Site Covalent Modification of Methionyl Peptides for the Production of FRET Complexes

Article abstract of DOI:10.1002/chem.201502699

Flax cyclic peptides (orbitides, linusorbs (LOs)) [1-8-NαC],[1-MetO₂]-linusorb B1 ([MetO₂]-LO1) and [1-9-NαC],[1-MetO₂]-linusorb B2 ([MetO₂]-LO2) are biologically active. These LOs lack active nuclei commonly used in peptide modification. We have developed reactions to activate methionine methyl sulphide to produce stable derivatives. In these reactions, LOs are converted to sulfonium intermediates and subsequently to derivatives containing active nuclei while preserving their fundamental structures. The reaction conditions preserved cyclic peptide fundamental structure and organic solvent solubility. [Met]-LO1 and [Met]-LO2 analogues containing activated groups (-CN, -COOEt, and -NH₂) in the form of methionine, methionine (S)-oxide, and methionine (S,S)-dioxide amino acids were synthesized and characterized by LCMS and 1D and 2D NMR spectroscopy. Coumarin orbitide complexes produced in this manner bind Eu³⁺ yielding FRET compounds that absorb energy through coumarin antennae and emit photons at lanthanide wavelengths.

Full text of DOI:10.1002/chem.201502699

DOI: 10.1002/chem.201502699
Full Paper
&
Structure Elucidation
Site Covalent Modification of Methionyl Peptides for the
Production of FRET Complexes
Pramodkumar D. Jadhav,*[a] Jianheng Shen,[a] Ramaswami Sammynaiken,[b] and
Martin J. T. Reaney*[a, c]
Abstract: Flax cyclic peptides (orbitides, linusorbs (LOs)) [1–
8-NaC],[1-MetO2]-linusorb B1 ([MetO2]-LO1) and [1–9-
NaC],[1-MetO2]-linusorb B2 ([MetO2]-LO2) are biologically
active. These LOs lack active nuclei commonly used in pep-
tide modification. We have developed reactions to activate
methionine methyl sulphide to produce stable derivatives. In
these reactions, LOs are converted to sulfonium intermedi-
ates and subsequently to derivatives containing active nuclei
while preserving their fundamental structures. The reaction
conditions preserved cyclic peptide fundamental structure
and organic solvent solubility. [Met]-LO1 and [Met]-LO2 ana-
logues containing activated groups (ÀCN, ÀCOOEt, and À
NH2) in the form of methionine, methionine (S)-oxide, and
methionine (S,S)-dioxide amino acids were synthesized and
characterized by LCMS and 1D and 2D NMR spectroscopy.
Coumarin orbitide complexes produced in this manner bind
Eu3+ yielding FRET compounds that absorb energy through
coumarin antennae and emit photons at lanthanide wave-
lengths.
Introduction
Lanthanide binding peptide ligands have been synthesized
by solid-phase peptide synthesis and used for fluorescence
studies in aqueous buffer solutions. Thermodynamic stability is
necessary if these compounds are to be used for in vitro and
in vivo applications.[10] Cyclic peptides and particularly orbitides
are more stable than linear peptides with the same sequence
and they typically resist protease degradation.[11] Cyclic pep-
tides with chromophores can be prepared using solid-phase
supports[12] or direct modification at reactive side chain amino
acids including serine, threonine, cysteine, and lysine.[13] The
latter approach is particularly efficient for peptides that have
few heteroatoms and, therefore, can be converted without the
use of protective groups. A further advantage of adduct forma-
tion is realized with mild reactions that preserve peptide chiral-
ity. This approach can help to generate families of analogues
that are suited for studies of structure/function activity rela-
tionships of biologically active peptides.[14]
Lanthanide complexes are used in various biomedical applica-
tions,[1] supramolecular chemistry,[2] and structural analysis.[3]
Lanthanide ions, Ln3+ show interesting luminescent properties
with sharp emission peaks, long luminescence lifetimes (micro-
second to milliseconds), large Stoke shifts and emissions rang-
ing from visible to near-IR.[4] Lanthanides have been incorpo-
rated into biomolecules such as peptides at calcium binding
sites,[5] attached at reactive side chains like cysteine,[6] or cou-
pled to modified amino acids.[7] However the inherent draw-
back of Ln3+ ions is low quantum yield, which can be over-
come in part by coupling Ln3+ with fluorophores such as tryp-
tophan or other fluorescent compounds.[7,8] Macrocyclic crypt-
and molecules such as crown ethers have been used as sub-
strates with coumarin as sensitizers. Molecules with greater
rigidity lose less energy in nonradiative emission.[9]
The systematic alkylation of methionine residues was previ-
ously reported for proteins using a-halo acetic acids and relat-
ed derivatives.[15] This reaction yielded a sulfonium salt, which
was converted by heating at 1008C to either homoserine or
homoserine lactone in which the product arises from the reac-
tion of a C-terminal methionine residue. It was also reported
that methionine can be regenerated from sulfonium salts
using b -mercaptoethanol.[16] This approach was used for isola-
tion of methionine peptides under acidic conditions.[17] The
synthesis of sulfonium polypeptides is also possible through
methionine alkylation.[18]
[a] Dr. P. D. Jadhav, Dr. J. Shen, Prof. M. J. T. Reaney
Plant Sciences, University of Saskatchewan
Saskatoon, SK (Canada)
E-mail: pramodkumar.jadhav@usask.ca
martin.reaney@usask.ca
[b] Dr. R. Sammynaiken
Saskatchewan Structural Sciences Centre
University of Saskatchewan
Saskatoon, SK (Canada)
[c] Prof. M. J. T. Reaney
Guangdong Saskatchewan Oilseed Joint Laboratory
Department of Food Science and Engineering
Jinan University, Guangzhou
Flax seed oil (Linum usitatisssimum L.) contains hydrophobic
orbitides (linusorbs, LOs) comprising eight to ten amino acids.
To date, 28 LOs, with molecular weights of approximately
1 kDa have been isolated from flaxseed.[19] Glycine and trypto-
Guangdong 510632 (People’s Republic of China)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201502699.
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Table 1. Amino acid sequences of glycine and tryptophan free LOs (1–7).
LO[a]
Previous
names
Amino acid sequence[b]
Molecular
formula
[1–9-NaC]-linusorb B3 (1)
[1–9-NaC]-linusorb B2 (2)
[1–9-NaC],[1-(Rs,Ss)-MetO][c]-linusorb B2 (3)
[1–9-NaC],[1-MetO2]-linusorb B2 (4)
[1–8-NaC]-linusorb B1 (5)
CLA
CLB
CLC
CLK
CLP
CLE
CLJ
NaC-(Ile-Leu-Val-Pro-Pro-Phe-Phe-Leu-Ile)
NaC-(Met-Leu-Ile-Pro-Pro-Phe-Phe-Val-Ile)
NaC-(MetO[d]-Leu-Ile-Pro-Pro-Phe-Phe-Val-Ile)
NaC-(MetO2[d]-Leu-Ile-Pro-Pro-Phe-Phe-Val-Ile)
NaC-(Met-Leu-Val-Phe-Pro-Leu-Phe-Ile)
NaC-(MetO-Leu-Val-Phe-Pro-Leu-Phe-Ile)
NaC-(MetO2-Leu-Val-Phe-Pro-Leu-Phe-Ile)
C57H85N9O9
C56H83N9O9S
C56H83N9O10S
C56H83N9O11S
C51H76N8O8 S
C51H76N8O9 S
[1–8-NaC],[1-(Rs,Ss)-MetO]-linusorb B1 (6)
[1–8-NaC],[1-MetO2]-linusorb B1 (7)
C51H76N8O10
S
[a] Linusorbs naming based on the proposed nomenclature of orbitides.[19d] [b] Homodetic cyclic peptides with N to C linkage between first and last amino
acid nomenclature convention.[22] [c] Methionine (S)-oxide has a chiral S. Epimers are designated S and R. [d] Designation of MetO and MetO2 describe me-
thionine (S)-oxide and methionine (S,S)-dioxide.
phan-free LOs are shown in Table 1. LOs 1 (LO3), 2 (LO2), 3
([MetO]-LO2), and 6 ([MetO]-LO1) have shown in vitro antitu-
mor activity against different cancer cell lines.[20] LO 1 inhibits
both cholate uptake into hepatocytes of rat liver (50% inhibi-
tion at 3 mm) and T-lymphocyte activation and proliferation.
Related compounds such as somatostatin and antanamide also
inhibit cholate uptake into hepatocytes.[21]
tion of LOs 3 and 6 through methionine residues while main-
taining their fundamental structure. To our knowledge modifi-
cation of cyclic peptides and LOs through methionine has not
been previously reported. A general synthetic protocol was de-
veloped that was used to prepare fluorescent LO adducts from
commercially available dyes. Spectroscopic analysis of fluores-
cent adducts was conducted in different solvents to under-
stand the photophysical behaviour of coumarin LO complexes.
LO conjugated coumarin complexes were also coupled with
transition metals to be used in LRET (lanthanide resonance
energy transfer) analysis. In addition, these modified LOs have
been characterized using spectrometric techniques including
1D and 2D NMR. Hence LOs tagged with a fluorescent label
can potentially be used in therapeutic monitoring and fluores-
cence energy transfer.
LO 1 inhibits concanavalin A induced T-cell proliferation (IC50
of 2.5 mgmLÀ1) with the largest immunosuppressive activity on
human peripheral blood lymphocytes of LOs tested. Flax LOs,
([MetO2]-LO2) (4) and ([MetO2]-LO1) (7) also possess immuno-
suppressive activity (Table 1).[23] The IC50 of 4 (25.2 mgmLÀ1) and
7 (28.1 mgmLÀ1) occurred at higher concentrations.[23] LOs 4
and 7 were synthesized in 75% isolated yield by oxone oxida-
tion of 3 and 6, respectively,[24] but were also detected in flax
meal heated at 1008C for 16 h and silica that has been in con-
tact with flax oil.[25]
Results and Discussion
The biological activity of 1 might arise from the Pro-Phe-Phe
tripeptide moiety. It was also found that the amino acid triad
Pro-Xxx1-Xxx2 (in which Xxx1 and Xxx2 are either hydrophobic
or aromatic amino acid residues) was also present in other im-
munosuppressant cyclic peptides such as cycloamanide B (Pro-
Ser-Phe), antamanide (Pro-Phe-Phe) and hymenistatin I (Pro-
Tyr-Val).[21a,26] Similar tripeptide fragments also occur in 4 and 7
in which Xxx1 is Phe in 4, but Leu in 7. Hence chemical modifi-
cations of 4 and 7 might display this bioactive tripeptide frag-
ment if these amino acids remain intact and separated from
the modified site.
