Biomimetic studies on the mechanism of stereoselective lanthionine formation

Article abstract of DOI:10.1039/b304945k

Selenocysteine derivatives are useful precursors for the synthesis of peptide conjugates and selenopeptides. Several diastereomers of Fmoc-3-methyl-Se-phenylselenocysteine (FmocMeSec(Ph)) were prepared and used in solid phase peptide synthesis (SPPS). Once incorporated into peptides, the phenylselenide functionality provides a useful handle for the site and stereospecific introduction of E- or Z-dehydrobutyrine residues into peptide chains via oxidative elimination. The oxidation conditions are mild, can be performed on a solid support, and tolerate functionalities commonly found in peptides, including variously protected cysteine residues. Dehydropeptides containing unprotected cysteine residues undergo intramolecular stereoselective conjugate addition to afford cyclic lanthionines and methyllanthionines, which have the same stereochemistry as found in lantibiotics, a family of ribosomally synthesized and post-translationally modified peptide antibiotics. The observed stereoselectivity is shown to originate from a kinetic rather than a thermodynamic preference.

Full text of DOI:10.1039/b304945k

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Biomimetic studies on the mechanism of stereoselective lanthionine
formation†
Yantao Zhu, Matt D. Gieselman, Hao Zhou, Olga Averin and Wilfred A. van der Donk*
Department of Chemistry, University of Illinois at Urbana-Champaign, 600 S. Mathews Ave,
Urbana, Illinois 61801, USA
Received 12th May 2003, Accepted 15th August 2003
First published as an Advance Article on the web 10th September 2003
Selenocysteine derivatives are useful precursors for the synthesis of peptide conjugates and selenopeptides. Several
diastereomers of Fmoc-3-methyl-Se-phenylselenocysteine (FmocMeSec(Ph)) were prepared and used in solid phase
peptide synthesis (SPPS). Once incorporated into peptides, the phenylselenide functionality provides a useful handle
for the site and stereospecific introduction of E- or Z-dehydrobutyrine residues into peptide chains via oxidative
elimination. The oxidation conditions are mild, can be performed on a solid support, and tolerate functionalities
commonly found in peptides, including variously protected cysteine residues. Dehydropeptides containing
unprotected cysteine residues undergo intramolecular stereoselective conjugate addition to afford cyclic lanthionines
and methyllanthionines, which have the same stereochemistry as found in lantibiotics, a family of ribosomally
synthesized and post-translationally modified peptide antibiotics. The observed stereoselectivity is shown to originate
from a kinetic rather than a thermodynamic preference.
Introduction
Lantibiotics are a class of post-translationally modified pep-
tides that belong to the family of bacteriocins. They possess
high antimicrobial activity against Gram-positive bacteria
and have been used for a variety of applications including food
preservation.1–3 Among the family members is the promising
antibacterial agent nisin that interacts with lipid II,4 the physio-
logical target of vancomycin. The proposed biosynthetic path-
way for lantibiotics is shown for nisin in Scheme 1. The pre-
cursor peptides that are the substrates for post-translational
modification consist of an N-terminal leader sequence that
remains unchanged during maturation (R1, Scheme 1) and a C-
terminal propeptide that undergoes site-specific dehydration
of serine and threonine residues catalyzed by a putative
dehydratase to give dehydroalanine (Dha) and Z-dehydro-
butyrine (Dhb) residues, respectively.1,5–8 Subsequent Michael
additions of cysteines to the Dha and Dhb residues catalyzed
by a putative cyclase in a regio- and stereoselective manner
produce thioethers with the -configuration at the newly
created stereogenic centers. The lantibiotics derive their name
from these cyclic structures, which are called lanthionines
when originating from Ser (e.g. ring A in nisin) and methyl-
lanthionines when formed from Thr (e.g. rings B–E, Scheme 1).
In the final step of maturation, the leader sequence is removed
by LanP-catalyzed proteolysis and the lantibiotic is released.9,10
We have been interested in devising chemical methods for
the incorporation of 1,2-didehydroamino acids into peptides
through the use of selenocysteine derivatives11 and have investi-
Scheme 1
gated the stereochemistry of non-enzymatic intramolecular
Michael additions that produce lanthionines and methyl-
lanthionines.12,13 These studies as well as those in other
laboratories14–16 have shown that biomimetic intramolecular
conjugate additions produce cyclic thioethers with the same
stereochemistry as that found in lantibiotics. It is unclear at
present whether the origin of this selectivity is thermodynamic
or kinetic, and only the regiochemistry of the formation of the
A-ring of nisin has been investigated.15
Our previous studies have shown that cyclization of peptide 1
containing a Z-Dhb residue stereoselectively produces methyl-
lanthionine 2 (Scheme 2),13 the B-ring found in subtilin, a close
1
relative of nisin. In the H NMR spectrum of 2, the amide
proton of residue 5 occurs upfield of the other NH protons
and undergoes very slow solvent exchange in D2O, indicating
that this proton is engaged in a rigid hydrogen bond, pre-
sumably to the carbonyl of residue 2 in a β-turn conformation
(Scheme 2).17 During the transition state of the conjugate
addition leading to the enolate intermediate, this hydrogen
bond may increase in strength and lower the energy barrier
of the reaction. It is tempting to speculate that the β-turn
† Electronic supplementary information (ESI) available: separation of
the diastereomers of 5; cleavage of peptides from resins; COSY NMR
spectrum of the product obtained from cyclization of both E-1 and 19.
See http://www.rsc.org/suppdata/ob/b3/b304945k/
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 3 0 4 – 3 3 1 5
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 3
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Scheme 2
conformation is also responsible for the intrinsic propensity
to form just one of four possible diastereomers, but other
explanations are possible. For instance, the observed high
stereoselectivity could have a thermodynamic rather than a
kinetic origin if the Michael addition is reversible under the
reaction conditions. In that scenario, reduction of the disulfides
of Z-1 and its geometric isomer E-1 would generate thiolate
isomers that would equilibrate with the same pair of enolates
and/or methyllanthionine products (Scheme 2). Hence, both
reactions should provide identical products. In this report, we
present new methodology to prepare E-Dhb residues within
peptides to provide insights into this question. Along the way,
we also explored the use of solid phase reaction conditions
for our oxidative elimination methodology to install dehydro
amino acids and investigated the regiochemistry of the
formation of the A and B rings of nisin.
Results and discussion
Synthesis of selenocysteine derivatives
In order to test the model in Scheme 2, an efficient synthesis
of E-Dhb-containing peptides was required. We previously
reported the preparation of Fmoc-(2R,3S)-3-methyl-Se-phenyl-
selenocysteine (anti-8)13 for use in solid phase peptide synthesis
(SPPS).18 Our design to utilize selenocysteine derivatives for
the preparation of stereodefined dehydrobutyrine containing
peptides rested on the supposition that selenides could be
selectively oxidized in the presence of unprotected function-
alities in peptides. Given the known syn-selectivity of the
oxidative elimination,19,20 we reasoned that the Z- or E-isomers
of dehydrobutyrine could be stereospecifically accessed from
anti-FmocMeSec(Ph) or syn-FmocMeSec(Ph), respectively.
To test this strategy, racemic syn-8 was accessed from -allo-
threonine in analogous manner to the preparation of anti-813 as
shown in Scheme 3. For the purpose of verifying clean in-
Scheme 3
version at C3 during the displacement of the sulfonate, anti-8
and syn-8 were transformed to their methyl esters anti- and syn-
9. A racemic mixture of all four possible diastereomers of 9
was also prepared via Michael addition of phenylselenolate
to Boc-protected dehydrobutyrine methyl ester 10. The latter
reaction afforded rac-9 with a diastereoselectivity of 10 : 1 in
favor of the syn-stereoisomers. The product mixture could be
separated into four near baseline-resolved peaks by HPLC on a
Whelk-O1 chiral stationary phase21 (ESI†). The first two eluting
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 3 0 4 – 3 3 1 5
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Table 1 Oxidation of Sec(Ph) and MeSec(Ph) containing peptidesa
Entry
Peptide
Dehydropeptideb
Solvent
Yield (%)c
1
2
3
4
5
6
[Boc-Sec(Ph)Cys-OMe]2
[Boc-DhaCys-OMe]2
MeOH
75
70
83
79
76
68
Fmoc-Cys(SEt)Sec(Ph)-ODpm
Ac-Gly-(2R,3S)MeSec(Ph)-OBn
Ac-Gly-syn-MeSec(Ph)-OBn
[A-(2R,3S)-MeSec(Ph)-PGCVA]2
Fmoc-Cys(SEt)Dha-ODpm
Ac-Gly-(Z)-Dhb-OBn
Ac-Gly-(E)-Dhb-OBn
[A-(Z)-Dhb-PGCVA]2
[A-(E)-Dhb-PGCVA]2
CH2Cl2/MeOH
CH2Cl2/MeOH
CH2Cl2/MeOH
H2O/MeCN
H2O/MeCN
d
d
[A-syn-MeSec(Ph)-PGCVA]2
a Dha = dehydroalanine, Dhb = dehydrobutyrine, Sec(Ph) = (Se)-phenylselenocysteine. b Conditions for entries 1-4: aq NaIO4 (4 equiv.) was added
at 25 ЊC to solutions of the peptides (final peptide concentrations 7-30 mM). For entries 5 and 6, the final concentrations of the peptides were
3 mM. c Yields are for HPLC purified products. d The symmetrical disulfide was formed by oxidation with I2 of the purified peptide containing
Acm-protected Cys.
compounds showed identical retention times as -syn-9 pre-
pared from allo-Thr according to Scheme 3. The last isomer to
elute was identified as (2R,3S)-9 (anti-9) by coinjection with the
material prepared from -Thr. This latter compound displayed
a single peak verifying that the nucleophilic displacement
of the p-toluenesulfonate in Scheme 3 occurred without loss
of stereochemical purity provided the stoichiometry of the
reagents and the order of addition are carefully controlled (see
experimental).
Preparation and use of dehydropeptides on solid phase
In principle, both oxidative elimination to form dehydro-
peptides and subsequent Michael addition should be amenable
to solid phase techniques. The feasibility of this approach has
been explored using a model tripeptide, Ac-Ala-Sec(Ph)-Ala,
bound to Wang resin. Both the oxidative elimination and
subsequent Michael addition of an external nucleophile were
monitored by on-resin magic angle spinning (MAS) NMR
spectroscopy24,25 with spin echo enhancement.26,27 Diagnostic
peaks were observed in the starting material at 8.25 ppm corre-
sponding to three overlapping amide protons, and at 3.27 ppm
associated with the methylene protons of Sec(Ph) (Fig. 1A).
Upon treatment with 5 equivalents of H2O2 in DMF, the three
amide protons resolved into three distinct resonances at 9.21,
8.84, and 8.42 ppm (Fig. 1B). In addition, the methylene peak
at 3.27 ppm disappeared and a new resonance at 6.40 ppm due
to one of the vinyl protons of the newly formed dehydroalanine
appeared. 2-Mercaptoethylamine was then used as a nucleo-
phile to add to the dehydropeptide on the resin (Fig. 1C).
During this reaction, the three amide proton resonances were
converted into two sets of diastereomeric amide proton signals
(8.67, 8.58, 8.41, and 8.36 ppm). Furthermore, the vinyl proton
of dehydroalanine disappeared, and a new group of peaks
between 3.00–3.40 ppm emerged that corresponded to the
newly generated methylene protons of the amino acid.
