Long-range intramolecular s → N acyl migration: A study of the formation of native peptide analogues via 13-, 15-, and 16-membered cyclic transition states

Article abstract of DOI:10.1021/jo2023125

The intramolecular long-range S → N acyl migration via 13-, 15-, and 16-membered cyclic transition states to form native tetra- and pentapeptide analogues was studied on S-acylcysteine peptides containing β- or γ-amino acids. The pH-dependency study of the acyl migration via a 15-membered cyclic transition state indicated that the reaction is favored at a pH range from 7.0 to 7.6. Experimental observations are supported by structural and computational investigations.

Full text of DOI:10.1021/jo2023125

Article
pubs.acs.org/joc
Long-Range Intramolecular S → N Acyl Migration: A Study
of the Formation of Native Peptide Analogues via 13-, 15-, and
16-Membered Cyclic Transition States
Khanh Ha,‡ Mamta Chahar,‡ Jean-Christophe M. Monbaliu,‡,# Ekaterina Todadze,‡ Finn K. Hansen,‡,§
Alexander A. Oliferenko,‡ Charles E. Ocampo,‡ David Leino,‡ Aaron Lillicotch,‡ Christian V. Stevens,#
and Alan R. Katritzky*,‡,¶
‡Center for Heterocyclic Compounds, Department of Chemistry, University of Florida, Gainesville, Florida 32611-7200, United States
#Department of Sustainable Organic Chemistry and Technology, Faculty of Bioscience Engineering, Ghent University, B-9000 Ghent,
Belgium
§Institut fur Pharmazeutische und Medizinische Chemie, Heinrich Heine Universitat Dusseldorf, Universitatsstrasse 1,
̈
̈
̈
̈
40225 Dusseldorf, Germany
̈
¶Chemistry Department, King Abdulaziz University, Jeddah, 21589 Saudi Arabia
S
* Supporting Information
ABSTRACT: The intramolecular long-range S → N acyl
migration via 13-, 15-, and 16-membered cyclic transition states
to form native tetra- and pentapeptide analogues was studied
on S-acylcysteine peptides containing β- or γ-amino acids. The
pH-dependency study of the acyl migration via a 15-membered
cyclic transition state indicated that the reaction is favored at a
pH range from 7.0 to 7.6. Experimental observations are
supported by structural and computational investigations.
followed by a long-range S → N acyl migration to afford a
native peptide linkage. SAL showed high sequence tolerance at
the ligation junction, therefore expanding the number of
glycoproteins potentially accessible.17−19
INTRODUCTION
■
Native chemical ligation (NCL), the most common form of
chemical ligation, has become a widely used chemoselective
technique to synthesize large peptides based on a capture/
rearrangement concept involving S-acylation by a peptide-
thioester of the N-terminal cysteine residue of another peptide,
followed by a S → N acyl shift to form a native amide linkage.1,2
NCL has been extensively studied in peptidic compounds
bearing a cysteine residue at the N-terminus.3 Modified cysteine
scaffolds have also been incorporated for the syntheses of novel
peptides and proteins4−8 and for surface immobilization.9 For
instance, N-acylcysteines10 have found utility as photoactivatable
analogues of glutathione11 and for the synthesis of oxytocin-like
peptides12 or glycopeptides.13−15
Recently, Haase and Seitz reported that the chemical ligation
of peptides bearing internal cysteine residues is significantly
accelerated in comparison to peptides lacking cysteine. In
particular, the highest ligation rates and yields were obtained
when the cysteine residue was incorporated at the fifth or sixth
position from the N-terminus of the C-terminal coupling
segment. However, the method required relatively long reaction
times (48−72 h) and the ligation products were neither isolated
nor characterized.16 Sugar-assisted ligation (SAL) has also been
introduced as an approach to overcome problems in the
synthesis of glycopeptides. The technique uses a glycopeptide
which undergoes thioester exchange with a peptide thioester,
Our group has recently studied the feasibility of chemical
ligation of S-acylated cysteine peptides via 5-, 8-, 11-, and 14-
membered cyclic transition states,20,21 leading to the correspond-
ing native di-, tetra-, and pentapeptides without the use of
auxiliaries. Additionally, this work was extended to peptides
containing nonterminal cysteine units, thus affording the synthesis
of diverse peptidic sequences.22 It was demonstrated that chemical
ligation of S-acyl peptides via an 8-membered cyclic transition state
is disfavored, whereas the 11- and 14-membered cyclic transition
states are relatively favored. Computational studies demonstrated
that hydrogen bonding provides additional stabilization and thus
facilitates ligation to form native peptides. The conformational
preorganization was demonstrated to be particularly effective on
tetra- and higher peptides.23
Unnatural amino acids have been used to prepare peptide
analogues with enhanced enzymatic stability,24,25 improved
pharmacodynamics,26 and bioavailability.27 β-Amino acid residues,
which are often encountered in natural products,28 have been used
for obtaining peptidomimetics29 and for affecting secondary and
Received: November 8, 2011
Published: January 20, 2012
© 2012 American Chemical Society
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Figure 1. Long-range S → N acyl migration to form native peptide analogues.
Scheme 1. Preparation of S-Acyl Tripeptide 13
tertiary structures of synthetic peptides.30 Peptides containing
γ-amino acids have shown antianxiety, anticonvulsive, and relaxing
effects.31,32
To investigate the further applicability of our methodology to
access peptide analogues,20−23 we study now long-range S → N
acyl migrations of S-acylated cysteine peptides containing β- or
γ-amino acid residues via 13-, 15-, and 16-membered cyclic
transition states (Figure 1), leading to the formation of the
corresponding native tetra- and pentapeptide analogues. The
impact of pH on the long-range S → N acyl migration is
exemplified using a S-acyl tetrapeptide. Reaction mechanisms and
governing parameters are discussed in our computational study.
migration via a 13-membered cyclic transition state is illustrated
in Scheme 1. Boc protection of γ-aminobutyric acid 1 was
carried out according to a reported procedure33 (Scheme 1),
affording N-Boc-γ-aminobutyric acid 3 in 87% yield. Com-
pound 3 was converted into the corresponding N-(Boc-
aminoacyl)benzotriazolide 4a (80%). Coupling of 4a with
L-cysteine (5) was carried out in a CH3CN/H2O mixture (8:2) in
the presence of Et3N during 3 h at 20 °C, leading to the N-Boc
protected cysteine dipeptide 6 in 82% yield. S-Acylation of
dipeptide 6 with Cbz-L-alanyl benzotriazole (7) in a
acetonitrile−water mixture (10:1) gave S-acyl dipeptide 8 in
74% yield. Boc deprotection of dipeptide 8 using HCl(g) in
methanol gave the HCl salt 9, which was neutralized with
triethylamine in DMF to afford S-acyl dipeptide 10. The final
coupling of unprotected S-acyl dipeptide 10 with Boc-Gly-
Bt 11, followed by Boc-deprotection gave S-acyl tripeptide
13 (65%).
RESULTS AND DISCUSSION
■
Demonstration of S → N Acyl Migration in S-Acyl
Tripeptide 13 via a 13-Membered Cyclic Transition State.
The preparation of starting tripeptide 13 for S → N acyl
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Table 1. Characterization Data of Ligation Product Mixtures
b
c
product ratio (%)
[M + H]+
a
d
entry
starting material
crude yield (%)
ligated peptide
transacylation product
ligated peptide
transacylation product
1
2
3
4
13
23a
23b
23c
88
92
94
82
28, 14
35, 24
64, 26a
57, 26b
72, 15
65, 25
36, 27a
43, 27b
935
1183
1257
1257
674
809
835
835
a
The combined crude yield of ligated product was calculated according to the following equation: combined crude yield = ([ligated peptide] + 2 ×
b
[transacylation product])/[ starting material]. The S-deacylated peptide side products were removed during the workup. Determined by HPLC−
MS semiquantitative. The area of ion-peak resulting from the sum of the intensities of the [M + H]+ and [M + Na]+ ions of each compound was
c
d
integrated. HPLC−MS (ESI). Analyzed as disulfide dimer.
a
Scheme 2. Chemical Ligation of S-Acyl Tripeptide 13
a
The ligation compound 14 is drawn as a monomer for clarity.
In our first set of ligation experiments, a solution of
tripeptide 13 in a mixture of NaH2PO4/Na2HPO4 buffer and
acetonitrile (pH = 7.8) was subjected to microwave irradiation
at 37 °C. HPLC−MS (ESI) analysis of the crude reaction
mixture revealed that the reaction does not complete after 3 h
(50% conversion of 13). However, when carried out at 50 W
and 50 °C, the ligation reaction proceeded essentially to
completion within 1 h. HPLC−MS (ESI) analysis demon-
strated that the expected ligation product 14 is formed together
with the intermolecular transacylation product 15 in a 28:72
ratio (Table 1, Scheme 2). The desired ligation product 14 was
detected as its disulfide dimer, producing a m/z 935 [M + H]+
ion which allows unambiguous distinction between the liga-
tion product 14 and the starting tripeptide 13 (molecular ion
[M + H]+, m/z = 469). It is well-known that small peptides
containing a Cys residue easily dimerize to the corresponding
disulfide dimers in solution in the absence of reducing agent.34
This product mixture was subsequently purified by semi-
preparative HPLC which enabled the isolation and the
characterization of the native peptide 14. Ligated product 14
was then further characterized by analytical HPLC and HRMS
analysis (Table 2).
Demonstration of S → N Acyl Migration in S-Acyl
Tetrapeptide 23a via a 15-Membered Cyclic Transition
State. For the S → N ligation via a 15-membered cyclic
transition state, the starting material 23a was obtained in a six-
step procedure starting from the L-cystine dimethyl ester
dihydrochloride (16). The protected dipeptide dimer 17a was
prepared in 67% yield from the mixed anhydride coupling of 16
with Boc-Gly-OH following a literature procedure (Scheme 3).35
The deprotection of 17a using HCl(g) in methanol gave 18a
in 84% yield. The coupling of 18a with Boc-protected
dipeptide Boc-β-Ala-L-Leu-OH (19a) provided the tetrapeptide
dimer 20a in 65% yield, which was subsequently treated with
tributylphosphine to afford the tetrapeptide monomer 21a in
75% yield. S-Acylation of N-Boc-protected cysteine tetrapeptide
21a with Cbz-L-Ala-Bt (7) in acetonitrile−water (10:1) in the
presence of triethylamine gave S-acyl tetrapeptide 22a in 86%
yield. Finally, compound 22a was Boc-deprotected using
HCl(g) in methanol to give the HCl salt 23a in 82% yield
(Scheme 3).
