Surugamides A-E, cyclic octapeptides with four D-amino acid residues, from a marine streptomyces sp.: LC-MS-aided inspection of partial hydrolysates for the distinction of D- and L-amino acid residues in the sequence

Article abstract of DOI:10.1021/jo400708u

Surugamides A-E (1-5), cyclic octapeptides with four d-amino acid residues, were isolated from the broth of marine-derived Streptomyces sp. Their planar structures were determined by analyses of spectroscopic data, and the absolute configuration of constituent amino acid residues was determined by the Marfey's method. Differentiation of d-Ile and l-Ile in the sequence was established by chiral analysis of fragment peptides obtained from the partial hydrolysate, whose identification was conducted by LC-MS/MS.

Full text of DOI:10.1021/jo400708u

Note
pubs.acs.org/joc
Surugamides A−E, Cyclic Octapeptides with Four D‑Amino Acid
Residues, from a Marine Streptomyces sp.: LC−MS-Aided Inspection
of Partial Hydrolysates for the Distinction of D- and L‑Amino Acid
Residues in the Sequence
Kentaro Takada,†,§ Akihiro Ninomiya,†,§ Masato Naruse,† Yi Sun,† Masayuki Miyazaki,‡ Yuichi Nogi,‡
Shigeru Okada,† and Shigeki Matsunaga*,†
†Laboratory of Aquatic Natural Products Chemistry, Graduate School of Agricultural and Life Sciences,
The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan
‡Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Natsushima, Yokosuka,
Kanagawa 237-0061, Japan
S
* Supporting Information
ABSTRACT: Surugamides A−E (1−5), cyclic octapeptides
with four D-amino acid residues, were isolated from the broth
of marine-derived Streptomyces sp. Their planar structures were
determined by analyses of spectroscopic data, and the absolute
configuration of constituent amino acid residues was
determined by the Marfey’s method. Differentiation of D-Ile
and L-Ile in the sequence was established by chiral analysis of
fragment peptides obtained from the partial hydrolysate, whose
identification was conducted by LC−MS/MS.
errestrial actinomycete bacteria are one of the most prolific
Tsources of secondary metabolites, from which a large
number of peptide antibiotics with a variety of bioactivities
have been isolated.1 Recent genome sequencing studies of
actinomycetes revealed that a significant number of peptides
of nonribosomal peptide synthetase (NRPS) or hybrid of
NRPS/polyketide synthase (PKS) origin are coded but have
not been isolated, indicating their higher metabolic potentials
than those inferred from the past records of natural products
discovery.2 In the course of our exploration of bioactive
metabolites from marine-derived actinomycetes, we found
five new cathepsin B inhibitory cyclic peptides, surugamides
A−E (1−5), from a marine Streptomyces sp. Four of eight
determined to be C48H81N9O8 by HRESIMS. 1H NMR spectrum
residues in surugamides are in the D-form. In this con-
tribution, we describe the structures of surugamides, which
comprise a new class of cyclic peptides, with particular
reference to the use of LC−MS to facilitate identification
of partial hydrolysis products. This process is indispensable
for establishing total configurational assignment of cyclic
peptides in which both D- and L-forms of one or more amino
acid residues are present.
The crude extract of the mycelia was subjected to solvent
partitioning, ODS flash chromatography, and reversed-phase
HPLC to afford surugamides A (1, 9.8 mg), B (2, 1.2 mg), C
(3, 1.2 mg), D (4, 1.3 mg), and E (5, 1.0 mg) (Figure S1 in
Supporting Information [SI]). The molecular formula of 1 was
in DMSO-d6 exhibited eight each of amide- and α-protons,
indicating the peptidic nature of the compound. Analyses of
COSY, TOCSY, and HSQC data revealed that 1 was composed
of eight proteinogenic amino acid residues: Ala (1 residue), Ile
or allo-Ile (designated as Ile unless specifically differentiated) (4),
Leu (1), Lys (1), and Phe (1) (Table S1 in SI). The presence
of eight amide bonds was deduced from the molecular for-
mula, which indicated 1 was cyclic form. Eight sequential
NOE correlations, δ 8.50 (NH, Ile1) /δ 4.27 (αH, Ala), δ 7.98
Received: April 3, 2013
© XXXX American Chemical Society
A
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The Journal of Organic Chemistry
Note
Figure 1. Total ion current (TIC) chromatogram of the partial hydrolysate of 1. Amino acid sequences of the fragments were determined on the basis of
the MS/MS data. For the HPLC conditions, see the Experimental Section.