LOs
4 [NaC-(MetO2-Leu-Ile-Pro-Pro-Phe-Phe-Val-Ile)] and 7
[NaC-(MetO2-Leu-Val-Phe-Pro-Leu-Phe-Ile)] are biologically
active[23] and heat stable.[25] Attempts to reduce MetO2 to Met
were unsuccessful (data not shown). The Met containing LOs 2
and 5 were, therefore, accessed through reduction of methio-
nine (S)-oxide (MetO) precursor LOs 3 and 6, respectively.
MetO containing LOs were readily reduced to Met containing
peptides using (NH4I/TFA/Me2S) and (TiCl4/NaI).[28] Modification
of resulting Met LOs with reactive iodomethylene groups is
then possible.[15a] Met, therefore, serves as point of attachment
for an activated side chain while keeping the fundamental
structure intact. Peptides containing reactive groups such as À
NH2, ÀCOOH, and ÀOH may thus be synthesized to allow selec-
tive coupling with other reactive functional groups present in
fluorescent labels.
LOs could be utilised as drugs or drug delivery agents as
they have biological activity, hydrophobicity and may form in-
clusion complexes.[27] LOs contain intrinsic fluorophores such
as Phe and Trp in their cyclic structure. LO 1 has been reported
to transfer energy to Tb3+ through Phe fluorescence.[27b] How-
ever natural amino acid fluorophores have limitations as excita-
tion occurs at wavelengths where background absorbance is
high for both the excitation and emission wavelengths, this
would make detection of a fluorescent signal in living tissues
difficult. Attaching a fluorescent dye to an orbitide would over-
come this but there are no prior reports of attachment of fluo-
rescent dyes to LOs.
LO 3 was reduced to 2 by using sodium borohydride in
presence of iodine (Scheme 1).[29] Met is reactive and can be re-
acted with various electrophilic groups such as haloalkyl or hal-
oacetyl to form additional compounds.[18a] The plausible mech-
anism for Met alkylation with an electrophilic group (RÀX) is
through formation of a sulfonium intermediate and subse-
quently release of MeX as a leaving group to form a SÀR com-
pound.[15a] LO 2 was subsequently treated with iodoacetonitrile
in the presence of DMF and N-methylpyrrolidone at 1008C for
20 h to form the nitrile, 8 (ÀSCH2CN). Similar treatment with io-
LOs contain amino acids such as Trp and Met that may be
suited for attachment of fluorescent dye or for producing
other modifications. This paper describes site-specific modifica-
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does not lead to elimination
products was sought. Amine 15
was obtained from ester 13 by
following its oxidation to a com-
paratively thermally stable 14.
Treatment of 14 with ethylene
diamine provided access to
amine 15 in 86% isolated yield.
Following successful synthesis of
amine 15 (Scheme 1), amine 18
was similarly prepared from LO
6 (Scheme 2).
LO fluorescent adducts
Amines 15 and 18 (Scheme 1
and 2) were used to prepare flu-
orescent LOs 19 and 20, respec-
tively (Scheme 3). The 7-methox-
ycoumarin-3-carboxylic acid suc-
cinimidyl ester was prepared as
previously reported[33] and was
coupled with 15 and 18 in the
presence
of
triethylamine
(Scheme 3). The resulting fluo-
rescent LO products 19 and 20
were subsequently purified by
preparative-HPLC chromatogra-
phy and were characterized by
MS/MS (Figure 1a,b) and NMR.
Scheme 1. Synthesis of amine derivative 15: a) NaBH4, I2, THF, RT; b) ICH2CN, DIPEA, NMP, 1008C; c) mCPBA, DCM,
RT; d) H2 (1 atm.), PtO2, AcOH, RT; e) H2 (1 atm), Pd/C, LiOH, dioxane/water, 1408C; f) NaIO4, THF: H2O;
g) ICH2COOEt, DIPEA, NMP, 1008C; h) mCPBA, DCM, RT; i) ethylenediamine, MeOH, reflux. mCPBA=meta-chloro-
perbenzoic acid; DIPEA=N,N-diisopropylethylamine.
doacetate yielded the ester, 13 (ÀSCH2COOEt). Met compounds
can be eliminated using Raney Ni to afford alkanes (a-amino
butyric acid)[30] or to produce an alkene at elevated tempera-
Characterization of 19 and 20
ture ((S)-vinyl glycine).[31] In our attempts to reduce nitrile 8 to
an amine led to elimination of mercaptoacetonitrile or hydro-
gen disulfide to form 9 under the experimental conditions.[32]
To avoid producing the elimination product, 8 was oxidized to
(S)-oxide nitrile (10) (ÀSOCH2CN). However, the (S)-oxide nitrile
10 underwent elimination under
The tandem mass fragmentation pattern (Figure 1a) and NMR
spectra (Experimental Section) of 19 showed remarkable simi-
larities to that of 4.[25] However, a fragment loss of m/z: 451 in
the MS/MS spectrum of 19 (Figure 1a) instead of loss of m/z:
reduction conditions (H2 at
1 atm., PdÀC, and LiOH at
1708C) to produce mixtures of
external and internal olefins
(data not shown). It was sur-
mised that oxidation of 8 to
a more stable (S,S)-dioxide 11 (À
SO2CH2CN) will provide access to
an amine 12 that resists elimina-
tion. Thus, mCPBA oxidation of 8
yielded 11, which was reduced
to amine 12 (ÀSO2CH2CH2NH2)
under 1 atm. of H2, PtO2, and
AcOH at room temperature in
low yield. Elimination products
were not detected under these
reaction conditions.
An alternative and better
Scheme 2. Synthesis of amine derivative 18: a) NaBH4, I2, THF, RT; g) ICH2COOEt, DIPEA, NMP, 1008C; h) mCPBA,
yielding route to amine 15 that DCM, RT; i) ethylenediamine, MeOH, reflux. NMP=N-methylpyrrolidone.
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tures of 19 and 20 were con-
firmed by MS/MS and NMR.
HPLC-MS
[M+H]+ ion peak at m/z:
1378.6751 corresponding to
molecular formula,
analysis
showed
a
a
C70H96N11O16S for LO 19. A m/z:
1281.6317 corresponded to the
molecular formula, C65H89N10O15S
of 20.
Scheme 3. Synthesis of fluorescent LOs 19 and 20.
Figure 1. CID MS/MS spectrums of 19 (A) and 20 (B) showing the fragmenta-
tion pattern of the MetO2 coumarin adduct.
Figure 2. Structure of 19 (A) and 20 (B). Double arrows show selected NOE
correlations. Half arrows show selected HMBC correlations.
163 (MetO2) in 4 indicated the presence of a 7-methoxycou-
marin-3-carboxylic acid conjugate of amine 15. The protons at-
tached to the heteroatoms were assigned from H NMR, and
Fluorescence studies
1
their coupling to amide protons and carbonyl carbon atoms
employed NOE and HMBC correlations (Figure 2 a,b). These
provided the amino acid sequence of the fluorescent LOs. Se-
quential assignment of b, g, and d protons was carried out by
The absorption and fluorescence studies were conducted in
four different solvents (methanol, ethanol, acetonitrile, and
DMF) at a concentration of 3”10À5 m. The samples were excit-
ed at 354 nm, and emission spectra were recorded. Coumar-
in 120 (7-amino-4-methyl-1, 2-benzopyrone) was used as a ref-
erence.[34]
1
using H-1H COSY and elucidation of attachments of these pro-
tons to carbon atoms was carried out by HMQC analysis. The
amino acid sequence of 19 was further established using
tandem mass spectrometry (Figure 1a). Fluorescent LO 20 was
similarly characterized. However, the 13C NMR spectrum
showed two sets of signals for each carbon atom correspond-
ing to two conformers of 20 similar to 7.[25] Signals from the
major conformer are shown in the Experimental Section. Struc-
7-Methoxycoumarin-3-carboxylic acid was used as a tag in
which the methoxy group acts as an electron donor and car-
bonyl as an acceptor. The fluorescence intensity of this cou-
marin dye is solvent-dependent and it increases with solvent
polarity with negligible emission shift.[35] It has been postulated
that, np* and pp* singlet states are close to each other in the
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Table 2. Spectroscopic analysis of 19 in different solvents.
Solvent
l max
l emission
f Quantum
[nm]
[nm]
yield
ethanol
methanol
acetonitrile
DMF
348
348
345
347
407
404
402
408
0.69
0.71
0.57
0.44
Table 3. Spectroscopic analysis of 20 in different solvents.
Solvent
l max
l emission
f Quantum
[nm]
[nm]
yield
ethanol
methanol
acetonitrile
DMF
348
347
345
347
403
403
405
406
0.62
0.68
0.59
0.42
nonpolar solvent, in which intersystem crossing takes place
and hence the fluorescence yield is lower. However, in polar
solvent the difference between these two states is greater
making intersystem crossing inefficient.[35b] Fluorescent LOs 19
and 20 absorb light at 347 nm and emit at 406 nm. Both show
the same trend with higher quantum yields in methanol and
ethanol than in either acetonitrile or DMF (Figures S37 and
S38, Supporting Information; Tables 2 and 3). The hydrogen
bonding of hydroxyl groups of alcohols has an extra effect
when compared to aprotic acetonitrile and DMF. However,
fluorescence in DMF occurs with very low quantum yield com-
pared to fluorescence in other solvents. Potentially nonradia-
tive decay or conformational changes occur in dyes that arise
from interaction with the LO in DMF lowering quantum yield.
Figure 3. MS spectrums of 21 (A) and 22 (B).
Fluorescence energy transfer from coumaryl–LO to metals
LO 19 was mixed with solutions of metal ions (TbCl3 and EuCl3)
to prepare LO metal adducts 21 (Tb) and 22 (Eu), respectively
(Scheme 4). The resulting LO metal complexes 21 and 22 were
characterized by MS (Figure 3a,b). The potential fluorescence
energy transfer from coumarin dye to a metal through LOs
was investigated in different solvents. In this experiment, the
Scheme 4. Synthesis of LO metal complexes 21 (Tb) and 22 (Eu).
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chromophore (coumarin) was excited with monochromatic
light (354 nm).