The intramolecular S → N acyl migration experiment 23a →
24 would proceed through a 15-membered-ring transition state.
Although attempted 13 to 14 acyl migration via a 13-
membered-ring transition state favored the intermolecular
transacylation reaction to give 15, precedents30−32 for
successful S → N acyl shift in sugar-assisted ligation (SAL)
proceeding (Figure 2) through transition states with 15-
membered rings encouraged us to pursue this approach.
The chemical ligation experiment was carried out at 2 mM
concentration of 23a under microwave irradiation at 50 °C and
50 W for 1 h at pH = 7.6 (Scheme 4). HPLC−MS (ESI)
analysis of the crude reaction mixture revealed the presence of
two major products in a 35:65 ratio (Table 1). The HPLC−MS
(ESI) analysis identified the major reaction products as the
desired ligation product 24 and the intermolecular transacylation
product 25 (Table 1). Compound 24 was isolated by semi-
Table 2. Characterization Data of the Isolated Ligation
Products 14, 24, 26a,b
after semipreparative
b
HPLC
HRMS [M + Na]+
ligation
product
isolated
yield (%)
retention
a
c
purity
calculated
found
time
14
24
>92%
>98%
>99%
>95%
18
31
42
37
957.3093
1183.4774
1279.4774
957.3117
1183.4733
1279.4718
1279.4775
16.95
19.42
20.89
18.64
26a
26b
1279.4774
a
b
Purity based on analytical HPLC. Analyzed as disulfide dimer.
c
1
preparative HPLC and fully characterized by H and 13C NMR
254 nm, MeOH/H2O (65:35), 0.15 mL/min.
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Scheme 3. Preparation of S-Acyl Tetrapeptide 23a
23a has four typical amide 13C signals with chemical shifts ranging
from δ 171.5 to 175.4 ppm and a very typical thioester carbonyl
13C observed at δ 203.2 ppm. In contrast, compound 24 showed
distinct 13C NMR resonances for five amide bonds at δ 169.1,
170.7, 170.8, 172.4, and 172.5 ppm (Figure 3). These results clearly
confirmed the S → N migration of Cbz-alanine to the N-terminus
(Scheme 4).
Figure 2. Proposed 15-membered cyclic transition states of (A) the
second-generation sugar-assisted ligation30 (SAL) and (B) the long-
range intramolecular acyl-migration.
An additional set of two reactions using different
concentrations in substrate 23a were carried out under the
same conditions. Interestingly, HPLC−MS (ESI) analysis
revealed that the concentration of the substrate does not
have a significant influence on the ligation/transacylation ratio.
At a 2 mM concentration of 23a, the measured ligation/
transacylation ratio was 45:55, while at 0.5 mM and 6 mM the
ratios were 43:57 and 55:45, respectively.
1
spectroscopy, HRMS (ESI), and analytical HPLC (Table 2). H
NMR spectra indicated the formation of the desired ligation
product 24 (appearance of five amide proton signals), while 13C
NMR provided further evidence. Indeed, S-aminoacyltetrapeptide
a
Scheme 4. Chemical Ligation of S-Acyl Tetrapeptide 23a
a
The ligation compound 24 is drawn as a monomer for clarity.
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was carried out under microwave irradiation at 50 °C and 50 W
for 1 h in NaH2PO4/Na2HPO4 buffer (1 M, pH 7.6) and
acetonitrile (8:1 mixture). HPLC−MS (ESI) analysis of the
crude ligation mixture revealed the major component (64%) to
be the expected ligation product 26a (molecular ion of the
disulfide dimer [M + H]+ m/z 1257 vs 630 for the [M + H]+
molecular ion of the starting S-(Pg-α-aminoacyl)tetrapeptide
23b). The HPLC−MS (ESI) confirmed that the second major
product is 27a formed by intermolecular trans-acylation (Table 1,
Scheme 6). Separation of 26a by semipreparative HPLC
allowed isolation of 42% of pure pentapeptide 26a, which was
further characterized by analytical HPLC and HRMS analysis
(Table 2).
Similarly, the acyl migration experiment of S-acylpeptide 23c
was carried out under the conditions described above. HPLC−
MS (ESI) analysis showed the presence of two main products
in a 57:43 ratio (Table 1, Scheme 7). As expected, the major
product of this experiment was the desired ligation product
26b. The subsequent separation of 26b by semipreparative
HPLC provided the purified ligation product 26b in 37% yield.
The product was characterized by HPLC analytical and HRMS
(Table 2).
Study of pH Dependence on the Ligation Experiment.
Chemical ligation is pH sensitive: ligation usually proceeds
rapidly around pH 7 but is rendered less efficient at pH < 5.5.32,36
In addition, in some cases the yields for traceless Staudinger
ligation in water increased at higher pH but decreased
drastically at pH ≥ 8.5.37 The ratio of the ligation product
24 versus the intermolecular transacylated compound 25 was
studied under different pH conditions (Table 3, Figure 4).
Chemical ligation experiments on 23a were carried out under
microwave irradiation at 50 °C and 50 W for 1 h in 1 M
phosphate buffer under pH values ranging from 6.2 to 8.2. After
workup, the crude ligation mixtures were subjected to HPLC−
MS (ESI) analysis. The results are summarized in Table 3 and
Figure 4. HPLC−MS (ESI) analysis of the crude ligation
mixture at pH = 6.2 showed the presence of the major
intermolecular transacylation product 25 and the desired
ligation product 24 in a 80:20 ratio. When the ligation
experiment was conducted at pH = 8.2, HPLC−MS (ESI)
analysis identified the same two products: the transacylated
product 25 and the desired ligated product 24 in a similar 85:15
ratio. In contrast, when ligation was carried out at pH 7.0 and at
pH 7.6, HPLC−MS (ESI) showed a significant increase in
ligation product 24 with calculated 24/25 ratios of 45:55 and
36:64, respectively. In the case of a lower pH, the starting
material 23a exists partially in the unreactive protonated form
and the acyl migration reaction is relatively slow, which favors
the intermolecular S-acylation of 24 by the excess of starting
material 23a to form the undesired product 25. At pH > 8, the
thiol group of the ligation product 24 is partially deprotonated,
which again favors the formation of the transacylation product
25 in a subsequent intermolecular reaction of 24 with
unreacted 23a. At pH = 7.3, a 24/25 ratio of 43:57 was
obtained. The S → N acyl migration in S-acyl tetrapeptide 23a
via a 15-membered cyclic transition state to give 24 is therefore
favored at a pH range from 7.0 to 7.6.
Figure 3. 13C NMR carbonyl signals in (A) ligated pentapeptide 24
and in (B) starting S-acylated tetrapeptide 23a.
Demonstration of S → N Acyl Migrations in S-Acyl
Tetrapeptides 23b and 23c Each via Distinct Isomeric
16-Membered Cyclic Transition States. To study the
chemical ligation via a 16-membered cyclic transition state, we
first prepared the tetrapeptide dimer 20b in 52% yield by
coupling amino-unprotected cystine dipeptide dimer 18b with
Boc-β-Ala-L-Phe-OH (19b). Then, tetrapeptide dimer 20b was
treated with tributylphosphine to release the tetrapeptide
monomer 21b in 68% yield. S-Acylation of N-Boc-protected
cysteine tetrapeptide 21b with Cbz-L-Ala-Bt (7) in a
acetonitrile−water mixture (10:1) in the presence of triethyl-
amine afforded S-acyl tetrapeptide 22b in 88% yield. Boc
deprotection of 22b using HCl(g) in methanol gave the
hydrochloride 23b in 81% yield (Scheme 5).
Similarly, 18b was coupled with Boc-GABA-L-Phe-OH (19c)
to afford Boc-protected tetrapeptide dimer 20c in 57% yield.
Cleavage of the disulfide bond in 20c furnished the monomer
21c (66%), which was S-acylated with Cbz-L-Ala-Bt (7) to
provide the Boc-protected S-acyl tetrapeptide 22c (92%). Final
Boc deprotection afforded the desired amino-unprotected
S-acylated tetrapeptide 23c in 87% yield (Scheme 5).
Computational Investigation. The elementary step of the
chemical ligation was investigated by computational means for
the S-acylated tripeptide 13 and for tetrapeptides 23a and 23c
(Cbz group was modeled by a methyl carbamate and the Phe
residue in tetrapeptide 23c was replaced by an Ala residue).
Tetrapeptide 23c was considered as a representative model for
We then investigated S → N acyl migrations for compounds
23b and 23c each of which proceeds via distinct isomeric 16-
membered cyclic transition states. Chemical ligation on 23b
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Scheme 5. Preparation of S-Acylated Tetrapeptides 23b and 23c
a
Scheme 6. Acyl Migration of S-Acyl Tetrapeptides 23b
a
The ligation product compound 26a is drawn as its monomer for clarity.
a
Scheme 7. Acyl Migration of S-Acyl Tetrapeptide 23c
a
The ligation product compound 26b is drawn as a monomer for clarity.
the 16-membered TS series. A full conformational search was
performed using the MMX force field by the PC Model program
package. Full geometry optimization and verification of the
Hessian have been performed by the G03 program package
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Table 3. Dependence of pH on the Product Ratio 24/25 in
the Ligation Reaction of 23a
Table 4. Bond Formation and Rupture (Å) among 13-, 15-,
and 16-Membered TSs
a
product ratio
TS
reactants
products
b
entry
pH
ligated peptide (24)
transacylation product (25)
ring size
N···C(O)
(OC)···S
(OC)−S
N−C(O)
1
2
3
4
5
6.2
7.0
7.3
7.6
8.2
20
45
43
36
15
80
55
57
64
85
13
15
16
1.56
1.56
1.56
2.42
2.49
2.35
1.79
1.78
1.79
1.35
1.35
1.35 h
a
Determined by HPLC−MS semiquantitation. The area of the ion-
peak resulting from the sum of the intensities of the [M + H]+ and
b
[M + Na]+ ions of each compound was integrated. Analyzed as disulfide
dimer.