(NH, Ile2) /δ 4.14 (αH, Ile1), δ 7.45 (NH, Lys) /δ 4.13 (αH,
Ile2), δ 7.91 (NH, Ile3) /δ 4.31 (αH, Lys), δ 8.45 (NH, Phe) /δ
3.82 (αH, Ile3), δ 7.72 (NH, Leu) /δ 4.35 (αH, Phe), δ 7.00
(NH, Ile4) /δ 4.20 (αH, Leu), and δ 7.73 (NH, Ala) /δ 4.06 (αH,
Ile4), permitted us to sequence 1 as cyclo[-Ile-Ile-Lys-Ile-Phe-
Leu-Ile-Ala-].
Ile in the fragments and set the hydrolysis time for 4 h on the
basis of the LC−MS data (Figure 1). The partial hydrolysate of 1
thus obtained was subjected to a LC−MS-guided fractionation,
in which 20% of the effluent was directed to the MS spectro-
meter, and the remaining portion was collected. The LC−MS/MS
data allowed us to identify fragments that contain Ile residues
of specific positions, i.e., Ile-Ile-Lys (fragment a), Lys-Ile-Phe
(fragment b), Ile-Ile (fragment c), and Leu-Ile (fragment d),
together with other fragments and several linear octapeptides
(Figure 1). Marfey’s analyses of fragments a and b indicated that
fragment a contained one residue each of D-Ile, L-Ile, and L-Lys and
fragment b contained L-Ile, D-Phe, and L-Lys (Figure S26 in SI).
Therefore, we had to determine the configurations of the
contiguous Ile residues. Due to the bulkiness of Ile we did not
find any fragment cleaved between these two Ile residues.
Therefore, we used the fraction, which was a mixture of fragments
c and d, to determine the configuration of Ile in question; we
were not able to separate these peptides nor distinguish them by
MS/MS data. Due to the scarcity of the material we sought to
differentiate optical isomers of fragments c and d chromato-
graphically. We first prepared all of their possible isomers (D-Ile-L-
Ile-OMe, D-Ile-D-Ile-OMe, L-Ile-L-Ile-OMe, L-Ile-D-Ile-OMe,
D-Leu-L-Ile-OMe, and D-Leu-D-Ile-OMe) and converted each
peptide to the methyl ester DAA derivative. The mixture of
fragments c and d was also derivatized in the same way and
subjected to the LC−MS analysis, which demonstrated that
fragments c and d were L-Ile-D-Ile-OMe and D-Leu-L-Ile-OMe,
respectively (Figure 2), allowing us to determine the position
of D-Ile in the sequence. From these observations we assigned
the structure of 1 as cyclo[-L-Ile-D-Ile-L-Lys-L-Ile-D-Phe-D-Leu-L-
Ile-D-Ala-].
Surugamides B (2)−E (5) all had the molecular formula of
C47H79N9O8 which was smaller than that of 1 by a CH2 unit.
TOCSY and HSQC data of 2−5 showed that one of the Ile
residues was replaced by a Val residue (Table S2 in SI).
Interpretation of the 2D NMR data revealed that Ile2, Ile3, Ile1,
or Ile4 in 2−5, respectively, was substituted for Val.
The Marfey’s analysis3 of the total hydrolysate of 1 showed
the presence of L-Lys, D-Phe, D-Leu, and D-Ala. DAA (2,4-
dinitrophenyl-5-L-alanine amide) derivatives of D-Ile and D-allo-
Ile were hardly separable in reversed-phase HPLC as were those
of L-Ile and L-allo-Ile. Therefore, we used 2,3,4,6-tetra-O-acetyl-
β-D-glucopyranosyl isothiocyanate (GITC)4 for the derivati-
zation of the hydrolysate and analyzed by LC−MS, allowing
us to conclude that 1 contained one residue of D-Ile and
three residues of L-Ile, but none of D-allo-Ile or L-allo-Ile
(Figure S24 in SI).
