Synthetic procedure for N-Fmoc amino acyl-N-sulfanylethylaniline linker as crypto-peptide thioester precursor with application to native chemical ligation

Article abstract of DOI:10.1021/jo3011107

N-Sulfanylethylanilide (SEAlide) peptides 1, obtainable using Fmoc-based solid-phase peptide synthesis (Fmoc SPPS), function as crypto-thioesters in native chemical ligation (NCL), yielding a wide variety of peptides/proteins. Their acylating potential with N-terminal cysteinyl peptides 2 can be tuned by the presence or absence of phosphate salts, leading to one-pot/multifragment ligation, operating under kinetically controlled conditions. SEAlide peptides have already been shown to be promising for use in protein synthesis; however, a widely applicable method for the synthesis of N-Fmoc amino acyl-N- sulfanylethylaniline linkers 4, required for the preparation of SEAlide peptides, is unavailable. The present study addresses the development of efficient condensation protocols of 20 naturally occurring amino acid derivatives to the N-sulfanylethylaniline linker 5. N-Fmoc amino acyl aniline linkers 4 of practical use in NCL chemistry, except in the case of the proline- or aspartic acid-containing linker, were successfully synthesized by coupling of POCl₃- or SOCl₂-activated Fmoc amino acid derivatives with sodium anilide species 6, without accompanying racemization and loss of side-chain protection. Furthermore, SEAlide peptides 7 possessing various C-terminal amino acids (Gly, His, Phe, Ala, Asn, Ser, Glu, and Val) were shown to be of practical use in NCL chemistry.

Full text of DOI:10.1021/jo3011107

Article
pubs.acs.org/joc
Synthetic Procedure for N‑Fmoc Amino Acyl‑N‑Sulfanylethylaniline
Linker as Crypto-Peptide Thioester Precursor with Application to
Native Chemical Ligation
Ken Sakamoto,† Kohei Sato,† Akira Shigenaga,† Kohei Tsuji,† Shugo Tsuda,†,‡ Hajime Hibino,‡
Yuji Nishiuchi,‡,§ and Akira Otaka*,†
†Institute of Health Biosciences and Graduate School of Pharmaceutical Sciences, The University of Tokushima,
Tokushima 770-8505, Japan
‡Saito Research Center, Peptide Institute, Inc., 7-2-9 Saito Ibaraki, Osaka 567-0085, Japan
§Department of Chemistry, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan
S
* Supporting Information
ABSTRACT: N-Sulfanylethylanilide (SEAlide) peptides 1,
obtainable using Fmoc-based solid-phase peptide synthesis
(Fmoc SPPS), function as crypto-thioesters in native chemical
ligation (NCL), yielding a wide variety of peptides/proteins.
Their acylating potential with N-terminal cysteinyl peptides 2
can be tuned by the presence or absence of phosphate salts,
leading to one-pot/multifragment ligation, operating under
kinetically controlled conditions. SEAlide peptides have
already been shown to be promising for use in protein synthesis; however, a widely applicable method for the synthesis of
N-Fmoc amino acyl-N-sulfanylethylaniline linkers 4, required for the preparation of SEAlide peptides, is unavailable. The present
study addresses the development of efficient condensation protocols of 20 naturally occurring amino acid derivatives to the
N-sulfanylethylaniline linker 5. N-Fmoc amino acyl aniline linkers 4 of practical use in NCL chemistry, except in the case of the
proline- or aspartic acid-containing linker, were successfully synthesized by coupling of POCl3- or SOCl2-activated Fmoc amino
acid derivatives with sodium anilide species 6, without accompanying racemization and loss of side-chain protection.
Furthermore, SEAlide peptides 7 possessing various C-terminal amino acids (Gly, His, Phe, Ala, Asn, Ser, Glu, and Val) were
shown to be of practical use in NCL chemistry.
INTRODUCTION
■
Peptide thioesters are indispensable synthetic intermediates for
native chemical ligations (NCLs), yielding much success in the
chemical syntheses of a wide variety of proteins.1,2 Practical
synthetic procedures, using Fmoc-based solid-phase peptide
synthesis (Fmoc SPPS), for peptide thioesters3−10 have been
extensively explored for two main reasons. One is that the method
has become increasingly popular because the experimental
procedures are simpler, and the synthetic materials more readily
available, than those used in Boc SPPS. The other is that optimized
synthetic protocols for Boc SPPS of peptide thioesters11 are
incompatible with Fmoc SPPS. In this context, we have developed
an N−S acyl-transfer-mediated synthetic methodology for peptide
thioesters in which the use of N-Fmoc amino acyl-N-
sulfanylethylaniline linkers 4 as a key synthetic unit guarantees
compatibility with Fmoc chemistry.7b,8 In the course of our
studies on N-sulfanylethylanilide peptides (referred as to SEAlide
peptides), we found that SEAlide peptides 1 function efficiently
as peptide thioesters in the presence of phosphate salts, without
preconversion to the corresponding thioester, to participate in
NCLs with an N-terminal cysteinyl peptide 2 (Figure 1),12 and
are of great use in kinetically controlled NCL reactions.6c,13
Figure 1. SEAlide peptides can function as peptide thioesters under
normal NCL conditions in the presence of phosphate salts.
Received: June 4, 2012
Published: July 20, 2012
© 2012 American Chemical Society
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We therefore realized that the development of a facile pre-
parative method for N-Fmoc amino acyl-N-sulfanylethylaniline
linkers 4 should lead to a further increase in the usefulness
of SEAlide peptides. In this study, the three issues shown in
Figure 2, i.e., coupling efficiency, racemization, and stability of
side-chain protections, are discussed.14
Table 1. Summary of Coupling of Fmoc Amino Acid
Derivatives with N-Sulfanylethylaniline Linker 5 under
Various Reaction Conditions
isolated yield of
Fmoc amino
acid
aniline linkers 4 (%)
a
entry
method
product
[racemization (%)]
1
Gly
A
4a
4a
4b
4b
4b
4c
4c
4d
4d
4e
4e
4f
94
2
Gly
D
A
B
65
3
Ala
80
4
Ala
75 [2.5]
67
5
Ala
D
A
D
A
D
A
D
A
D
A
D
6
Val
98
7
Val
87
8
Ile
88
9
Ile
87
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
Leu
74
Leu
88
Figure 2. Issues addressed in the preparation of N-Fmoc amino acyl-
N-sulfanylethylaniline linker 4.