Figure 5. Pictures of the 13-, 15-, and 16-membered TSs associated
with the long-range intramolecular S → N acyl transfer for the model
peptides 13, 23a, and 23c.
reactions are exothermic, the enthalpy of reaction ranging from
53.9 to 67.6 kJ mol−1.
The computed activation barriers for the 13-, 15-, and 16-
membered TSs associated with the long-range intramolecular
S → N acyl transfer for the model peptides 13, 23a, and 23c are
shown in Table 5. Values obtained using Tomasi’s PCM model
Table 5. Computed Activation Barriers for 13-, 15-, and 16-
TSs (kJ mol−1)
Figure 4. Effect of pH on S → N acyl migration in S-acyl tetrapep-
tide 23a.
ΔE⧧ (gas)
13-TS
15-TS
16-TS
165.0
224.8
186.0
(revision E.01).38 Transition states were optimized and
localized at the HF/6-31G* level with zero point energy
correction and were verified by frequency and IRC calculations.
All reactants were optimized in the gas phase at the HF/6-
31G* level. For the 13-membered cyclic case, TSs were also
isolated at the HF/6-31+G* and B3LYP/6-31G*. Neither the
inclusion of diffuse functions nor electronic correlation
fundamentally altered the nature of the TS. Single point
calculations were carried out for some representative examples
using Tomasi’s Polarizable Continuum (PCM) with dielectric
constants of 78.4 (water phase), 36.6 (acetonitrile), and 7.6
(tetrahydrofuran) to provide an indication of the sensitivity of
the reaction toward solvent polarity.
A full conformational analysis was performed on tripeptide
13 plus tetrapeptides 23a and 23c using the MMX force field.
In complete agreement with our previous study,23 the most
stable conformers of compounds 13, 23a, and 23b are folded
into a cyclic ready-to-ligate structure. The transition states
(TSs) for the long-range intramolecular S → N acyl migration
via 13-, 15-, and 16-membered cyclic transition states were
located and studied. As a common feature (Table 4 and Figure 5),
these TSs are late, in the Hammond postulate sense, i.e.,
product-like with a net transfer of the alanine residue from the
S-cysteine terminus (average bond length in rupture, 2.42 Å) to
the N-terminus (average bond length in formation, 1.56 Å).
The average advancement of the 13-, 15-, and 16-membered
TSs is about 90% among the reaction coordinate. These
are indicative of the reaction sensitivity to solvent polarity: the
activation barriers obtained for the tripeptide 13 leveled off at
122.7, 137.5, and 136.5 kJ mol−1 in water, acetonitrile, and
tetrahydrofuran, respectively.
For the long-range intramolecular S → N acyl transfer of
model peptide 13, we were also able to isolate a TS including
two molecules of water involved in an “active” H-bonding
network (Figure 6): the first one stabilizes the sulfur anion in
formation while the second one seems to interact with the N−H
terminus in order to facilitate the proton transfer and hence
acylation of the N terminus (see the Supporting Information
for details).
The activation barriers presented in Table 5 show some
trends. The lowest activation barrier was computed for the
chemical ligation of tripeptide 13 (13-membered cyclic TS),
even though the observed yield of the corresponding ligation
product was the lowest of the series. This result is probably
linked to the formation of a tight zwitterionic pair between the
N-terminus and the free carboxylic acid on the cysteyl residue
(tripeptide 13 is the only of the series to bear a free carboxylic
acid) that competes with the intramolecular acylation and
hence gives the desired ligation production in low yield (Table 1).
Notably, the TSs associated with the chemical ligation of
tripeptide 13 (13-TS) and tetrapeptide 23c (16-TS) display
stabilizing hydrogen bonding (short contacts ranging from 1.87
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EXPERIMENTAL SECTION
■
General Methods. Melting points were determined on a capillary
point apparatus equipped with a digital thermometer. H NMR and
1
13C NMR spectra were recorded in CDCl3, DMSO-d6, acetone-d6, or
CD3OD-d4 using a 300 or 500 MHz spectrometer (with TMS as an
internal standard). The following abbreviations are used to describe
spin multiplicity: s = singlet, d = doublet, t = triplet, q = quartet, m =
multiplet, brs = broad singlet, dd = doublet of doublets, ddd = doublet
of doublets of doublets. All microwave assisted reactions were carried
out with a single mode cavity Discover Microwave Synthesizer (CEM
Corporation, NC). The reaction mixtures were transferred into a 10 mL
glass pressure microwave tube equipped with a magnetic stirrer bar.
The tube was closed with a silicon septum and the reaction mixture
was subjected to microwave irradiation (Discover mode; run time:
60 s.; PowerMax-cooling mode). HPLC−MS analyses were
performed on a reverse phase gradient using 0.2% acetic acid in
H2O/methanol as mobile phases; wavelength = 254 nm; and mass
spectrometry was done with electrospray ionization (ESI). Ether
refers to diethyl ether.
Figure 6. Pictures of the 13-membered TSs for the long-range
intramolecular S → N acyl transfer catalyzed by water for the model
peptide 13. Distances are indicated in Å.
Boc-GABA-OH (3). Di-tert-butyl dicarbonate (4.37 g, 20 mmol)
was added to a stirred solution of 4-aminobutyric acid (20 mmol, 2.06 g)
in 1,4-dioxane (60 mL), NaOH (1 M, 60 mL), and H2O (60 mL) at
0 °C. The reaction mixture was stirred for 1 h at room temperature and
was concentrated under reduced pressure. The residual solution was
acidified with a 10% aqueous citric acid to pH 2−3 in an ice bath. This
aqueous phase was extracted with EtOAc (4 × 60 mL), and the
organic layers were combined and washed with H2O (2 × 50 mL) and
saturated brine (20 mL). The organic solution was dried over
anhydrous Na2SO4, filtered from drying agent, and concentrated under
reduced pressure. Recrystallization from EtOAc gave Boc-GABA-OH.
White microcrystals, 3.54 g, 87% yield; mp 54−56 °C. 1H NMR
(CDCl3, 300 MHz): δ 1.44 (s, 9H), 1.82 (quint, J = 7.1 Hz, 2H), 2.39
(t, J = 7.2 Hz, 2H), 3.10−3.25 (m, 2H), 4.77 (br s, 1H), 10.60 (br s,
1H). 13C NMR (CDCl3, 75 MHz): δ 25.3, 28.6, 31.5, 40.0, 79.7, 156.4,
178.5. Anal. Calcd for C9H17NO4: C 53.19, H 8.43, N 6.89. Found: C
53.26, H 8.73, N 6.77.
to 2.20 Å observed between the CO and NH of the GABA
residue), while the TS associated to tetrapeptide 23a (15-TS)
does not include this feature. The absence of an electrostatic
stabilizing interaction in the latter is consistent with the higher
computed activation barrier (Table 5) and, hence, the lower
yield observed experimentally (Table 1). In the 16-TS, the
above-mentioned short-contact not only folds the structure-
bringing the reactive moiety to a closer proximity but also
increases the nucleophilicity (increased negative NBO charge)
of the N-terminus, in agreement with the superior ligation yield
experimentally observed for this series (Table 1). These results
are in good agreement with the experimental observations and
strongly suggest that the intramolecular long-range acyl
migration of 23c is favored compared to chemical ligation for
compounds 13 and 23a.
Boc-GABA-Bt (4a). A solution of N-Boc-GABA-OH (3.23 g, 15.9
mmol) in CH2Cl2 (30 mL) was added to a solution of
dicyclohexylcarbodiimide (3.27 g, 15.9 mmol) and 1H-benzotriazole
(1.89 g, 15.9 mmol) in CH2Cl2 (10 mL). The reaction mixture was
stirred at room temperature for 1.5 h. The precipitate was filtered, and
the solution was passed through 6.00 g of Celite. The solvent was
evaporated under reduced pressure, and the crude mixture obtained
was dissolved in EtOAc (50 mL). The organic layer was washed with
saturated Na2CO3 (3 × 30 mL), brine (20 mL) and dried over
MgSO4. Recrystallization from CH2Cl2−hexanes gave Boc-GABA-Bt.
CONCLUSIONS
■
In this paper, we successfully apply our methodology to
synthesize peptide analogues and also provide mechanistic
evidence for the ligation process. A series of novel S-acyl
peptides containing β- and/or γ-amino acid residues, which are
useful intermediates in various synthetic and biological
applications, were synthesized according to original protocols.
The ligation step was investigated under MW heating and was
found to be sensitive to pH. The observed variation in ligation
yield allows for reactivity scaling. The native peptides obtained
after chemical ligation of tripeptide 13 and tetrapeptides 23a−c
via 13-, 15-, and 16-membered cyclic transition states were
isolated in modest to good yields. Interestingly, the ligation
products 26a and 26b derived through isomeric 16-membered
cyclic TSs were obtained in similar yields. In a representative
example, the native peptide was isolated and fully characterized.
The experimental results show that the extent of chemical
ligation follows the following scale: 13-TS ≤ 15-TS ≪ 16-TS.
Our computational study revealed that the chemical ligation
associated with 13-TS has the lowest activation barrier; but in
this case the formation of a tight zwitterionic pair may hamper
the long-range intramolecular S → N acyl transfer thus
decreasing the yield. The chemical ligation proceeding through
16-TS appeared to be highly favored in comparison with the
15-TS. The importance of stabilization by intramolecular short
contacts has been illustrated.
1
White microcrystals, 4.68 g, 97% yield; mp 108−110 °C. H NMR
(CDCl3, 300 MHz): δ 1.41 (br s, 9H), 2.12 (p, J = 7.0 Hz, 2H), 3.29−
3.36 (m, 2H), 3.49 (t, J = 7.2 Hz, 2H), 4.72 (br s, 1H), 7.52 (ddd, J =
8.2, 7.1, 1.1 Hz, 1H), 7.67 (ddd, J = 8.2, 7.2, 1.0 Hz, 1H), 8.13 (ddd,
J = 8.2, 0.8, 0.8 Hz, 1H), 8.29 (ddd, J = 8.6, 1.1, 0.8 Hz, 1H). 13C
NMR (CDCl3, 75 MHz): δ 25.0, 28.5, 32.9, 39.9, 79.5, 114.6, 120.3,
126.3, 130.6, 131.3, 146.3, 156.2, 172.2. Anal. Calcd for C15H20N4O3:
C 59.20, H 6.62, N 18.41. Found: C 59.34, H 6.71, N 18.39.