In order to complete the structure elucidation of 1, we had to
distinguish between D- and L-Ile in the amino acid sequence. This
process is, in general, the most time- and sample-consuming
step in structure elucidation of peptides.5 Isolation of suitable
fragments by partial hydrolysis is prerequisite for the distinction
of optical isomers of amino acids in a peptide sequence,6 and it is
hard to predict whether desirable fragments can be generated
and acquirable under certain hydrolysis conditions. Therefore,
in order to facilitate this step of structure elucidation we used
LC−MS/MS analysis, which worked out successfully to set up
proper hydrolysis conditions and to identify peptide fragments.
Because most of constituent amino acids in 1 were nonpolar
and bulky, 1 was expected to be sluggish toward acid hydrolysis.
From this inspection, we chose harsher conditions of 4 N HCl
at 100 °C and optimized the reaction time (Figure S25 in SI).
We hoped to isolate smaller peptides to determine the chirality of
The absolute configurations of 2−5 were determined in the
same manner. The Marfey’s analysis of their total hydrolysates
showed that the configurations of L-Lys, D-Phe, D-Leu, and D-Ala
were conserved among 2−5. The configuration of 2 was assigned
from the Marfey’s analysis alone, because remaining amino acids
were one D-Val and three L-Ile. However, 3−5 contained one
D-Ile and two L-Ile residues, necessitating the determination of
B
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Note
Figure 2. LC−MS analyses of DAA derivatives of the partial hydrolysate and synthetic standards. For the HPLC conditions, see the Experimental
Section.
the position of D-Ile residue. The positions of D-Ile in 3 and 5
were determined as described above: LC−MS analyses of DAA
derivative of fragments c and d obtained by partial hydrolysis. In
the case of 4, we anticipated that the Ile residue in the Val-Ile
fragment was in the D-form and prepared L-Val-D-Ile-OMe and
L-Val-L-Ile-OMe. Their presence in the partial hydrolysate was
examined by the LC−MS analysis after converting to the methyl
ester DAA derivative. This analysis demonstrated the presence
of L-Val-D-Ile, permitting us to locate the position of D-Ile in 4
(Figure S27 in SI).
The total asymmetry of the molecule was conserved
throughout the five peptides. We speculated that the forma-
tion of the minor congeners 2−5 was due to the permissive
specificities of the adenylation domain of NRPS. In fact,
replacements of Ile by Val (or vice versa) are observed in
nonribosomal peptides such as actinomycins from Streptomyces
sp.7 and surfactins from Bacillus subtilis.8 Surugamides 1−5
showed inhibitory activity against bovine cathepsin B, a cysteine
protease implicated in invasion of metastatic tumor cells,9 with
IC50 values of 21 μM, 27 μM, 36 μM, 18 μM, and 16 μM,
respectively.
A large number of cyclic octapeptides have been reported from
marine sponges10 and terrestrial plants.11 They are remarkably
different from surugamides, because almost all of them contain
one or more Pro residues and composed only of L-amino acids.
Cyclic octapeptides are rare among microbial secondary
metabolites.12 To the best of our knowledge surugamides
comprise a new class of cyclic peptide natural products. In the
course of LC−MS analyses of our extract library of actinomycetes
culture broths, we found that several strains of Streptomyces sp.
isolated from deep sea sediments collected at diverse locations
around Japan including Suruga Bay (see Experimental Section)
produced surugamide A (1). 16S rRNA phylogenetic analyses
indicated that all the strains were Streptomyces sp. The partial
16S rRNA sequences (1410 bp) of three of them were 100%
identical to those of S. coelicolor NBRC12854 and S. somaliensis
NBRC12916. It is interesting to note that biosynthetic gene
cluster of 1 is widely distributed in marine Streptomyces species
collected around Japan.
EXPERIMENTAL SECTION
■
General Experimental Procedure. Optical rotation and UV
spectra were measured in MeOH. NMR spectra were recorded on a
600 MHz NMR spectrometer at 293 K for all compounds. 1H and 13
C
NMR chemical shifts were referenced to the solvent peaks: δH 3.30 and
δC 49.0 for CD3OD, δH 2.49 and δC 40.0 for DMSO-d6.
The Strains Producing Surugamides. Deep-sea sediment was
collected by the unmanned ROV HYPER-DOLPHIN system from
Kinko Bay (−106 m) in 2005. The sediment sample was stored in a
sterilized sampler and frozen with liquid nitrogen. Then the sample was
transported to the laboratory, where it was kept frozen until processed.