Phe
83
Phe
4f
88
Pro
4g
4g
4h
4h
4h
4h
4h
4h
4i
48
In our previous investigation, the loading of aliphatic Fmoc
derivatives of amino acids such as Gly and Ala derivatives on an
aniline linker 5 was achieved by coupling Fmoc amino acyl
chlorides with sodium anilide 6, produced by treating aniline 5
with NaH (Method A, Figure 3). Here, the requisite acyl
Pro
39
Ser(t-Bu)
Ser(t-Bu)
Ser(t-Bu)
Ser(t-Bu)
Ser(t-Bu)
Ser(t-Bu)
Asn(Tr)
Asn(Tr)
Asn(Tr)
Asp(Ot-Bu)
Asp(Ot-Bu)
Thr(t-Bu)
Glu(Ot-Bu)
Gln(Tr)
Cys(Tr)
Met
modified A
89 [0.3]
48 [33]
B
b c
C
51 [n.d. ]
d
C
65
D
90 [1.8]
e
D
91 [0.4]
f
modified A

C
4i
88
89
D
4i
e
modified A
4j

D
D
D
D
D
D
D
D
D
D
D
D
4j
68
84
72
93
4k
4l
4m
4n
4o
4p
4q
4r
4s
4t
b
72 [n.d. ]
80
Tyr(t-Bu)
His(Tr)
His(MBom)
Lys(Boc)
Arg(Pbf)
Trp
86
Figure 3. Preparation of Fmoc-Gly- (or Fmoc-Ala)-incorporated
aniline linker (4a or 4b) using coupling of acyl chlorides with sodium
anilide 6 (Method A).
41 [23]
88 [0.4]
80
95
4u
92
chlorides were prepared by a standard protocol using SOCl2
in CH2Cl2 in the presence of a catalytic amount of DMF.
Application of this procedure (Method A) to other Fmoc
amino acids, possessing acid-labile protecting groups such as
t-Bu, Boc or Tr group, is expected to induce loss of the side-
chain protections and fail to afford the desired linkers.15
In this study, we investigate versatile procedures compatible
with the coupling of the aniline linker 5 with naturally
occurring proteogenic Fmoc amino acid derivatives. We discuss
SOCl2- and POCl3-mediated procedures. The applicability of
the resulting aniline linkers 4 to NCL protocols, including the
synthesis of SEAlide peptides, is also addressed.
a
Method A: Fmoc amino acid chlorides, prepared from reaction of
Fmoc amino acids with SOCl2 in CH2Cl2 in the presence of a catalytic
amount of DMF, were reacted with sodium anilide 6 at ambient
temperature. Modified Method A: Instead of Fmoc amino acids as in
Method A, the corresponding triethylammonium salts were used.
Method B: Fmoc amino acids were coupled with the aid of POCl3 in
pyridine solvent. Method C: An equimolar mixture of Fmoc amino
acids, Et3N, and POCl3 in CH2Cl2 was used for the coupling with
aniline 5. Method D: An equimolar mixture of Fmoc amino acids,
Et3N, and POCl3 in THF (5-fold excess over 6) was coupled with
sodium anilide 6 at ambient temperature for 24 h at 0.3 M
b
concentration of activated acyl components. n.d. = not detected.
c
d
3-fold excess of Fmoc amino acid was used. 5-fold excess of Fmoc
e
f
RESULTS AND DISCUSSION
amino acid was used. Coupling reaction was conducted at 4 °C. No
desired products were obtained.
■
Initially, in addition to Gly and Ala derivatives (4a and 4b),
other aliphatic amino acids such as Val, Ile, Leu, and Phe were
successfully incorporated to yield the aniline linkers 4 (4c Val,
4d Ile, 4e Leu, and 4f Phe) using Method A, although the yield
of the Pro-incorporated linker 4g was medium. These results
are summarized in Table 1 (entries 6, 8, 10, 12, and 14), as
are the coupling data obtained from other coupling procedures
mentioned later. The syntheses of aniline linkers (4h, 4i, and
4j) possessing the acid-labile Fmoc amino acids Ser(t-Bu),
Asn(Tr), and Asp(Ot-Bu), respectively, were examined using a
slightly refined version of Method A (referred to as modified
Method A), which included the use of triethylammonium salts
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of Fmoc amino acids for the preparation of acyl chlorides with
SOCl2. We assumed that the modified conditions would suppress
the loss of acid-labile protecting groups to yield the requisite acyl
chlorides, and subsequent coupling with the sodium anilide 6
would give the desired materials.16 The reaction of Fmoc-Ser(t-
Bu)-OH using modified Method A gave the desired Ser-
incorporated aniline linker 4h in 89% isolated yield, without
loss of optical purity (entry 16). However, this was not the case
for the syntheses of the Asn(Tr)- and Asp(Ot-Bu)-incorporated
linkers (4i and 4j) (entries 23 and 25). Activation of Fmoc-
Asn(Tr)-OH with SOCl2 in the presence of Et3N afforded an
N-Tr cyclic imide derivative. Activation of Fmoc-Asp(Ot-Bu)-OH
also failed to yield the corresponding acyl chloride; loss of the
t-Bu group, probably attributable to formation of a five-
membered-ring compound, was confirmed by NMR measure-
ments.15 Although bis(trichloromethyl)carbonate seems to be a
potential reagent of choice, its applicability to the synthesis of the
aniline linker 4 remains in doubt.17
Next, we investigated the usefulness of POCl3 in the coupling
of amino acid derivatives with weakly nucleophilic aromatic
amines.18 The applicability of the POCl3-mediated protocol was
examined using Fmoc-Ala-OH as a representative amino acid.
On the basis of previous reports in the literature, POCl3-
mediated coupling in pyridine solvent (Method B) was
employed. The reaction, using a 2-fold excess of Fmoc-Ala-
OH and POCl3 over the aniline 5 in pyridine for 1.5 h at 4 °C,
afforded the desired aniline linker 4b in 48% isolated yield.