In solvents (methanol, ethanol, and DMF) with exchangeable
protons that participate in H-bonds and quenching no emis-
sions or fluorescence (Tb, Eu bands) were observed. Eu com-
plexes with LOs dissolved in acetonitrile in different concentra-
tions produced a fluorescence emission spectrum (Eu band)
that indicated possible fluorescence energy transfer from the
fluorophore to Eu (Figure 4a). No similar emissions were ob-
served for Tb complexes in acetonitrile. These results show
that fluorescence energy transfer from coumarin to Eu occurs
in acetonitrile (this is attributed to the lower solvation effects
of acetonitrile).
The effects of Eu on coumarin quantum yield in acetonitrile
were further investigated. As the number of equivalents of Eu
increased, the quantum yield of LO–coumarin complexes de-
creased (Figure 4b). At higher ratios of Eu to LO–coumarin
complex fluorescence energy is transferred between coumarin
dye and Eu.
FRET utility of LOs
To determine FRET ability of LO 22 in biological environments,
it was examined in living cells. A low concentration (476 nm) of
LO 22 was administered to immortalized human dermal fibro-
blasts and incubated for 15 min. Cells were imaged by confocal
fluorescence microscope by using 350 nm excitation (Figure 5).
No fluorescence was seen in the cytoplasm of the control,
whereas LO 22-treated cells show red emission at 600 nm. Pre-
liminary results show that LO 22 was accumulated in the cyto-
plasm and also confirms the ability of coumarin to excite Eu
through the peptide.
Figure 4. Fluorescence spectrum of 22 (A) and effect of Eu equivalents on
the quantum yield of coumarin and Eu (B).
LO metal adducts with coumarin might also have potential
for use in organic light-emitting diodes (OLEDs). Previously,
a LO analogue with tryptophan
sensitizer was used to excite
Tb3+ with an energy band gap
of 2.9 eV. Charge transfer be-
tween LO and the complexed
metal did not occur but the
band gap was favourable to en-
courage future investigations.[36]
Hence LO 22 with coumarin sen-
sitizer might lower the band gap
required for the construction of
more efficient OLEDs.
Conclusion
In summary, we have described
a methodology for derivatization
of LOs (3 and 6) using methio-
Figure 5. LO 22 localizes to the cytoplasm of human cells. NB1 hTERT immortalized human dermal fibroblasts
nine as a point of attachment to grown on coverslips were either untreated (control) or cells (LO 22) with 476 nm LO 22 for 15 min. Coverslips
were then placed on 500 mL of cell culture media and live cells imaged. For both control and LO 22 treated condi-
give modified fluorescent LOs 19
tions, cells were counterstained with the DNA specific dye Hoechst 33342 (green). Peptide localization is shown in
and 20 that were utilized in fluo-
red. For both control and LO 22 treated conditions a phase-contrast (grey scale) image was acquired and all chan-
rescence studies. We have also
nels merged to show localization. The red signal in the nucleus of control and peptide treated cells is due to the
described methods for increas- overlap in excitation of both the peptide and the Hoechst dye. Scale bar=10 mm.
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Mass spectra and MS/MS of 19 and 20 were acquired using a Micro-
TOF-Q II Mass Spectrometer (Bruker Daltonik GmbH, Bremen, Ger-
many) equipped with APCI source (Parameters: dry temp, 2008C;
Vaporizer temp, 4508C; dry gas, 8 LminÀ1; nebulizer; 1.6 bar; capil-
lary voltage, 4000 V). Additional HR HPLC-QTOF MS analyses were
performed with an Agilent 1100 HPLC connected to QSTAR XL
spectrometer (Mass Spectrometer Hybrid Quadrupole-TOF LCMS/
MS, Applied Biosystems, Toronto, Canada) with an electrospray ion-
ization (ESI) source. Chromatographic separation was achieved at
room temperature using a Hypersil ODS C-18 (Hewlett-Packward,
Germany, 5 mm particle size silica, 100”2.1 mm) column. The
mobile phase consisted of a linear gradient of solvent A, H2O con-
taining 0.1% formic acid, and solvent B, acetonitrile containing
0.1% formic acid. Elution was performed using a linear gradient
from 55% of B to 90% of B within 25 min at a flow rate of
ing the stability of such compounds to heat, reduction, and ox-
idation. This strategy is suitable for making analogues of LOs
for use in different applications. The structures of 20 deriva-
tives (ester, nitrile, amine, and alcohol) were fully elucidated
using HR HPLC-MS-ESI, tandem mass spectrometry, 1D (1H and
13C), and 2D NMR (NOE, COSY, HSQC and HMBC) spectroscopic
methods. Fluorescence studies of coumarin complexes were
conducted in different solvents and both compounds show
high quantum yield in alcohols. This is the first report of their
complete characterization. Eu complex 22 has shown fluores-
cence energy transfer from dye to metal. We also have suc-
cessfully demonstrated FRET utility as a cytoplasm specific
stain. These structures will probably provide a better under-
standing of the mechanism(s) of their biological activity and
can possibly be used as fluorescent biosensors in biological re-
search and drug discovery.
0.25 mLminÀ1
.
Fluorescence excitation and emission spectra were acquired with
a p*-180 PiStar stopped-flow spectrometer (Applied Photophysics,
Surrey, UK), both slit widths were set at 4 nm. The relative quan-
tum yield of standard coumarin 120 (7-amino-4-methyl-1, 2-benzo-
pyrone) in ethanol (0.98 in EtOH) was used to calculate quantum
yield. Absorbance of all solutions between 200 and 700 nm was
below 0.7 AU. Fluorescence spectra details: Intensity, 3 s, Scan
time, 1, Boxcar width, 0, Range, 200–1000 nm. All solutions were
excited at 354 nm.
Experimental Section
General
All chemicals were purchased from Sigma–Aldrich and HPLC grade
solvents were used. Compounds 3 and 6 were obtained from flax
oil.[37] All other reagents were purchased from commercial sources
Human dermal fibroblasts (NB1) immortalized with human telo-
merase (hTERT) as described previously,[38] were grown on cover-
slips in 5% CO2 in DMEM media for 24 h at 378C. For LO treat-
ments, 50 mL acetonitrile containing 10 mm LO 22 was added to
1 mL of media containing Hoescht 33342 (H33342; 1:500 dilution
of 10 mgmLÀ1) and added to the cells. Control and treated cells
were then imaged using a Zeiss 710 LSM 2-photon confocal invert-
ed microscope and Chameleon Coherent laser as a light source
(Chameleon Vision laser system). For imaging, excitation wave-
lengths were set and 350 nm for H33342 dye and emission wave-
lengths acquired at 405 nm. For LO 22 imaging excitation was set
at 350 nm and the emission wavelength set to 600 nm. Images
were imported into Image J/Fiji imaging software (http://fiji.sc/Fiji)
and processed using identical exposure times and contrast in both
treated and control cells.
1
and were used without further purification. The H (500 MHz) and
13C NMR (125.8 MHz) spectra were recorded on a Bruker Avance
spectrometer (Bruker Biospin, Germany, Inverse triple resonance
probe, TXI, 5 mm, XWIN-NMR 3.5 software). Spectra acquired at
320 K had improved resolution when compared to spectra ob-
tained at lower temperatures (data not shown). Subsequent
2D NMR spectra were also acquired at 320 K. Proton and corre-
sponding carbon signals were assigned using 1H–1H COSY and
HMQC correlations. The correlations between amide protons, car-
bonyl carbons and a-CH protons were determined from NOE and
1
HMBC experiments. H NMR spectra were taken in CDCl3 and refer-
enced to appropriate solvent signals. Chemical shifts are reported
in ppm units downfield from tetramethylsilane. FTIR spectra were
recorded on a Bio-Rad FTS-40 spectrometer (Win-IR software, Ver-
sion-4.14) using diffuse reflectance mode on samples dispersed in
KBr (DRIFT); only diagnostic and/or intense peaks are reported.
Synthesis
HPLC-DAD analysis was performed with an Agilent 1200 series
HPLC system equipped with a quaternary pump, autosampler, de-
gasser and diode array detector (Agilent G1315C/D, 1024-element
photodiode array, wavelength range 190–950 nm). The peaks were
detected at wavelengths of 214 nm with a 10 nm bandwidth
against a reference signal at 300 nm. Chromatography was con-
ducted using a Chromolith SpeedRod RP-18e column (2 mm meso-
porous size, 50”4.6 mm I.D.) equipped with an online filter (Chro-
molith RP-18e guard cartridge, 5”4.6 mm). The mobile phase con-
sisted of H2O/acetonitrile (70:30 to 30:70 in 4 min, 10:90 in 0.5 min,
70:30 in 0.5 min and equilibrated to 70:30 for 1 min) at a flow rate
[Met]-LO2 (2): Sodium borohydride (1 equiv, 0.07 g) and iodine
(1.5 equiv, 0.35 g) were added to a stirred solution of LO 3 (2 g,
1.9 mmol) in THF (25 mL). The resulting mixture was stirred at
room temperature for 1 h and was subsequently treated with
water and was extracted with CH2Cl2. The solvent was removed
under reduced pressure using rotary evaporator and 2 was purified
1
by flash column chromatography (1.8 g). H NMR (500 MHz, CDCl3,
COSY, NOESY): Met1: d=4.27 (1H, dd, 8.4, 8.2 Hz, a), 1.99 (2H, m,
b), 2.57 (1H, m, g), 2.49 (1H, m, g), 2.07 (3H, s, eMe), 6.52 ppm (1H,
d, 6.4 Hz, NH); Leu2: d=3.65 (1H, m, a), 1.63 (2H, m, b), 1.99 (1H,
m, g), 0.94 (3H, d, 6.6 Hz, dMe), 0.92 ppm (3H, d, 6.6 Hz, dMe); Ile3:
d=4.44 (1H, m, a), 1.92 (1H, m, b), 1.78 (2H, m, g), 1.05 (3H, d,
6.4 Hz, gMe), 0.84 (3H, t, 7.5 Hz, dMe), 7.34 ppm (1H, br s, NH);
Pro4: d=4.03 (1H, m, a), 2.24 (1H, m, b), 1.92 (1H, m, b), 2.10 (1H,
m, g), 1.99 (1H, m, g), 4.19 (1H, m, d), 3.72 (1H, m, d); Pro5: d=
3.94 (1H, m, a), 1.92 (2H, m, b), 1.50 (1H, m, g), 0.74 (1H, m, g),
3.34 (1H, m, d), 3.00 ppm (1H, m, d); Phe6: d=4.74 (1H, m, a), 3.35
(1H, m, b), 3.00 (1H, m, b), 7.26 (2H, m, d), 7.18 (1H, m, e), 7.10
(2H, m, z), 7.95 ppm (1H, m, NH); Phe7: d=3.95 (1H, m, a), 3.21
(1H, m, b), 2.79 (1H, m, b),7.26 (2H, m, d), 7.18 (1H, m, e), 7.10 (2H,
of 2 mLminÀ1 [32]
A Chromolith SemiPrep RP-18e column (2 mm par-
.
ticle size, 100”10 mm I.D.) in series with a matching guard column
was used for preparative liquid chromatographic separations. The
mobile phase consisted of H2O/acetonitrile (80:20 for 0.5 min fol-
lowed by a linear gradient to 10:90 in 4.5 min, 80:20 in 0.25 min
and equilibrated to 80:20 for 0.75 min) at
5 mLminÀ1
a flow rate of
.
