Effective synthesis of kynurenine-containing peptides via on-resin ozonolysis of tryptophan residues: Synthesis of cyclomontanin B

Article abstract of DOI:10.1039/c3ob41631c

The preparation of Kyn-containing peptides is difficult, owing to the low reactivity of Kyn in the coupling reaction. In this report, Kyn-containing peptides were efficiently obtained via on-resin ozonolysis of the corresponding Trp-containing peptide. In

Full text of DOI:10.1039/c3ob41631c

Organic &
Biomolecular Chemistry
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Effective synthesis of kynurenine-containing peptides
via on-resin ozonolysis of tryptophan residues:
synthesis of cyclomontanin B†
Cite this: Org. Biomol. Chem., 2013, 11,
7616
Clarence T. T. Wong,‡ Hiu Yung Lam‡ and Xuechen Li*
The preparation of Kyn-containing peptides is difficult, owing to the low reactivity of Kyn in the coupling
reaction. In this report, Kyn-containing peptides were efficiently obtained via on-resin ozonolysis of the
corresponding Trp-containing peptide. In addition, a Kyn-containing cyclic peptide, cyclomontanin B,
has been synthesized by this strategy in the combination with serine/threonine ligation (STL)-mediated
cyclization.
Received 9th August 2013,
Accepted 11th September 2013
DOI: 10.1039/c3ob41631c
www.rsc.org/obc
Introduction
Kynurenine (Kyn) is a metabolite amino acid produced biologi-
cally from the degradation of tryptophan by a series of enzymatic
reactions.1 The enzymes, including tryptophan 2,3-dioxygenase
(TDO) and indoleamine 2,3-dioxygenase (IDO), use superoxide
to perform oxidative metabolism on tryptophan. These
enzymes are important in the regulation of cell growth, di-
vision and inflammation.2,3 Kyn itself plays a central role in the
kynurenine pathway, which includes three distinct pathways to
form kynurenic acid, 3-hydroxy-L-kynurenine and anthranilic
acid using a series of tightly controlled enzymes. As a result,
variations of the Kyn concentration in the body could indicate
diseases, including cancers, HIV, rheumatoid arthritis, di-
abetics and CNS disorders.4–8 On the other hand, Kyn itself
was reported to be a neuro-protective molecule on the CNS but
at the same time immunosuppressive.9
Although tryptophan and Kyn are highly similar in terms of
size and hydrophobicity, the occurrence of Kyn in natural pro-
ducts suggests that Kyn could have specificity toward their
receptors or targets. For instance, daptomycin (Fig. 1), an FDA
approved cyclic Kyn-containing lipodepsipeptide isolated from
Streptomyces roseoporus, has been used in treatment of skin
infections caused by Gram-positive pathogens. It has been
shown that the mutation of Kyn to Trp causes decreased anti-
bacterial activity.10,11 Moreover, Malik et al. have discovered a
Kyn-containing decapeptide hormone from Vietnamese stick
insects that has hypertrehalosemic and cardioacceleratory
Fig. 1 The structure of daptomycin and cyclomontanin B.
activities.12 Bowie and co-workers have isolated a Kyn-contain-
ing opioid tetrapeptide from the skin of the Australian red tree
frog.13 In addition, a cyclic Kyn-containing peptide, cyclomon-
tanin B, isolated from seed of Annona montana has exhibited
promising anti-inflammatory activity (Fig. 1).14 Therefore, the
presence of Kyn residues in peptide sequences could possibly
alter the properties and therapeutic index of the peptides.
As a result, an efficient synthesis method for Kyn-containing
peptides and proteins would be valuable to perform functional
and structural studies as well as screening of novel Kyn bio-
active peptides. An earlier preparation of Kyn compounds
relied on ozonolysis of Trp-OH to produce Kyn(CHO)-OH;
however, the yield from this transformation was very low
(∼20–33%).15 Hoffmann and co-workers reported the synthesis
of Kyn from Trp via oxindolealanine (Oia) kynurenine with an
overall yield of 35%.16 Another multistep preparation of Kyn
via β-3-oxindolyl-alanine was recently developed.17
Recently, we have disclosed an easy solution to the problem
of Kyn preparation, where an N-Boc-protected Trp building
block [e.g., Fmoc-Trp(Boc)-OH] was efficiently ozonolyzed to
Department of Chemistry, The University of Hong Kong, Hong Kong, P. R. China.