Boc-GABA-L-Cys-OH (6). Boc-GABA-Bt (0.50 g, 1.6 mmol) was
suspended in acetonitrile (20 mL), and a solution of L-cysteine (0.20 g,
1.6 mmol) in water containing triethylamine (0.19 mL, 1.5 mmol) was
added. The mixture was stirred at 20 °C until the TLC revealed
complete consumption of the starting materials. The solvent was
removed under reduced pressure, and the residue was taken in ethyl
actetate. Then the solution was washed with 3 N HCl (3 × 15 mL)
and brine (10 mL). Recrystallization from ethyl acetate−hexanes
yielded Boc-GABA-L-Cys-OH. Sticky white solid, 0.41 g, 84% yield. 1H
NMR (DMSO-d6, 300 MHz): δ 1.37 (br s, 9H), 1.54−1.62 (m, 2H),
1.99 (s, 1H), 2.10 (t, J = 7.4 Hz, 2H), 2.85−2.94 (m, 3H), 3.12 (dd,
J = 13.7, 4.7 Hz, 1H), 4.43−4.50 (m, 1H), 6.80 (br s, 1H), 8.25 (d, J =
8.2 Hz, 1H). 13C NMR (CDCl3, 75 MHz): δ 26.9, 28.5, 32.7, 40.0,
53.8, 80.0, 156.9, 172.9, 173.8. Anal. Calcd for C12H22N2O5S: C 47.04,
H 7.24, N 9.14. Found: C 47.04, H 6.97, N 9.38.
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Boc-GABA-Cys(S-L-Cbz-Ala)-OH (8). To a solution of Boc-
GABA-L-Cys-OH (1.0 g, 3.26 mmol) in acetonitrile (15 mL),
KHCO3 (0.32 g, 6.5 mmol) in water was added dropwise at room
temperature. After 15 min L-Cbz-Ala-Bt (1.05 g, 3.26 mmol) was
added slowly to the reaction mixture. The solution was further stirred
at room temperature for 5 h. Acetonitrile was removed under reduced
pressure, and the residue formed was taken in ethyl acetate (30 mL)
and washed with 3 N HCl (3 × 15 mL) and brine (10 mL). Then, the
solution was concentrated under reduced pressure, and hexane was
added until the solution was turbid and the compound was left to
crystallize in the freezer. The solid obtained was filtered and dried to
give Boc-GABA-Cys(S-L-Cbz-Ala)-OH. White microcrystal, 1.23 g,
74% yield; mp 70−72 °C. 1H NMR (DMSO-d6, 300 MHz): δ 1.25 (d,
J = 7.0 Hz, 3H), 1.37 (s, 9H), 1.52−1.62 (m, 2H), 2.08 (t, J = 7.2 Hz,
2H), 2.84−2.92 (m, 2H), 3.02 (dd, J = 13.4, 8.6 Hz, 1H), 3.27 (dd, J =
13.6, 5.0 Hz, 1H), 4.15−4.24 (m, 1H), 4.26−4.36 (m, 1H), 4.90−5.10
(m, 2H), 6.80 (br s, 1H), 7.22−7.46 (m, 5H), 8.07 (d, J = 7.3 Hz, 1H),
8.20 (d, J = 8.0 Hz, 1H). 13C NMR (DMSO-d6, 75 MHz): δ 17.3, 25.8,
28.3, 29.6, 32.6, 51.3, 56.6, 65.7, 77.4, 127.7, 128.4, 136.7, 155.7, 155.8,
171.7, 171.9, 201.7. Anal. Calcd for C23H33N3O8S: C 54.00, H 6.50,
N 8.21. Found: C 53.63, H 6.59, N 8.45.
158.5, 172.7, 173.3, 175.5, 203.3. Anal. Calcd for C25H36N4O2S: C
52.80, H 6.38, N 9.85. Found: C 53.12, H 6.65, N 10.20.
Gly-GABA-L-Cys(S-L-Cbz-Ala)-OH Hydrochloride (13). Boc-
Gly-GABA-Cys(S-L-Cbz-Ala)-OH (0.3 g, 0.52 mmol) was dissolved
in dioxane/HCl(g) solution (8 mL), and the reaction mixture was
stirred at room temperature. After 1 h, the solvent was removed under
reduced pressure, and the residue was crystallized from methanol−
diethyl ether to yield the title compound, Gly-GABA-Cys(S-L-Cbz-
Ala)-OH hydrochloride. White solid, 0.17 g, 65% yield; mp 80−83 °C.
1H NMR (CD3OD, 300 MHz): δ 1.35 (d, J = 6.7 Hz, 3H), 1.80−1.88
(m, 2H), 2.23−2.32 (m, 2H), 3.19 (dd, J = 13.8, 7.6 Hz, 1H), 3.28 (br
s, 2H), 3.50 (dd, J = 13.7, 3.5 Hz, 1H), 3.71 (br s, 2H), 4.28−4.30 (m,
1H), 4.60−4.64 (m, 1H), 5.09 (br s, 2H), 7.28−7.34 (m, 5H), 7.53−
7.54 (m, 3H), 7.90−7.92 (m, 3H). 13C NMR (CD3OD, 75 MHz): δ
18.4, 26.8, 31.2, 34.4, 40.4, 42.1, 53.6, 58.8, 68.3, 128.1, 129.1, 130.0,
138.4, 158.8, 167.7, 173.6, 175.8, 203.8. Anal. Calcd for
C20H29ClN4O7S·0.5H2O: C 46.74, H 5.88, N 10.90. Found: C 46.68,
H 5.96, N 10.85.
Boc-β-Ala-Bt (4b). A solution of N-Boc-β-Ala-OH (3.00 g, 15.9
mmol) in CH2Cl2 (30 mL) was added to a solution of
dicyclohexylcarbodiimide (3.27 g, 15.9 mmol) and 1H-benzotriazole
(1.89 g, 15.9 mmol) in CH2Cl2 (10 mL). The reaction mixture was
stirred at room temperature for 1.5 h. The precipitate was filtered, and
the solution was passed through 6.00 g of Celite. The solution was
evaporated under reduced pressure, and the crude mixture obtained
was dissolved in EtOAc (50 mL). The organic layer was washed with
saturated Na2CO3 (3 × 30 mL) and brine (20 mL) and dried over
MgSO4. Concentration under reduced pressure produced the final
product. Recrystallization from CH2Cl2−hexanes gave Boc-β-Ala-Bt.
GABA-L-Cys(S-L-Cbz-Ala)-OH Hydrochloride (9). HCl(g) was
passed through a solution of Boc-GABA-Cys(S-L-Cbz-Ala)-OH (0.5 g,
0.97 mmol) in methanol (8 mL) for 30 min at room temperature. The
solvent was then evaporated under reduced pressure, and the residue
was recrystallized from methanol−diethyl ether to yield the product
GABA-L-Cys(S-L-Cbz-Ala)-OH hydrochloride. Sticky white solid, 0.41 g,
1
89% yield. H NMR (DMSO-d6, 300 MHz): δ 1.25 (d, J = 7.3 Hz,
3H), 1.72−1.84 (m, 2H), 2.17−2.26 (m, 2H), 2.71−2.82 (m, 2H),
3.04 (dd, J = 13.5, 8.5 Hz, 1H), 3.28 (dd, J = 13.7, 5.2 Hz, 1H), 4.15−
4.25 (m, 1H), 4.28−4.37 (m, 1H), 5.06 (s, 2H), 7.26−7.40 (m, 5H),
7.99 (br s, 3H), 8.10 (d, J = 7.4 Hz, 1H), 8.4 (d, J = 8.0 Hz, 1H). 13C
NMR (DMSO-d6, 75 MHz): δ 17.3, 23.1, 29.5, 31.8, 38.3, 51.5, 56.7,
65.7, 127.7, 127.9, 128.4, 136.8, 155.8, 171.4, 171.6, 201.8. Anal. Calcd
for C18H26ClN3O6S·1.5H2O: C 45.52, H 6.15, N 8.85. Found: C
45.19, H 6.59, N 8.95.
1
White microcrystals, 3.46 g, 63% yield; mp 112−115 °C. H NMR
(CDCl3, 300 MHz): δ 1.43 (s, 9H), 3.66−3.70 (m, 4H), 5.11 (br s,
1H), 7.53 (t, J = 7.6 Hz, 1H), 7.68 (t, J = 7.6 Hz, 1H), 8.14 (d, J = 8.2
Hz, 1H), 8.28 (d, J = 8.2 Hz, 1H). 13C NMR (CDCl3, 75 MHz): δ
28.5, 35.8, 36.6, 79.8, 114.4, 120.4, 126.4, 130.7, 131.1, 146.3, 155.9,
171.5. Anal. Calcd for C14H18N4O3: C 57.92, H 6.25, N 19.30. Found:
C 58.00, H 6.33, N 19.31.
GABA-L-Cys(S-L-Cbz-Ala)-OH (10). Triethylamine (0.24 g, 2.4
mmol) was added dropwise to a solution of GABA-L-Cys(S-L-Cbz-
Ala)-OH hydrochloride (0.5 g, 1.2 mmol) in DMF (7 mL), and the
reaction mixture was stirred at room temperature for 30 min. A white
precipitate was filtered, washed with diethyl ether, and dried under
vacuum to give pure GABA-L-Cys(S-L-Cbz-Ala)-OH. White micro-
crystal, 0.38 g, 1.6 mmol, 77% yield; mp 110−114 °C. 1H NMR
(DMSO-d6, 300 MHz): δ 1.25 (d, J = 7.3 Hz, 3H), 1.76−1.84 (m,
2H), 2.18−2.24 (m, 2H), 2.74−2.86 (m, 2H), 3.00 (dd, J = 12.8,
7.8 Hz, 1H), 3.33 (dd, J = 13.0, 4.7 Hz, 1H), 3.23−3.44 (m, 2H),
4.08−4.12 (m, 1H), 4.12−4.20 (m, 1H), 5.01−5.09 (m, 2H), 7.30−
7.40 (m, 5H), 7.90 (d, J = 7.3 Hz, 1H), 7.99 (d, J = 7.6 Hz, 1H). 13C
NMR (DMSO-d6, 75 MHz): δ 17.5, 23.4, 31.2, 32.4, 45.1, 52.7, 56.6,
65.7, 127.7, 127.8, 128.4, 136.8, 155.7, 170.9, 172.4, 202.1. Anal. Calcd
for C18H25N3O6S·0.5H2O: C 51.42, H 6.23, N 9.99. Found: C 51.04,
H 6.18, N 9.65.