Reverse-phase enrichment of LO reaction mixtures were also per-
formed using Sep-Pak vac 20cc (5 g) tc18 cartridges (37–55 mm par-
ticle size, 125 Š, Waters, Ireland).
Chem. Eur. J. 2015, 21, 17023 – 17034
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17029
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Full Paper
m, z), 7.45 (1H, d, 6.4, NH); Val8: d=3.89 (1H, m, a), 2.24 (1H, m,
b), 1.05 (3H, d, 6.6 Hz, gMe), 1.00 (3H, d, 6.6 Hz, gMe), 8.09 ppm
(1H, brs, NH); Ile9: d=4.20 (1H, m, a), 1.99 (1H, m, b), 1.50 (2H, m,
g), 0.95 (3H, d, 6.6 Hz, gMe), 0.89 (3H, t, 7.5 Hz, dMe), 6.82 ppm
(1H, br s, NH); 13C NMR (125 MHz, CDCl3, HMQC, HMBC): Met1: d=
58.6 (a), 25.4 (b), 31.3 (g), 15.3 (eMe), 170.7 ppm (C=O); Leu2: d=
58.6 (a), 28.9 (b), 25.2 (g), 23.4 (dMe), 21.4 (dMe), 171.4 (C=O); Ile3:
d=56.2 (a), 37.2 (b), 25.4 (g), 16.1 (gMe), 11.0 (dMe), 171.5 ppm
(C=O); Pro4: d=60.9 (a), 31.5 (b), 21.5 (g), 46.8 (d), 170.0 ppm (C=
O); Pro5: d=54.8 (a), 37.5 (b), 24.7 (g), 48.3 (d), 174.7 ppm (C=O);
Phe6: d=60.9 (a), 36.0 (b), 137.2 (g), 129.7 (d), 129.0 (e), 127.4 (z),
172.5 ppm (C=O); Phe7: d=55.6 (a), 37.7 (b), 136.4 (g), 129.1 (d),
128.9 (e), 126.8 (z), 175.0 ppm (C=O); Val8: d=62.4 (a), 29.6 (b),
19.6 (gMe), 18.3 (gMe), 172.4 ppm (C=O); Ile9: d=54.0 (a), 35.0 (b),
25.4 (g), 15.4 (gMe), 10.8 (dMe), 171.5 ppm (C=O).
(2H, m, z); Phe7: d=4.23 (1H, m, a), 3.34 (2H, m, b), 7.30 (2H, m,
d), 7.24 (1H, m, e), 7.15 (2H, m, z), 7.96 (1H, d, 5, NH); Val8: d=
3.81 (1H, m, a), 2.19 (1H, m, b), 1.01 (3H, d, 7 Hz, gMe), 0.98 (3H,
d, 7 Hz, gMe), 7.67 ppm (1H, brs, NH); Ile9: d=4.29 (1H, m, a), 2.06
(1H, m, b), 1.65 (2H, m, g), 0.95 (3H, d, 6.4 Hz, gMe), 0.87 (3H, t,
7.5 Hz, dMe), 6.51 ppm (1H, brs, NH); 13C NMR (125 MHz, CDCl3,
HMQC, HMBC): Met1: d=53.4 (a), 25.2 (b), 28.9 (g), 17.1 (SCH2),
116.7 (CN), 170.8 (C=O); Leu2: d=62.7 (a), 29.6 (b), 25.4 (g), 23.4
(dMe), 21.4 (dMe), 171.6 ppm (C=O); Ile3: d=55.8 (a), 37.4 (b), 24.8
(g), 15.3 (gMe), 11.0 (dMe), 172.0 (C=O); Pro4: d=54.9 (a), 37.4 (b),
24.7 (g), 48.3 (d), 170.1 ppm (C=O); Pro5: d=61.0 (a), 31.6 (b), 21.7
(g), 47.0 (d), 174.6 ppm (C=O); Phe6: d=55.4 (a), 37.7 (b), 137.0 (g),
129.6 (d), 129.0 (e), 127.4 (z), 172.2 ppm (C=O); Phe7: d=58.7 (a),
35.8 (b), 136.4 (g), 129.1 (d), 128.9 (e), 127.0 (z), 175.1 ppm (C=O);
Val8: d=58.6 (a), 29.6 (b), 19.5 (gMe), 18.4 (gMe), 172.2 ppm (C=O);
Ile9: d=58.7 (a), 35.3 (b), 24.8 (g), 16.2 (gMe), 11.1 (dMe),
172.1 ppm (C=O).
[Met]-LO1 (5): Similarly LO 6 (2 g, 2.0 mmol) was reduced with
sodium borohydride (1 equiv, 0.08 g) and iodine (1.5 equiv, 0.39 g)
to give 5 (1.8 g) using the procedure above for LO 2 (1.81 g).
1H NMR (500 MHz, CDCl3, COSY, NOESY): Met1: d=4.69 (1H, m, a),
2.38 (1H, m, b), 1.83 (1H, m, b), 2.47 (2H, m, g), 2.06 (3H, s, eMe),
2.11 (3H, s, eMe minor), 7.38 ppm (1H, m, NH); Leu2: d=4.25 (1H,
m, a), 2.27 (1H, m, b), 1.81 (1H, m, b), 1.92 (1H, m, g), 0.90 (3H, m,
dMe), 0.87 ppm (3H, m, dMe); Val3: d=4.45 (1H, m, a), 2.07 (1H,
m, b), 0.97 (3H, d, 6.4 Hz, gMe), 0.94 (3H, d, 6.4 Hz, gMe), 7.04 ppm
(1H, d, 7.5, NH); Phe4: d=4.29 (1H, m, a), 3.00 (2H, m, b), 7.39–
7.17 (5H, m, d, e, z), 6.86 ppm (1H, brs, NH); Pro5: d=4.31 (1H, m,
a), 1.65 (1H, m, b), 1.52 (1H, m, b), 1.66 (1H, m, g), 1.46 (1H, m, g),
3.37 ppm (2H, m, d); Leu6: d=3.79 (1H, m, a), 2.21 (1H, m, b), 1.83
(1H, m, b), 1.57 (1H, m, g), 0.94 (3H, m, dMe), 0.90 (3H, m, dMe),
7.59 ppm (1H, m, NH); Phe7: d=4.32 (1H, m, a), 3.16 (1H, m, b),
3.02 (1H, m, b), 7.39–7.17 (5H, m, d, e, z), 7.57 ppm (1H, m, NH);
Ile8: d=4.09 (1H, m, a), 1.83 (1H, m, b), 1.57 (2H, m, g), 0.81 (3H,
d, 6.4 Hz, gMe), 0.87 (3H, m, dMe), 6.07 ppm (1H, brs, NH).
13C NMR (125 MHz, CDCl3, HMQC, HMBC): Met1: d=52.4 (a), 29.8
(b), 29.9 (g), 15.5 (eMe), 15.6 (eMe (minor)), 171.1 ppm (C=O); Leu2:
d=63.1 (a), 36.3 (b), 25.3 (g), 23.4 (dMe), 22.1 (dMe), 172.1 ppm
(C=O); Val3: d=57.5 (a), 31.7 (b), 19.4 (gMe), 18.2 (gMe), 171.7 (C=
O); Phe4: d=51.3 (a), 38.8 (b), 135.4 (g), 129.2 (d), 129.2 (e), 127.6
(z), 171.4 ppm (C=O); Pro5: d=58.5 (a), 37.2 (b), 25.2 (g), 46.5 (d),
170.6 ppm (C=O); Leu6: d=55.3 (a), 30.9 (b), 25.1 (g), 23.3 (dMe),
21.4 (dMe), 173.1 ppm (C=O); Phe7: d=53.4 (a), 37.4 (b), 135.8 (g),
129.6 (d), 129.5 (e), 127.9 (z), 174.1 ppm (C=O); Ile8: d=60.3 (a),
35.8 (b), 25.2 (g), 16.3 (gMe), 11.7 (dMe), 172.3 ppm (C=O).
1-2-Aminobutanoic acid LO2 (9): Compound
8
(30 mg,
0.03 mmol) was dissolved in dioxane: H2O (3:1 v/v, 1.2 mL). Pd-C
and LiOH were added to the reaction mixture that was stirred
overnight at 1408C under H2 (1 atm). The reaction mixture was fil-
tered then neutralized with a solution of sat. NaHCO3. The reaction
mixture was extracted with ethyl acetate then concentrated to
obtain the crude product (9). HPLC-MS analyses indicated
a [M+H]+ ion at m/z: 1012.6275, corresponding to a molecular for-
mula of C55H81N9O9.
S4.1-Cyanomethylhomocysteine (S)-oxide LO3 (10): Compound 8
(30 mg, 0.03 mmol) was dissolved in THF/H2O (5:2 v/v, 1.2 mL) and
then cooled to 08C before addition of aqueous solution of NalO4
(0.8 mL, excess). The reaction mixture was stirred overnight and
then filtered. After removal of THF under reduced pressure, EtOAc
was added to the resulting aqueous solution, which was then
washed successively with water and brine. The organic layer was
evaporated to yield a (S)-oxide nitrile 10 (25 mg). HPLC-MS analy-
ses indicated a [M+H]+ ion at m/z: 1099.5958, corresponding to
a molecular formula of C57H83N10O10S. The structure was further
confirmed by LCMS/MS analyses.