E-mail: xuechenl@hku.hk; Fax: +852 28571586
†Electronic supplementary information (ESI) available: Details of experimental
procedure. See DOI: 10.1039/c3ob41631c
provide Fmoc-Kyn(Boc, CHO)-OH in
a quantitative yield
(Fig. 2a).18 However, we also observed an abnormally low reac-
tivity of Fmoc-Kyn(Boc)-OH in the coupling reaction.18 Herein,
‡CTTW and HYL with equal contribution.
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Fig. 2 The synthesis of Kyn building blocks from tryptophan via ozonolysis.
we report an efficient strategy to synthesize Kyn-containing
peptides, including cyclic peptides.
Results and discussion
To overcome the low reactivity of the Kyn moiety in the coup-
ling reaction, we sought to examine the feasibility of on-resin
ozonolysis of Trp-containing peptides to obtain the corres-
ponding Kyn moieties. To this end, we first synthesized, via
the standard Fmoc-SPPS either on a rink amide resin or a
2-chlorotrityl chloride resin, an array of Trp-containing pep-
tides composed of 4–5 amino acids (Table 1). The resulting
resin-bound peptides were directly subjected to ozonolysis con-
ditions (O3, −78 °C, DCM, 5 min; subsequently, Me2S, 1 h, rt),
followed by TFA cleavage. The LCMS analysis indicated that all
the peptides could be converted into Kyn containing peptides
directly from tryptophan via ozonolysis without noticeable
side-products (Table 1). For peptides containing methionine,
under our conditions with ozone treatment, approximately
26% of the amino acid was converted into sulfone, 54%
became sulfoxide and less than 12% remained unreacted.
Other ozone-sensitive amino acids reported by Sharma and
Berlett,19 including the conversion of histidine into 2-amino-
N-formylureido-succinimic acid and oxidation of tyrosine to
2-amino-3-(3,4-dioxo-cyclohexa-1,5-dienyl)-propionic acid, were
not observed under our reaction conditions.
Fig. 3 Retrosynthetic strategies of cyclomontanin B.
chloride resin to be preloaded with Fmoc-Gly-OH using the
standard conditions. Then the functionalized resin was sub-
jected to Fmoc-SPPS to assemble the linear peptide. The result-
ing peptide was released from the resin with a mild acid
mixture (TFE–DCM–AcOH), whilst the side-chain protecting
groups were kept intact. With this protected linear peptide pre-
cursor in hand, we then applied some conditions commonly
used for peptide lactamization. Under the conditions
attempted (PyBOP, DIEA, DMF, and DEPET, DIEA, DMF), only
traces of the desired cyclic product together with dimerized
and trimerized cyclic peptides were observed by LC-MS analy-
sis. Instead of probing other coupling reagents or other cycliza-
tion sites, we decided to take up an alternative strategy
(Fig. 3b).
Recently, we have reported a serine/threonine ligation (STL)
enabling protein synthesis and peptide cyclization, in which a
peptide with a C-terminal salicylaldehyde (SAL) ester could
chemoselectively react with another or the same peptide con-
taining an N-terminal serine or threonine residue to form an
N,O-benzylidene acetal linkage intermediate, affording the
natural peptide linkage Xaa-Ser/Thr upon acidolysis.18,20
According to the retrosynthesis of cyclomontanin B, the cycli-
zation could be performed at the conjunction of Kyn-Thr using
threonine ligation. As observed previously, Kyn-OH was resist-
ant to the coupling reaction;18 thus cyclization at the Kyn-Thr
site could be likely challenging, and could serve as a good
model to test the constraint of serine/threonine ligation (STL).
We planned to use Boc-SPPS together with the on-resin ozo-
nolysis approach to prepare the requisite linear peptide precur-
sor (3) amenable to cyclization via threonine ligation-mediated
cyclization. As shown in the retrosynthetic analysis (Fig. 3b),
we envisioned that the peptide could be assembled on a resin
linked with 2-vinylphenol via Boc-SPPS. Thus synthesized
peptide precursor (4) was subjected to on-resin ozonolysis; the
peptide would be released from the resin. At the same time,
Having demonstrated the effectiveness of the on-resin ozo-
nolysis of Kyn-containing peptides, we next undertook the syn-
thesis of a Kyn-containing cyclic peptide, cyclomontanin B.
Two synthetic strategies were devised (Fig. 3).