Boc-Gly-GABA-L-Cys(S-L-Cbz-Ala)-OH (12). Triethylamine (0.02
g, 0.29 mmol) was added dropwise to a solution of GABA-L-Cys(S-L-
Cbz-Ala)-OH (0.10 g, 0.24 mmol) in acetonitrile−water (10:2) at
room temperature. After 10 min, Boc-Gly-Bt (0.06 g, 0.24 mmol) was
added slowly to the reaction mixture. The solution was further stirred
at room temperature for 4 h. Acetonitrile was removed under reduced
pressure, and the residue was taken in ethyl acetate (30 mL) and
washed with 3 N HCl (2 × 15 mL) and brine (10 mL). Then ethyl
acetate was removed under reduced pressure, and the compound was
recrystallized from ethyl acetate−hexanes. The solid obtained was
filtered and dried to give Boc-Gly-GABA-Cys(S-L-Cbz-Ala)-OH.
White solid, 0.09 g, 66% yield; mp 110−112 °C. 1H NMR
(CD3OD, 300 MHz): δ 1.35 (d, J = 7.2 Hz, 3H), 1.45 (s, 9H),
1.74−1.84 (m, 2H), 2.24 (t, J = 7.3 Hz, 2H), 3.12−3.26 (m, 3H), 3.50
(dd, J = 13.9, 4.8 Hz, 1H), 3.67 (s, 2H), 4.25−4.33 (m, 1H), 4.56−
4.65 (m, 1H), 5.12 (s, 2H), 7.26−7.40 (m, 5H), 7.44−7.52 (m, 2H),
7.80−7.98 (m, 2H). 13C NMR (CD3OD, 75 MHz): δ 18.0, 26.6, 28.8,
31.0, 34.1, 39.9, 44.8, 53.2, 58.4, 67.9, 80.9, 128.9, 129.1, 129.6, 138.2,
Boc-β-Ala-L-Leu-OH (19a). A solution of L-leucine (0.81 g, 6.20
mmol) and triethylamine (0.63 g, 6.20 mmol) in acetonitrile (10 mL)
and water (5 mL) was added to the suspension of Boc-β-Ala-Bt (1.50 g,
5.17 mmol) in acetonitrile (30 mL). The solution was stirred for 15 h,
and then the solvent was evaporated under reduced pressure. The
crude product was dissolved in ethyl acetate. The organic phase was
washed with 2 N HCl (3 × 10 mL) and brine (10 mL). The solution
was dried over MgSO4, filtered, and then concentrated under vacuum.
The solid was recrystallized from ethyl acetate−hexanes to yield Boc-
β-Ala-L-Leu-OH. White microcrystals, 1.13 g, 72% yield; mp 124−
127 °C. 1H NMR (DMSO-d6, 300 MHz): δ 0.84 (d, J = 6.4 Hz, 3H), 0.88
(d, J = 6.4 Hz, 3H), 1.37 (s, 9H), 1.45−1.53 (m, 2H), 1.54−1.68 (m,
1H), 2.21−2.32 (m, 2H), 3.10 (dd, J = 13.3, 7.2 Hz, 2H), 4.15−4.23
(m, 1H), 6.68 (t, J = 5.2 Hz, 1H), 8.11 (d, J = 7.9, Hz 1H), 12.50 (br s,
1H). 13C NMR (DMSO-d6, 75 MHz): δ 21.3, 22.9, 24.3, 28.2, 35.5,
36.7, 39.9, 50.1, 77.6, 155.4, 170.4, 174.2. Anal. Calcd for C14H26N2O5:
C 55.61, H 8.67, N 9.26. Found: C 55.98, H 8.91, N 9.22.
(Boc-Gly-L-Cys-OCH3)2 (17a). A solution of Boc-Gly-OH (1.13 g,
6.4 mmol) in dry THF (10 mL) under argon was cooled to −15 °C in
an ice bath with stirring. N-methylmorpholine (0.65 g, 6.4 mmol),
followed by isobutyl chloroformate (0.84 g, 6.4 mmol) were added.
After 5 min, a solution of L-cystine dimethyl ester dihydrochloride
(1.00 g, 2.9 mmol) and N-methylmorpholine (0.65 g, 6.4 mmol) in
DMF (5 mL) were added. The ice bath was removed after 5 min, and
the solution was allowed to stir for 24 h at room temperature. The
solution was concentrated under vacuum; the residue was taken up in
ethyl acetate (20 mL) and 2 N HCl (5 mL). The organic phase was
washed successively with saturated Na2CO3 (3 × 10 mL) and 2 N HCl
(3 × 10 mL). The solution was dried over dry MgSO4, filtered, and
then concentrated under vacuum. The peptide was recrystallized from
diethyl ether−hexanes to give (Boc-Gly-L-Cys-OCH3)2. White micro-
crystals, 1.13 g, 66% yield; mp 51−54 °C. 1H NMR (DMSO-d6, 300 MHz):
δ 1.38 (br s, 18H), 2.96 (dd, J = 13.9, 8.3 Hz, 2H), 3.12 (dd, J = 13.4,
4.8 Hz, 2H), 3.58 (d, J = 5.8 Hz, 4H), 3.65 (s, 6H), 4.55−4.65 (m, 2H),
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6.94 (t, J = 5.8 Hz, 2H), 8.35 (d, J = 7.9 Hz, 2H). 13C NMR (DMSO-
d6, 75 MHz): δ 28.2, 39.1, 42.9, 51.2, 52.2, 78.1, 155.7, 169.6, 170.8.
Anal. Calcd for C22H38N4O10S2: C 45.35, H 6.57, N 9.62. Found: C
45.64, H 6.76, N 9.45.
crystallize overnight at −20 °C. The solid obtained was filtered,
washed with diethyl ether (3 mL) and CH2Cl2 (3 mL), and dried to
give the corresponding Boc-β-Ala-L-Leu-Gly-L-Cys(S-L-Cbz-Ala)-
OCH3. White microcrystals, 739 mg, 87% yield; mp 100−105 °C.
1H NMR (DMSO-d6, 300 MHz): δ 0.83 (d, J = 6.4 Hz, 3H), 0.83 (d,
J = 6.4 Hz, 3H), 1.25 (d, J = 7.3 Hz, 3H), 1.36 (s, 9H), 1.44 (t, J = 7.2
Hz, 2H), 1.52−1.64 (m, 1H), 2.28 (t, J = 6.5 Hz, 2H), 3.06−3.15 (m,
3H), 3.24 (dd, J = 14.1, 6.4 Hz, 1H), 3.62 (s, 3H), 3.70 (t, J = 6.1 Hz,
2H), 4.14−4.26 (m, 2H), 4.36−4.43 (m, 1H), 5.06 (s, 2H), 6.70 (t, J =
5.0 Hz, 1H), 7.30−7.42 (m, 5H), 8.07 (t, J = 6.8 Hz, 1H), 8.18 (t, J =
5.6 Hz, 1H), 8.33 (d, J = 7.6 Hz, 1H). 13C NMR (acetone-d6, 75
MHz): δ 18.0, 22.2, 23.4, 25.4, 28.7, 37.8, 41.2, 43.2, 52.7, 53.3, 57.9,
67.1, 78.8, 128.7, 129.3, 138.0, 156.7, 156.9, 169.9, 171.2, 173.0, 173.4,
202.0. Anal. Calcd for C31H47N5O10S: C 54.61, H 6.95, N 10.27.
Found: C 54.58, H 6.80, N 10.08.
β-Ala-L-Leu-Gly-L-Cys(S-L-Cbz-Ala)-OCH3 Hydrochloride
(23a). HCl gas was passed through a solution of Boc-β-Ala-L-Leu-
Gly-L-Cys(S-L-Cbz-Ala)-OCH3 (600 mg, 0.88 mmol) dissolved in
methanol for 1.5 h at rt. The solvent was then evaporated under
reduced pressure, and the solid was recrystallized from methanol−
ether to yield β-Ala-L-Leu-Gly-L-Cys(S-L-Cbz-Ala)-OCH3 hydro-
chloride. White microcrystals, 0.45 g, 82% yield; mp 174−185 °C.
1H NMR (DMSO-d6, 300 MHz): δ 0.86 (dd, J = 12.9, 6.4 Hz, 6H),
1.25 (d, J = 7.2 Hz, 3H), 1.42−1.50 (m, 2H), 1.54−1.64 (m, 1H),
2.92−3.00 (m, 2H), 3.09 (dd, J = 13.4, 7.4 Hz, 1H), 3.23 (dd, J = 14.6,
6.7 Hz, 1H), 3.62 (br s, 3H), 3.65 (br s, 2H), 3.70−3.76 (m, 2H),
4.14−4.22 (m, 1H), 4.29 (dd, J = 15.0, 7.5 Hz, 1H), 4.32 (q, J = 6.8
Hz, 1H), 5.06 (s, 2H), 7.30−7.42 (m, 5H), 7.90 (br s, 3H), 8.10 (d,
J = 7.6 Hz, 1H), 8.26 (t, J = 5.4 Hz, 1H), 8.33−8.37 (m, 1H), 8.40 (d,
J = 7.3 Hz, 1H). 13C NMR (CD3OD, 75 MHz): δ 18.0, 22.2, 23.5,
26.0, 30.7, 32.8, 37.2, 41.4, 41.9, 43.3, 53.3, 54.0, 58.4, 67.9, 128.8,
129.2, 129.6, 138.2, 158.4, 171.5, 172.0, 172.9, 175.4, 203.2. Anal.