Increasing the amounts of reagents (5-fold excess) raised the
reaction yield (75%). However, removal of the Fmoc group of
the resulting linker 4b, followed by coupling with Boc-L-Leu-
OH, gave a diastereomeric mixture of dipeptides, indicating
racemization of Fmoc-L-Ala-OH during the condensation step
using Method B (ca. 2.5% racemization) (entry 4). This
problem was more pronounced in the condensation of Fmoc-L-
Ser(t-Bu)-OH (33% racemization) (entry 17). The extent of
racemization observed in the coupling of Fmoc-Ser(t-Bu)-OH
was determined with high sensitivity by Fmoc-removal from the
resulting linker 4h, followed by condensation with Boc-L-Leu-
OH or Boc-D-Leu-OH, and subsequent analysis using reversed-
phase HPLC. In this analysis, the major component generated
from coupling with Boc-D-Leu-OH corresponds to a D-Leu-L-
Ser derivative, an enantiomeric dipeptide of Boc-L-Leu-D-Ser
compound derived from racemized Ser material (ent-4h).
Coupling of Boc-Ala-OH with a weak nucleophilic hetero-
aromatic amine in pyridine with the aid of POCl3 at −15 °C for
30 min was reported to induce no racemization. However, our
case involves the use of an N-substituted aniline, and the larger
decrease in nucleophilicity requires harsher conditions
(reaction temperature: 4 °C vs −15 °C; reaction time 1.5 h
vs 30 min) for the coupling reaction to proceed, leading to the
observed racemization. The use of POBr3 in pyridine also did
not improve the coupling efficacy.
which is a problematic coupling as mentioned above (modified
Method A), proceeded to yield the desired material in 88%
isolated yield (entry 23). In the coupling of Ser derivatives, a
5-fold excess of an activated species was used to improve the
chemical yield. The attempted reaction of Fmoc-L-Ser(t-Bu)-
OH with aniline 5 in the presence of Et3N and POCl3 was
incomplete even after 26 h, and gave the corresponding aniline
linker 4 in 65% isolated yield (entry 19).
We therefore examined the effects of increasing the
nucleophilicity by the use of sodium anilide 6 (Method D).
The reaction of a 5-fold excess of the activated species resulting
from Fmoc-L-Ser(t-Bu)-OH, Et3N, and POCl3 in THF with the
preformed sodium anilide 6 in THF at ambient temperature
proceeded to completion within 24 h to furnish the desired
material 4h in 90% isolated yield with 1.8% racemization
(entry 20). Furthermore, conducting the coupling reaction at
4 °C suppressed racemization to less than 1%, without loss of
coupling yield (entry 21). Encouraged by these results, we used
the above reaction conditions (Method D) in the synthesis of
proteogenic amino acids incorporating aniline linkers 4. The
results are summarized in Table 1.
As shown in Table 1, the reactions performed using Method
D, except for the coupling of Pro and His(Tr) (entries 15 and
33), proceeded with reasonable efficiency to give the
corresponding aniline linkers 4 in isolated yields of over 65%.
Further investigations aimed at improving the coupling of Pro
or His(Tr) were suspended because of the unsuitability of the
Pro-thioester for NCL reactions20 or for reasons mentioned
later in the case of His(Tr). Because Cys and His, like Ser, are
well-known to have high susceptibility to racemization during
coupling reactions with amines, the extent of racemization of
the synthesized Fmoc-Cys(Tr)- and Fmoc-His(Tr)-incorpo-
rated linkers (4n and 4q) were also examined using Fmoc
deprotection followed by coupling with Boc-L-Leu-OH or
Boc-D-Leu-OH, as used previously for Ser(t-Bu). In the case of
Cys(Tr), no racemization was detected (entry 30). However,
use of the His(Tr) derivative resulted in 23% racemization
(entry 33). Suppression of the loss of optical purity of His
during the coupling step with sodium anilide 6 was achieved by
employing a newly developed His derivative. Very recently, we
reported that a p-methoxybenzyloxymethyl (MBom) group on
the π-nitrogen of the imidazole ring suppresses racemiza-
tion during coupling steps more efficiently than does an Nτ-
protecting group such as Tr.21 Coupling of Fmoc-His(MBom)-
OH using Method D proceeded efficiently to afford the desired
anilide linker 4r in 88% isolated yield with negligible amounts
of racemization product (<0.4%) (entry 34). The coupling
efficiencies of Gly and Ala using Method D were lower than
those using Method A (entry 1 vs 2; entry 3 vs 4). One possible
explanation for these results is that acylation of the carbamoyl
nitrogen of the less-hindered Fmoc amino acids derived from
Gly or Ala occurs under Method D conditions to prevent the
desired acylation to give the aniline linker.22 Subjection of
Fmoc-Gly-OH to a reagent mixture consisting of POCl3 and
Et3N in CH2Cl2 led to partial formation of the envisioned acyl-
carbamoyl species, which was also detected in TLC analysis of
the reaction mixture of Fmoc-Gly-OH in Method D. On the
basis of these results, we concluded that the POCl3-mediated
coupling conditions (Method D) are widely compatible with
the incorporation of naturally occurring amino acid derivatives,
except for Gly and Ala, to the aniline linker 5. The sterically
less-hindered Gly and Ala derivatives are more efficiently
incorporated in the linker using Method A; this method is also
We next examined whether the excess amount of pyridine is
involved in the racemization. Instead of pyridine, an equimolar
amount of Et3N to POCl3 was used in CH2Cl2 (Method C).19
The reaction of an equimolar mixture (3-fold excess over
aniline) of Fmoc-L-Ser(t-Bu)-OH, POCl3, and Et3N in CH2Cl2
with aniline 5 at 4 °C for 1 h afforded the desired aniline linker
4 in 51% isolated yield, although complete consumption of the
starting aniline was not observed (entry 18). It is worth noting
that the pronounced racemization, detected in the reaction with
POCl3 in pyridine, was completely suppressed (<0.1%). Using
similar reaction conditions, the reaction of Fmoc-Asn(Tr)-OH,
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Figure 4. Summary of NCL reaction of SEAlide peptides 7 with N-terminal cysteinyl peptide 8. (A) Reaction conditions of attempted NCL
reactions. (B) Progress of NCL reactions: the fraction ligated was determined by HPLC separation and integration of ligated peptides 9 (integ. 9)
detected at 220 nm as a fraction of the sum of the unreacted N-terminal cysteinyl peptide (integ. 8) and integ. 9. (C) Table for NCL reactions: n.d. =
not determined.
excellent for the coupling of Fmoc amino acids without pro-
tected side-chain functionalities (entries 6, 8, 10, and 12).