The Streptomyces sp. JAMM992 was isolated from this sample. The
taxonomy of the strains was determined by 16S rRNA phylogenetic
analyses using universal 27F and 1492R primers. The Streptomyces sp.
JAMM1350, JAMM2700, and JAMM2709 were isolated from the
deep-sea sediment collected from Sagami Bay, Japan in 2006 and 2008.
The Streptomyces sp. ACT198 was isolated from the deep-sea sediment
collected by the unmanned ROV KAIKO system from Suruga Bay
(−2409 m) in 2001. The strains JAMM992, JAMM2700, and JAMM2709
had the same 16S rRNA sequences (1410 bp). The sequences from
JAMM1350 and ACT198 were 99.8 and 99.4% identical with that from
JAMM992, respectively.
Fermentation, Extraction, and Isolation. Fermentation of
the strain Streptomyces sp. JAMM992 was performed in PC-1 medium
(1.0% starch, 1.0% polypeptone, 1.0% meat extract, 1.0% molasses,
pH 7.2, 5 L) for 5 days at 30 °C with agitation and aeration. After the
filtration of this culture, the mycelia were extracted with acetone and
MeOH. The dried extract was purified by RP-HPLC (Cosmosil ARII ϕ
10 mm × 250 mm) after the ODS flash chromatography to afford
surugamides A (1, 9.8 mg), B (2, 1.2 mg), C (3, 1.2 mg), D (4,1.2 mg),
and E (5, 1.0 mg)
Surugamide A (1): pale yellow; [α]20 −0.8 (c 0.1, MeOH); UV
D
1
(MeOH) λmax (log ε) 210 (3.98); H NMR data (DMSO-d6), see
Table S1 in SI; 13C NMR data (DMSO-d6), see Table S1 in SI; HRMS
(ESI-TOF)m/z: [M + Na]+ Calcd for C48H81N9NaO8 934.6106; Found
934.6089, Δ −1.7 mmu.
Surugamide B (2): pale yellow; [α]20 +3.3 (c 0.1, MeOH); UV
D
1
(MeOH) λmax (log ε) 207 (3.85); H NMR data (DMSO-d6), see
Table S2 in SI; 13C NMR data (DMSO-d6), see Table S2 in SI; HRMS
(ESI-TOF) m/z: [M + Na]+ Calcd for C47H79N9NaO8920.5949; Found
920.5940, Δ −1.0 mmu.
Surugamide C (3): pale yellow; [α]20 −5.2 (c 0.1, MeOH); UV
D
1
(MeOH) λmax (log ε) 207 (3.84); H NMR data (DMSO-d6), see
Table S2 in SI; 13C NMR data (DMSO-d6), see Table S2 in SI; HRMS
(ESI-TOF) m/z: [M + Na]+ Calcd for C47H79N9NaO8 920.5949; Found
920.5923, Δ −2.1 mmu.
C
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The Journal of Organic Chemistry
Note
Surugamide D (4): pale yellow; [α]20 −2.2 (c 0.1, MeOH); UV
Et3N (200 μL) and stirred at rt for 1h. The product was diluted with
H2O and extracted with EtOAc. The EtOAc layer was dried, dissolved in
TFA (0.5 mL), and left at rt for 1 h. The solvent was removed to afford
L-Ile-L-Ile-OMe which was used without further purification. Boc-DL-Ile,
Boc-D-Leu, DL-Ile-L-Ile-OMe, L-Leu-L-Ile-OMe. L-Ile-DL-Ile-OMe, L-Val-
DL-Ile-OMe, and L-Val-L-Ile-OMe were prepared in the same manner.
LC−MS Analyses of DAA Derivatives of Dipeptide Methyl
Esters. A mixture of fragments c and d (Ile-Ile and Leu-Ile) was treated
with TMS diazomethane. The reaction mixture was dried under a stream
of N2 and converted to the DAA derivatives which were analyzed by
RP-HPLC (Cosmosil 2.5C18-MSII 2.0 mm × 100 mm) using a gradient
elution of 20 to 100% MeCN containing 0.5% AcOH. Retention times
for standard dipeptide methyl esters (min): L-Ile-D-Ile-OMe (25.6),
L-Ile-L-Ile-OMe (25.8), D-Ile-L-Ile-OMe (27.0), D-Leu-L-Ile-OMe
(27.2). Retention times of the derivative from partial hydrolysate of 1:
25.6, 27.2. Each partial hydrolysate of 2−5 was treated in the same
way. Each product was analyzed by RP-HPLC using a gradient elution
from H2O−75% MeCN containing 0.5% AcOH. Retention times of
standard dipeptide methyl esters (min): L-Val-L-Ile-OMe (33.5), L-Val-
D-Ile-OMe (35.3), L-Ile-D-Ile-OMe (37.0), L-Ile-L-Ile-OMe (37.4), D-Ile-
L-Ile-OMe (38.5), D-Leu-L-Ile-OMe (38.7). Retention times of the
pertinent derivatives from partial hydrolysates (min): 3, 37.1 and 38.7;
4, 35.4; 5, 37.0.