Calcd for C26H40ClN5O8S·3H2O: C 46.46, H 6.90, N 10.42. Found: C
46.53, H 6.72, N 10.54.
(Gly-L-Cys-OCH3)2 Hydrochloride (18a). HCl gas was passed
through a solution of (Boc-Gly-L-Cys-OCH3)2 (0.58 g, 1.0 mmol) in
methanol (15 mL) for 30 min. The methanol solution was
concentrated under vacuum, and ether was added. The turbid solution
was left to crystallize in the freezer overnight. The solid formed was
filtered and washed with dry diethyl ether (10 mL) and dried to give
the corresponding (Gly-L-Cys-OCH3)2 hydrochloride. White micro-
crystals, 0.38 g, 83% yield; mp 185−189 °C. 1H NMR (DMSO-d6, 300
MHz): δ 2.98 (dd, J = 13.9, 8.5 Hz, 2H), 3.15 (dd, J = 13.7, 5.0 Hz,
2H), 3.60 (s, 4H), 3.66 (s, 6H), 4.57−4.68 (m, 2H), 8.36 (s, 6H), 9.31
(d, J = 7.4 Hz, 2H). 13C NMR (DMSO-d6, 75 MHz): δ 39.9, 51.5,
52.4, 166.3, 170.4. Anal. Calcd for C12H24Cl2N4O6S2·H2O: C 30.45, H
5.54, N 11.84. Found: C 30.33, H 5.53, N 11.98.
(Boc-β-Ala-L-Leu-Gly-L-Cys-OCH3)2 (20a). A solution of Boc-β-
Ala-L-Leu-OH (0.91 g, 3 mmol) in dry THF (10 mL) under argon was
cooled to −15 °C in an ice bath with stirring. N-Methylmorpholine
(0.33 g, 3.2 mmol), followed by isobutyl chloroformate (0.45 g, 3.2
mmol) were added. After 4 min, a solution of (Gly-L-Cys-OCH3)2
hydrochloride (0.68 g, 1.5 mmol) and N-methylmorpholine (0.7 g, 1.6
mmol) in DMF (5 mL) was added. The ice bath was removed after
5 min, and the solution was allowed to stir for 12 h at room temperature.
The solution was concentrated under vacuum; the residue was taken
up in ethyl acetate (30 mL) and water (5 mL). The organic phase was
washed successively with saturated Na2CO3 (2 × 15 mL), water (10
mL), 2 N HCl (15 mL), and water (10 mL). The solution was dried
over MgSO4, filtered, and then concentrated under vacuum. The
peptide was recrystallized from ethyl acetate−hexanes to give (Boc-β-
Ala-L-Leu-Gly-L-Cys-OCH3)2. White microcrystals, 0.93 g, 65% yield;
mp 105−111 °C. 1H NMR (DMSO-d6, 300 MHz): δ 0.83 (d, J = 6.4
Hz, 6H), 0.88 (d, J = 6.6 Hz, 6H), 1.36 (s, 18H), 1.44 (t, J = 7.1, 4H),
1.53−1.63 (m, 2H), 2.25−2.31 (m, 4H), 2.95 (dd, J = 13.8, 8.5 Hz,
2H), 3.07−3.16 (m, 6H), 3.65 (s, 6H), 3.70−3.75 (m, 4H), 4.19−4.26
(m, 2H), 4.58 (dd, J = 13.3, 8.2 Hz, 2H), 6.70 (t, J = 5.5 Hz, 2H), 8.07
(d, J = 7.3 Hz, 2H), 8.22 (t, J = 5.6 Hz, 2H), 8.30 (d, J = 7.9 Hz, 2H).
13C NMR (acetone-d6, 75 MHz): δ 19.4, 22.2, 23.4, 25.4, 28.7, 36.9,
37.8, 40.6, 41.3, 43.5, 52.8, 53.4, 78.8, 156.7, 170.2, 171.5, 173.2, 174.1.
Anal. Calcd for C40H70N8O14S2: C 50.51, H 7.42, N 11.78. Found: C
50.35, H 7.56, N 11.39.
Boc-β-Ala-L-Leu-Gly-L-Cys-OCH3 (21a). (Boc-β-Ala-L-Leu-Gly-L-
Cys-OCH3)2 (1.36 g, 1.43 mmol) was treated with P(t-Bu)3 (0.58 g,
0.71 mL, 2.86 mmol) in 20 mL of 9:1 MeOH:water for 2 h at rt under
argon gas. The reaction mixture was concentrated under vacuum and
the residue was taken up in ethyl acetate (15 mL). The solution was
dried over MgSO4, and then concentrated under vacuum. The peptide
was recrystallized from ethyl acetate:hexane to give Boc-β-Ala-L-Leu-
Gly-L-Cys-OCH3. White microcrystals, 1.02 g, 75% yield; mp 140−
142 °C. 1H NMR (DMSO-d6, 300 MHz): δ 0.83 (d, J = 6.4 Hz, 3H), 0.88
(d, J = 6.4 Hz, 3H), 1.25 (br s, 1H), 1.37 (s, 9H), 1.44 (t, J = 7.3 Hz,
2H), 1.53−1.65 (m, 1H), 2.23−2.34 (m, 2H), 2.74−2.90 (m, 2H),
3.11 (dd, J = 12.9, 7.0 Hz, 2H), 3.65 (s, 3H), 3.73 (d, J = 4.6 Hz, 2H),
4.16−4.24 (m, 1H), 4.46−4.53 (m, 1H), 6.72 (t, J = 6.1 Hz, 1H),
8.08−8.13 (m, 2H), 8.32 (t, J = 6.1 Hz, 1H). 13C NMR (acetone-d6, 75
MHz): δ 22.2, 23.4, 25.4, 26.7, 28.7, 36.9, 37.8, 41.1, 43.5, 52.6, 53.6,
55.5, 78.8, 156.7, 169.9, 171.3, 173.2, 173.8. Anal. Calcd for
C20H36N4O7S: C 50.40, H 7.61, N 11.76. Found: C 50.45, H 7.85,
N 11.38.
Boc-β-Ala-L-Leu-Gly-L-Cys(S-L-Cbz-Ala)-OCH3 (22a). (Boc-β-
Ala-L-Leu-Gly-L-Cys-OCH3 (500 mg, 1.25 mmol) was suspended in
acetonitrile (15 mL), and a solution of Cbz-L-Ala-Bt 7 (596 mg, 1.25
mmol) in acetonitrile−water (10 mL of 4:1) containing an equivalent
amount of triethylamine (127 mg, 1.26 mmol) was added. The mixture
was stirred at 25 °C for 3 h until completion. Acetonitrile was removed
under reduced pressure, and the residue was taken in ethyl acetate (30
mL) and extracted with 2 N HCl (2 × 20 mL), water (15 mL), and
brine (10 mL). Ethyl acetate was concentrated under reduced
pressure, and hexane was added; the turbid solution was left to
Boc-β-Ala-L-Phe-OH (19b). The compound was prepared
according to the method for preparation of Boc-β-Ala-L-Leu-OH
1
(19a). White microcrystals, 1.02 g, 65% yield; mp 154 −157 °C. H
NMR (DMSO-d6, 300 MHz): δ 1.37 (br s, 9H), 2.19−2.25 (m, 2H),
2.84 (dd, J = 13.7, 9.5, Hz, 1H), 3.00−3.11 (m, 3H), 4.37−4.44 (m,
1H), 6.63 (br s, 1H), 7.17−7.30 (m, 5H), 8.23 (d, J = 8.0 Hz, 1H). 13C
NMR (DMSO-d6, 75 MHz): δ 28.3, 35.5, 36.6, 36.7, 53.4, 77.6, 126.4,
128.1, 129.1, 137.7, 155.4, 170.3, 173.0. Anal. Calcd for C17H24N2O5:
C 60.70, H 7.19, N 8.33. Found: C 60.29, H 7.22, N 8.19.
(Boc-β-Ala-L-Cys-OCH3)2 (17b). The compound was prepared
according to the method for preparation of (Boc-Gly-L-Cys-OCH3)2
1
(17a). White microcrystals, 1.27 g, 72% yield; mp 117−119 °C. H
NMR (DMSO-d6, 300 MHz): δ 1.37 (s, 18H), 2.29 (t, J = 7.3 Hz,
4H), 2.92 (dd, J = 13.8, 8.8 Hz 2H), 3.06−3.13 (m, 6H), 3.64 (s, 6H),
4.53 (dd, J = 13.2, 8.1 Hz, 2H), 6.72 (br s, 2H), 8.46 (d, J = 7.4 Hz,
2H). 13C NMR (DMSO-d6, 75 MHz): δ 28.2, 35.4, 36.5, 51.1, 52.2,
77.6, 155.4, 170.6, 170.9. Anal. Calcd for C24H42N4O10S2: C 47.20, H
6.93, N 9.17. Found: C 47.33, H 7.12, N 9.01.
(β-Ala-L-Cys-OCH3)2 hydrochloride (18b). The compound was
prepared according to the method for preparation of (Gly-L-Cys-
OCH3)2 hydrochloride (18a). White microcrystals, 0.20 g, 82% yield;
mp 87−92 °C. 1H NMR (DMSO-d6, 300 MHz): δ 2.58 (t, J = 7.4 Hz,
4H), 2.92−3.00 (m, 6H), 3.12 (dd, J = 14.1, 4.9 Hz, 2H), 3.66 (br s,
6H), 4.53−4.60 (m, 2H), 8.04 (br s, 6H), 8.81 (d, J = 7.7 Hz, 2H). 13C
NMR (DMSO-d6, 75 MHz): δ 32.6, 32.7, 51.9, 52.9, 170.3, 171.4.
Anal. Calcd for C14H28Cl2N4O6S2·0.75H2O: C 33.84, H 5.98, N 11.27.
Found: C 34.06, H 6.01, N 10.88.