Having established facile synthetic procedures for the
N-Fmoc amino acyl-N-sulfanylethylaniline linkers 4, we next
examined the usefulness of SEAlide peptides 7 with C-terminal
diversity by subjecting various SEAlide peptides 7 to NCLs
with an N-terminal cysteinyl peptide 8. Dawson and co-workers
reported that analyses of the reaction rates of conventional
NCL for X-Cys ligation sites, where X is any of the 20 naturally
occurring amino acids, using model ligations indicated that the
amino acids can be listed in descending order of preference as
follows: Gly (G), Cys (C), His (H) > Phe (F), Met (M), Tyr
(Y), Ala (A), Trp (W) > Asn (N), Ser (S), Asp (D), Gln (Q),
Glu (E), Lys (K), Arg (R) > Leu (L), Thr (T) > Val (V), Ile
(I) > Pro (P).23 Referring to these results, we chose G, H, F, A,
N, S, E, and V as representative amino acids to examine the
applicability of SEAlide peptides to NCL. Aspartic acid (D) was
also added to the list of examined amino acids because NCL at
the Asp-Cys site resulted in the formation of a non-negligible
amount of the β-backbone isoform.24
Model peptides H-VQGSX-CFGRK-NH2 (9) were eval-
uated for possible involvement of SEAlide peptides 7 in NCL
reactions. The SEAlide peptides H-VQGSX-NAr-L-NH2 (X =
G (7a), A (7b), V (7c), F (7f), S (7h), N (7i), D (7j), E (7l),
and H (7r)) were synthesized using standard Fmoc-based
protocols. Removal of the allyl group of the aniline linkers 4
was achieved using Pd(0) (0.1 equiv) in the presence of
N-methylaniline in THF to give allyl-deprotected Fmoc amino
acyl aniline linkers Fmoc-X-NAr-OH 10 (X = G (10a), A
(10b), V (10c), F (10f), S(t-Bu) (10h), N(Tr) (10i), D(Ot-
Bu) (10j), E(Ot-Bu) (10l), and H(MBom) (10r)), which were
then coupled on leucyl-Rink-amide-type NovaSyn TGR resin
using O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium
hexafluorophosphate (HATU) in the presence of diisopropyle-
thylamine (DIPEA), with the aim of achieving high coupling
efficiency, followed by Fmoc synthesis.8 The completed resins
were treated with TFA−thioanisole−m-cresol−1,2-ethanedithiol
(EDT)−H2O (80:5:5:5:5 or 90:2.5:2.5:2.5:2.5, v/v) at ambient
temperature for 90−120 min. Except for the Asp-containing
SEAlide peptide 7j, all the SEAlide peptides were obtained in
high purity using this TFA-based deprotection followed by
reversed-phase HPLC purification. Each of the successfully
obtained SEAlide peptides (1 mM) was subjected to model NCL
reactions with H-CFGRK-NH2 8 (1 mM) in 3 M guanidine
(Gn)·HCl−0.5 M sodium phosphate buffer in the presence of
30 mM 4-mercaptophenylacetic acid (MPAA)25 and 40 mM
tris(carboxyethyl)phosphine hydrochloride (TCEP) (pH 7.3) at
37 °C. The time courses of the reactions are summarized in
Figure 4B. The attempted NCLs, except for those with 7c and 7l,
in the presence of phosphate salts went almost to completion
within 24−36 h to yield the desired ligated peptides. Ligation of
the Val- and Glu-peptides (7c and 7l) required more time to
complete the reaction; however, the desired NCL products (9c
and 9l) were obtained in reasonable isolated yields after 72 and
48 h, respectively (Figure 4C). For the NCLs using Ala-, Ser-, or
His-peptide (7b, 7h, or 7r), the extent of racemization was
checked by comparative HPLC analysis of the L- and D-amino
acid-containing peptides (Figure 4C). Under standard ligation
conditions for SEAlide peptides, racemization of Ser-peptide
(ca. 7%) was detected, as is the case in other NCL protocols
such as the cysteinyl-prolyl-ester ligation.7l Suitable conditions
for tuning the ligation conditions to suppress epimerization of
the Ser-peptide have yet to be found.
Exposure of the Asp-containing peptide resin to a TFA-based
reagent cocktail (TFA−thioanisole−m-cresol−EDT−H2O) at
room temperature for 120 min gave a mixture of two compounds
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(major:minor = 88:12, HPLC peak area) with the desired mass
number but different retention times on HPLC.
The later-eluted component (minor) disappeared on treat-
ment with sodium mercaptoethylsulfonate (MESNA) under
neutral conditions (Figure 5). HPLC analysis of the reaction
the Asp-Pro linkage is susceptible to hydrolysis under acidic
conditions;26 i.e., involvement of the side-chain carboxylic acid
functionality as an acid−base catalyst in the N−S acyl transfer
of the Asp-N-(sulfanylethyl)aniline linkage via seven-membered
ring formation could be attributable to the observed isomer-
ization to the thioester peptide 7j-thioester (Figure 6). This
Figure 6. Plausible reaction mechanism for thioester formation from
SEAlide peptide 7j under acidic conditions.
assumption prompted us to speculate that HPLC purification
under neutral conditions, to keep the Asp side-chain carboxyl
anion form, would be effective for Asp-SEAlide peptide 7j.
HPLC purification of the crude materials containing anilide 7j
and thioester peptides 7j-thioester with a 10 mM aqueous
ammonium acetate (NH4OAc) (pH 6.1)−CH3CN system,
followed by lyophilization, yielded pure 7j.
Having obtained pure Asp-SEAlide 7j, we next subjected
this material to model ligation with the N-terminal cysteinyl
peptide 8. However, the attempted reaction using the SEAlide
peptide 7j as a crypto-thioester resulted in failure and gave
a mixture of α- and β-peptides (9j and 9j-β) (Figure 5), as
reported for Asp thioester peptides.24 NCL reactions at the
Asp-Cys site still need to be addressed.