Preparation of dehydropeptides
We have recently shown that the oxidative elimination is com-
patible with oxidation sensitive amino acids, such as methionine,
trityl or disulfide protected cysteines, and tryptophan
unprotected at the indole moiety.12 Synthetic trityl protected
cysteine residues can be chemoselectively oxidized with I2 to the
corresponding symmetrical disulfides without modification of
the selenide. Conversely, the phenylselenide can be oxidatively
eliminated with NaIO4 without effecting trityl or disulfide
protected cysteines (e.g. Table 1, entries 1 and 2). The ability to
selectively oxidize cysteines to disulfides or selenocysteines to
dehydroalanines proved extremely useful for the preparation of
lanthionines (vide infra). As envisioned, oxidative elimination
of (2R,3S)-MeSec(Ph) produced only the Z-isomer of dehydro-
butyrine (entry 3). This outcome could either reflect the prefer-
ential elimination of the more acidic α-proton over the γ-methyl
protons, or alternatively it could be the result of initial elimi-
nation of a γ-proton to provide vinylglycine, which subsequently
rearranged to the thermodynamically more stable Z-dehydro-
butyrine. This latter possibility was ruled out by the formation
of E-dehydrobutyrine when the experiment was performed with
a peptide containing syn-MeSec(Ph) (entry 4).
The versatility of this chemoselective and site-specific
methodology is demonstrated with longer peptides prepared by
SPPS. As with cysteine derivatives, caution must be taken to
avoid racemization during coupling of the Sec-derivatives in
SPPS. This was achieved using DIC/HOBt as coupling reagents
thereby avoiding the use of tertiary amine bases.22 Furthermore,
higher yields of purified peptides were obtained when depro-
tection protocols with piperidine were kept short. With longer
exposure times, elimination of phenylselenide occurs to some
extent as evidenced by detection of β-piperidylalanine contain-
ing peptides (M-71) in the crude product after cleavage from
the resin. With these precautions, our yields of purified Sec
containing peptides were comparable to those obtained for
other peptides of similar length prepared in our laboratory. The
oxidations of the purified peptides in entries 5 and 6 proceeded
slowly as monitored by RP-HPLC, with MS analysis indicating
a rapid initial oxidation to the selenoxide and a slow subsequent
elimination (5 hours). Typical functionalities found in peptides
such as amines, amides, guanidines, alcohols, and acids do not
interfere when left unprotected during the oxidation.12 How-
ever, when an unprotected N-terminal Ser or Thr is present,
H2O2 must be used as oxidant since periodate would oxidize
these amino acids.23
Fig. 1 MAS spin echo enhanced NMR spectroscopy of (A) Ac-Ala-
Sec(Ph)-Ala bound to Wang resin, (B) after treatment for 45 min with
H2O2, and (C) after addition of 2-mercaptoethylamine. Arrows indicate
characteristic resonances discussed in the text.
In addition to NMR spectroscopy, the Michael addition
reaction was also monitored by quantitative ninhydrin test
of the free amine group that is added during the transform-
ation.15,28 Fig. 2A shows the increase in absorbance at 570 nm
corresponding to the ninhydrin product versus time. Also
depicted is the time dependent increase in the integration of the
resonance at 3.26 ppm of the product. Non-linear regression
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thiolate isomers would equilibrate with the same pair of
enolates and/or methyllanthionine products (Scheme 2), and
both reactions should provide identical products. Similar to
our previous report on the cyclization of Z-1,13 the reaction
with E-1 indeed produced one major peak when analyzed by
1
HPLC. However, the H NMR spectrum of the product indi-
cated that it consisted of a different MeLn isomer than was
obtained with Z-1. This rules out a thermodynamic origin of
the observed stereoselectivity. The product formed with Z-1
therefore originates from preferential Si-face attack on the Z-
Dhb residue to provide 6, followed by protonation of the
enolate from the least hindered side to give 2 (Scheme 2). On the
other hand, the Michael addition with E-1 must provide 7 since
otherwise the same final product should have been obtained.
Hence, both Michael additions involve preferential attack of
the thiolate on the bottom face (as drawn in Scheme 2) of either
geometric isomer of dehydrobutyrine.
In analogy to 6, we anticipated that the protonation of
enolate 7 in the cyclization of E-1 would occur anti to the
methyl group on the β-carbon to produce (2R,3R,6R)-3-methyl-
lanthionine 5. Authentic methyllanthionines 4 and 5 were
required to confirm this hypothesis. It was envisioned that these
targets could be obtained by performing the Michael reaction
in the reverse direction with stereodefined 3-methylcysteine
residues at the second position of the peptide and a dehydro-
alanine at the fifth position. Such transformations could give
rise to two diastereomeric methyllanthionine products with
either the  or -configuration at the newly formed α-stereo-
center of the fifth residue, only the former of which can be
formed according to Scheme 2. Four diastereomeric peptides
13a–d were prepared as shown in Scheme 5. Conjugate addition
of benzylthiol to Boc-protected Z-dehydrobutyrine methyl
ester 10 followed by protecting group exchange produced a
1 : 1 mixture of diastereomers 14 that were debenzylated and
converted to the corresponding disulfides 15. The mixture
of diastereomers of 15 was used in SPPS to produce four
diastereomeric peptides 13 that were separated by HPLC and
oxidized to the corresponding Dha-containing disulfides
(Scheme 6). One of the diastereomers was identified as 16 by
comparison of its NMR spectrum with authentic material
prepared in our previous work.13 A second diastereomer was
assigned structure 17 by use of ( )-syn-14, which resulted in a
mixture of peptides 16 and 17. Reduction of the disulfide
of dehydropeptide 17 followed by increasing the pH of the
reaction mixture led to the formation of two methyllanthionine
diastereomers, neither of which coeluted with the product
formed from peptide E-1. Hence, contrary to our expectation,
(2R,3R,6R)-3-methyllanthionine 5 was not produced in the
cyclization of E-1.
Fig. 2 Kinetics of Michael addition of 2-mercaptoethylamine to Ala-
Dha-Ala on Wang resin determined by quantitative ninhydrin test (A)
and 1H-MAS-NMR spectroscopy (B).
analysis of the data using a single exponential equation pro-
vided rate constants of (8.5 1.0) × 10Ϫ3 minϪ1 (Fig. 2A) and
(11.0 1.0) × 10Ϫ3 minϪ1 (Fig. 2B). In both cases, the graphs
indicate that the Michael addition appears to be complete
after 6 h. Thus, phenylselenocysteines can be transformed into
dehydroalanines under mild oxidizing conditions on the solid
phase, and the product can be used for subsequent Michael
addition. To evaluate the generality of these results, a solid
phase conjugate ligation was used to synthesize glycopeptide 12
(Scheme 4). Oxidative elimination of resin-bound peptide 11,
subsequent Michael ligation and deprotection and cleavage
from the resin provided glycoconjugate 12 after HPLC purifi-
cation in 32–45% overall yield based on resin loading.29 This
represents a significant improvement compared to performing
the oxidation and Michael addition in solution,30 in part as a
result of decreasing the number of HPLC purifications from
three to one.
Scheme 4
Mechanistic studies on the biomimetic formation of
methyllanthionines
Scheme 5
The synthesis of E-1 was accomplished by incorporation of
the precursor syn-8 into the peptide by SPPS and subsequent
oxidation with NaIO4. The E-stereochemistry of the Dhb
The two remaining diastereomers of 13 were therefore
also oxidized to the corresponding Dha-containing disulfides
(Scheme 7). Both peptides were subjected to the conditions for
cyclization resulting in a ∼3 : 1 mixture of methyllanthionine
diastereomers for both reactions. HPLC analysis showed that in
one reaction neither product corresponded to the methyl-
1
generated was confirmed by H NMR spectroscopy. The Z-
isomer of peptide 1 was prepared as previously described.13 If
the cyclization reaction were under thermodynamic control,
then upon reduction of the disulfides of Z-1 and E-1 both
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 3 0 4 – 3 3 1 5
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from the final diastereomeric peptide 19 showed that one of
1
the two methyllanthionine isomers displayed an identical H
NMR spectrum as that formed from E-1 and the two com-
pounds also coeluted in HPLC experiments. Since only 4 can
be formed in the cyclizations of both 19 and E-1, we conclude
that enolates 6 and 7 are both protonated from the Re-face
irrespective of the configuration at C3 of the methyl-
lanthionine. Because the face selectivity of the initial attack
by the thiolate and the protonation of the resulting enolate are
the same for both isomers of 1, the observed stereochemistry in
these reactions is dominated by the conformation of the peptide
backbone.
Given the stereoselective non-enzymatic formation of the
natural diastereomers of individual lanthionines and methyl-
lanthionines shown in this and previous work,12–16 we decided
to investigate to what extent the regio- and stereoselectivity is
governed by the dehydropeptide itself. To this end, using
our synthetic methodology we prepared dehydropeptide 21
(Scheme 8), a potential precursor to the A and B rings of nisin
Scheme 6
Scheme 8
(Scheme 1). The prospect of preparing an N-terminal fragment
of nisin by biomimetic cyclization is intriguing given the
reported importance of this region for binding to its lipid II
target.31 In whole cell assays, nisin 1–12 acts as an antagonist
for binding of wild-type nisin.32 The biosynthetic machinery
does not allow non-conservative mutations in this region of the
antibiotic,33,34 and hence a biomimetic synthetic route would be
extremely useful for structure–activity relationship studies.
Peptide 20 was prepared by SPPS and the two Dha and
two Dhb residues were oxidatively unmasked in 27% yield after
HPLC purification. Although the overall yield is low, four
dehydro amino acids were installed in one step. The intra-
molecular disulfide was reduced and upon addition of base,
Scheme 7
lanthionine formed from E-1. On the basis of this result,
the cyclization precursor was assigned structure 18 since its
products would be different from those that can be accessed
from E-1. Indeed, the NMR spectra of the cyclization products
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cyclization took place as evidenced by HPLC and MS-MS. 1H
NMR analysis of the crude reaction mixture showed that the
product did not consist of the A and B rings of nisin. Instead,
the resonances of the vinyl protons of the two dehydro-
butyrines were still present in the product, whereas the signals
for the vinyl protons of the two Dha residues were absent.
Hence, the much faster cyclization rate for lanthionines
compared to methyllanthionines13 prevents the biomimetic
cyclization in which one Ln (A-ring) and one MeLn (B-ring)
would have been formed (Scheme 1). At present, we tentatively
assign the structure drawn in Scheme 8 to the product. Bradley
and coworkers have shown that Cys7 reacts preferentially with
Dha3 over Dha5 to give a five-residue lanthionine ring as a
3 : 1 mixture of diastereomers.15 We postulate that after
this initial cyclization, Cys11 reacts with Dha5 to form a
six-residue lanthionine ring. LC-MS analysis showed that at
least two product isomers are formed, which could be either
diastereomers or constitutional isomers. Since the cyclization
did not lead to the formation of the rings found in nisin, no
further attempts were made to elucidate the exact structures
of the products. The important conclusion with respect to the
biosynthesis of lantibiotics drawn from these studies is that
lantibiotics biosynthesis requires enzymatic control over the
regioselectivity and/or processivity of the cyclization reactions.
Illinois. For larger peptides, matrix assisted laser desorption
ionization (MALDI) MS was carried out on an Applied Bio-
systems Voyager DE STR or electrospray ionization (ESI) MS
was used employing a Finnigan LCQ Deca XP or a Micromass
Quattro. Fast atom bombardment (FAB) ionization was
used for smaller peptides and organic molecules employing
a Micromass 70-VSE instrument for low resolution and a
Micromass 70-SE-4F for high resolution measurements.
CHN elemental analysis was performed by the Microanalysis
Laboratory, University of Illinois with a Leeman Labs Inc.
Model DE 440 elemental analyzer. Described in the electronic
supplementary information† are the procedures used to deter-
mine the stereochemical purity of anti-8 and a COSY spectrum
of compound 4.
Fmoc-allo-( )-Thr-ODpm
Fmoc-( )-allo-threonine (1.65 g, 4.85 mmol) was placed in a
round bottom flask with diphenylmethyl hydrazone (1.86 g,
9.70 mmol), and the mixture was suspended in CH2Cl2 (16 mL).