First, we examined the feasibility of traditional cyclization
at the Gly–Leu conjunction, followed by in-solution ozonolysis
to convert Trp into Kyn (Fig. 3a). It started with 2-chlorotrityl
Table 1 Synthetic Kyn-containing peptide models and the percentages of
conversion
a
Entry
Sequence
Obs. mass
%
1
2
3
4
5
H-S-Kyn-E-Q-F-OH
Ac-H-Y-Kyn-F-P-NH2
H-H-Kyn-Y-A-OH
H-F-P-Kyn-L-NH2
H-R-V-N-Kyn-M(O)-NH2
701.01
794.00
580.02
564.92
723.91
75
89
99
99
54
a Conversion based on LC-MS analysis.
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the Kyn moiety would be obtained from the corresponding
Trp.
To enable Boc-SPPS synthesis, Boc-Trp(CHO)-OH needs to
be used. Thus, we first studied the effectiveness of ozonolysis
of Boc-Trp(CHO)-OH. Indeed, the reaction proceeded equally
well as Fmoc-Trp(Boc)-OH (Fig. 2b). Next, an aminomethyl
resin derivatized with a 2-hydroxylphenyl-acrylamide linker (6)
was subjected to Boc-SPPS, with Boc-Trp(CHO)-OH, Boc-Ala-OH,
Boc-Asn(Xan)-OH, Boc-Leu-OH, Boc-Gly-OH, Boc-Pro-OH, and
Boc-Thr(Bzl)-OH, sequentially. After the resin bound peptide
(7) was synthesized, TMSOTf/TFA/thioanisole is used to
remove the side chain protecting groups while keeping the
peptide intact on the resin. Then, on-resin ozonolysis (O3,
−78 °C, DCM/TFA) released the peptide from the resin with the
generation of the required peptide salicylaldehyde ester; at the
same time, the embedded Trp(CHO) residue was converted
into Kyn(CHO, CHO). This time, the formyl groups on Kyn fell
off to afford peptide 8 (72%) together with mono-formylated
Kyn product (28%), due to the presence of TFA during on-resin
ozonolysis (Fig. 4).
With linear cyclomontanin B precursor in hand, we conti-
nued to examine the peptide cyclization via a threonine lig-
ation. Towards the end, the peptide SAL ester was dissolved in
a pyridine–acetic acid mixture (1 : 2, mole : mole) at a concen-
tration of 1 mM. Gratifyingly, the peptide SAL ester underwent
cyclization smoothly to completion within 4 hours. Then, after
acidolysis with TFA/H2O to remove the N,O-benzylidene acetal,
followed by the basic treatment to remove the CHO group on
Kyn, the desired cyclomontanin B was obtained in 16% yield
after reverse-phase HPLC purification based on the resin
loading (Fig. 5). It is worthwhile to note that Kyn possesses
very low reactivity in the coupling reaction; however, the cycli-
zation at Kyn-Thr via threonine ligation is apparently not
retarded, demonstrating the efficiency of STL.
Fig. 5 Analytical HPLC traces of the peptide cyclization to synthesize cyclomon-
taninin B. (a) The crude peptide SAL ester after on-resin ozonolysis; (b) threonine
ligation-mediated cyclization; (c) the crude product after acidolysis and removal
of the formyl group. X = Kyn.
Conclusions
In summary, we have presented a new approach to the syn-
thesis of Kyn-containing peptides via on-resin ozonolysis of
the corresponding suitably protected Trp-containing peptides.
This strategy is found to be very effective with easy operation
in this regard, allowing the Kyn-containing peptide to be
accessed readily. Furthermore, we have combined this strategy
and STL-mediated cyclization to prepare a Kyn-containing
cyclic peptide, cyclomontanin B.
Experimental
General
All commercial materials (Aldrich, Chemimpex and GL
Biochem) were used without further purification. All solvents
were of reagent grade or HPLC grade (RCI or DUKSAN). Dry
dichloromethane (CH2Cl2) was distilled from calcium hydride
(CaH2). The following Boc amino acids were purchased from
GL Biochem and Chemimpex and used in the solid phase syn-
thesis: Boc-Ala-OH, Boc-Asn(Xan)-OH, Boc-Leu-OH, Boc-Phe-
OH, Boc-Thr(Bzl)-OH, Boc-Pro-OH, Boc-Trp(For)-OH, Boc-Gly-
OH. Fmoc amino acids were purchased from GL Biochem and
used in solid phase synthesis: Fmoc-Ala-OH, Fmoc-Asn(Trt)-
OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Glu(OtBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Ile-OH, Fmoc-
Leu-OH, Fmoc-Met-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH,
Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH,
Fmoc-Val-OH. All separations involved a mobile phase of
0.05% TFA (v/v) in acetonitrile (solvent A)/0.05% TFA (v/v) in
water (solvent B). HPLC separations were performed with a
Fig. 4 Synthetic scheme of cyclomontanin B.