(Boc-β-Ala-L-Phe-β-Ala-L-Cys-OCH3)2 (20b). The compound was
prepared according to the method for preparation of (Boc-β-Ala-L-
Leu-Gly-L-Cys-OCH3)2 (20a). White microcrystals, 0.45 g, 52% yield;
1
mp 187−197 °C. H NMR (DMSO-d6, 300 MHz): δ 1.35 (s, 18H),
2.13−2.23 (m, 4H), 2.28 (t, J = 7.5 Hz, 4H), 2.73 (dd, J = 13.6, 9.6 Hz,
2H), 2.91−3.03 (m, 8H), 3.10 (dd, J = 13.7, 5.3 Hz, 2H), 3.16−3.27
(m, 4H), 3.63 (s, 6H), 4.38−4.45 (m, 2H), 4.52−4.59 (m, 2H), 6.61
(t, J = 5.3 Hz, 2H), 7.12−7.27 (m, 10H), 8.09 (bs, 2H), 8.15 (d,
J = 8.5 Hz, 2H), 8.57 (d, J = 7.6 Hz, 2H). 13C NMR (DMSO-d6,
2646
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Article
75 MHz): δ 28.2, 34.8, 35.2, 35.5, 36.6, 37.8, 51.2, 52.2, 54.0, 77.6,
126.2, 127.9, 129.1, 138.0, 155.3, 170.1, 170.6, 170.9, 171.1. Anal.
Calcd for C48H70N8O14S2: C 55.05, H 6.74, N 10.70. Found: C 55.00,
H 7.15, N 10.72.
Gly-L-Cys-OCH3 (21a). White microcrystals, 0.27 g, 66% yield; mp
84−90 °C. 1H NMR (DMSO-d6, 300 MHz): δ 0.88 (dd, J = 13.5, 7.1
Hz, 1H), 1.37 (br s, 9H), 1.42−1.51 (m, 2H), 2.02 (t, J = 7.4 Hz, 2H),
2.71−2.88 (m, 5H), 3.02 (dd, J = 13.5, 4.2 Hz, 1H), 3.65 (br s, 3H),
3.69−3.83 (m, 2H), 4.46−4.53 (m, 2H), 6.75 (br s, 1H), 7.15−7.26
(m, 5H), 8.15 (d, J = 8.0 Hz, 1H), 8.19 (d, J = 7.9 Hz, 1H), 8.38 (d,
J = 5.4 Hz, 1H). 13C NMR (DMSO-d6, 75 MHz): δ 25.4, 25.7, 28.3,
32.6, 37.3, 41.8, 51.2, 54.2, 54.5, 77.4, 126.2, 128.0, 129.1, 138.0, 155.6,
16.9, 170.5, 171.9, 172.2. Anal. Calcd for C24H36N4O7S: C 54.95, H
6.92, N 10.68. Found: C 54.84, H 6.59, N 10.67.
Boc-GABA-L-Phe-Gly-L-Cys(S-L-Cbz-Ala)-OCH3 (22c). The com-
pound was prepared according to the method for preparation of Boc-
β-Ala-L-Leu-Gly-L-Cys(S-L-Cbz-Ala)-OCH3 (22a). White microcrys-
tals, 0.27 g, 87% yield; mp 91−96 °C. 1H NMR (DMSO-d6, 300
MHz): δ 1.26 (d, J = 7.2 Hz, 3H), 1.37 (br s, 9H), 1.42−1.50 (m, 2H),
2.01 (t, J = 7.4 Hz, 2H), 2.70−2.82 (m, 3H), 3.02 (dd, J = 14.0, 3.8 Hz,
1H), 3.10 (dd, J = 13.7, 7.7 Hz, 1H), 3.25 (dd, J = 12.9, 5.0 Hz, 1H),
3.63 (br s, 3H), 3.65−3.82 (m, 2H), 4.15−4.25 (m, 1H), 4.38−4.54
(m, 2H), 5.06 (br s, 2H), 6.74 (br s, 1H), 7.14−7.40 (m, 10H), 8.08−
8.11 (m, 2H), 8.29 (t, J = 5.4 Hz, 1H), 8.40 (d, J = 7.5 Hz, 1H). 13C
NMR (acetone-d6, 75 MHz): δ 18.1, 26.9, 28.8, 33.4, 37.9, 40.3, 52.8,
56.3, 57.9, 67.1, 78.7, 127.3, 128.7, 129.2, 129.3, 130.1, 138.0, 138.8,
156.9, 157.2, 169.9, 171.3, 172.6, 174.0, 202.1. Anal. Calcd for
C35H47N5O10S: C 57.60, H 6.49, N 9.60. Found: C 57.49, H 6.66,
N 9.46.
GABA-L-Phe-Gly-L-Cys(S-L-Cbz-Ala)-OCH3 Hydrochloride
(23c). The compound was prepared according to the method for
preparation of β-Ala-L-Leu-Gly-L-Cys(S-L-Cbz-Ala)-OCH3 hydrochlor-
ide (23a). White microcrystals, 0.17 g, 87% yield; mp 125−131 °C. 1H
NMR (CD3OD, 300 MHz): δ 1.36 (d, J = 7.2 Hz, 3H), 1.79−1.88 (m,
2H), 2.24−2.41 (m, 2H), 2.76−2.85 (m, 2H), 2.91 (dd, J = 13.7, 9.4
Hz, 1H), 3.16−3.27 (m, 2H), 3.43 (dd, J = 13.8, 5.3 Hz, 1H), 3.67−
3.74 (m, 5H), 4.29 (q, J = 7.3 Hz, 1H), 4.56−4.72 (m, 2H), 5.06−5.16
(m, 2H), 7.18−7.36 (m, 10H). 13C NMR (CD3OD, 75 MHz): δ 18.0,
24.3, 30.7, 33.5, 38.6, 40.3, 43.3, 53.4, 56.7, 58.4, 68.0, 128.0, 128.8,
129.2, 129.6, 130.4, 138.2, 138.6, 158.5, 171.4, 171.9, 17.3, 174.8,
203.3. Anal. Calcd for C30H40ClN5O8S·3H2O: C 50.03, H 6.44, N
9.72. Found: C 50.06, H 6.24, N 9.55.
General Procedure for Long-Range Acyl-Migration of N-
Terminus-Unprotected S-(Pg-α-aminoacyl)-tripeptide 13 and
S-(Pg-α-aminoacyl)tetrapeptide 23a,b,c to Form Native Pep-
tides Analogues 14, 24, 26a,b. The N-terminus unprotected S-(Pg-
α-aminoacyl)peptide (13, 23a−c; 0.02 mmol) was suspended in
degassed phosphate buffer (NaH2PO4/Na2HPO4) (1 M, pH = 7.8 for
13, 6.2−8.2 for 23a, and 7.6 for 26a,b; 9.6 mL), and acetonitrile (0.4
mL) was added dropwise until the starting material was dissolved. The
mixture was subjected to microwave irradiation (50 °C, 50 W, 1 h).
The reaction was allowed to cool to rt, and acetonitrile was removed
under reduced pressure and the residue was acidified with 2 N HCl to
pH = 1. The mixture was extracted with ethyl acetate (3 × 10 mL), the
combined organic extracts were dried over MgSO4, and the solvent
was removed under reduced pressure. Native peptide analogues 14,
24, 26a, or 26b were subsequently isolated as disulfide dimers by
semipreparative HPLC.
Boc-β-Ala-L-Phe-β-Ala-L-Cys-OCH3 (21b). The compound was
prepared according to the method for preparation of Boc-β-Ala-L-Leu-
Gly-L-Cys-OCH3 (21a). White microcrystals, 0.21 g, 68% yield; mp
137−142 °C. 1H NMR (DMSO-d6, 300 MHz): δ 0.84−0.94 (m, 1H),
1.36(br s, 9H), 2.10−2.25 (m, 2H), 2.31 (t, J = 7.2 Hz, 2H), 2.68−
2.86 (m, 3H), 2.93−3.04 (m, 3H), 3.15−3.30 (m, 2H), 3.64 (s, 3H),
4.38−4.49 (m, 2H), 6.62 (bs, 1H), 7.17−7.27 (m, 5H), 8.04 (bs, 1H),
8.12 (d, J = 8.6 Hz, 1H), 8.43 (d, J = 7.6 Hz, 1H). 13C NMR (CD3OD,
75 MHz): δ 26.7, 28.9, 36.3, 37.1, 37.2, 38.0, 39.1, 53.2, 56.3, 56.4,
80.3, 127.9, 129.6, 130.4, 138.7, 158.3, 172.3, 173.7, 173.9. Anal. Calcd
for C24H36N4O7S: C 54.95, H 6.92, N 10.68. Found: C 55.02, H 7.01,
N 11.00.
Boc-β-Ala-L-Phe-β-Ala-L-Cys(S-L-Cbz-Ala)-OCH3 (22b). The
compound was prepared according to the method for preparation of
Boc-β-Ala-L-Leu-Gly-L-Cys(S-L-Cbz-Ala)-OCH3 (22a). White micro-
crystals, 0.25 g, 81% yield; mp 158−162 °C. 1H NMR (DMSO-d6, 300
MHz): δ 1.24 (d, J = 7.2 Hz, 3H), 1.35 (br s, 9H), 2.12−2.30 (m, 4H),
2.71 (dd, J = 13.6, 9.2 Hz, 1H), 2.91−3.10 (m, 4H), 3.16−3.26 (m,
3H), 3.61 (s, 3H), 4.14−4.22 (m, 1H), 4.34−4.48 (m, 2H), 5.05 (s,
2H), 6.61 (br s, 1H), 7.16−7.30 (m, 5H), 7.30−7.42 (m, 5H), 7.98−
8.02 (m, 1H), 8.07−8.12 (m, 2H), 8.47 (d, J = 7.6 Hz, 1H). 13C NMR
(CD3OD, 75 MHz): δ 18.0, 28.9, 30.7, 36.2, 37.1, 37.3, 38.0, 39.1,
53.3, 53.4, 56.4, 58.4, 67.9, 80.3, 127.9, 128.9, 129.2, 129.6, 130.4,
138.2, 138.7, 158.4, 172.2, 173.8, 203.3. Anal. Calcd for
C35H47N5O10S: C 57.60, H 6.49, N 9.60. Found: C 57.42, H 6.70,
N 9.44.