A 1% (w/v) solution of I2 in CH2Cl2 (1.0 mL) was added to
the mixture at 0 ЊC. Iodobenzenediacetate (3.12 g, 9.70 mmol)
was added slowly over 1.5 h as the mixture was stirred under
Ar. The reaction was stirred at 0 ЊC for 3 h. The solvent was
evaporated and the resulting yellow oil was dissolved in EtOAc
(25 mL). The organic layer was washed with H2O (20 mL),
saturated aqueous NaHCO3 (3 × 20 mL), and H2O again
(20 mL). The organic layer was dried over MgSO4 and the sol-
vent was evaporated to give a yellow oil. The oil was dissolved
in EtOAc and loaded onto a silica plug. Impurities were washed
through with 8 : 1 hexanes : EtOAc, then the plug was washed
with 30 : 1 CH2Cl2 : CH3OH to elute the product (2.20 g, 89%).
1H NMR (500 MHz, CDCl3): δ ppm 1.09 (d, J = 6.57 Hz, 3H),
1.19–1.23 (m, 2H), 4.23 (t, J = 7.05 Hz, 1H), 5.61 (br s, 1H), 5.69
(d, J = 7.22 Hz, 1H), 6.93 (s, 1H), 7.27–7.43 (m, 13H), 7.46–7.48
Summary
We demonstrated the use of syn- and anti-8 for the stereo-
specific introduction of E- and Z-dehydrobutyrine residues into
peptides. The method installs the dehydro amino acids after
global deprotection in the ultimate step of the synthesis of
dehydropeptides, minimizing possible side reactions, and is
fully compatible with all physiological amino acids including
appropriately protected cysteines. The oxidation step can be
carried out on a solid support and can be conveniently moni-
tored by solid state magic angle spinning NMR spectroscopy.
The utility of the methodology is demonstrated by the solution
phase biomimetic syntheses of lanthionines and methyl-
lanthionines. These studies show that while the precursor
peptides to the lantibiotics have an inherent propensity to
cyclize to the naturally occurring stereoisomers, enzymatic
control must be exerted to ensure proper regioselectivity and
to overcome the much lower reactivity of dehydrobutyrines
compared to dehydroalanines. Our mechanistic studies indicate
that the observed highly selective stereochemical outcome in the
intramolecular Michael additions has a kinetic rather than a
thermodynamic origin.
(m, 1H), 7.58 (d, J = 5.54 Hz, 2H), 7.76 (d, J = 7.71 Hz, 2H); 13
C
NMR (125 MHz, CDCl3): δ ppm 20.1, 47.4, 59.5, 67.5, 68.3,
78.6, 120.2, 125.4, 127.3, 128.0, 128.4, 128.9, 139.6, 141.5,
143.9, 144.1, 157.0, 170.5; FAB-HRMS m/z calcd for C32H29
O5NNa (M ϩ Na)ϩ 530.1943, found 530.1945.
Fmoc-allo-( )-Thr(OTs)-ODpm
Fmoc-allo-( )-threonine-ODpm (2.8 g, 5.52 mmol) was dis-
solved in pyridine (20 mL), cooled to 0 ЊC, and TsCl (2.6 g,
13.8 mmol) was added. The mixture was stirred at 0 ЊC for 2 d.
The mixture was diluted with EtOAc (100 mL) and washed
with 5% KHSO4 (50 mL × 5). The organic layer was dried with
MgSO4 and concentrated. Crystallization from ether afforded
Experimental
1
2.9 g (79%) of a white solid. H NMR (500.0 MHz, CDCl3):
δ ppm 1.35 (d, J = 6.44 Hz, 3H), 2.36 (s, 3H), 4.17 (t, J =
7.09 Hz, 1H), 4.26–4.35 (m, 2H), 4.56 (d, J = 7.39 Hz, 1H), 4.98
(m, 1H), 5.60 (d, J = 8.52 Hz, 1H), 6.90 (s, 1H), 7.27–7.43 (m,
16H), 7.57 (d, J = 7.40 Hz, 2H), 7.62 (d, J = 8.07 Hz, 2H), 7.67
(d, J = 7.46 Hz, 2H); 13C NMR (125 MHz, CDCl3): δ ppm
18.19, 22.02, 47.46, 58.26, 68.01, 78.70, 79.36, 120.24, 125.43,
127.24, 127.39, 127.54, 128.01, 128.15, 128.49, 128.56, 128.85,
130.08, 139.20, 141.50, 143.94, 145.23, 155.80, 167.44.
General details
Protected amino acids were purchased from either Advanced
ChemTech or Chem-Impex. Wang resins pre-loaded with the
C-terminal Fmoc-protected amino acid were obtained from
Advanced ChemTech. MeOH and EtOH were purified via
treatment with Mg and I2 followed by distillation, THF was
distilled from sodium/benzophenone, CH2Cl2 was distilled from
CaH2, and pyridine was distilled from CaH2 onto 4 Å molecular
sieves and stored under Ar. Automated SPPS were performed
on a Rainin model PS3 peptide synthesizer. RP-HPLC purifi-
cation was performed on either a Rainin Dynamax system or
a Beckman System Gold with Vydac C18 analytical or pre-
Fmoc-( )-syn-MeSec-ODpm
KOH (0.426 g, 7.60 mmol) was dissolved in MeOH (10 mL),
and PhSeH (1.37 g, 8.76 mmol) was added under Ar. The
mixture was stirred at room temperature for 10 min. The
solvent was removed to afford an off-white solid. Fmoc-allo-
( )-Thr(OTs)-ODpm (2.52 g, 3.80 mmol) in DMF (12 mL) was
cooled to 0 ЊC and transferred to the above solid. The orange
mixture was stirred at room temperature for 4 h. Ice-cold 5%
aqueous KHSO4 (100 mL) was added, and the mixture was
extracted with EtOAc (250 mL). The organic layer was washed
with saturated NaHCO3 (50 mL), brine (50 mL), dried, and
parative columns. H and 13C NMR data were obtained on
1
either a Varian U400 or U500 spectrophotometer in CDCl3 or
D2O. Optical rotations were recorded on a JASCO DIP-360
Digital Polarimeter. Melting points were measured on a Fisher
melting apparatus without correction. Infrared (IR) spectra
were obtained using a Perkin Elmer Spectrum BX. Mass spec-
trometry of peptides was performed by the Mass Spectrometry
Laboratory, School of Chemical Sciences, University of
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 3 0 4 – 3 3 1 5
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concentrated. Purification by flash chromatography35 (hexanes
: EtOAc = 5 : 1) afforded a white solid (1.71 g, 70%; Rf = 0.36,
5 : 1 hexanes : EtOAc). H NMR (500 MHz, CDCl3): δ ppm
CH2Cl2/TFA (4 mL, 50/50) for 30 min at rt. The solvent was
evaporated under reduced pressure, and DIEA (4 mL) and
CH3CN (10 mL) were added to the flask. BocSec(Ph)-OH12
(0.31 g, 0.92 mmol) and benzotriazol-1-yloxytris(dimethyl-
amino)phosphonium hexafluorophosphate (BOP) (0.40 g,
0.92 mmol) were added. The mixture was stirred at 25 ЊC for
2 h, and the solvent was evaporated. The crude product was
then dissolved in EtOAc and washed with 3 M HCl, saturated
aqueous NaHCO3 and brine. The organic phase was dried over
MgSO4. The residue obtained after filtration and concentration
of the filtrate was purified by flash column chromatography
1
1.50 (d, J = 7.09 Hz, 3H), 3.82 (qd, J = 7.12 Hz, 3.81 Hz, 1H),
4.23 (t, J = 7.07 Hz, 1H), 4.39 (m, 2H), 4.79 (dd, J = 8.86 Hz,
3.65 Hz, 1H), 5.60 (d, J = 9.00 Hz, 1H), 6.77 (s, 1H), 7.20 (t, J =
7.49 Hz, 2H), 7.27–7.42 (m, 10H), 7.48 (d, J = 7.15 Hz, 2H),
7.59 (d, J = 7.48 Hz, 2H), 7.79 (d, J = 7.41 Hz, 2H); 13C NMR
(125 MHz, CDCl3): δ ppm 20.46, 42.11, 47.36, 59.82, 67.69,
79.17, 120.25, 125.41, 127.30, 127.33, 127.59, 127.96, 128.35,
128.42, 128.72, 128.81, 129.33, 135.79, 139.56, 139.67, 141.57,
156.58, 170.02.
eluting with CH2Cl2/MeOH (40/1) to afford the desired com-
20
pound (0.65 g, 85%) as a white solid. M.p.: 52-54 ЊC. [α]D
=
Ϫ20.2 (c = 0.52, CHCl3). IR (CHCl3): 3325, 2977, 1744, 1676,
1491, 1167 cmϪ1. 1H NMR (500 MHz) δ ppm 1.41 (s, 9 H), 2.62
(d, 2 H, J = 4.03), 3.15–3.30 (m, 2 H), 3.70 (s, 3 H), 4.33 (br,
1 H), 4.39–4.44 (m, 1 H), 5.20 (br, 1 H), 6.63 (d, 1 H, J = 8.21),
7.19–7.53 (m, 20 H). 13C NMR (125 MHz) δ ppm 28.5, 30.2,
33.8, 51.5, 52.8, 53.2, 54.5, 67.1, 127.2, 127.7, 128.3, 129.5,
129.7, 133.3, 144.5, 155.4, 170.3, 170.6. FAB-HRMS m/z calcd
for C37H41N2O5S80Se (M ϩ 1)ϩ 705.1901, found 705.1904. Anal.
Calcd for C37H40N2O5SSe C 63.15, H 5.73, N 3.98, found C
62.84, H 5.57, N 3.59%.
Fmoc-( )-syn-MeSec-OH (syn-8)
Fmoc-( )-syn-MeSec-ODpm (1.20 g, 1.86 mmol) was dissolved
in 10 mL of CH2Cl2/TFA (1/1), and triisopropylsilane was
dropped in until the mixture turned colorless. The solution
was stirred at room temperature for 1 h, and the solvent was
removed by rotary evaporation. Purification by flash chromato-
graphy (CH2Cl2 : MeOH = 20 : 1) afforded a solid (0.737 g,
83%). 1H NMR (500 MHz, CDCl3): δ ppm 1.55 (d, J = 7.19 Hz,
3H), 3.88 (qd, J = 7.14 Hz, 3.44 Hz, 1H), 4.28 (t, J = 7.30 Hz,
1H), 4.46 (d, J = 7.33 Hz, 2H), 4.69 (dd, J = 9.17 Hz, 3.37 Hz,
1H), 5.63 (d, J = 9.52 Hz, 1H), 7.23–7.29 (m, 5H), 7.36 (t,
J = 7.33 Hz, 3H), 7.44 (td, J = 7.27 Hz, 2.04 Hz, 2H), 7.62 (dd,
J = 12.33 Hz, 7.40 Hz, 4H), 7.81 (d, J = 7.42 Hz, 2H); 13C NMR
(125 MHz, CDCl3): δ ppm 20.18, 41.27, 47.28, 59.33, 67.61,
120.21, 125.40, 127.34, 127.97, 128.56, 129.35, 135.90, 141.52,
143.86, 144.07, 156.75, 175.39.
BocDhaCys(Trt)OMe
BocSec(Ph)Cys(Trt)OMe (30 mg, 0.04 mmol) placed in a 10 mL
round bottom flask was dissolved in 0.6 mL of THF. NaIO4
(34 mg, 0.16 mmol) dissolved in a few drops of water was
added. The mixture was stirred at 0 ЊC for 6 h, then CH2Cl2
was added. The organic solution was washed with water and
aqueous NaHCO3, then dried over MgSO4. The title compound
(19 mg, 82%) was obtained after removal of solvent and puri-
fied by flash column chromatography eluting with hexane/
EtOAc (85/15). M.p.: 43–45 ЊC. [α]D20 = Ϫ1.6 (c = 0.72, CHCl3).