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Waters HPLC system equipped with a photodiode array detec- (4.0 equiv.) and DIPEA (8.0 equiv.) in anhydrous DMF (10 mL)
tor (Waters 2996) at wavelength 190–400 nm using a Vydac was added to the resin. The mixture was gently agitated for
218TP™ C18 column (5 µm, 300 Å, 4.6 × 250 mm) at a flow 45 min followed by sequential washing with DMF (10 mL × 3)
rate of 0.6 mL min−1 for analytical HPLC and XBridge™ and a and CH2Cl2 (10 mL × 3). Next, a mixture of TMSOTf–TFA–
Prep C18 10 µm OBD™ column (10 µm, 300 Å, 30 × 250 mm) thioanisole (1 : 8.5 : 0.5, v/v/v) was added to the resin bound
at a flow rate of 15 mL min−1 for preparative HPLC. Low- peptide at 0 °C and the mixture was gently agitated for 1 h at
resolution mass spectral analyses were performed using a 0 °C. The resin was then washed with CH2Cl2 (10 mL × 6).
Waters 3100 mass spectrometer.
Then, the resin bound peptide was swollen in CH2Cl2–TFA
(95 : 5, v/v) at −78 °C. After the mixture was treated with O3 at
−78 °C for 5 min, dimethyl sulfide (10.0 equiv. relative to the
resin capacity) was then added at −78 °C. The reaction mixture
Typical procedure for solid phase peptide synthesis of linear
peptides
Fmoc-rink-amide resin: the Fmoc-rink-amide resin (GL was allowed to warm to room temperature over 1 h. The
Biochem, loading 0.49 mmol g−1, 300–500 mg) was swollen in mixture was then filtered and the filtrate was concentrated
CH2Cl2 for 30 min followed by deprotection of the Fmoc group under vacuum to afford crude NH2-Thr-Pro-Gly-Leu-Asn-Ala-
using 20% piperidine in DMF. The resin was washed with Kyn-SAL ester. ESI calcd for C41H56N9O12 [M + H]+ m/z =
DMF (10 ml × 3) and CH2Cl2 (10 ml × 3). A solution of Fmoc- 866.40; found 866.54 (containing 28% NH2-Thr-Pro-Gly-Leu-
Xaa-OH (4.0 equiv. relative to the resin capacity), HATU Asn-Ala-Kyn (CHO)-SAL ester). ESI calcd for C42H56N9O13
(4.0 equiv.) and DIPEA (8.0 equiv.) in anhydrous DMF (10 mL) [M + H]+ m/z = 894.95; found 895.43.
was added to the resin. The mixture was gently agitated for
Peptide cyclization. Crude NH2-Thr-Pro-Gly-Leu-Asn-Ala-Kyn
45 min followed by sequential washing with DMF (10 mL × 3) (For)-SAL ester (130 mg, 0.15 mmol) obtained from the above
and CH2Cl2 (10 mL × 3). 2-Chlorotrityl chloride resin: 2-chloro- was dissolved in 150 mL pyridine–acetic acid (1 : 2, mole :
trityl chloride resin (GL Biochem, loading 0.79 mmol g−1
,
mole) at a concentration of 1 mM at room temperature. The
300–500 mg) was swollen in CH2Cl2 for 15 min. The first Fmoc reaction mixture was stirred at room temperature for 4 h. The
amino acids (2.0 equiv.) were mixed with DIPEA (4.0 equiv.) for solvent was removed under vacuum. The crude cyclized
5 min in CH2Cl2. Then the solution and the resin were mixed product was then treated with 95% TFA for 30 min to give the
and gently agitated for 1 h. The unreacted resin was capped by native cyclic peptide. The solvent was removed by a stream of
CH3OH. A solution of Fmoc-Xaa-OH (4.0 equiv. relative to the condensed air. Next, the crude reaction mixture was treated
resin capacity), HATU (4.0 equiv.) and DIPEA (8.0 equiv.) in with Tris-HCl buffer (pH 8) for 4 h to remove the formyl group
anhydrous DMF (10 mL) was added to the resin. The mixture on Kyn. Preparative HPLC purification (10–60% CH3CN–H2O
was gently agitated for 45 min followed by sequential washing over 30 min) followed by lyophilization afforded cyclomonta-
1
with DMF (10 mL × 3) and CH2Cl2 (10 mL × 3).