Figure 5. Formation of anhydride from thioester-type peptide
7j-thioester followed by hydrolysis or generation of α- and β-peptides
(9j and 9j-β).
indicated the presence of the hydrolyzed aniline linker 11,
which is formed by thiolysis of the possibly formed thioester-
type peptide 7j-thioester (later-eluted component) with
MESNA or direct conversion to anhydride 13 from 7j-
thioester under neutral conditions. Under neutral conditions,
the thioester-type peptides 7j-thioester and 12 were not
detected in HPLC analysis because of facile nucleophilic attack
of the β-carboxylate anion to thioester moieties leading to
formation of 13. In order to obtain pure amide-type SEAlide
peptide 7j, the early eluted component (major) was fractionated
by reversed-phase HPLC using an eluent containing 0.1% TFA.
Analytical HPLC showed that the pooled fraction contained a
homogeneous peptide 7j; however, lyophilization of the pooled
solution resulted in the formation of a mixture of the anilide 7j
and the thioester-types peptide 7j-thioester. Such N−S acyl
transfer during TFA-mediated deprotection or lyophilization
steps has not been observed for other amino acid-containing
SEAlide materials.
CONCLUSION
■
Efficient synthetic protocols for the preparation of the Fmoc
amino acyl aniline linkers 4 were developed in this study. The
developed protocols have wide applicability to naturally occurring
amino acid derivatives, which can be appropriately employed in
NCL reactions. SEAlide peptides, as crypto-thioesters, have
proved to be very useful in NCL chemistry, including one-pot/
multifragment condensations operating under kinetic conditions,
so a preparative methodology applicable to a wide variety of
SEAlide peptides will open up new avenues in the use of SEAlide
peptides. Applications of the SEAlide unit to complex protein
synthesis and in chemical biology will be reported in due course
from our laboratory.
These results indicate that the Asp-SEAlide moiety tends to
isomerize to the corresponding thioester under acidic
conditions, which keep the side-chain carboxylic acid in the
undissociated form. This could be rationalized by the fact that
EXPERIMENTAL SECTION
Representative Experimental Procedure for Method A. To a
solution of aniline 5 (100 mg, 0.21 mmol) in THF (2.0 mL)
was added by portions 55% NaH (suspendend in mineral oil)
■
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(10 mg, 0.23 mmol) at 4 °C. The mixture was stirred for 30 min at
ambient temperature. In another flask, Fmoc-Leu-OH (148 mg,
0.42 mmol) was treated with SOCl2 (0.31 mL, 4.2 mmol) in CH2Cl2
(1.5 mL) in the presence of a drop of DMF at room temperature.
After being stirred at this temperature for 3 h, the reaction was
concentrated under reduced pressure to dryness. The residue was
added the above-mentioned sodium anilide 6 solution at 4 °C. After
15 h reaction at ambient temperature, the reaction was quenched by
the addition of saturated NaHCO3 aqueous solution (saturated
NaHCO3 aq). Resulting mixture was extracted with EtOAc. The
organic phase was successively washed with saturated NaHCO3 aq,
10% citric acid aq, and brine and dried over MgSO4. After removal of
the solvent under reduced pressure, chromatographic purification on
silica gel using EtOAc/n-hexanes gave desired Fmoc-Leu-incorporated
linker (allyl ester) 4e in 74% (126 mg) isolated yield.
Representative Experimental Procedure for Modified
Method A. A mixture of Fmoc-Ser(t-Bu)-OH (480 mg, 1.3 mmol)
and Et3N (174 μL, 1.4 mmol) in CH2Cl2 (12 mL) was treated with
SOCl2 (137 μL, 2.0 mmol) in the presence of a drop of DMF at
refluxing temperature. After 2 h reaction, solvent was removed to
dryness under reduced pressure. The residue was redissolved with THF
(3 mL), and the formed salts were filtered off. To the resulting filtrate
was added sodium anilide (0.63 mmol) in THF (6 mL) added at room
temperature. After being stirred for 20 h at ambient temperature, the
reaction was quenched by the addition of saturated NaHCO3 aq.
Resulting mixture was extracted with EtOAc. The organic phase was
successively washed with saturated NaHCO3 aq, 10% citric acid aq, and
brine and dried over MgSO4. Removal of the solvent under reduced
pressure, followed by chromatographic purification on silica gel using
EtOAc/n-hexanes gave desired Fmoc-Ser(t-Bu)-incorporated linker
(allyl ester) 4h in 89% (469 mg) isolated yield.
Representative Experimental Procedure for Method B. To a
mixture of Fmoc-Ser(t-Bu)-OH (399 mg, 1.0 mmol) and aniline 5
(100 mg, 0.21 mmol) in pyridine (2.0 mL) was added POCl3 (97 mL,
1.0 mmol) at −15 °C. After being stirred at 4 °C for 3 h, the reaction
was quenched by the addition of saturated NaHCO3 aq. Procedures
identical to those employed in modified Method A afforded desired
material 4h in 48% (85 mg) isolated yield.
Representative Experimental Procedure for Method C. To a
stirred mixture of Fmoc-Ser(t-Bu)-OH (2.0 g, 5.2 mmol) and Et3N
(0.73 mL, 5.2 mmol) in CH2Cl2 (17 mL) were successively added
aniline 5 (500 mg, 1.0 mmol) and POCl3 (0.49 mL, 5.2 mmol) at
4 °C. After being stirred at 4 °C for 26 h, the reaction was quenched
by the addition of saturated NaHCO3 aq. Procedures identical to those
employed in modified Method A afforded 571 mg of desired material
4h in 65% isolated yield.
126.7, 127.0, 127.7, 127.8, 128.4, 129.5, 129.8, 131.1, 131.9, 141.3, 143.8,
144.5, 145.0, 155.9, 165.3, 171.6; HRMS (ESI-TOF) m/z calcd for
C51H49N2O5S [M + H]+ 801.3362, found 801.3358.
Allyl 4-[(Fmoc-L-Ile-2-tritylsulfanylethyl)amino]benzoate (4d).