IR (CHCl3) 3401, 1731, 1667, 1632, 1494, 1444, 1392, 1217,
Fmoc-( )-syn-MeSecOMe ( -syn-9)
An Aldrich Mini Diazald Apparatus was used. A solution of
diazald (0.044 g, 0.21 mmol) in 3 mL of Et2O was used to
generate diazomethane, which was condensed and dropped into
a solution of Fmoc-( )-syn-MeSec-OH (0.020 g, 0.042 mmol)
dissolved in 5 mL of ethyl ether in the receiving flask cooled in
an ice/salt bath at Ϫ15 ЊC. After the solvent was evaporated, the
1
1159 cmϪ1. H NMR (400 MHz) δ ppm 1.48 (s, 9 H), 2.64
(dd, 1 H, J = 18, 4.5 Hz), 2.72 (dd, 1 H, J = 18, 5.68 Hz), 3.73
(s, 3 H), 4.56–4.60 (m, 1 H), 5.09 (t, 1 H, J = 1.7 Hz), 6.05 (s,
1 H), 6.60 (d, 1 H, J = 7.46 Hz), 7.17 (br, 1 H), 7.20–7.39 (m, 15
H). 13C NMR (125 MHz) δ ppm 28.5, 33.8, 51.8, 53.1, 67.3,
80.8, 98.9, 127.2, 128.3, 129.7, 134.5, 144.4, 152.9, 163.8, 170.7.
1
product was obtained (0.020 g, 98%). H NMR (500 MHz,
CDCl3): δ ppm 1.52 (d, J = 7.13 Hz, 3H), 3.33 (s, 3H), 3.86
(qd, J = 7.20 Hz, 3.60 Hz, 1H), 4.25 (t, J = 7.21 Hz, 1H), 4.39
(d, J = 7.09 Hz, 2H), 4.64 (dd, J = 9.28 Hz, 3.44 Hz, 1H), 5.64
(d, J = 9.24 Hz, 1H), 7.29–7.42 (m, 5H), 7.41 (m, 2H), 7.61 (m,
4H), 7.78 (d, J = 7.19 Hz, 2H); 13C NMR (125 MHz, CDCl3):
δ ppm 20.63, 42.58, 47.46, 52.68, 58.96, 67.67, 120.30, 125.49,
127.43, 128.05, 128.49, 129.37, 135.79, 141.60, 144.06, 144.24,
156.64, 170.90; HRMS-ESI for C26H26NO4Se (M ϩ H) calcd.
496.1027, found 496.1046.
ϩ
The product fragments during mass spectrometry give CPh3
(detected by positive FAB-LRMS) and (M Ϫ CPh3)Ϫ (detected
by negative FAB-LRMS).
[BocSec(Ph)CysOMe]2
BocSec(Ph)Cys(Trt)OMe (0.128 g, 0.178 mmol) in 4 mL of
MeOH was dropped into a solution of I2 in MeOH (0.0452 g,
0.178 mmol, 2 mL MeOH). The mixture was stirred at rt for
2 h. An aqueous solution of Na2S2O3 was dropped into the
reaction to remove the orange color. Water (30 mL) was added
to the reaction, and EtOAc (60 mL) was used to extract the
organic compounds. The organic layer was dried over MgSO4
and filtered. The solution was concentrated to afford the crude
product. Purification by flash chromatography with 20 : 1
CH2Cl2 : MeOH provided the product (Rf = 0.32, 20 : 1 CH2Cl2 :
BocCys(Trt)OMe
The procedure for the synthesis of compound -syn-9 was
employed. Starting with 1.50 g of Boc-Cys(Trt)-OH, the
reaction produced 1.34 g (86%) of Boc-Cys(Trt)-OMe. M.p.:
20
41-42 ЊC. [α]D = ϩ18.8 (c = 0.55, CHCl3). IR (CHCl3): 3419,
2977, 1748, 1717, 1492, 1166 cmϪ1. 1H NMR (400 MHz) δ ppm
1.43 (s, 9 H, tBu), 2.57 (d, 2 H, J = 5.52, CH2), 3.70 (s, 3 H,
CH3), 4.28 (m, 1 H, CH ), 5.01 (d, 1 H, J = 6.5, NH ), 7.20–7.40
(m, 15 H, trityl). 13C NMR (100 MHz) δ ppm 28.5 (CH3/CH,
C(CH3)3), 34.3 (C/CH2, CH2), 52.6 (CH3/CH, OCH3), 52.7
(CH3/CH, CH), 67.0 (C/CH2, CPh3), 80.2 (C/CH2, CMe3),
127.1 (CH3/CH, Ph), 128.2 (CH3/CH, Ph), 129.7 (CH3/CH, Ph),
144.5 (C/CH2, Ph), 155.2 (C/CH2, CO amide), 171.5 (C/CH2,
CO ester). FAB-HRMS m/z calcd for C28H32NO4S (M ϩ 1)ϩ
478.2052, found 478.2050. Anal. Calcd for C28H31NO4S
C 70.41, H 6.54, N 2.93, found C 70.67, H 6.24, N 2.71%.
20
MeOH; yield 81%). M.p.: 145–146 ЊC. [α]D = ϩ2.9 (c = 0.46,
CHCl3). IR (CHCl3): 3308, 2977, 1744, 1664, 1508, 1165 cmϪ1.
1H NMR (500 MHz, CDCl3) δ ppm 1.45 (9H, s), 3.06–3.16
(m, 2H), 3.27 (m, 2H), 3.74 (s, 3H), 4.48 (br, 1H), 4.73–4.78 (dt,
1H, J = 7.4, 5.4), 5.60 (d, 1H, J = 7.81), 7.22–7.27 (m, 3H), 7.30–
7.35 (br s, 1H), 7.53–7.55 (m, 2H); 13C NMR (126 MHz,
CDCl3) δ ppm 28.5, 30.0, 40.8, 52.2, 53.0, 54.5, 80.6, 127.63,
129.4, 129.7, 133.4, 155.8, 170.5, 171.1; FAB-HRMS m/z calcd
for C36H51N4O10S280Se2 (M ϩ 1)ϩ 923.1377, found 923.1377.
Anal. Calcd. for C36H50N4O10S2Se2 C 46.95, H 5.47, N 6.08,
found C 46.55, H 5.45, N 5.98%.
BocSec(Ph)Cys(Trt)OMe
Boc-Cys(Trt)-OMe (0.44 g, 0.92 mmol) was treated with
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 3 0 4 – 3 3 1 5
3310

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[BocDhaCys(OMe)]2
8H), 7.55–7.57 (m, 2H); 13C NMR (125 MHz, CDCl3): δ ppm
18.2, 23.1, 40.5, 43.4, 57.2, 67.7, 128.4, 128.5, 128.7, 128.8,
128.9, 129.5, 135.3, 135.5, 169.2, 170.0, 171.0; IR (CHCl3):
3300, 3056, 2927, 1738, 1658, 1520, 1375, 1266, 743, 703 cmϪ1;
HRMS (FABϩ): calcd [M ϩ H] C21H25N2O4Se1 449.0980, found
449.0980.
[BocSec(Ph)CysOMe]2 (29.3 mg, 0.055 mmol) in 2 mL of
MeOH was mixed with NaIO4 (95.0 mg, 0.444 mmol) in a
few drops of water at 0 ЊC. The solution was warmed to rt
and stirred overnight. EtOAc was added into the reaction,
and the resulting solution was washed with water, aqueous
NaHCO3 and dried over MgSO4. Filtration and concentration
of the filtrate gave the crude product. Purification by flash
Ac-Gly-Z-Dhb-OBn
column chromatography eluting with CH2Cl2/MeOH (20/1)
Ac-Gly-(2R,3S)-MeSec(Ph)-OBn (0.052 g, 0.140 mmol) was
dissolved in 6.0 mL of CH2Cl2 and 6.0 mL of MeOH. NaIO4
(0.120 g, 0.560 mmol) was dissolved in 3.0 mL of water. The
NaIO4 solution was added to the dipeptide solution at room
temperature and the mixture was stirred for 0.5 h. Water
(10 mL) was added and the mixture was extracted with CH2Cl2
(80 mL). The organic layer was washed with water (30 mL),
brine (30 mL), and dried with MgSO4. The solvent was
removed under reduced pressure. Purification over silica gel
(CH2Cl2 : MeOH = 20 : 1) afforded the desired product (Yield:
83%; Rf = 0.29, CH2Cl2 : MeOH = 20 : 1). 1H NMR (500 MHz,
CDCl3): δ ppm 1.76 (d, J = 7.24 Hz, 3H), 2.00 (s, 3H), 4.06 (d,
J = 5.28 Hz, 2H), 5.17 (s, 2H), 6.63 (t, J = 5.20 Hz, 1H), 6.85
(q, J = 7.21 Hz, 1H), 7.31–7.37 (m, 5H), 7.87 (s, 1H); 13C NMR
(125 MHz, CDCl3): δ ppm 14.7, 23.1, 43.8, 67.4, 126.2, 128.4,
128.6, 128.8, 135.3, 135.8, 164.3, 170.0, 171.3; IR (CHCl3):
3286, 3034, 1722, 1662, 1520, 1386, 1269, 1145, 1070, 1029, 968,
737, 699 cmϪ1; HRMS (FABϩ): calcd. for [M ϩ H] C15H19N2O4
291.1345, found 291.1345.
20
afforded 25.5 mg (75%) of the product. M.p.: 62–65 ЊC. [α]D
=
ϩ3.6 (c = 1.00, CHCl3). IR (CHCl3): 3392, 1731, 1663, 1630,
1499, 1368, 1247, 1160. 1H NMR (400 MHz) δ ppm 1.47
(s, 9 H), 3.23 (d, 2 H, J = 5.16 Hz), 3.78 (s, 3 H), 4.89 (dt, 1 H,
J = 7, 5 Hz), 5.21 (t, 1 H, J = 1.7 Hz), 6.07 (s, 1 H), 6.99 (d, 1 H,
J = 7.29 Hz), 7.21 (s, 1 H). 13C NMR (125 MHz) δ ppm
28.4, 40.6, 52.2, 53.2, 80.9, 99.2, 134.5, 153.0, 164.1, 170.6.
FAB-HRMS m/z calcd for C24H39N4O10S2 (M ϩ 1)ϩ 607.2107,
found 607.2115.
Boc-(2R,3S )-MeSec(Ph)-OBn
The procedure described by Wakamiya et al.36 was followed
starting with (2S,3R)-2-tert-butoxycarbonylamino-3-(phenyl-
20
selanyl)butyric acid benzyl ester13 giving 67% yield. [α]D
=
19.2Њ (c = 1.79 w/v%, CHCl3);1H NMR (500 MHz, CDCl3):
δ ppm 1.40 (d, J = 6.89 Hz, 3H), 1.43 (s, 9H), 3.61 (qd, J = 7.05
Hz, 4.60 Hz, 1H), 4.56 (dd, J = 8.66 Hz, 4.29 Hz, 1H), 5.15 (AB,
Jab = 12.2 Hz, 2H), 5.25 (d, J = 8.46 Hz, 1H), 7.25–7.38 (m, 8H),
7.58 (dd, J = 7.58 Hz, 1.37 Hz, 2H); 13C NMR (125 MHz,
CDCl3): δ ppm 18.6, 28.5, 41.5, 58.4, 67.5, 128.3, 128.5, 128.8,
129.4, 135.4, 135.6, 156.4, 170.8; IR (CHCl3): 3432, 3057, 2982,
2931, 1732, 1498, 1478, 1456, 1373, 1266, 1161, 1047, 732,
704 cmϪ1; HRMS (FABϩ): calcd for [M ϩ H] C22H27N1O4Se1
449.1105, found 449.1106.