S4.1-Cyanomethylhomocysteine (S,S)-dioxide LO4 (11): Compound
8 (9.4 mg, 0.009 mmol) was dissolved in dichloromethane (1 mL) to
which mCPBA (3 equiv, 6.4 mg) was added. The reaction mixture
was stirred at room temperature for 1 h, and was sequentially
washed with a solution of 10% Na2S2O3 (5 mL), saturated aq.
NaHCO3, and H2O to yield sulfonyl nitrile 11 (10 mg). 1H NMR
S4.1-Cyanomethylhomocysteine LO2 (8): Compound 2 (50 mg,
0.05 mmol) was dissolved in N-methylpyrrolidone (2.5 mL) at room
temperature. Diisopropylethylamine (2.5 equiv, 20 mL) and iodoace-
tonitrile (7 equiv, 25 mL) were sequentially added to the solution
and the resulting mixture was heated to 1008C for 20 h. The reac-
tion mixture was extracted with EtOAc and combined organic ex-
tracts were washed with water and subsequently concentrated
under high vacuum (3 mbar) to yield a crude mixture. This crude
mixture was purified using Sep-Pak cartridges and analyzed by
(500 MHz, CDCl3, COSY, NOESY): MetO2 : d=4.85 (1H, m, a), 2.69
1
(1H, m, b), 2.35 (1H, m, b), 3.67 (1H, m, g), 3.53 (1H, m, g), 4.04
(2H, s, SO2CH2), 7.35 ppm (1H, m, NH); Leu2: d=4.02 (1H, m, a),
2.12 (2H, m, b), 1.65 (1H, m, g), 0.95 (3H, d, 6.4 Hz, dMe), 0.93 (3H,
d, 6.4 Hz, dMe), 7.78 ppm (1H, brs, NH); Ile3: d=4.48 (1H, m, a),
1.93 (1H, m, b), 1.70 (2H, m, g), 1.11 (3H, d, 6.4 Hz, gMe), 0.87 (3H,
m, dMe), 8.13 ppm (1H, brs, NH); Pro4: d=3.87 (1H, m, a), 2.27
(1H, m, b), 1.70 (1H, m, b), 2.05 (1H, m, g), 1.96 (1H, m, g), 3.93
(1H, m, d), 3.67 (1H, m, d); Pro5: d=4.12 (1H, m, a), 1.99 (2H, m,
b), 1.47 (1H, m, g), 1.24 (1H, m, g), 3.35 ppm (2H, m, d); Phe6: d=
4.89 (1H, m, a), 3.04 (2H, dd, 4.2, 12.5 Hz, b), 7.26 (3H,m, d, e), 7.16
(2H, m, z), 7.19 ppm (1H, m, NH); Phe7: d=4.07 (1H, m, a), 3.39
(2H, m, b), 7.34 (2H, m, d), 7.26 (1H, m, e), 7.16 (2H, m, z),
7.61 ppm (1H, brs, NH); Val8: d=3.87 (1H, m, a), 2.12 (1H, m, b),
0.95 (3H, m, gMe), 0.94 (3H, m, gMe), 6.47 ppm (1H, brs, NH); Ile9:
d=4.58 (1H, m, a), 1.99 (1H, m, b), 2.16 (2H, m, g), 0.97 (3H, m,
gMe), 0.84 (3H, m, dMe), 6.73 ppm (1H, brs, NH); 13C NMR
1
HPLC. LO nitrile 8 was obtained (0.027 g). H NMR (500 MHz, CDCl3,
COSY, NOESY): Met1: d=4.35 (1H, m, a), 2.41 (1H, m, b), 2.06 (1H,
m, b), 2.86 (1H, m, g), 2.78 (1H, m, g), 3.28 (2H, s, SCH2); Leu2: d=
3.81 (1H, m, a), 1.62 (2H, m, b), 2.16 (1H, m, g), 0.95 (3H, d, 6.4 Hz,
dMe), 0.93 ppm (3H, d, 6.4 Hz, dMe); Ile3: d=4.53 (1H, m, a), 1.92
(1H, m, b), 2.21 (2H, m, g), 1.06 (3H, d, 6.8 Hz, gMe), 0.89 ppm (3H,
t, 7.5, dMe); Pro4: d=3.98 (1H, m, a), 2.22 (1H, m, b), 1.91 (1H, m,
b), 2.03 (1H, m, g), 1.89 (1H, m, g), 4.11 (1H, m, d), 3.74 ppm (1H,
m, d); Pro5: d=4.02 (1H, m, a), 1.93 (2H, m, b), 1.51 (1H, m, g),
0.87 (1H, m, g), 3.34 (1H, m, d), 3.06 ppm (1H, m, d); Phe6: d=4.72
(1H, m, a), 3.16 (1H, m, b), 2.77 (1H, m, b), 7.24 (3H, m, d, e), 7.07
1
(125 MHz, CDCl3, HMQC, HMBC): MetO2 : d=51.7 (a), 25.4 (b), 50.8
(g), 42.5 (SO2CH2), 110.2 (CN), 171.1 ppm (C=O); Leu2: d=61.0 (a),
38.4 (b), 25.4 (g), 22.9 (dMe), 21.9 (dMe), 171.1 (C=O); Ile3: d=56.3
Chem. Eur. J. 2015, 21, 17023 – 17034
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(a), 36.6 (b), 24.8 (g), 15.6 (gMe), 11.2 (dMe), 171.8 (C=O); Pro4: d=
61.8 (a), 28.5 (b), 25.1 (g), 48.1 (d), 170.4 ppm (C=O); Pro5: d=61.0
(a), 32.1 (b), 21.9 (g), 47.4 (d), 173.8 ppm (C=O); Phe6: d=53.4 (a),
35.1 (b), 136.1 (g), 129.7 (d), 128.9 (e), 127.5 (z), 173.8 ppm (C=O);
Phe7: d=57.8 (a), 36.7 (b), 135.4 (g), 129.1 (d), 128.9 (e), 127.3 (z),
175.1 ppm (C=O); Val8: d=59.1 (a), 29.3 (b), 19.6 (gMe), 18.2 (gMe),
173.0 (C=O); Ile9: d=58.3 (a), 36.8 (b), 24.8 (g), 16.1 (gMe), 11.3
(dMe), 171.8 ppm (C=O).
S4.1-(2-Aminoethyl)homocysteine (S,S)-dioxide LO4 (12): Com-
pound 11 (8 mg, 0.007 mmol) was dissolved in acetic acid (0.5 mL)
under argon and PtO2 was added. The reaction mixture was stirred
at rt under H2 (1 atm) overnight. The reaction mixture was filtered
and then neutralized with sat. NaHCO3. The reaction mixture was
extracted with ethyl acetate then concentrated to give the crude
product (12). HPLC-MS analyses indicated a [M+H]+ ion at m/z:
1119.6275, corresponds to a molecular formula of C57H83N10O10S.
S4.1-Ethoxycarbonylmethylhomocysteine LO2 (13): Compound 2
(0.3 g, 0.28 mmol) was dissolved in N-methylpyrrolidone (10 mL) at
room temperature. Diisopropylethylamine (2.5 equiv, 0.12 mL) and
iodoacetate (7 equiv, 0.23 mL) were sequentially added to the solu-
tion and the resulting mixture was heated to 1008C with stirring
for 20 h. The reaction mixture was extracted with EtOAc and the
combined organic extracts were washed with water and subse-
quently concentrated under high vacuum to yield a crude mixture
that was purified using Sep-Pak cartridges and analyzed by HPLC.
The ester (ÀCOOEt) derivative 13 was obtained (0.27 g). 1H NMR
(500 MHz, CDCl3, COSY, NOESY): Met1: d=4.27 (1H, dd, 8, 8.4 Hz,
a), 1.95 (2H, m, b), 2.63 (1H, m, g), 2.44 (1H, m, g), 3.19 (2H, s,
SCH2), 4.16 (2H, q, 7 Hz, SCH2COOCH2), 1.26 (3H, t, 7 Hz, S-
CH2COOCH2CH3), 6.44 ppm (1H, brs, NH); Leu2: d=3.61 (1H, m, a),
2.17 (2H, m, b), 1.98 (1H, m, g), 0.93 (3H, d, 7 Hz, dMe), 0.91 (3H,
d, 7 Hz, dMe); Ile3: d=4.42 (1H, m, a), 1.91 (1H, m, b), 2.22 (2H, m,
g), 1.00 (3H, d, 6.6 Hz, gMe), 0.83 (3H, t, 7 Hz, dMe), 7.44 ppm (1H,
brs, NH); Pro4: d=4.03 (1H, brs, a), 2.22 (1H, m, b), 1.91 (1H, m,
b), 2.02 (1H, m, g), 1.87 (1H, m, g), 4.16 (1H, brs, d), 3.71 (1H, m,
d); Pro5: d=3.95 (1H, m, a), 1.95 (2H, m, b), 1.52 (1H, m, g), 0.85
(1H, m, g), 3.33 (1H, m, d), 2.98 ppm (1H, m, d); Phe6: d=4.71 (1H,
m, a), 3.21 (1H, m, b), 2.77 (1H, m, b), 7.25 (2H, m, d), 7.19 (1H, m,
e), 7.09 (2H, m, z) 7.45 ppm (1H, brs, NH); Phe7: d=4.03 (1H, m,
a), 3.36 (2H, m, b), 7.25 (2H, m, d), 7.19 (1H, m, e), 7.09 (2H, m, z);;
Val8: d=3.87 (1H, m, a), 2.22 (1H, m, b), 1.05 (3H, d, 7.3 Hz, gMe),
1.03 (3H, d, 7.3 Hz, gMe), 8.09 ppm (1H, brs, NH); Ile9: d=4.16 (1H,
m, a), 2.10 (1H, m, b), 1.61 (2H, m, g), 0.94 (3H, d, 7 Hz, gMe), 0.87
(3H, t, 7 Hz, dMe), 6.79 ppm (1H, brs, NH).