nin B as a white powder (35 mg, 16% from resin loading). H
NMR and 13C NMR spectra were recorded and were found to
be the same as the natural cyclomontanin B.14 ESI calcd for
Typical procedure for on-resin ozonolysis
The resin bound peptide was swollen in CH2Cl2 at −78 °C. C34H50N9O10 [M + H]+ m/z = 744.36; found 743.67. 1H NMR
After the mixture was treated with O3 at −78 °C for 5 min, (400 MHz, pyridine-d5) δ 10.26 (1H, m), 9.27–9.28 (2H, m),
dimethyl sulfide (10.0 equiv. relative to the resin capacity) was 9.00 (1H, d, J = 8.0 Hz), 8.76 (1H, d, J = 9.4 Hz), 8.60–8.64 (1H,
then added at −78 °C. The reaction mixture was allowed to m), 8.14 (1H, d, J = 9.4 Hz), 7.72 (1H, d, J = 7.2 Hz), 7.08–7.12
warm to room temperature over 1 h. The solvent was drained (2H, m), 6.80–6.82 (2H, m), 6.35–6.39 (2H, m), 5.86–5.90 (1H,
and the resin was washed with DMF (10 ml × 3) and CH2Cl2 m), 5.25–5.29 (2H, m), 4.94–4.95 (1H, m), 4.65–4.72 (1H, m),
(10 ml × 3). A mixture of TFA–H2O–TIS (9 : 0.5 : 0.5, v/v/v) was 4.25–4.62 (2H, m), 4.33 (1H, t, J = 7.8 Hz), 3.52–4.02 (7H, m),
used for global deprotection of the peptide for 1.5 h at room 1.77–1.95 (3H, m), 1.60–1.69 (3H, m), 1.50 (3H, d, J = 6.2 Hz),
temperature. The peptide was precipitated with Et2O and was 1.22–1.23 (4H, m), 0.76–0.79 (6H, m). 12C NMR (100 MHz, pyri-
confirmed by analytical LCMS (5–95% ACN–H2O over 15 min).
dine-d5) δ 200.6, 175.9, 173.9, 173.8, 173.7, 173.5, 170.9, 170.8,
165.5, 153.5, 135.7, 133.0, 118.9, 118.6, 116.2, 70.5, 62.9, 59.1,
55.5, 54.2, 52.6, 52.3, 49.7, 45.4, 44.9, 43.5, 37.3, 30.7, 26.0,
Synthesis of cyclomontanin B
Synthesis of NH2-Thr-Pro-Gly-Leu-Asn-Ala-Kyn-SAL ester. 25.9, 23.6, 23.5, 21.2, 18.1.
The aminomethyl resin derivatized with a 2-hydroxylphenyl-
acrylamide linker (6) (loading, 0.9–1.0 mmol g−1) was washed
with DMF (10 mL × 3). A solution of the first amino acid [Boc-
Trp(CHO)-OH] to be coupled (4.0 equiv. relative to the resin
Acknowledgements
capacity), PyBOP (4.0 equiv.) and DIPEA (8.0 equiv.) in anhy- We thank the National Basic Research Program of China (973
drous DMF (10 mL) was added. The mixture was gently agi- Program, 2013CB836900), the General Research Fund
tated for 12 h. The Boc group was removed with neat TFA (2 × (HKU703811P, 707412P) of the Research Grants Council of
5 min) followed by sequential washing with CH2Cl2 (10 mL × 3), Hong Kong, the Peacock Program-Project Development Fund
DMF (10 mL × 3) and CH2Cl2 (10 mL × 3). A solution of (KQC201109050074A) and Seed Funding from the University
Boc-Xaa-OH (4.0 equiv. relative to the resin capacity), HATU of Hong Kong (201111159010) for financial support.
This journal is © The Royal Society of Chemistry 2013
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