, we synthesized protected amino acids and model
hexapeptides containing sulfide (
Me2Met(O), and -(SS)-Me2Met(O)], or sulfone [
eties. The (SR)- and (SS)-stereochemistries of -Me2Met(O) were ste-
L-Me2Met), sulfoxides [L-(SR)-
21. Kuroda, K.; Caputo, G. A.; DeGrado, W. F. Chem. Eur. J. 2009, 15, 1123.
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23. In contrast to these data, the 1H NMR chemical shifts of two protected amino
L
L-Me2Met(O2)] moi-
L
acids
9 and 10 were almost identical in the amide proton region. See
reoselectively introduced by asymmetric oxidation of the sulfide.
Spectral and biological analyses using model hexapeptides proved
that the one stereochemical perturbation of the sulfoxide of the L-
Me2Met(O) moiety has an important structural role in controlling
the overall physicochemical and therefore functional properties
of the model hexapeptides. Further structural and biological anal-
Supplementary data for detail.
24. Jiang, L.; Burgess, K. J. Am. Chem. Soc. 2002, 124, 9028.
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Biochemistry 1983, 22, 1264.
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S., Rappoport, Z., Stirling, C. J. M., Eds.; Wiley: New York, 1988; p 541.
27. Hill, R. B.; DeGrado, W. F. Structure 2000, 8, 471.