Cathepsin B Inhibitory Assay. Cathepsin B inhibitory assay was
performed according to a modification of the method of Hisawa et al.13
The enzyme (cathepsin B from bovine spleen, Sigma C6286) was
stocked at 1 unit/mL in 50 mM MES pH 6.0 and 0.1% Brij-35.The
enzyme solution was diluted by 100 times with the buffer before use.
The mixture of 4 μL test sample solution, 100 μL of the enzyme solution,
and 50 μL of 25 μM fluorescent substrate (Z-Arg-Arg-AMC, Peptide
Institute, Inc.) in DMSO was incubated at 37 °C for 30 min. The
fluorescence of the liberated AMC was measured with an excitation at
345 nm and emission at 440 nm. The experiments were conducted in
triplicate. IC50 values (μM) of 1, 2, 3, 4, 5, and E-64 (>98% pure, Peptide
Institute, Osaka) were as follows: 21 1, 27 2, 36 3, 18 1, 16 2,
and 0.014 0.002, respectively.
D
1
(MeOH) λmax (log ε) 208 (3.87); H NMR data (DMSO-d6), see
Table S2 in SI; 13C NMR data (DMSO-d6), see Table S2 in SI; HRMS
(ESI-TOF) m/z: [M + Na]+ Calcd for C47H79N9NaO8 920.5949; Found
920.5928, Δ −2.1 mmu.
Surugamide E (5): pale yellow; [α]20 +5.6 (c 0.1, MeOH); UV
D
1
(MeOH) λmax (log ε) 209 (3.92); H NMR data (DMSO-d6), see
Table S2 in SI; 13C NMR data (DMSO-d6), see Table S2 in SI; HRMS
(ESI-TOF) m/z: [M + Na]+ Calcd for C47H79N9NaO8 920.5949; Found
920.5923, Δ −2.6 mmu.
Total Hydrolysis and Derivatization with FDAA and GITC. A
portion of 1 (100 μg) was hydrolyzed in 6 N HCl at 110 °C overnight.
The solution was dried under a stream of N2 and dissolved in H2O
(100 μL). To a half portion of the solution were added 100 μL of 1%
FDAA in acetone and 20 μL of 1 M NaHCO3 and allowed to react at
50 °C for 30 min. The mixture was quenched with 10 μL of 2 N HCl.
To the other half was added 10 μL 6% triethylamine and 1% GITC in
acetone and allowed to react at rt for 10 min. The mixture was quenched
with 10 μL of 5% AcOH. Surugamides B−E (2−5) were also
hydrolyzed, and the hydrolysates were derivatized with FDAA in the
same manner.