White amorphous solid, yield: 87% (848 mg); [α]21 114.9 (c 1.00,
D
1
CHCl3); H NMR (CDCl3, 400 MHz) δ = 0.67 (3H, d, J = 6.6 Hz),
0.73 (3H, t, J = 7.2 Hz), 0.84−0.98 (1H, m), 1.31−1.45 (1H, m),
1.45−1.56 (1H, m), 2.28 (1H, ddd, J = 13.3, 9.0, and 5.4 Hz), 2.55
(1H, ddd, J = 13.3, 8.3, and 5.1 Hz), 3.37 (1H, ddd, J = 14.3, 8.3, and
5.4 Hz), 3.60 (1H, ddd, J = 14.3, 9.0, and 5.1 Hz), 4.08 (1H, dd, J =
8.8 and 7.5 Hz), 4.20 (1H, t, J = 7.0 Hz), 4.31 (1H, dd, J = 10.5 and
7.0 Hz), 4.38 (1H, dd, J = 10.5 and 7.0 Hz), 4.85 (2H, d, J = 5.6 Hz),
5.30 (1H, d, J = 8.8 Hz), 5.33 (1H, d, J = 10.5 Hz), 5.44 (1H, d, J =
17.4 Hz), 6.06 (1H, ddt, J = 17.4, 10.5, and 5.6 Hz), 7.06 (2H, d, J =
8.0 Hz), 7.10−7.36 (17H, m), 7.40 (2H, t, J = 7.4 Hz), 7.59 (2H, dd,
J = 7.2 and 2.5 Hz), 7.76 (2H, d, J = 7.5 Hz), 8.04 (2H, d, J = 8.0 Hz);
13C NMR (CDCl3, 75 MHz) δ = 11.2, 15.6, 23.9, 29.2, 38.2, 47.2, 49.0,
55.8, 65.9, 66.8, 66.9, 118.6, 119.9, 125.1, 125.1, 126.6, 127.0, 127.7,
127.8, 128.4, 129.5, 129.6, 129.8, 131.1, 132.0, 141.3, 143.8, 144.5,
145.0, 155.8, 165.3, 171.7; HRMS (ESI-TOF) m/z calcd for
C52H51N2O5S [M + H]+ 815.3519, found 815.3549.
Allyl 4-[(Fmoc-L-Leu-2-tritylsulfanylethyl)amino]benzoate (4e).
White amorphous solid, yield: 88% (75 mg); [α]18 97.4 (c 1.00,
D
1
CHCl3); H NMR (CDCl3, 400 MHz) δ = 0.37 (3H, d, J = 6.0 Hz),
0.72 (3H, d, J = 6.2 Hz), 1.08−1.20 (1H, m), 1.32−1.49 (2H, m), 2.30
(1H, ddd, J = 12.9, 9.3, and 5.4 Hz), 2.53 (1H, ddd, J = 12.9, 8.3, and
5.3 Hz), 3.28 (1H, ddd, J = 13.8, 8.3, and 5.4 Hz), 3.61 (1H, ddd, J =
13.8, 9.3, and 5.3 Hz), 4.15−4.40 (4H, m), 4.86 (2H, d, J = 5.6 Hz),
5.27 (1H, d, J = 9.2 Hz), 5.33 (1H, d, J = 10.5 Hz), 5.43 (1H, d, J =
17.1 Hz), 6.06 (1H, ddt, J = 17.1, 10.5, and 5.6 Hz), 7.05−7.35 (19H,
m), 7.39 (2H, t, J = 7.4 Hz), 7.58 (2H, dd, J = 7.2 and 4.8 Hz), 7.76
(2H, d, J = 7.4 Hz), 8.06 (2H, d, J = 8.1 Hz); 13C NMR (CDCl3, 75
MHz) δ = 20.8, 23.2, 24.3, 29.2, 42.3, 47.1, 48.9, 50.2, 65.9, 66.9, 66.9,
118.6, 119.9, 119.9, 125.1, 126.6, 127.0, 127.7, 127.8, 128.4, 129.5,
129.8, 131.1, 131.9, 141.3, 143.8, 143.9, 144.5, 144.9, 156.0, 165.2,
172.5; HRMS (ESI-TOF) m/z calcd for C52H50N2NaO5S [M + Na]+
837.3338, found 837.3304.
Allyl 4-[(Fmoc-L-Phe-2-tritylsulfanylethyl)amino]benzoate (4f).
White amorphous solid, yield: 88% (883 mg); [α]19 63.8 (c 1.00,
D
1
CHCl3); H NMR (CDCl3, 400 MHz) δ = 2.21−2.41 (2H, m), 2.71
(1H, dd, J = 12.9 and 6.1 Hz), 2.85 (1H, dd, J = 12.9 and 8.8 Hz),
3.25−3.45 (2H, m), 4.15 (1H, t, J = 7.1 Hz), 4.21−4.40 (1H, m), 4.32
(2H, d, J = 7.1 Hz), 4.85 (2H, d, J = 5.5 Hz), 5.33 (1H, d, J = 10.4 Hz),
5.38 (1H, d, J = 8.9 Hz), 5.43 (1H, d, J = 17.3 Hz), 6.05 (1H, ddt, 17.3,
10.4, and 5.5 Hz), 6.88 (2H, d, J = 7.2 Hz), 7.09−7.34 (22H, m), 7.39
(2H, t, J = 7.4 Hz), 7.55 (2H, dd, J = 7.2 and 4.4 Hz), 7.76 (2H, d, J =
7.4 Hz), 7.88 (2H, d, J = 8.1 Hz); 13C NMR (CDCl3, 75 MHz) δ =
29.0, 39.8, 47.1, 48.9, 52.9, 65.9, 66.9, 66.9, 118.6, 119.9, 125.1, 125.1,
126.7, 127.0, 127.7, 127.8, 128.0, 128.5, 129.5, 129.7, 130.9, 132.0, 132.0,
135.8, 141.3, 143.7, 143.8, 144.5, 155.2, 165.3, 170.9; HRMS (ESI-TOF)
m/z calcd for C55H49N2O5S [M + H]+ 849.3362, found 849.3356.
Allyl 4-[(Fmoc-L-Pro-2-tritylsulfanylethyl)amino]benzoate (4g).