[Ac-Gly-( )-syn-MeSec(Ph)-OBn]
Fmoc-( )-syn-MeSec-OBn (0.049 g, 0.086 mmol) was dissolved
in 1 mL of CHCl3 and treated with 1 mL of 4-amino-
methylpiperidine (4-AMP) at rt for 30 min. The mixture was
washed with 10% NaH2PO4 (5 × 20 mL) and concentrated. The
residue was dissolved in 2 mL of CH2Cl2. Ac-Gly-OH (0.0096
g, 0.082 mmol) and BOP (0.036 g, 0.082 mmol) were added
followed by diisopropylethylamine (0.032 g, 0.25 mmol). The
mixture was stirred overnight and diluted with CH2Cl2 (80 mL),
washed with 1 M HCl (30 mL), sat. NaHCO3 (30 mL), water
and brine. The organic layer was dried and concentrated.
Purification over silica gel (CH2Cl2 : MeOH = 20 : 1) provided
the product (0.037 g, 96%; Rf = 0.25, CH2Cl2 : MeOH = 20 : 1).
1H NMR (500 MHz, CDCl3): δ 1.48 (d, J = 7.29 Hz, 3H), 2.07
(s, 3H), 3.87 (qd, J = 7.24 Hz, 3.71 Hz, 1H), 4.00 (qd, J = 16.77
Hz, 5.18 Hz, 2H), 4.58 (B of AB, Jab = 12.19 Hz, 1H), 4.93 (dd,
J = 8.83 Hz, 3.61 Hz, 1H), 4.95 (A of AB, Jab = 12.19 Hz, 1H),
6.36 (br, 1H), 6.81 (d, J = 8.94 Hz, 1H), 7.21–7.36 (m, 8H),
7.57–7.59 (m, 2H); 13C NMR (125 MHz, CDCl3): δ 20.33,
23.15, 42.33, 56.99, 57.76, 67.59, 127.95, 128.56, 128.65, 128.76,
128.80, 129.39, 135.03, 135.88, 169.46, 169.86, 170.81; FAB-
HRMS calcd for C21H25N2O480Se [M ϩ H] 449.0982, found
449.0980.
Fmoc-( )-syn-MeSec-OBn
The title compound was prepared using a similar procedure
as for Boc-(2R,3S)-MeSec(Ph)-OBn. Yield: 88%; 1H NMR
(500 MHz, CDCl3): δ ppm 1.54 (d, J = 7.19 Hz, 3H), 3.88 (qd,
J = 7.22 Hz, 3.10 Hz, 1H), 4.26 (t, J = 7.32 Hz, 1H), 4.41 (d,
J = 7.52 Hz, 2H), 4.62 (d, J = 12.19 Hz, 1H), 4.72 (dd, J = 9.23,
3.40 Hz, 1H), 4.99 (d, J = 12.20 Hz, 1H), 5.69 (d, J = 9.32 Hz,
1H), 7.24–7.45 (m, 12H), 7.62 (m, 4H), 7.80 (d, J = 7.14 Hz,
2H); 13C NMR (125 MHz, CDCl3): δ ppm 20.52, 42.49, 47.34,
59.05, 67.53, 67.56, 120.25, 125.39, 125.43, 127.35, 127.97,
128.11, 128.48, 128.64, 128.73, 128.79, 129.38, 129.72, 135.12,
135.85, 135.89, 141.55, 143.92, 144.15, 156.64, 170.32; FAB-
HRMS calcd for C32H30N1O4Se1 [M ϩ H] 572.1340, found
572.1340.
Ac-Gly-(2R,3S )-MeSec(Ph)-OBn
Boc-(2R,3S)-MeSec(Ph)-OBn (0.056 g, 0.15 mmol) was dis-
solved in 9 mL of CH2Cl2 : TFA (2 : 1) and stirred at room
temperature for 1 h. The solvent was removed by rotary evapor-
ation. The residue obtained was dissolved in 3 mL of CH3CN.
Ac-Gly-OH (0.018 g, 0.15 mmol) and benzotriazol-1-yloxy-
Ac-Gly-E-Dhb-OBn
Ac-Gly-( )-syn-MeSec(Ph)-OBn (0.018 g, 0.038 mmol) was
dissolved in 2.0 mL of CH2Cl2 and 2.0 mL of MeOH. NaIO4
(0.041 g, 0.154 mmol) was dissolved in 1.0 mL of water. The
NaIO4 solution was added to the dipeptide solution at room
temperature and the mixture was stirred for 0.5 h. Water
(10 mL) was added and the mixture was extracted with EtOAc
(140 mL). The organic layer was washed with water (40 mL),
brine (30 mL), and dried with MgSO4. The solvent was
removed under reduced pressure. Purification over silica gel
(CH2Cl2 : MeOH = 20 : 1) afforded the desired product (0.0092
tris(dimethylamino)phosphonium
hexafluorophosphate
(0.067 g, 0.15 mmol), were added followed by diisopropylethyl-
amine (0.039 g, 0.30 mmol). The mixture was stirred for 1 h and
diluted with CH2Cl2 (80 mL), washed with 1 M HCl (30 mL),
sat. NaHCO3 (30 mL), water and brine. The organic layer was
dried and concentrated. Purification over silica gel (CH2Cl2 :
MeOH = 40 : 1) provided the product (0.052 g, 92%; Rf = 0.26,
CH2Cl2 : MeOH = 40 : 1). 1H NMR (500 MHz, CDCl3): δ ppm
1.40 (d, J = 7.26 Hz, 3H), 1.99 (s, 3H), 3.68 (qd, J = 7.13 Hz,
4.25 Hz, 1H), 3.88 (B of ABX, Jab = 18.41 Hz, Jbx = 8.66 Hz,
1H), 3.91 (A of ABX, Jab = 18.41 Hz, Jax = 8.44 Hz, 1H), 4.85
(dd, J = 8.40 Hz, 4.32 Hz, 1H), 5.14 (AB, Jab = 12.22 Hz, 2H),
6.48 (X of ABX, 1H), 7.13 (d, J = 8.39 Hz, 1H), 7.25–7.37 (m,
1
g, 79%; Rf = 0.29, CH2Cl2 : MeOH = 20 : 1). H NMR (500
MHz, CDCl3): δ 2.02 (s, 3H), 2.10 (d, J = 7.73 Hz, 3H), 3.99
(d, J = 6.26 Hz, 2H), 5.27 (s, 2H), 6.31 (br, 1H), 7.16 (q, J =
7.39 Hz, 1H), 7.37–7.42 (m, 5H), 7.92 (s, 1H); 13C NMR (125
MHz, CDCl3): δ 14.70, 23.06, 44.22, 67.70, 125.41, 128.67,
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 3 0 4 – 3 3 1 5
3311

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128.81, 128.93, 129.89, 135.35, 164.21, 167.62, 171.01; FAB-
HRMS calcd for C15H19N2O4 [M ϩ H] 291.1345, found
291.1345.
echo enhancement was applied with a delay of 4 ms between
180Њ pulses (26 µs). A recycle delay of 9.5 s was used between
acquisitions. MAS- 1H NMR (DMF-d7, 500 MHz) δ ppm 1.36
(6H, s, βCH3), 1.96 (3H, s, CH3-Ac), 3.28 (2H, s, βCH2), 4.39
(2H, s, αCH-Ala), 4.72 (1H, s, αCH-PhSec), 8.25 (3H, s, NH ).
The resin bound peptide (0.72 g, 0.43 mmol) was swollen in
DMF (11 mL) for 30 min, and then 30% H2O2 (0.25 mL, 2.16
mmol) was added. The resin mixture was agitated every 10 s by
purging the reaction with N2. Small aliquots of resin were
removed, washed with DMF, EtOH, and CH2Cl2 every 15 min
Fmoc-( )-syn-S-benzyl-3-methylcysteine (14)
Boc-syn-( )-S-benzyl-3-methylcysteine methyl ester (0.60 g,
1.8 mmol) was treated with 10 mL of TFA/CH2Cl2 at rt for 1 h.
The mixture was then concentrated under reduced pressure.
The residue was treated with 12 M HCl at 65 ЊC for 5 h, then
concentrated. NaHCO3 (0.16 g, 1.9 mmol) in 18 mL of H2O
and 12 mL of acetone was added to the residue. FmocOSu was
added to the mixture. The reaction was kept at pH ∼ 9 and
stirred at rt overnight. The reaction was diluted with EtOAc,
and washed with 1 M HCl. The aqueous layer was extracted
with EtOAc (2 × 50 mL). The organic layer was dried over
MgSO4, concentrated and purified by silica gel chromatography
to afford 0.64 g (81%, Rf = 0.32, 15 : 1 CH2Cl2 : MeOH) of the
product. 1H NMR (500 MHz, CDCl3): δ ppm 1.33 (d, J = 7.12
Hz, 3 H), 3.40 (qd, J = 7.88 Hz, 3.92 Hz, 1 H), 3.77 (B of AB,
J = 13.38 Hz, 1 H), 3.81 (A of AB, J = 13.38 Hz, 1 H), 4.27
(t, J = 6.99 Hz, 1 H), 4.44 (d, J = 7.16 Hz, 2 H), 4.60 (dd, J =
8.85 Hz, 3.08 Hz, 1 H), 5.61 (d, J = 8.92 Hz, 1 H), 7.25–7.33 (m,
7 H), 7.42 (m, 2 H), 7.63 (t, J = 6.85 Hz, 2 H), 7.79 (d, J = 7.13
Hz, 2 H); 13C NMR (125 MHz, CDCl3): δ ppm 19.94, 36.14,
42.20, 47.35, 58.64, 67.70, 120.31, 125.40, 127.41, 127.49,
127.60, 128.05, 128.88, 129.15, 137.77, 141.59, 143.87, 144.06,
156.90, 175.91; FAB-HRMS calcd for C26H25N1O4S1Na1 [M ϩ
Na] 470.1402, found 470.1403. Fmoc-( )-anti-S-benzyl-3-
methylcysteines were synthesized similarly.
1
for 3 h. H NMR spectroscopy showed the reaction to be
complete in 45 min. A small amount of resin was subjected to
cleavage conditions (5% TIS in TFA, 90 min), and FAB MS
was obtained on the crude cleavage product. MAS-1H NMR
(DMF-d7, 500 MHz) δ ppm 1.36 (3H, s, βCH3-Ala), 1.44 (3H,
s, βCH3-Ala), 1.97 (3H, CH3-Ac), 4.54 (2H, s, αCH ), 6.39 (1H,
s, CH2-vinyl), 8.42 (1H, s, NH ), 8.84 (1H, s, NH ), 9.21 (1H, s,
NH ); FAB-MS 274 (M ϩ 1)ϩ.
Michael addition to Ac-Ala-Dha-Ala-Wang resin
The Ac-Ala-Dha-Ala-Wang resin (0.42 g, 0.25 mmol) was
swollen in DMF (7 mL) for 30 min. 2-Mercaptoethylamine
hydrochloride salt (0.29 g, 2.53 mmol) and N-methyl morpho-
line (0.69 mL, 6.32 mmol) were dissolved in DMF (3 mL)
and added to the mixture. The resin was agitated every 10 s by
purging with N2 for 7.5 h. Small aliquots of resin were with-
drawn every 10 min, washed with DMF, CH2Cl2, and EtOH
1
and dried in a vacuum dessicator. H NMR spectroscopy and
quantitative ninhydrin tests showed the reaction to be complete
after 6 h. The product resin was subjected to cleavage con-
ditions (5% TIS/TFA, 90 min) and the product was purified by
preparative RP-HPLC (Vydac C-18 column) to give the peptide
Fmoc-syn-3-methylcystines (15)
SiCl4 (2.86 g, 16.82 mmol) and Ph2SO (0.85 g, 4.20 mmol) in 12
mL of TFA were added to Fmoc-syn-( )-S-benzyl-3-methyl-
cysteine (0.37 g, 0.84 mmol) at 0 ЊC and the resulting mixture
was stirred for 1 h. The reaction mixture was diluted with 40
mL of Et2O. The solution was treated with saturated aq.