LC−MS Analyses of the FDAA and GITC Derivatives of Amino
Acids. The DAA derivatives were analyzed by reversed-phase HPLC
(Cosmosil 2.5C18-MSII 2.0 mm× 100 mm) using a gradient elution of
10 to 50% MeCN containing 0.5% AcOH for 30 min. Retention times
for standard amino acids (min): L-Ala (10.5), D-Ala (12.4), L-Val (13.6),
D-Val (16.1), L-Ile (15.6), D-Ile (18.2), L-allo-Ile (15.6), D-allo-Ile (18.2),
L-Leu (16.1), D-Leu (18.5), L-Phe (16.1), D-Phe (18.1), L-Lys (17.2),
D-Lys (18.5). Retention times (Rt) for the acid hydrolysate of 1 (min):
12.4 (m/z 342, Ala), 15.6 (m/z 384, Ile), 17.1 (m/z 651, Lys), 18.0 (m/z
418, Phe), 18.2 (m/z 384, Ile), 18.4 (m/z 384, Leu); 2: 12.2 (m/z 342,
Ala), 15.6 (m/z 384, Ile), 16.1 (m/z 372, Val), 17.1 (m/z 651, Lys), 18.0
(m/z 418, Phe), 18.3 (m/z 384, Leu); 3: 12.2 (m/z 342, Ala), 13.6 (m/z
372, Val), 15.5 (m/z 384, Ile), 17.1 (m/z 651, Lys), 18.0 (m/z 418, Phe),
18.1 (m/z 384, Ile), 18.3 (m/z 384, Leu); 4: 12.2 (m/z 342, Ala), 13.5
(m/z 372, Val), 15.5 (m/z 384, Ile), 17.0 (m/z 651, Lys), 18.0 (m/z 418,
Phe), 18.1 (m/z 384, Ile), 18.3 (m/z 384, Leu); 5: 12.2 (m/z 342, Ala),
13.6 (m/z 372, Val), 15.5 (m/z 384, Ile), 17.1 (m/z 651, Lys), 17.9 (m/z
418, Phe), 18.1 (m/z 384, Ile), 18.3 (m/z 384, Leu)
The GITC derivatives were analyzed by RP-HPLC (Cosmosil
2.5C18-MSII 2.0 mm × 100 mm) using a gradient elution of 21 to 26%
MeCN containing 0.5% AcOH for 120 min. Retention times for
standard amino acids (min): L-Ile (31.9), D-Ile (42.6), L-allo-Ile (30.7),
D-allo-Ile (42.3). Retention times of the pertinent derivatives from the
acid hydrolysate of 1 (min): 31.9 (L-Ile), 42.6 (D-Ile).
LC−MS-Guided Fractionation of the Partial Hydrolysate. An
aliquot of 1 (200 μg) was hydrolyzed in 4 N HCl at 100 °C for 4 h. The
dried hydrolysate was redissolved in 20 μL of MeOH. A 15 μL portion
was separated by RP-HPLC (Cosmosil 2.5C18-MSII 2.0 mm × 100 mm)
using a gradient elution from 0 to 35% MeCN containing 0.5% AcOH.
The eluate was split with a microsplitter bulb (GL Science) located
between the UV detector and the MS detector. One fifth of the eluate was
directed to ESIMS for the MS/MS analyses, and the remaining effluent
was collected every 12 s for 48 min into 96-well deep plates. After
the assignments of peptide sequences by extensive MS/MS analyses,
the fractions containing Ile-Ile-Lys, Lys-Ile-Phe, a mixture of Ile-Ile and
Leu-Ile, and linear octapeptides were identified.
ASSOCIATED CONTENT
* Supporting Information
■
S
NMR data for 1−5 and the results of Marfey analyses. This
material is available free of charge via the Internet at http://pubs.
acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: assmats@mail.ecc.u-tokyo.ac.jp. Tel.: 81-3-5841-5297.
Fax: 81-3-5841-8166.
■
Author Contributions
§K.T. and A.N. contributed equally to this paper.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by Grant-in-Aid for Scientific Research
on Innovative Areas “Chemical Biology of Natural Products”
(23102007) from MEXT.
Marfey’s Analyses of Ile-Ile-Lys and Lys-Ile-Phe. A fraction
containing a peptide was hydrolyzed in 6 N HCl at 110 °C overnight.
The solution was dried, converted to the methyl ester DAA derivative,
and analyzed as described above. Retention times for standard amino
acids (min): L-Ile (15.7), D-Ile (18.2), L-Phe (16.1), D-Phe (18.1), L-Lys
(17.2), D-Lys (18.5). Retention times of the derivatives from the
hydrolysate (min): fragment a (Ile-Ile-Lys): 15.8 (m/z 384, Ile), 17.1
(m/z 651, Lys), 18.2 (m/z 384, Ile); fragment b (Lys-Ile-Phe): 15.6
(m/z 384, Ile), 17.1 (m/z 651, Lys), 18.0 (m/z 418, Phe).
Synthesis of Dipeptide Methylesters. L-Ile (50 mg) was
dissolved in 10% HCl−MeOH (1 mL) and heated at 100 °C for 2 h
to yield L-Ile-OMe which was dissolved in DMF (1 mL). To the solution
were added Boc-L-Ile (75 mg), WSCI (75 mg), HOBt (50 mg), and
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