13C NMR (125 MHz, CDCl3, HMQC, HMBC): Met1: d=58.1 (a), 25.4
(b), 29.8 (g), 33.7 (SCH2), 60.9 (SCH2COOCH2), 14.3
(SCH2COOCH2CH3), 169.8 (COOCH2CH3), 170.7 ppm (C=O); Leu2: d=
58.5 (a), 28.8 (b), 25.1 (g), 23.5 (dMe), 21.3 (dMe), 171.2 ppm (C=O);
Ile3: d=56.1 (a), 37.1 (b), 25.3 (g), 16.1 (gMe), 11.1 (dMe), 171.5 (C=
O); Pro4: d=54.6 (a), 37.4 (b), 24.7 (g), 48.3 (d), 170.6 ppm (C=O);
Pro5: d=61.5 (a), 31.5 (b), 21.5 (g), 46.8 (d), 174.8 ppm (C=O); Phe6:
d=55.7 (a), 37.7 (b), 137.2 (g), 129.6 (d), 128.9 (e), 127.4 (z),
172.4 ppm (C=O); Phe7: d=61.5 (a), 35.7 (b), 136.4 (g), 129.0 (d),
128.8 (e), 126.7 (z), 175.1 ppm (C=O); Val8: d=62.4 (a), 29.5 (b),
19.6 (gMe), 18.3 (gMe), 172.4 ppm (C=O); Ile9: d=53.7 (a), 34.9 (b),
25.3 (g), 15.2 (gMe), 10.7 (dMe), 172.0 ppm (C=O).
SCH2COOCH2CH3), 7.42 ppm (1H, d, 9, NH); Leu2: d=4.31 (1H, m,
a), 2.30 (1H, m, b), 1.75 (1H, m, b), 1.95 (1H, m, g), 0.86 ppm (6H,
d, 6.6 Hz, dMe); Val3: d=4.46 (1H, m, a), 2.06 (1H, m, b), 0.96 (3H,
d, 6.4 Hz, gMe), 0.94 (3H, d, 6.4 Hz, gMe), 7.03 ppm (1H, d, 8.6,
NH); Phe4: d=4.32 (1H, m, a), 3.01 (2H, m, b), 7.38–7.15 (5H, m, d,
e, z), 6.72 (1H, brs, NH); Pro5: d=4.31 (1H, m, a), 1.57 (1H, m, b),
1.45 (1H, m, b), 1.64 (1H, m, g), 1.50 (1H, m, g), 3.37 ppm (2H, m,
d); Leu6: d=3.79 (1H, m, a), 2.22 (1H, m, b), 1.84 (1H, m, b), 1.57
(1H, m, g), 0.87 (6H, m, dMe), 7.53 ppm (1H, d, 7 Hz, NH); Phe7:
d=4.30 (1H, m, a), 3.16 (1H, m, b), 3.04 (1H, m, b), 7.38–7.15 (5H,
m, d, e, z), 7.59 ppm (1H, brs, NH); Ile8: d=4.11 (1H, m, a), 1.84
(1H, m, b), 1.57 (2H, m, g), 0.80 (3H, d, 6.4 Hz, gMe), 0.87 (3H, m,
dMe), 6.08 ppm (1H, brs, NH); 13C NMR (125 MHz, CDCl3, HMQC,
HMBC): Met1: d=52.2 (a), 31.1 (b), 29.6 (g), 33.8 (SCH2), 61.4
(SCH2COOCH2), 14.3 (SCH2COOCH2CH3), 170.5 (S-CH2COOCH2CH3),
171.1 (C=O); Leu2: d=51.3 (a), 29.9 (b), 30.8 (g), 23.2 (dMe), 22.1
(dMe), 173.1 ppm (C=O); Val3: d=57.4 (a), 31.6 (b), 19.4 (gMe), 18.1
(gMe), 171.7 ppm (C=O); Phe4: d=53.3 (a), 38.8 (b), 135.4 (g), 129.2
(d), 129.1 (e), 127.6 (z), 171.3 (C=O); Pro5: d=63.2 (a), 38.2 (b), 25.3
(g), 46.5 (d), 170.8 ppm (C=O); Leu6: d=55.3 (a), 37.2 (b), 25.1 (g),
22.8 (dMe), 21.9 (dMe), 173.1 ppm (C=O); Phe7: d=58.4 (a), 37.4
(b), 135.9 (g), 129.6 (d), 129.5 (e), 127.9 (z), 174.1 ppm (C=O); Ile8:
d=60.2 (a), 35.8 (b), 25.3 (g), 16.3 (gMe), 11.7 (dMe), 172.3 ppm
(C=O).
S4.1-Ethoxycarbonylmethylhomocysteine (S,S)-dioxide LO4 (14):
Compound 13 (0.15 g, 0.13 mmol) was dissolved in dichlorome-
thane (5 mL) to which mCPBA (3 equiv, 0.1 g) was added. The reac-
tion mixture was stirred at room temperature for 1 h, and then se-
quentially washed with solutions of 10% Na2S2O3 (5 mL), saturated
aq. NaHCO3, and H2O to yield methionine (S,S)-dioxide 14 (0.16 g).
1
1H NMR (500 MHz, CDCl3, COSY, NOESY): MetO2 : d=4.61 (1H, m,
a), 2.46 (2H, m, b), 3.46 (2H, m, g), 3.95 (2H, s, SO2CH2), 4.23 (2H,
q, 7 Hz, SO2CH2COOCH2), 1.29 (3H, t, 7 Hz, SO2CH2COOCH2CH3),
7.20 ppm (1H, d, 7, NH); Leu2: d=4.06 (1H, d, 7.9 Hz, a), 2.16 (2H,
m, b), 1.63 (1H, m, g), 0.95 (3H, d, 6.4 Hz, dMe), 0.93 (3H, d, 6.4 Hz,
dMe), 7.63 ppm (1H, d, 6 Hz, NH); Ile3: d=4.48 (1H, m, a), 1.93
(1H, m, b), 1.50 (2H, m, g), 1.07 (3H, d, 6.8 Hz, gMe), 0.84 (3H, t,
6.8 Hz, dMe), 6.60 (1H, brs, NH); Pro4: d=3.81 (1H, m, a), 2.16 (1H,
m, b), 1.67 (1H, m, b), 2.17 (1H, m, g), 1.95 (1H, m, g), 4.01 (1H, m,
d), 3.46 ppm (1H, m, d); Pro5: d=4.06 (1H, m, a), 2.16 (1H, m, b),
1.94 (1H, m, b), 1.49 (1H, m, g), 0.96 (1H, m, g), 3.29 ppm (2H, m,
d); Phe6: d=4.85 (1H, m, a), 3.09 (2H, dd, 4.2, 12.5 Hz, b), 7.24 (2H,
m, d), 7.20 (1H, m, e), 7.12 (2H, m, z); Phe7: d=3.99 (1H, m, a),
3.67 (2H, m, b), 7.30 (3H, m, d, e), 7.12 (2H, m, z) 7.72 ppm (1H,
brs, NH); Val8: d=3.87 (1H, m, a), 2.16 (1H, m, b), 0.95 (3H, d,
6.4 Hz, gMe), 0.93 (3H, d, 6.4 Hz, gMe); Ile9: d=4.48 (1H, m, a),
1.95 (1H, m, b), 2.16 (2H, m, g), 0.92 (3H, d, 6.4 Hz, gMe), 0.90 ppm
(3H, t, 6.4 Hz, dMe); 13C NMR (125 MHz, CDCl3, HMQC, HMBC):
1
MetO2 : d=52.4 (a), 25.3 (b), 51.2 (g), 57.6 (SO2CH2), 62.7
(SO2CH2COOCH2), 14.1 (SO2CH2COOCH2CH3), 162.8 (COOCH2CH3),
170.6 ppm (C=O); Leu2: d=61.0 (a), 37.9 (b), 25.4 (g), 23.2 (dMe),
21.7 (dMe), 171.4 (C=O); Ile3: d=56.3 (a), 36.2 (b), 24.8 (g), 15.6
(gMe), 11.1 (dMe), 171.7 ppm (C=O); Pro4: d=62.0 (a), 28.6 (b), 25.1
(g), 48.1 (d), 170.5 ppm (C=O); Pro5: d=56.6 (a), 31.9 (b), 21.7 (g),
47.1 (d), 174.1 ppm (C=O); Phe6: d=54.2 (a), 36.1 (b), 136.4 (g),
129.8 (d), 129.0 (e), 127.4 (z), 173.0 ppm (C=O); Phe7: d=58.3 (a),
36.2 (b), 136.3 (g), 128.9 (d), 127.3 (e), 127.3 (z), 175.2 ppm (C=O);
Val8: d=58.8 (a), 29.4 (b), 19.6 (gMe), 18.3 (gMe), 172.8 ppm (C=O);
Ile9: d=58.3 (a), 36.7 (b), 25.0 (g), 16.1 (gMe), 11.3 (dMe),
171.8 ppm (C=O).