Representative Experimental Procedure for Method D. To a
solution of Fmoc-Ser(t-Bu)-OH (400 mg, 1.05 mmol) in THF
(2.4 mL) were successively added Et3N (146 μL, 1.05 mmol) and
POCl3 (98 μL, 1.05 mmol) at 4 °C. Instantly, the resulting solution
was added to the sodium amide 6 solution in THF (0.21 mmol,
1.1 mL), prepared according to Method A, at 4 °C with additional
stirring at ambient temperature for 24 h. For all other coupling, 0.3 M
concentration of Fmoc amino acid species was used. Procedures
identical to those employed in modified Method A afforded 159 mg of
desired material 4h in 90% isolated yield.
White amorphous solid, yield: 39% (66 mg); [α]19 62.0 (c 1.00,
D
CHCl3); 1H NMR (CDCl3, 400 MHz) δ = 1.56−2.07 (4.3H, m), 2.13−
2.34 (1.2H, m), 2.42−2.54 (0.5H, m), 3.12−3.34 (1H, m), 3.34−3.51
(2H, m), 3.51−3.72 (1H, m), 3.87−4.05 (1H, m), 4.10−4.40 (3H, m),
4.67−4.81 (2H, m), 5.20−5.30 (1H, m), 5.31−5.41 (1H, m), 5.91−6.05
(1H, m) 6.50−6.78 (1H, m), 6.95−7.57 (22H, m), 7.64−7.74 (2H, m),
7.76−7.88 (1H, m), 7.91−8.10 (1H, m); 13C NMR (CDCl3, 75 MHz)
δ = 23.3, 24.4, 29.3, 30.2, 31.4, 46.9, 47.1, 47.3, 47.5, 48.5, 48.9, 57.0,
57.5, 65.8, 65.9, 66.8, 67.0, 67.3, 118.5, 118.6, 119.9, 125.0, 125.1, 125.3,
126.6, 126.9, 127.0, 127.0, 127.1, 127.6, 127.6, 127.8, 127.9, 128.4, 128.6,
129.4, 129.5, 129.6, 131.0, 131.9, 131.9, 141.2, 143.8, 144.0, 144.1, 144.4,
144.6, 145.2, 145.7, 154.2, 154.7, 165.2, 165.4, 171.5, 171.9; HRMS
(ESI-TOF) m/z calcd for C51H46N2NaO5S [M + Na]+ 821.3025, found
821.3010.
Allyl 4-[(Fmoc-L-Val-2-tritylsulfanylethyl)amino]benzoate (4c).
White amorphous solid, yield: 87% (831 mg); [α]21 100.5 (c 1.00,
D
1
CHCl3); H NMR (CDCl3, 400 MHz) δ = 0.68 (3H, d, J = 6.5 Hz),
0.76 (3H, d, J = 6.5 Hz), 1.75 (1H, oct, J = 6.5 Hz), 2.28 (1H, ddd, J =
12.8, 9.0, and 5.5 Hz), 2.55 (1H, ddd, J = 12.8, 8.4, and 5.4 Hz), 3.34
(1H, ddd, J = 14.2, 8.4, and 5.5 Hz), 3.58 (1H, ddd, J = 14.2, 9.0, and
5.4 Hz), 4.05 (1H, dd, J = 9.0 and 6.5 Hz), 4.20 (1H, t, J = 7.0 Hz),
4.30 (1H, dd, J = 10.4 and 7.0 Hz), 4.38 (1H, dd, J = 10.4 and 7.0 Hz),
4.85 (2H, d, J = 5.6 Hz), 5.33 (1H, d, J = 10.4 Hz), 5.34 (1H, d, J =
9.0 Hz), 5.44 (1H, d, J = 17.2 Hz), 6.06 (1H, ddt, J = 17.2, 10.4, and
5.6 Hz), 7.06 (2H, d, J = 8.2 Hz), 7.09−7.36 (17H, m), 7.39 (2H, t, J =
7.3 Hz), 7.58 (2H, dd, J = 7.4 and 2.8 Hz), 7.76 (2H, d, J = 7.3 Hz),
8.05 (2H, d, J = 8.2 Hz); 13C NMR (CDCl3, 75 MHz) δ = 17.2, 19.5,
29.2, 31.5, 47.2, 49.0, 56.4, 65.9, 66.9, 66.9, 118.7, 120.0, 125.1, 125.1,
Allyl 4-[(Fmoc-L-Ser(t-Bu)-2-tritylsulfanylethyl)amino]benzoate
(4h). White amorphous solid, yield: 90% (159 mg); [α]20 52.1 (c
D
1
1.00, CHCl3); H NMR (CDCl3, 400 MHz) δ = 1.06 (9H, s), 2.35−
2.54 (2H, m), 3.18−3.28 (1H, br m), 3.32 (1H, t, J = 7.8 Hz), 3.42
6953
dx.doi.org/10.1021/jo3011107 | J. Org. Chem. 2012, 77, 6948−6958

The Journal of Organic Chemistry
Article
(1H, ddd, J = 13.5, 8.3, and 5.2 Hz), 3.54 (1H, ddd, J = 13.5, 8.3, and
5.8 Hz), 4.17 (1H, t, J = 7.0 Hz), 4.30 (2H, d, J = 7.0 Hz) 4.25−4.38
(1H, m), 4.85 (2H, d, J = 5.4 Hz), 5.32 (1H, d, J = 10.4 Hz), 5.36−
5.48 (1H, m), 5.43 (1H, d, J = 17.2 Hz), 6.04 (1H, ddt, J = 17.2, 10.4,
and 5.4 Hz), 7.05−7.43 (21H, m), 7.57 (2H, dd, J = 7.2 and 3.6 Hz),
7.75 (2H, d, J = 7.4 Hz), 8.03 (2H, d, J = 8.3 Hz); 13C NMR (CDCl3,
75 MHz) δ = 27.2, 29.2, 47.1, 49.2, 51.6, 62.5, 65.8, 67.0, 73.5, 118.5,
119.9, 125.1, 126.6, 127.0, 127.6, 127.8, 128.4, 129.5, 129.7, 130.9,
132.0, 141.2, 143.8, 143.8, 144.5, 145.1, 155.5, 165.3, 170.3; HRMS
(ESI-TOF) m/z calcd for C53H53N2O6S [M + H]+ 845.3624, found
845.3643.