β-Ala-L-Phe-β-Ala-L-Cys(S-L-Cbz-Ala)-OCH3 Hydrochloride
(23b). The compound was prepared according to the method for
preparation of β-Ala-L-Leu-Gly-L-Cys(S-L-Cbz-Ala)-OCH3 hydrochlor-
ide. White microcrystals, 0.16 g, 81% yield; mp 158−162 °C. 1H NMR
(DMSO-d6, 300 MHz): δ 1.24 (d, J = 7.2 Hz, 3H), 2.24−2.30 (m,
2H), 2.40 (dd, J = 15.4,7.7 Hz, 2H), 2.74 (dd, J = 13.5, 9.6 Hz, 1H),
2.82−2.88 (m, 2H), 2.97 (dd, J = 13.7, 4.7 Hz, 1H), 3.08 (dd, J = 13.6,
8.0 Hz, 1H), 3.18−3.29 (m, 4H), 3.62 (br s, 3H), 4.13−4.23 (m, 1H),
4.34−4.48 (m, 2H), 5.06 (s, 2H), 7.16−7.28 (m, 5H), 7.28−7.37 (m,
5H), 7.91 (br s, 3H), 8.10 (d, J = 7.3 Hz, 1H), 8.17 (t, J = 5.4 Hz, 1H),
8.43 (d, J = 8.5 Hz, 1H), 8.53 (d, J = 7.7 Hz, 1H). 13C NMR (CD3OD,
75 MHz): δ 17.9, 30.6, 32.8, 36.1, 36.9, 37.1, 38.9, 53.2, 56.5, 58.4,
67.8, 127.9, 128.8, 129.1, 129.5, 130.3, 138.1, 138.5, 158.4, 172.1,
173.8, 203.5. Anal. Calcd for C30H40ClN5O8S·H2O: C 52.66, H 6.19,
N 10.24. Found: C 52.64, H 6.62, N 10.13.
Boc-GABA-L-Phe-OH (19c). The compound was prepared
according to the method for preparation of Boc-β-Ala-L-Leu-OH
(19a). White microcrystals, 1.82 g, 68% yield; mp 78−81 °C. 1H NMR
(DMSO-d6, 300 MHz): δ 1.37 (br s, 9H), 1.49 (p, J = 7.2 Hz, 2H),
1.98−2.05 (m, 2H), 2.79−2.87 (m, 3H), 3.04 (dd, J = 13.9, 5.0 Hz,
1H), 4.36−4.44 (m, 1H), 6.76 (t, J = 5.5 Hz, 1H), 7.16−7.30 (m, 5H),
8.15 (d, J = 8.2 Hz, 1H). 13C NMR (DMSO-d6, 75 MHz): δ 26.0, 28.4,
32.7, 36.9, 53.5, 77.7, 126.5, 128.3, 129.2, 137.8, 155.7, 172.1, 173.3.
Anal. Calcd for C18H26N2O5: C 61.70, H 7.48, N 7.99. Found: C
61.32, H 7.55, N 7.59.
Cbz-L-Ala-Gly-GABA-L-Cys-OH (14). Colorless oil, 1.7 mg, 18%
yield. ESI-MS m/z: 935 (M + H). HRMS (ESI) calcd for
C40H54N8O14S2Na [M + Na]+ 957.3093, found 957.3117.
(Boc-GABA-L-Phe-Gly-L-Cys-OCH3)2 (20c). The compound was
prepared according to the method for preparation of (Boc-β-Ala-L-
Leu-Gly-L-Cys-OCH3)2 (20a). White microcrystals, 0.33 g, 52% yield;
mp 110−115 °C. 1H NMR (DMSO-d6, 300 MHz): δ 1.37 (br s, 18H),
1.41−1.52 (m, 4H), 2.02 (t, J = 7.2 Hz, 4H), 2.70−2.82 (m, 6H),
2.93−3.06 (m, 4H), 3.15 (dd, J = 13.7, 4.3 Hz, 2H), 3.65 (br s, 6H),
3.72 (dd, J = 16.5, 5.0 Hz, 2H), 3.82 (dd, J = 17.0, 5.8 Hz, 2H), 4.43−
4.54 (m, 2H), 4.55−4.64 (m, 2H), 6.74 (br s, 2H), 7.14−7.32 (m,
10H), 8.12 (d, J = 8.2 Hz, 2H), 8.33 (br s, 2H), 8.40 (d, J = 7.7 Hz,
2H). 13C NMR (DMSO-d6, 75 MHz): δ 25.7, 28.3, 32.6, 37.4, 41.6,
51.3, 52.3, 54.1, 77.4, 126.2, 128.0, 129.1, 138.0, 155.5, 169.0, 170.7,
171.7, 172.0. Anal. Calcd for C48H70N8O14S2: C 55.05, H 6.74, N
10.70. Found: C 55.12, H 6.46, N 10.56.
Cbz-L-Ala-β-Ala-L-Leu-Gly-L-Cys-OCH3 (24). The compound was
prepared from β-Ala-L-Leu-Gly-L-Cys(S-L-Cbz-Ala)-OCH3 hydro-
chloride according to the general procedure for long-range acyl
migration. Cbz-L-Ala-β-Ala-L-Leu-Gly-L-Cys-OCH3 was subsequently
isolated as a disulfide dimer by semipreparative HPLC. Colorless oil,
3.1 mg, 31% yield. 1H NMR (DMSO-d6, 300 MHz): δ 0.83−0.88 (m,
12H), 1.17 (d, J = 7.2 Hz, 6H), 1.45 (t, J = 7.2 Hz, 4H), 1.56−1.64 (m,
2H), 2.26−3.36 (m, 4H), 2.97 (dd, J = 13.8, 8.4 Hz, 2H), 3.13 (dd, J =
13.9, 5.1 Hz, 2H), 3.21−3.28 (m, 4H), 3.65 (br s, 6H), 3.69−3.80 (m,
4H), 3.95−4.01 (m, 2H), 4.25 (q, J = 7.4 Hz, 2H), 4.57−4.61 (m,
2H), 4.97−5.05 (m, 4H), 7.28−7.32 (m, 2H), 7.32−7.38 (m, 10H),
7.87 (t, J = 5.8 Hz, 2H), 8.09 (d, J = 7.4 Hz, 2H), 8.21 (t, J = 5.8 Hz,
2H), 8.32 (d, J = 7.7 Hz, 2H). 13C NMR (DMSO-d6, 75 MHz): δ 18.2,
Boc-GABA-L-Phe-Gly-L-Cys-OCH3 (21c). The compound was
prepared according to the method for preparation of Boc-β-Ala-L-Leu-
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21.5, 23.0, 24.2, 35.0, 35.3, 40.5, 41.5, 50.0, 51.2, 51.3, 52.2, 65.4,
127.7, 127.8, 128.3, 137.0, 155.6, 169.1, 170.7, 170.8, 172.4, 172.5. ESI-
MS m/z: 1183 (M + Na). HRMS (ESI) calcd for C52H76N10O16S2Na
[M + Na]+ 1183.4774, found 1183.4733.
(12) Photaki, I. J. Am. Chem. Soc. 1966, 88, 2292−2299.
(13) Payne, R. J.; Wong, C.-H. Chem. Commun. 2010, 46, 21−43.
(14) Wehner, J. W.; Lindhorst, T. K. Synthesis 2010, 18, 3070−3082.
(15) Guranda, D. T.; Shapovalova, I. V.; Svedas, V. K. Russ. J. Bioorg.
Chem. 2004, 30, 403−408.
Cbz-L-Ala-β-Ala-L-Phe-β-Ala-L-Cys-OCH3 (26a). The compound
was prepared from β-Ala-L-Phe-β-Ala-L-Cys(S-L-Cbz-Ala)-OCH3
hydrochloride according to the general procedure for long-range acyl
migration. Cbz-L-Ala-β-Ala-L-Phe-β-Ala-L-Cys-OCH3 was subsequently
isolated as the disulfide dimer by semipreparative HPLC. Colorless oil,
2.2 mg, 42% yield. ESI-MS m/z: 1256 (M + H). HRMS (ESI) calcd
for C60H76N10O16S2Na [M + Na]+ 1279.4774, found 1279.4718.
Cbz-L-Ala-GABA-L-Phe-Gly-L-Cys-OCH3 (26b). The compound
was prepared from GABA-L-Phe-Gly-L-Cys(S-L-Cbz-Ala)-OCH3 hydro-
chloride according to the general procedure for long-range acyl
migration. Cbz-L-Ala-GABA-L-Phe-Gly-L-Cys-OCH3 was subsequently
isolated as a disulfide dimer by semipreparative HPLC. Colorless oil,
1.9 mg, 42% yield. ESI-MS m/z: 1256 (M + H). HRMS (ESI) calcd
for C60H76N10O16S2Na [M + Na]+ 1279.4774, found 1279.4775.
(16) Haase, C.; Seitz, O. Eur. J. Org. Chem. 2009, 2096−2101.
(17) Ficht, S.; Payne, J. R.; Brik, A.; Wong, C.-H. Angew. Chem., Int.
Ed. 2007, 46, 5975−5979.
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S
* Supporting Information
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2009, 424, 69−78.
Detailed experimental procedures, copies of 1H and 13C NMR for
all the novel compounds, copies of the chromatograms from the
HPLC experiments, and computational details. This material is
available free of charge via the Internet at http://pubs.acs.org.
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2010, 16, 3185−3203.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: Katritzky@chem.ufl.edu.
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Notes
(30) Hagihara, M.; Anthony, N. J.; Stout, T. J.; Clardy, J.; Schreiber,
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The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
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(32) Foster, A. C.; Kemp, J. A. Curr. Opin. Pharmacol. 2006, 6, 7−17.
(33) He, H. T.; Xu, C. R.; Song, X.; Siahaan, T. J. J. Pept. Res. 2003,
61, 331−342.
This work was supported by the Belgian American Educational
Foundation, Inc. (B.A.E.F.), by the Research Foundation-
Flanders (FWO-Vlaanderen, Belgium), by the Kenan Founda-
tion (University of Florida), and the King Abdulaziz University
(Saudi Arabia). The authors are grateful to Dr. C. D. Hall for
reading the manuscript, Dr. Jodie Johnson (University of
Florida) for his help in HLPC−MS analyses, Dr. M. C. A.
Dancel (University of Florida) for her help with HRMS
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