NaHCO3. The aqueous layer was then acidified to pH ∼ 1,
extracted with EtOAc, dried over MgSO4 and concentrated to
afford 0.29 g (98%) of the title compound. FAB-LRMS calcd
for C38H36N2O8S2 [M ϩ H] 713.8, found 714.2. The material
was used without further purification. Fmoc-anti-S-benzyl-3-
methylcystines were synthesized similarly.
1
as an oil (66 mg, 75%). H NMR (DMF-d7, 500 MHz) δ ppm
1.38 (6H, s, βCH3-Ala), 1.96 (3H, s, CH3-Ac), 3.05 (2H, s, CH2),
3.13 (1H, br m, CH2), 3.26 (2H, s, βCH2), 4.44 (2H, s, αCH-
Ala), 4.74 (2H, br m, αCH ), 8.41 (1H, br m, NH ), 8.58
(1H, s, NH-diastereomer 1), 8.67 (1H, s, NH-diastereomer 2);
FAB-MS 349 (100%) (M ϩ 1)ϩ, 371 (42%) (M ϩ Na)ϩ, 387
(14%) (M ϩ K)ϩ. The stock solutions and procedures used
for the quantitative ninhydrin tests were those described by
Merrifield and coworkers.28 The dried resin (3–4 mg) was sub-
jected to the reaction conditions at 100 ЊC for 10 min. The resin
was then washed and the mother liquor diluted as described.
The color yield of the reaction was monitored at 570 nm by
UV/VIS spectrophotometry.
Solid phase peptide synthesis
Peptides were synthesized in an automated peptide synthesizer
using standard Fmoc protocols.37 Resins (generally 0.6 mmol
g
Preparation of Ac-Gly-Ala-Cys(ꢀ-glucosyl)-Ala-Ser-Thr-Ser-
OH (12) on solid phase
Ϫ1 loading capacity) were swollen in DMF (3 × 6 mL, 10 min).
Fmoc groups were removed with 20% v/v piperidine in DMF
(2 × 6 mL, 7 min). After deprotection, the resins were rinsed
with DMF (6 × 6 mL, 30 s). Amino acids were activated by
reaction with O-benzotriazole-N,N,NЈ,NЈ-tetramethyluronium
hexafluorophosphate (HBTU) and 0.4 M N-methylmorpholine
(3 mL, 30 s). The activated solutions were transferred into
the reaction vessel. The amino acid cartridges were rinsed with
DMF (3 mL, 30 s) and this solution was also transferred to the
reaction vessel. Coupling of the amino acids in general pro-
ceeded for 30 min or until complete as determined by a Kaiser
test.38 For every coupling, four equivalents of each amino acid
and HBTU with respect to the resin loading capacity were
used. Following each coupling, the resins were rinsed with
DMF (3 × 6 mL, 30 s).
Ac-Gly-Ala-Sec-Ala-Ser(OtBu)-Thr(OtBu)-Ser(OtBu)-Wang
resin (11, 0.576 g, 0.29 mmol) was obtained by SPPS. A small
quantity of beads (10 mg) was cleaved in 3 mL of TFA/CH2Cl2
(95/5) with 3 drops of TIS for 1 h. Removal of TFA by rotary
evaporation afforded an oily residue, which turned to a white
solid upon addition of ether. MALDI-MS m/z calcd for
C29H43N7O12SeNa (M ϩ Na)ϩ 784.2, found 783.9. Ac-Gly-Ala-
Sec-Ala-Ser(OtBu)-Thr(OtBu)-Ser(OtBu)-Wang resin (0.251 g,
0.127 mmol) was swelled in DMF (7 mL) for 0.5 h. H2O2 (30%
aq., 71 µL) was added to the resin. The mixture was purged
periodically with N2 for 2 h. Ac-Gly-Ala-Dha-Ala-Ser(OtBu)-
Thr(OtBu)-Ser(OtBu)-Wang resin (0.226 g) was washed with
DMF, MeOH and CH2Cl2, and dried under reduced pressure.
A small quantity of beads (3 mg) was cleaved in 3 mL of TFA/
CH2Cl2 (95/5) with 3 drops of TIS for 1 h. Removal of TFA by
rotary evaporation afforded an oily residue, which turned to a
white solid upon addition of ether. MALDI-MS m/z calcd for
C23H37N7O12Na (M ϩ Na)ϩ 626.2, found 626.1. The sodium
salt of 1-thio-β-glucosylpyranose (50 mg, 0.23 mmol) was
passed through an HCl-regenerated cation exchange column
and collected into 0.1 M aqueous Et3N. The solution was
Ac-Ala-Dha-Ala-Wang resin
The precursor peptide was synthesized on a 0.4 mmol scale
1
using standard Fmoc protocols. MAS H NMR spectra were
obtained using a Varian nanoprobe on a Varian UI500WB
instrument. Resin loaded with the peptide (3–5 mg) was placed
in the nanoprobe cell and swollen in 40 µL of DMF-d7. Spin
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 3 0 4 – 3 3 1 5
3312

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lyophilized to give a white foam, which was dissolved in 0.1 mL
of DMF and transferred to 40 mg (0.022 mmol) of Ac-Gly-
Ile-(2R,3S )-Me-Sec(Ph)-Sec(Ph)-Ile-Sec(Ph)-Leu-Cys(Acm)-
(2R,3S )-methyl-Sec(Ph)-Pro-Gly-Cys(Acm)-Lys-Ala (23)
Ala-Dha-Ala-Ser(OtBu)-Thr(OtBu)-Ser(OtBu)-Wang
resin.
The title compound was prepared on solid phase. Fmoc-Ala-
Wang resin (0.167 g, 0.1 mmol) was swollen in DMF.
Fmoc-(2R,3S)-methyl-Sec(Ph)-OH, Fmoc-Sec(Ph)-OH and
Fmoc-Cys(Acm)-OH were coupled manually with HOBT
and DIC. Couplings were allowed to proceed for 2 h each. The
completion of coupling was monitored by bromophenyl blue.
The peptide was cleaved from the resin with water (0.5 mL),
anisole (0.5 mL), thioanisole (0.5 mL), triisopropylsilane
(0.3 mL) and TFA (10 mL). TFA was removed by rotary
evaporation and ice-cold Et2O was added to precipitate the
peptide, which was purified by RP-HPLC (41.1 mg, 21%).
LRMS (MALDI): calculated C84H122N16O16S2Se4 1994.53,
found 1995.50 [M ϩ H].
The mixture was stirred for 2 d. The resin was filtered and
washed with DMF, MeOH and CH2Cl2. The peptide was
cleaved from the resin (60 mg) with 10 mL of 95/3/2 TFA/
CH2Cl2/TIS. Removal of TFA by rotary evaporation afforded
an oily residue, which turned to a white solid upon addition of
ether. The peptide was purified by HPLC to provide 8.0 mg
(overall 45%) of the desired peptide. MALDI-MS m/z calcd for
C29H49N7O17SNa (M ϩ Na)ϩ 822.2, found 821.9.
[A-syn-MeSec(Ph)-PGCVA]2
The HPLC-purified peptide A-syn-MeSec(Ph)-PGC(Acm)VA
(0.049 g, 0.048 mmol) was dissolved in 4 mL of 50% aq. MeOH.
Acetic acid (0.2 mL) was added followed by a 0.05 M solution
of I2 in 1.5 mL of MeOH. The reaction mixture was stirred at
room temperature for 45 min and quenched with 10% aq.
sodium thiosulfate until the mixture was colorless. The mixture
was concentrated by evaporation under reduced pressure and
purified by preparative RP-HPLC (0.027 g). Yield: 74%; FAB-
MS calcd for C62H93N14O16S2Se2 [M ϩ H] 1513.46, found
1513.64.
Ile-(2R,3S )-Me-Sec(Ph)-Sec(Ph)-Ile-Sec(Ph)-Leu-
Cys-(2R,3S )-methyl-Sec(Ph)-Pro-Gly-Cys-Lys-Ala (20)
Compound 23 (8 mg, 4.0 µmol) was placed in a 50 mL, round
bottomed flask and dissolved in 80% aq. HOAc (3 mL). I2
(30.6 mg, 120 µmol) was dissolved in 24 mL of 80% aq. HOAc
and added to the peptide solution. The reaction mixture was
stirred at rt for 25 min. Water (50 mL) was added and the
aqueous layer was washed with CCl4 (4 × 30 mL) to remove
excess I2. The aqueous layer was lyophilized and then purified
by RP-HPLC. Yield: 54%. LRMS (MALDI): calculated
C78H110N14O14S2Se4 1850.44, found 1851.76 [M ϩ H].
(A-E-DhbPGCVA)2 (E-1)
Peptide [A-syn-MeSec(Ph)-PGCVA]2 (5.0 mg, 3.3 µmol) was
dissolved in 6 mL of H2O and NaIO4 (0.008 g, 0.037 mmol) in
0.6 mL of H2O was added. The reaction mixture was stirred at
room temperature for 5.5 h and purified by semi-preparative
Ile-Dha-Dha-Ile-Dha-Leu-Cys-Dhb-Pro-Gly-Cys-Lys-Ala (21)
and cyclization product 22
1
RP-HPLC. Yield: 2.7 mg, 68%. H NMR (D2O, 500 MHz):
δ ppm 0.78 (d, 3H, J = 6.81 Hz, Val-CH3), 0.79 (d, 3H, J = 7.53
Hz, Val-CH3), 1.23 (d, 3H, J = 7.24 Hz, Ala-CH3), 1.41 (d, 3H,
J = 7.03 Hz, Ala-CH3), 1.59 (d, 3H, J = 7.36 Hz, Dhb-CH3),
1.77–1.97 (m, 5H, Pro-2Hγ ϩ Pro-Hβ ϩ Val-Hβ), 2.23 (m,
1H, Pro-Hβ), 2.81 (B of ABX, 1H, J = 13.92 Hz, 8.74 Hz,
Cys-Hβ), 3.03 (A of ABX, 1H, J = 13.92 Hz, 4.74 Hz, Cys-Hβ),
3.40–3.57 (m, 2H, Pro-2Hδ), 3.84 (s, 2H, Gly-H), 3.97 (q, 1H,
J = 4.74 Hz, Ala-Hα), 4.00 (d, J = 6.74 Hz, 1H, Val-Hα), 4.12
(q, 1H, J = 6.78 Hz, Ala-Hα), 4.36 (t, 1H, J = 6.48 Hz, Pro-Hα),
4.56 (m, 1H, Cys-Hα), 5.58 (q, 1H, J = 7.20 Hz, Dhb-Hβ);
FAB-MS calcd. for C50H81N14O16S2 [M ϩ H] 1197.53, found
1197.58.
A similar procedure was conducted as for compound E-1.
LRMS (MALDI): calculated C54H86N14O14S2 1218.59, found
1219.28 [M ϩ H]. The same procedure was conducted for the
cyclization of 21 as described for methyllanthionine 4. LRMS
(MALDI): calculated C54H88N14O14S2 1220.60, found 1221.13
[M ϩ H]. 1H NMR analysis showed the presence of the
quartets corresponding to the vinyl protons of the two Dhb
residues.
A-MeCys-PGU(Ph)VA (13a–d)
The mixture of peptides was synthesized on an automated
peptide synthesizer by standard Fmoc chemistry using either
syn-15 or a mixture of syn/anti-15. Fmoc-Ala-Wang resin
(0.16 g, 0.10 mmol) was swollen in DMF for 30 min. Couplings
were allowed to proceed for 40 min for Fmoc-Val-OH and
Fmoc-Gly-OH, 60 min for Fmoc-Ala-OH and Fmoc-Pro-OH,
and 120 min for Fmoc-MeCystine-OH and FmocSec(Ph)-OH.