S4.1-Ethoxycarbonylmethylhomocysteine LO1 (16): Similarly
5
(1 g, 1.0 mmol) was activated with iodoacetate (7 equiv, 1.55 g,
0.21 mL) and diisopropylethylamine (2.5 equiv, 0.36 g, 0.45 mL) in
N-methylpyrrolidone (8 mL) using the above procedure to give 16
1
(1.1 g). H NMR (500 MHz, CDCl3, COSY, NOESY): Met1: d=4.69 (1H,
S4.1-Ethoxycarbonylmethylhomocysteine (S,S)-dioxide LO7 (17):
Similarly 16 (0.7 g, 0.68 mmol) was oxidized with mCPBA (3 equiv,
0.35 g) in dichloromethane (15 mL) using the above procedure to
m, a), 2.45 (1H, m, b), 1.84 (1H, m, b), 2.62 (2H, m, g), 3.19 (2H, s,
SCH2), 4.14 (2H, q, 7 Hz, SCH2COOCH2), 1.25 (3H, t, 7 Hz,
Chem. Eur. J. 2015, 21, 17023 – 17034
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17031
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

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1
give 17 (0.74 g). 1H NMR (500 MHz, CDCl3, COSY, NOESY): MetO2 :
S4.1-[2-(2-Aminoethylamino)-2-oxo-ethyl]homocysteine (S,S)-diox-
ide LO7 (18): Similarly 17 (0.3 g, 0.29 mmol) was coupled with eth-
ylene diamine (excess) in methanol (6 mL) to give 18 (0.27 g) using
the procedure described above. 1H NMR (500 MHz, CDCl3, COSY,
d=4.71 (1H, m, a), 2.58 (1H, m, b), 2.18 (1H, m, b), 3.52 (1H, m, g),
3.17 (1H, m, g), 3.92 (2H, dd, 14.6, 14.6, SO2CH2), 4.23 (2H, q, 7 Hz,
SO2CH2COOCH2), 1.29 (3H, t, 7 Hz, SO2CH2COOCH2CH3), 7.69 ppm
(1H, brs, NH); Leu2: d=4.33 (1H, m, a), 2.32 (1H, m, b), 1.72 (1H,
m, b), 1.95 (1H, m, g), 0.88 (3H, m, dMe), 0.81 (3H, m, dMe), 6.83
(1H, brs, NH); Val3: d=4.47 (1H, m, a), 2.05 (1H, m, b), 0.97 (3H, d,
6.8 Hz, gMe), 0.94 (3H, d, 6.8 Hz, gMe), 6.97 ppm (1H, brs, NH);
Phe4: d=4.34 (1H, m, a), 3.02 (2H, m, b), 7.39–7.18 (5H, m, d, e, z),
6.66 ppm (1H, brs, NH); Pro5: d=4.12 (1H, m, a), 1.72 (1H, m, b),
1.42 (1H, m, b), 1.65 (1H, m, g), 1.47 (1H, m, g), 3.36 ppm (2H, m,
d); Leu6: d=3.79 (1H, m, a), 2.18 (1H, m, b), 1.86 (1H, m, b), 1.54
(1H, m, g), 0.94 (3H, d, 6.8 Hz, dMe), 0.88 (3H, m, dMe), 7.61 ppm
(1H, d, 7 Hz, NH); Phe7: d=4.31 (1H, m, a), 3.17 (1H, m, b), 3.07
(1H, m, b), 7.39–7.18 (5H, m, d, e, z), 7.69 ppm (1H, brs, NH); Ile8:
d=4.07 (1H, m, a), 1.86 (1H, m, b), 1.54 (2H, m, g), 0.79 (3H, m,
gMe), 0.86 (3H, m, dMe), 6.12 ppm (1H, brs, NH); 13C NMR
1
NOESY) MetO2 : d=4.92 (1H, m, a), 2.58 (1H, m, b), 2.20 (1H, m,
b), 3.43 (2H, m, g), 3.41 (2H, m, SO2CH2), 3.06 (2H, m,
NHCH2CH2NH2), 3.43 (2H, m, NHCH2CH2 NH2), 4.36 (2H, brs,
NHCH2CH2NH2), 6.81 ppm (1H, m, NH); Leu2: d=4.01 (1H, m, a),
1.74 (1H, m, b), 1.65 (1H, m, b), 2.38 (1H, m, g), 0.84 (3H, d, 6.6 Hz,
dMe), 0.78 (3H, d, 6.6 Hz, dMe), 8.97 ppm (1H, brs, NH); Val3: d=
4.19 (1H, m, a), 2.38 (1H, m, b), 0.95 (6H, m, gMe), 6.49 ppm (1H,
brs, NH); Phe4: d=4.81 (1H, m, a), 3.22 (1H, m, b), 3.12 (1H, m, b),
7.25–7.06 (5H, m, d, e, z), 7.02 ppm (1H, m, NH); Pro5: d=4.47 (1H,
m, a), 2.38 (1H, m, b), 1.75 (1H, m, b), 2.04 (1H, m, g), 1.95 (1H, m,
g), 3.73 (1H, m, d), 3.52 ppm (1H, m, d); Leu6: d=3.45 (1H, m, a),
2.05 (1H, m, b), 1.95 (1H, m, b), 1.75 (1H, m, g), 0.92 (6H, m, dMe),
8.11 ppm (1H, m, NH); Phe7: d=5.24 (1H, m, a), 3.07 (2H, m, b),
7.25–7.06 (5H, m, d, e, z), 7.52 ppm (1H, brs, NH); Ile8: d=4.08
(1H, m, a), 1.28 (1H, m, b), 1.46 (1H, m, g), 1.41 (1H, m, g), 1.04
(3H, m, gMe), 0.91 ppm (3H, m, dMe); 13C NMR (125 MHz, CDCl3,
1
(125 MHz, CDCl3, HMQC, HMBC): MetO2 : d=51.3 (a), 24.8 (b), 51.4
(g), 57.5 (SO2CH2), 62.7 (SO2CH2COOCH2), 14.1 (SO2CH2COOCH2CH3),
162.9 (SO2CH2COOCH2CH3), 170.9 ppm (C=O); Leu2: d=51.2 (a),
37.0 (b), 25.4 (g), 23.3 (dMe), 21.8 (dMe), 171.6 (C=O); Val3: d=57.5
(a), 32.0 (b), 19.4 (gMe), 18.2 (gMe), 171.6 ppm (C=O); Phe4: d=
53.1 (a), 38.9 (b), 135.5 (g), 129.2 (d), 129.1 (e), 127.6 (z), 171.2 ppm
(C=O); Pro5: d=54.2 (a), 31.0 (b), 25.2 (g), 46.5 (d), 170.4 ppm (C=
O); Leu6: d=55.3 (a), 29.9 (b), 29.3 (g), 23.0 (dMe), 21.4 (dMe),
173.4 ppm (C=O); Phe7: d=58.4 (a), 37.3 (b), 135.6 (g), 129.6 (d),
129.5 (e), 127.9 (z), 174.6 ppm (C=O); Ile8: d=60.2 (a), 35.7 (b), 25.0
(g), 16.3 (gMe), 11.7 (dMe), 172.5 ppm (C=O).
1
HMQC, HMBC): MetO2 : d=50.3 (a), 25.0 (b), 50.8 (g), 60.4 (SO2CH2),
40.4
(NHCH2CH2NH2),
40.7
(NHCH2CH2NH2),
163.1
(CONHCH2CH2NH2), 170.5 ppm (C=O); Leu2: d=56.4 (a), 39.8 (b),
29.6 (g), 23.0 (dMe), 22.3 (dMe), 172.9 ppm (C=O); Val3: d=59.7 (a),
30.5 (b), 19.6 (gMe), 17.6 (gMe), 171.8 ppm (C=O); Phe4: d=54.5
(a), 36.7 (b), 138.0 (g), 129.0 (d), 128.7 (e), 126.7 (z), 172.9 ppm (C=
O); Pro5: d=63.7 (a), 29.8 (b), 25.0 (g), 48.4 (d), 173.9 ppm (C=O);
Leu6: d=59.7 (a), 25.6 (b), 25.0 (g), 22.5 (dMe), 22.0 (dMe),
173.4 ppm (C=O); Phe7: d=50.8 (a), 35.9 (b), 137.3 (g), 128.8 (d),
128.3 (e), 126.6 (z), 172.3 ppm (C=O); Ile8: d=54.5 (a), 25.3 (b), 39.8
(g), 17.6 (gMe), 10.2 (dMe), 172.3 ppm (C=O).
S4.1-[2-(2-Aminoethylamino)-2-oxo-ethyl]homocysteine (S,S)-diox-
ide LO4 (15): Compound 14 (0.1 g, 0.09 mmol) was dissolved in
methanol and ethylene diamine (excess) was added. The resulting
reaction mixture was refluxed overnight. The organic solvents were
evaporated and the resulting crude product was purified using
Sep-Pak cartridges and analyzed by HPLC. LO 15 was obtained
7-Methoxycoumarin-3-carboxylic succinimidyl ester: 7-Methoxy-
coumarin-3-carboxylic succinimidyl ester was prepared as previous-
ly described.[33] 7-Methoxycoumarin-3-carboxylic acid (0.22 g,
1 mmole) was dissolved in DMF (2.5 mL) and N-hydroxy succini-
mide ester (1 equiv, 0.12 g) was added. The reaction mixture was
cooled to 08C, DCC (1.1 equiv, 0.23 g) was added and the mixture
was stirred for 30 min at 08C and then warmed to RT and stirred
for an additional 2 h. The solution was filtered to remove DCC and
a solvent mixture of isopropanol/hexane (1:20) was added to the
1
(0.09 g). 1H NMR (500 MHz, CDCl3, COSY, NOESY): MetO2 : d=4.72
(1H, m, a), 2.50 (1H, m, b), 2.40 (1H, m, b), 3.48 (1H, m, g), 3.41
(1H, m, g), 3.90 (2H, s, SO2CH2), 2.87 (4H, m, NHCH2CH2NH2),
7.41 ppm (1H, brs, NH); Leu2: d=3.88 (1H, brs, a), 2.17 (2H, brs,
b), 1.64 (1H, m, g), 0.92 (6H, d, 6.6 Hz, dMe), 7.75 ppm (1H, brs,
NH); Ile3: d=4.47 (1H, m, a), 1.92 (1H, m, b), 1.50 (2H, m, g), 1.08
(3H, d, 6.6 Hz, gMe), 0.86 (3H, t, 7 Hz, dMe), 8.03 ppm (1H, brs,
NH); Pro4: d=3.79 (1H, brs, a), 2.17 (1H, m, b), 1.64 (1H, m, b),
2.04 (1H, m, g), 1.94 (1H, m, g), 3.97 (1H, brs, d), 3.65 ppm (1H, m,
d); Pro5: d=4.11 (1H, m, a), 2.12 (1H, m, b), 1.95 (1H, m, b), 1.50
(1H, m, g), 0.96 (1H, m, g), 3.31 ppm (2H, m, d); Phe6: d=4.86 (1H,
m, a), 3.10 (1H, m, b), 3.00 (1H, m, b), 7.24 (3H, m, d, e), 7.15 ppm
(2H, m, z); Phe7: d=3.97 (1H, m, a), 3.31 (2H, m, b), 7.32 (2H, m,
d), 7.24 (1H, m, e), 7.15 (2H, m, z) 7.67 ppm (1H, brs, NH); Val8:
d=3.88 (1H, m, a), 2.17 (1H, m, b), 0.92 ppm (6H, d, 6.6 Hz, gMe);
Ile9: d=4.53 (1H, m, a), 1.98 (1H, m, b), 1.64 (2H, m, g), 0.96 (3H,
d, 6.4 Hz, gMe), 0.84 (3H, t, 6.4 Hz, dMe), 6.78 ppm (1H, brs, NH);
1
filtrate to give product (0.27 g). H NMR (500 MHz, CDCl3): d=8.76
(1H, s), 7.57 (1H, d), 6.95 (1H, d, J=9.0 Hz), 6.85 (1H, s), 3.95 (3H,
t), 2.90 ppm (4H, s).
LO coumarin complex 19: A mixture of LO 15 (0.2 g, 0.05 mmol)
and 7-methoxycoumarin-3-carboxylic succinimidyl ester (1.5 equiv,
0.08 g) was dissolved in a solution of 30% methanol/acetonitrile
and triethylamine (1 equiv, 0.02 g) was added. The reaction mixture
was stirred at room temperature for 2 h. The organic solvents were
evaporated and the crude product was purified using preparative-
1
HPLC to give 19 (0.24 g). H NMR (500 MHz, CDCl3, COSY, NOESY):
1
MetO2 : d=4.76 (1H, m, a), 2.51 (1H, m, b), 2.39 (1H, m, b), 3.40
1
13C NMR (125 MHz, CDCl3, HMQC, HMBC): MetO2 : d=57.2 (a), 25.1
(2H, m, g), 3.87 (2H, s, SO2CH2), 2.87 (4H, m, NHCH2CH2NH), 7.79
(b), 50.7 (g), 58.6 (SO2CH2), 42.9 (NHCH2CH2NH2), 41.2
(NHCH2CH2NH2), 171.9 ppm (C=O); Leu2: d=61.8 (a), 29.9 (b), 25.5
(g), 23.1 (dMe), 21.9 (dMe), 171.9 (C=O); Ile3: d=56.3 (a), 36.7 (b),
25.4 (g), 15.6 (gMe), 11.2 (dMe), 171.9 ppm (C=O); Pro4: d=59.1 (a),
28.7 (b), 25.1 (g), 48.2 (d), 170.9 ppm (C=O); Pro5: d=61.2 (a), 32.0
(b), 21.7 (g), 47.3 (d), 173.6 ppm (C=O); Phe6: d=54.2 (a), 35.6 (b),
136.4 (g), 129.8 (d), 129.0 (e), 127.4 (z), 173.6 ppm (C=O); Phe7: d=
61.2 (a), 38.4 (b), 135.9 (g), 129.1 (d), 128.9 (e), 127.3 (z), 175.2 ppm
(C=O); Val8: d=59.1 (a), 29.4 (b), 19.7 (gMe), 18.3 (gMe), 173.2 ppm
(C=O); Ile9: d=58.3 (a), 36.7 (b), 25.4 (g), 16.1 (gMe), 11.3 (dMe),
173.0 ppm (C=O).
(1H,
brs,
NHCH2CH2NHCoumarin),
8.90
(1H,
m,
NHCH2CH2NHCoumarin), 3.91 (3H, s, OCH3), 8.79 (1H, s), 7.57 (1H,
d, 8.6 Hz), 6.92 (1H, d, 8.6 Hz), 6.85 (1H, s), 7.31 ppm (1H, m, NH);
Leu2: d=3.83 (1H, m, a), 1.93 (2H, m, b), 1.62 (1H, m, g), 0.92 ppm
(6H, d, 7 Hz, dMe); Ile3: d=4.47 (1H, m, a), 1.90 (1H, m, b), 1.49
(2H, m, g), 1.07 (3H, d, 6.8 Hz, gMe), 0.82 ppm (3H, t, 6.8 Hz, dMe);
Pro4: d=3.78 (1H, brs, a), 2.15 (1H, m, b), 1.64 (1H, m, b), 1.98
(1H, m, g), 1.94 (1H, m, g), 3.86 (1H, brs, d), 3.61 ppm (1H, m, d);
Pro5: d=4.11 (1H, m, a), 1.93 (2H, m, b), 1.49 (1H, m, g), 0.95 (1H,
m, g), 3.31 ppm (2H, m, d); Phe6: d=4.86 (1H, m, a), 3.08 (1H, m,
b), 2.98 (1H, m, b), 7.25 (3H, m, d, e), 7.14 ppm (2H, m, z); Phe7:
Chem. Eur. J. 2015, 21, 17023 – 17034
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ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
d=3.92 (1H, m, a), 3.31 (2H, m, b), 7.30 (2H, m, d), 7.25 (1H, m, e),
7.14 (2H, m, z) 7.71 ppm (1H, brs, NH); Val8: d=3.86 (1H, m, a),
2.16 (1H, m, b), 0.90 ppm (6H, d, 7 Hz, gMe); Ile9: d=4.55 (1H, m,
a), 1.98 (1H, m, b), 1.51 (2H, m, g), 0.95 (3H, d, 6.4 Hz, gMe), 0.84
(3H, t, 6.4 Hz, dMe), 6.78 ppm (1H, brs, NH); 13C NMR (125 MHz,
complex was confirmed by an HPLC-MS peak at 764.8 [M+Eu]2+
corresponding to a molecular formula, C70H94N11O16SEu of the Eu
complex and C70H97N11O16SCl2Eu [M+EuCl2]+: 1600.5.
1
CDCl3, HMQC, HMBC): MetO2 : d=52.1 (a), 25. 5 (b), 50.6 (g), 58.4
Acknowledgements
(SO2CH2), 40.5 (NHCH2CH2NHCoumarin), 39.5 (NHCH2CH2NH2), 56.2
(OCH3), 165.3, 163.2, 161.8, 161.2, 157.0, 148.6, 131.3, 114.9, 114.2,
112.6, 100.6, 171.0 ppm (C=O); Leu2: d=61.8 (a), 38.4 (b), 25.8 (g),
23.1 (dMe), 21.9 (dMe), 171.7 ppm (C=O); Ile3: d=56.3 (a), 36.6 (b),
25.1 (g), 15.6 (gMe), 11.1 (dMe), 171.9 ppm (C=O); Pro4: d=56.2 (a),
28.6 (b), 25.5 (g), 48.1 (d), 170.9 ppm (C=O); Pro5: d=61.1 (a), 32.0
(b), 21.7 (g), 47.3 (d), 173.7 (C=O); Phe6: d=54.1 (a), 35.5 (b), 136.3
(g), 129.7 (d), 129.0 (e), 127.3 (z), 173.2 ppm (C=O); Phe7: d=57.3
(a), 36.9 (b), 135.9 (g), 129.1 (d), 128.9 (e), 127.3 (z), 175.4 ppm (C=
O); Val8: d=56.2 (a), 29.4 (b), 19.7 (gMe), 18.3 (gMe), 173.2 ppm
(C=O); Ile9: d=59.1 (a), 36.8 (b), 25.1 (g), 16.1 (gMe), 11.3 (dMe),
173.0 ppm (C=O).
The authors thank Dr. C. Eskiw (University of Saskatchewan) for
preparing and analyzing FRET samples in the cell culture. Fi-
nancial support was provided by Genome Canada, TUFGEN
(Total Utilization Flax Genomics), and the Agricultural Develop-
ment Fund from the Saskatchewan Ministry of Agriculture.
Keywords: alkylation · FRET · lanthanides · methionine ·
natural products · peptides
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LO coumarin complex 20: Similarly 18 (0.2 g, 0.18 mmol) was cou-
pled with 7-methoxycoumarin-3-carboxylic succinimidyl ester
(1.5 equiv, 0.09 g) in the presence of triethylamine (1 equiv,
0.19 mg, 0.025 mL) in a solution of 30% methanol/acetonitrile to
1
give 20 (0.25 g) by using the above procedure. H NMR (500 MHz,
1
CDCl3, COSY, NOESY): MetO2 : d=4.63 (1H, m, a), 2.52 (1H, m, b),
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2.17 (1H, m, b), 3.29 (1H, m, g), 3.13 (1H, m, g), 3.72 (2H, s,
SO2CH2), 3.60 (2H, m, NHCH2CH2NHCoumarin), 3.49 (2H, m,
NHCH2CH2NHCoumarin), 8.76 (1H, brs, NHCH2CH2NHCoumarin),
8.96 (1H, m, NHCH2CH2NHCoumarin), 3.87 (3H, s, OCH3), 8.81 (1H,
s), 7.63 (1H, d, 9 Hz), 6.86 (1H, d, 9 Hz), 6.80 (1H, s), 7.74 ppm (1H,
m, NH); Leu2: d=4.33 (1H, m, a), 2.33 (1H, m, b), 1.54 (1H, m, b),
1.84 (1H, m, g), 0.83 ppm (6H, m, dMe); Val3: d=4.42 (1H, m, a),
2.17 (1H, m, b), 1.02 (6H, m, gMe), 7.56 ppm (1H, brs, NH); Phe4:
d=4.42 (1H, m, a), 3.13 (1H, m, b), 3.06 (1H, m, b), 7.30–7.10 ppm
(5H, m, d, e, z); Pro5: d=4.12 (1H, m, a), 1.46 (2H, m, b), 1.59 (1H,
m, g), 1.46 (1H, m, g), 3.29 ppm (2H, m, d); Leu6: d=3.60 (1H, m,
a), 1.70 (2H, m, b), 1.39 (1H, m, g), 0.93 (6H, m, dMe), 7.95 ppm
(1H, d, 7.5, NH); Phe7: d=4.97 (1H, m, a), 3.02 (2H, m, b), 7.30–
7.10 ppm (5H, m, d, e, z); Ile8: d=3.90 (1H, m, a), 1.76 (1H, m, b),
1.60 (2H, m, g), 0.83 (3H, m, gMe), 0.93 (3H, m, dMe), 7.67 ppm
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1
(1H, brs, NH); 13C NMR (125 MHz, CDCl3, HMQC, HMBC): MetO2 :
d=52.0 (a), 25.4 (b), 50.7 (g), 48.2 (SO2CH2), 40.3
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(g), 22.7 (dMe), 21.6 (dMe), 171.3 ppm (C=O); Phe7: d=53.5 (a),
37.6 (b), 137.7 (g), 129.0 (d), 128.4 (e), 126.5 (z), 171.9 ppm (C=O);
Ile8: d=58.7 (a), 35.9 (b), 25.2 (g), 16.1 (gMe), 11.5 (dMe),
171.3 ppm (C=O).
Tb complex of 19 (21): Compound 19 was co-dissolved with one
equivalent of TbCl3 in methanol and then refluxed overnight. Final-
ly, the solvent was removed to obtain Tb complex (21). Putative Tb
complex was confirmed by HPLC-MS. HPLC-MS analysis showed
a peak at 767.8 [M+Tb]2+ corresponding to a molecular formula,
C70H95N11O16STb of the Tb complex. Other compounds identified
were C70H97N11O16SCl2Tb [M+TbCl2]+: 1608.5; C70H95N11O16SNa
[M+Na]+: 1400.7
Eu complex of 19 (22): Compound 19 was co-dissolved with one
equivalent of EuCl3 in methanol and then refluxed overnight. Final-
ly, solvent was removed to obtain Eu complex (22). Putative Eu
Chem. Eur. J. 2015, 21, 17023 – 17034
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Chem. Eur. J. 2015, 21, 17023 – 17034
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17034
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