Coupling of syn-Fmoc-MeCystine-OH (15, 0.071 g, 0.1 mmol)
was carried out in the presence of HOBt (0.027 g, 0.2 mmol)
and DIC (0.032 mL, 0.2 mmol). The resin was isolated by
filtration, washed with DMF, ethanol and CH2Cl2, and dried in
vacuo in a desiccator. The peptide was cleaved from the resin
using 10 mL of 95% TFA and 5% CH2Cl2 for 1.5 h, and the
solvent was removed (10 mg, 13%). MALDI-MS for [M ϩ H]
calcd. 1513.6, found 1513.5. The mixture of diastereomeric
disulfides (4.2 mg, 0.0028 mmol) was dissolved in 4 mL of H2O.
Zn powder (41 mg, 0.63 mmol) and 0.1 mL of TFA were added
to the mixture. The reaction was stirred at rt for 5 h and purified
on RP-HPLC (13a, 1.0 mg, 24%, 13b, 1.2 mg, 29%). FAB-
HRMS for C31H48N7O8S80Se [M ϩ H] calcd. 758.2450, found
Methyllanthionine 4
Peptide E-1 (1.0 mg, 0.8 µmol) was dissolved in 0.52 mL of
degassed CH3CN/20 mM aq. NH4OAc (1 : 1). A solution of
TCEP (1.7 mg, 6.0 µmol) in 20 mM NH4OAc (0.09 mL) was
added and the reaction mixture was stirred at room temperature
until complete as determined by RP-HPLC. Aqueous NH4OAc
(20 mM, 3.0 mL) was added to the mixture and the pH of
the mixture was adjusted to 8–9 by the addition of Et3N.
The cyclization was complete in about 10 h as determined by
RP-HPLC. The mixture was purified by RP-HPLC to give the
1
product 4 (0.7 mg). Yield: 70%. H NMR (D2O, 500 MHz):
δ ppm 0.78 (d, J = 6.62 Hz, 3H, Val-CH3), 0.79 (d, J = 6.46 Hz,
3H, Val-CH3), 1.13 (d, J = 9.57 Hz, 3H, Cys(Me)-CH3), 1.16 (d,
J = 8.07 Hz, 3H, Ala-CH3), 1.39 (d, J = 6.46 Hz, 3H, Ala-CH3),
1.74 (m, 1H, Pro-Hβ), 1.83 (m, 1H, Pro-Hβ), 1.91 (m, 1H,
Val-Hβ), 2.02 (m, 1H, Pro-2Hγ), 2.26 (m, 1H, Pro-Hα), 2.65 (AB
of ABX, 1H, J = 4.78 Hz, Cys-Hβ), 2.89 (qd, 1H, J = 6.89, 3.22,
MeLan-Hβ), 3.12 (AB of ABX, J = 3.09 Hz, 1H, MeLan-Hβ),
3.40 (m, 2H, Pro-Hδ), 3.82 (q, 2H, J = 6.69 Hz, Gly-Hα), 3.92 (q,
1H, J = 6.12 Hz, Ala-Hα), 3.95 (d, 1H, J = 8.03 Hz, Val-Hα), 3.99
(q, 1H, J = 7.08 Hz, Ala-Hα), 4.80 (d, 1H, J = 9.75 Hz, Pro-Hα),
4.84 (t, J = 3.78 Hz, 1H, Cys-Hα), 4.90 (d, J = 3.22 Hz, 1H,
MeLan-Hα); FAB-HRMS calcd for C25H42N7O8S [M ϩ H]
600.2816, found 600.2818.
1
758.2450. Compound 13a H NMR: (D2O, 500 MHz): δ ppm
0.71 (d, J = 6.83 Hz, 3 H, Val-CH3), 0.74 (d, J = 6.76 Hz, 3 H,
Val-CH3), 1.20 (d, J = 7.07 Hz, 3 H, Ala-CH3), 1.21 (d, J = 6.23
Hz, 3 H, MeCys-CH3), 1.42 (d, J = 7.17 Hz, 3 H, Ala-CH3),
1.83–1.92 (m, 4 H, Pro-Hβ, Pro-2Hγ ϩ Val-Hβ), 2.14–2.19 (m,
1 H, Pro-Hβ), 3.09 (B of ABX, Jab = 13.67 Hz, Jbx = 9.45 Hz,
1 H, Sec-CH2), 3.20 (A of ABX, Jab = 13.67 Hz, Jax = 3.91 Hz,
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 3 0 4 – 3 3 1 5
3313

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1 H, Sec-CH2), 3.22 (m, 1 H, MeCys-Hβ), 3.65 (AB, Jab = 17.10
Hz, 2 H, Gly-CH2), 3.61–3.71 (m, 2 H, Pro-Hδ), 3.87 (d, J = 7.10
Hz, 1 H, Val-Hα), 3.99-4.01 (m, 2 H, Ala-Hα), 4.28 (dd, J = 8.35,
4.75 Hz, 1 H, Pro-Hα), 4.36-4.39 (m, 1 H, Sec-Hα), 4.62 (m, 1 H,
MeCys-Hα), 7.17–7.22 (m, 3 H, Ph), 7.40–7.43 (m, 2 H, Ph).
Compound 13b: 1H NMR: (D2O, 500 MHz): δ ppm 0.71 (d, J =
6.73 Hz, 3 H, Val-CH3), 0.74 (d, J = 6.76 Hz, 3 H, Val-CH3),
1.20 (d, J = 7.11 Hz, 3 H, Ala-CH3), 1.23 (d, J = 6.96 Hz, 3 H,
MeCys-CH3), 1.35 (d, J = 7.08 Hz, 3 H, Ala-CH3), 1.74–1.83
(m, 1 H, Pro-Hβ), 1.83–1.91 (m, 2 H, Pro-Hγ ϩ Val-Hβ), 1.91–
1.99 (m, 1 H, Pro-Hγ), 2.14–2.22 (m, 1 H, Pro-Hβ), 3.10 (B of
ABX, Jab = 14.30 Hz, Jbx = 7.34 Hz, 1 H, Sec-CH2), 3.21 (A
of ABX, Jab = 14.30 Hz, Jax = 6.56 Hz, 1 H, Sec-CH2), 3.30 (m,
1 H, MeCys-Hβ), 3.55–3.60 (m, 1 H, Pro-Hδ), 3.67–3.71 (m,
1 H, Pro-Hδ), 3.68 (AB, Jab = 17.10 Hz, 2 H, Gly-CH2), 3.87
(d, J = 7.40 Hz, 1 H, Val-Hα), 3.98–4.02 (m, 2 H, Ala-Hα), 4.25
(t, J = 7.40 Hz, 1 H, Pro-Hα), 4.39–4.42 (m, 1 H, Sec-Hα), 4.62
(m, 1 H, MeCys-Hα), 7.19–7.20 (m, 3 H, Ph), 7.41–7.43 (m,
2 H, Ph). A-(2R,3S)-MeCys-PGU(Ph)VA (13c) and A-(2S,3R)-
MeCys-PGU(Ph)VA (13d) were prepared similarly using the
four diastereomers of Fmoc-( )-MeCystine-OH (15). The
corresponding disulfides were obtained in 20% yield (138 mg,
0.45 mmol synthesis) and after reduction with TCEP the four
diastereomers were separated by RP-HPLC (C18 column,
gradient 2–100% MeCN in H2O, 0.1% TFA, elution order 13b,
13a, 13c, 13d). A-(2R,3S)-MeCys-PGU(Ph)VA was obtained in
19% yield, FAB-HRMS for C31H48N7O8S80Se [M ϩ H] calcd.
758.2450, found 758.2447 and A-(2S,3R)-MeCys-PGU(Ph)VA
was obtained in 32% yield, FAB-HRMS for C31H48N7O8S80Se
[M ϩ H] calcd. 758.2450, found 758.2447. 13c 1H NMR: (D2O,
500 MHz): δ ppm 0.73 (d, J = 6.64 Hz, 3 H, Val-CH3), 0.75 (d,
J = 6.81 Hz, 3 H, Val-CH3), 1.19 (d, J = 6.84 Hz, 3 H, Ala-CH3),
1.26 (d, J = 7.48 Hz, 3 H, MeCys-CH3), 1.34 (d, J = 7.03 Hz,
3 H, Ala-CH3), 1.83–1.95 (m, 4 H, Pro-Hβ, Pro-2Hγ ϩ Val-Hβ),
2.16–2.18 (m, 1 H, Pro-Hβ), 3.09 (B of ABX, Jab = 14.74 Hz,
Jbx = 8.77 Hz, 1 H, Sec-CH2), 3.21 (A of ABX, Jab = 14.74 Hz,
Jax = 6.64 Hz, 1 H, Sec-CH2), 3.15 (m, 1 H, MeCys-Hβ), 3.69
(AB, Jab = 16.97 Hz, 2 H, Gly-CH2), 3.61–3.71 (m, 2 H, Pro-Hδ),
3.87 (d, J = 7.66 Hz, 1 H, Val-Hα), 3.97 (q, J = 7.32 Hz, 1 H,
Ala-Hα), 4.11 (q, J = 7.56 Hz, 1 H, Ala-Hα), 4.28 (m, 1 H, Pro-
Hα), 4.40 (m, 1 H, Sec-Hα), 4.62 (m, 1 H, MeCys-Hα), 7.17–7.26
(m, 3 H, Ph), 7.40–7.46 (m, 2 H, Ph). 13d 1H NMR: (D2O, 500
MHz): δ ppm 0.73 (d, J = 6.70 Hz, 3 H, Val-CH3), 0.75 (d, J =
6.99 Hz, 3 H, Val-CH3), 1.20 (d, J = 6.91 Hz, 3 H, Ala-CH3),
1.25 (d, J = 7.40 Hz, 3 H, MeCys-CH3), 1.40 (d, J = 7.07 Hz,
3 H, Ala-CH3), 1.83–1.95 (m, 4 H, Pro-Hβ, Pro-2Hγ ϩ Val-Hβ),
2.16–2.18 (m, 1 H, Pro-Hβ), 3.08–3.12 (m, 2 H, Sec-CH2 and
MeCys-Hβ), 3.19–3.22 (m, 1 H, Sec-CH2), 3.67 (AB, Jab = 16.89
Hz, 2 H, Gly-CH2), 3.68–3.79 (m, 2 H, Pro-Hδ), 3.87 (d, J = 7.61
Hz, 1 H, Val-Hα), 3.95 (q, J = 7.13 Hz, 1 H, Ala-Hα), 4.10
(q, J = 7.15 Hz, 1 H, Ala-Hα), 4.33 (m, 1 H, Pro-Hα), 4.39 (m,
1 H, Sec-Hα), 4.62 (m, 1 H, MeCys-Hα), 7.19–7.23 (m, 3 H, Ph),
7.40-7.46 (m, 2 H, Ph).
Pro-2Hδ), 3.88 (d, J = 7.90 Hz, 1 H, Val-Hα), 3.98–4.02 (m,
2 H, Ala-Hα), 4.22 (t, J = 7.40 Hz, 1 H, Pro-Hα), 4.39–4.42 (m,
1 H, Sec-Hα), 7.16–7.20 (m, 3 H, Ph), 7.34–7.45 (m, 2 H, Ph).
[A-(2S,3S)-MeCys-PGU(Ph)VA]2 was prepared similarly from
13a (1.0 mg, 0.0013 mmol). The product was obtained in 40%
yield (0.4 mg). FAB-HRMS for [M ϩ H] calcd. 1513.4666,
1
found 1513.4666. H NMR: (D2O, 500 MHz): δ ppm 0.71 (d,
J = 6.70 Hz, 3 H, Val-CH3), 0.74 (d, J = 6.69 Hz, 3 H, Val-CH3),
1.18 (d, J = 6.97 Hz, 3 H, Ala-CH3), 1.22 (d, J = 7.22 Hz, 3 H,
MeCys-CH3), 1.39 (d, J = 6.95 Hz, 3 H, Ala-CH3), 1.81–1.95
(m, 4 H, Pro-Hβ, Pro-2Hγ ϩ Val-Hβ), 2.10-2.18 (m, 1 H, Pro-
Hβ), 3.08–3.14 (m, 1 H, Sec-CH2), 3.17–3.24 (m, 2 H, Sec-CH2
ϩ MeCys-Hβ), 3.65 (s, 2 H, Gly-CH2), 3.61–3.70 (m, 2 H, Pro-
2Hδ), 3.86 (d, J = 7.20 Hz, 1 H, Val-Hα), 3.96 (q, J = 7.40, 1 H,
Ala-Hα), 4.04 (q, J = 7.59, 1 H, Ala-Hα), 4.25 (dd, J = 8.45, 4.85
Hz, 1 H, Pro-Hα), 4.38 (m, 1 H, Sec-Hα), 4.74 (d, J = 7.70, 1 H,
MeCys-Hα), 7.16–7.23 (m, 3 H, Ph), 7.37–7.43 (m, 2 H, Ph).
[A-(2R,3S)-MeCys-PGU(Ph)VA]2 was prepared from 13c
(7.4 mg, 0.010 mmol) in 84% yield (6.2 mg). MS-HRESI for
1
[M ϩ H]2ϩ calcd. 757.2375, found 757.2357. H NMR: (D2O,
500 MHz): δ ppm 0.73 (d, J = 6.85 Hz, 3 H, Val-CH3), 0.76 (d,
J = 6.73 Hz, 3 H, Val-CH3), 1.11 (d, J = 7.07 Hz, 3 H, MeCys-
CH3), 1.25 (d, J = 7.33 Hz, 3 H, Ala-CH3), 1.37 (d, J = 7.12 Hz,
3 H, Ala-CH3), 1.80 (m, 1 H, Pro-Hβ), 1.81–1.95 (m, 2 H, Pro-
2Hγ), 1.95–1.98 (m, 1 H, Val-Hβ) 2.16–2.18 (m, 1 H, Pro-Hβ),
3.07 (B of ABX, Jab = 13.31 Hz, Jbx = 7.48 Hz, 1 H, Sec-CH2),
3.19 (A of ABX, Jab = 13.31 Hz, Jax = 6.58 Hz, 1 H, Sec-CH2),
3.45 (m, 1 H, MeCys-Hβ), 3.59 (m, 1 H, Pro-Hδ), 3.69 (s, 2 H,
Gly-CH2), 3.74 (m, 1 H, Pro-Hδ), 3.89 (d, J = 7.28 Hz, 1 H,
Val-Hα), 4.02 (q, J = 7.16 Hz, 1 H, Ala-Hα), 4.10 (q, J = 6.95 Hz,
1 H, Ala-Hα), 4.28 (t, J = 7.55 Hz, 1 H, Pro-Hα), 4.40 (m, 1H,
Sec-Hα), 5.11 (d, J = 3.60 Hz, 1H, MeCys-Hα), 7.14–7.22 (m,
3H, Ph), 7.35–7.44 (m, 2 H, Ph). [A-(2S,3R)-MeCys-PGU-
(Ph)VA]2 was prepared from 13d (7.6 mg, 0.010 mmol) in 90%
yield (6.8 mg). MS-HRESI for [M ϩ H]2ϩ calcd. 757.2375,
found 757.2353. 1H NMR: (D2O, 500 MHz): δ ppm 0.73 (d, J =
6.76 Hz, 3 H, Val-CH3), 0.76 (d, J = 6.83 Hz, 3 H, Val-CH3),
1.14 (d, J = 6.95 Hz, 3 H, MeCys-CH3), 1.25 (d, J = 7.32 Hz,
3 H, Ala-CH3), 1.41 (d, J = 7.09 Hz, 3 H, Ala-CH3), 1.83–1.95
(m, 4 H, Pro-Hβ, Pro-2Hγ ϩ Val-Hβ), 2.11–2.16 (m, 1 H, Pro-
Hβ), 3.12 (B of ABX, Jab = 13.22 Hz, Jbx = 7.34 Hz, 1 H, Sec-
CH2), 3.22 (A of ABX, Jab = 13.22 Hz, Jax = 6.26 Hz, 1 H,
Sec-CH2), 3.29 (m, 1 H, MeCys-Hβ), 3.69 (AB, Jab = 17.05 Hz,
2 H, Gly-CH2), 3.64–3.76 (m, 2 H, Pro-Hδ), 3.88 (d, J = 7.05 Hz,
1 H, Val-Hα), 4.01 (q, J = 7.17 Hz, 1 H, Ala-Hα), 4.10 (q, J =
7.30 Hz, 1 H, Ala-Hα), 4.33 (dd, J = 8.30, 5.10 Hz, 1 H, Pro-Hα),
4.39 (m, 1 H, Sec-Hα), 5.05 (d, J = 5.60 Hz, 1 H, MeCys-Hα),
7.18–7.23 (m, 3 H, Ph), 7.39-7.43 (m, 2 H, Ph).
[A-(2R,3R)-MeCys-PGDhaVA]2 (17) and isomers 16, 18 and 19
[A-(2R,3R)-MeCys-PGU(Ph)VA]2 (0.6 mg, 0.0004 mmol) was
dissolved in 1 mL of H2O. H2O2 (30%, 10 µL) was added and
the reaction was stirred for 30 min. Another 10 µL of H2O2
was added and the mixture was stirred for another 30 min.
The mixture was then purified by RP-HPLC (0.3 mg, 64%).
[A-(2R,3R)-MeCys-PGU(Ph)VA]2
1
A-(2R,3R)-MeCys-PGU(Ph)VA 13b (1.2 mg, 0.0016 mmol)
was dissolved in 1 mL of H2O. A solution of I2 in MeOH
(0.0028 M) was dropped into the reaction until the yellow color
was sustained (after the 3rd drop). A drop of saturated Na2S2O3
was added to quench excess I2. The mixture was concentrated
and purified by RP-HPLC (0.6 mg, 50%). FAB-HRMS for
MALDI-MS for [M ϩ H] calcd. 1197.5, found 1197.7. H
NMR: (D2O, 500 MHz): δ ppm 0.79 (d, J = 7.90 Hz, 3 H,
Val-CH3), 0.82 (d, J = 6.85 Hz, 3 H, Val-CH3), 1.18 (d, J = 7.31
Hz, 3 H, Ala-CH3), 1.25 (d, J = 7.01 Hz, 3 H, MeCys-CH3), 1.34
(d, J = 6.99 Hz, 3 H, Ala-CH), 1.79–2.02 (m, 4 H, Pro-Hβ ϩ
Pro-2Hγ ϩ Val-Hβ), 2.13–2.20 (m, 1 H, Pro-Hβ), 3.06 (m, 1 H,
MeCys-Hβ), 3.63 (m, 1 H, Pro-Hδ), 3.73 (m, 1 H, Pro-Hδ), 3.86
(s, 2 H, Gly-CH2), 3.97–4.04 (m, 3 H, Val-Hα ϩ 2 Ala-Hα), 4.30
(m, 1 H, Pro-Hα), 5.51 (d, J = 3.80 Hz, 2 H, vinyl-H). [A-
(2S,3S)-MeCys-PGDhaVA]2 (16) was prepared similarly from
[A-(2S,3S)-MeCys-PGU(Ph)VA]2 (0.4 mg, 0.00026 mmol).
MALDI-MS for [M ϩ H] calcd. 1197, found 1197.7. 1H NMR:
(D2O, 500 MHz): δ ppm 0.79 (d, J = 6.75 Hz, 3 H, Val-CH3),
0.82 (d, J = 6.76 Hz, 3 H, Val-CH3), 1.19 (d, J = 6.72 Hz, 3 H,
1
[M ϩ H] calcd. 1513.4666, found 1513.4666. H NMR: (D2O,
500 MHz): δ ppm 0.70 (d, J = 6.87 Hz, 3 H, Val-CH3), 0.74 (d,
J = 6.85 Hz, 3 H, Val-CH3), 1.20 (d, J = 6.15 Hz, 3 H, Ala-CH3),
1.26 (d, J = 7.08 Hz, 3 H, MeCys-CH3), 1.34 (d, J = 7.27 Hz,
3 H, Ala-CH3), 1.76–1.92 (m, 3 H, Pro-Hβ ϩ Pro-Hγ ϩ Val-Hβ),
1.92–1.98 (m, 1 H, Pro-Hγ), 2.12–2.17 (m, 1 H, Pro-Hβ), 3.05–
3.13 (m, 2 H, Sec-CH2 ϩ MeCys-Hβ), 3.18 (m, 1 H, Sec-CH2),
3.65 (AB, Jab = 17.10 Hz, 2 H, Gly-CH2), 3.67–3.81 (m, 2 H,
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 3 0 4 – 3 3 1 5
3314

View Article Online
Ala-CH3), 1.23 (d, J = 7.37 Hz, 3 H, MeCys-CH3), 1.41 (d, J =
7.06 Hz, 3 H, Ala-CH3), 1.87–1.94 (m, 3 H, Pro-Hβ ϩ Pro-2Hγ),
1.95–2.02 (m, 1 H, Val-Hβ), 2.13–2.20 (m, 1 H, Pro-Hβ), 3.20
(m, 1 H, MeCys-Hβ), 3.65 (m, 1 H, Pro-Hδ), 3.73 (m, 1 H, Pro-
Hδ), 3.85 (s, 2 H, Gly-CH2), 3.96 (q, J = 7.13 Hz, 1 H, Ala-Hα),
4.03 (d, J = 7.90 Hz, 1 H, Val-Hα), 4.07 (q, J = 7.33 Hz, 1 H,
Ala-Hα), 4.30 (m, 1 H, Pro-Hα), 5.51 (d, J = 7.82 Hz, 2 H,
vinyl-H). [A-(2R,3S)-MeCys-PGDhaVA]2 (18) was prepared
from [A-(2R,3S)-MeCys-PGU(Ph)VA]2 (6.2 mg, 0.0041 mmol)
in 86% yield (4.2 mg). MS-ESI for [M ϩ H]2ϩ calcd. 599.3,
References
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found 599.7. H NMR: (D2O, 500 MHz): δ ppm 0.78 (d, J =
6.76 Hz, 3 H, Val-CH3), 0.80 (d, J = 6.81 Hz, 3 H, Ala-CH3),
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1
product of E-1 and showed identical H and COSY NMR
spectra.
Acknowledgements
This work was supported by grants from the Beckman Founda-
tion, the National Institutes of Health (GM58822), and the
Research Corporation (R10187). YZ acknowledges support
from a Zeneca Graduate Fellowship. NMR spectra were
obtained in the Varian Oxford Instrument Center for
Excellence, funded in part by the W.M. Keck Foundation,
NIH (PHS 1 S10 RR10444), and NSF (CHE 96-10502). The
purchase of the mass spectrometers was funded in part by the
National Institutes of Health (Voyager, RR 11966; Quattro,
RR 07141; Micromass 70-SE-4F, GM 27029; Micromass 70
VSE, RR 04648). Any opinions, findings, and conclusions
expressed in this publication are those of the authors and do
not necessarily reflect the views of the Beckman Foundation,
NIH, NSF, or the Research Corporation.
37 Rainin PS3 Automated Solid Phase Peptide Synthesizer: User Guide,
Rainin Instrument Company, Woburn, MA 01888, 1998.
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O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 3 0 4 – 3 3 1 5
3315