130.0, 131.3, 131.9, 141.2, 143.7, 143.8, 144.5, 144.6, 155.7, 165.2, 171.1,
171.8; HRMS (ESI-TOF) m/z calcd for C55H54N2NaO7S [M + Na]+
909.3549, found 909.3569.
Allyl 4-[(Fmoc-L-Gln(Tr)-2-tritylsulfanylethyl)amino]benzoate
(4m). White amorphous solid, yield: 93% (1032 mg); [α]19 48.3 (c
D
1
1.00, CHCl3); H NMR (CDCl3, 400 MHz) δ = 1.64−1.88 (2H, m),
2.00−2.22 (2H, m), 2.28 (1H, ddd, J = 13.5, 9.0, and 5.3 Hz), 2.46
(1H, ddd, J = 13.5, 8.0, and 5.3 Hz), 3.29 (1H, ddd, J = 14.1, 8.0, and
5.3 Hz), 3.48 (1H, ddd, J = 14.1, 9.0, and 5.3 Hz), 4.06−4.23 (2H, m),
4.24−4.41 (2H, m), 4.84 (2H, d, J = 5.5 Hz), 5.31 (1H, d, J = 10.4 Hz),
5.42 (1H, d, J = 17.2 Hz), 5.62 (1H, d, J = 8.2 Hz), 6.04 (1H, ddt, J =
17.2, 10.4, and 5.5 Hz), 6.47 (1H, s), 7.00−7.43 (36H, m), 7.56 (2H, d,
J = 6.6 Hz), 7.74 (2H, dd, J = 7.6 and 2.8 Hz), 7.98 (2H, d, J = 8.2 Hz);
13C NMR (CDCl3, 75 MHz) δ = 27.8, 29.2, 32.7, 47.1, 49.0, 51.3, 65.8,
66.8, 67.0, 70.5, 118.6, 119.9, 125.1, 126.6, 127.0, 127.0, 127.7, 127.8,
127.9, 128.3, 128.6, 129.5, 129.6, 130.0, 131.3, 131.9, 141.2, 141.3,
143.7, 143.8, 144.5, 155.8, 165.2, 170.4, 171.0; HRMS (ESI-TOF) m/z
calcd for C70H62N3O6S [M + H]+ 1072.4359, found 1072.4370.
Allyl 4-[(Fmoc-L-Cys(Tr)-2-tritylsulfanylethyl)amino]benzoate
Allyl 4-[(Fmoc-L-Asn(Tr)-2-tritylsulfanylethyl)amino]benzoate
(4i). White amorphous solid, yield: 89% (980 mg); [α]19 2.4
D
(c 0.40, CHCl3); 1H NMR (CDCl3, 400 MHz) δ = 2.37 (2H, t, J = 7.4
Hz), 2.50 (1H, br d, J = 15.2 Hz), 2.60 (1H, dd, J = 15.2 and 5.2 Hz),
3.35 (2H, t, J = 7.4 Hz), 4.05 (1H, t, J = 6.3 Hz), 4.17 (2H, d, J = 6.3
Hz), 4.38−4.55 (1H, m), 4.76 (2H, d, J = 5.4 Hz), 5.26 (1H, d, J =
10.4 Hz), 5.37 (1H, d, J = 17.4 Hz), 5.43−5.56 (1H, m), 5.97 (1H,
ddt, J = 17.4, 10.4, and 5.4 Hz), 6.70 (1H, s), 6.91 (2H, d, J = 7.9 Hz),
7.06−7.34 (32H, m), 7.38 (2H, t, J = 7.3 Hz), 7.49 (2H, d, J = 6.7 Hz),
7.75 (2H, d, J = 7.2 Hz), 7.89 (2H, d, J = 7.9 Hz); 13C NMR (CDCl3,
75 MHz) δ = 28.9, 36.0, 39.4, 46.9, 49.3, 49.4, 50.2, 65.6, 66.9, 67.1,
70.4, 74.0, 118.4, 119.8, 119.9, 124.8, 124.9, 124.9, 125.0, 126.5, 126.8,
126.9, 127.0, 127.4, 127.6, 127.8, 127.9, 128.4, 128.6, 129.3, 129.5, 130.9,
131.8, 141.1, 141.1, 141.8, 143.4, 143.5, 143.6, 143.6, 144.3, 144.4, 155.0,
155.6, 165.1, 168.2, 170.2, 172.9, 174.8; HRMS (ESI-TOF) m/z calcd
for C69H59N3NaO6S [M + Na]+ 1080.4022, found 1080.4039.
(4n). White amorphous solid, yield: 72% (78 mg); [α]18 16.8 (c
D
1.00, CHCl3); 1H NMR (CDCl3, 400 MHz) δ = 2.06 (1H, dd, J = 12.4
and 6.8 Hz), 2.20−2.34 (2H, m), 2.38−2.51 (1H, m), 3.26−3.50 (2H,
m), 4.19 (1H, t, J = 7.3 Hz), 4.22−4.36 (3H, m), 4.86 (2H, d, J = 5.6
Hz). 5.34 (1H, d, J = 10.4 Hz), 5.45 (1H, d, J = 17.1 Hz), 5.38−5.50
(1H, m), 6.06 (1H, ddt, J = 17.1, 10.4, and 5.6 Hz), 6.86 (2H, d, J =
7.4 Hz), 7.08−7.34 (32H, m), 7.39 (2H, t, J = 7.4 Hz), 7.59 (2H, d, J =
7.2 Hz), 7.76 (2H, d, J = 7.4 Hz), 7.95 (2H, d, J = 8.3 Hz); 13C NMR
(CDCl3, 75 MHz) δ = 29.1, 34.0, 47.0, 49.2, 50.6, 65.9, 66.5, 66.9,
67.1, 118.6, 119.9, 125.2, 126.6, 126.7, 127.1, 127.7, 127.8, 127.9,
128.1, 129.4, 129.5, 129.9, 131.2, 131.9, 141.2, 143.7, 143.8, 144.2,
144.4, 144.5, 155.3, 165.1, 169.6; HRMS (ESI-TOF) m/z calcd for
C68H58N2NaO5S2 [M + Na]+ 1069.3685, found 1069.3671.
Allyl 4-[(Fmoc-L-Asp(Ot-Bu)-2-tritylsulfanylethyl)amino]benzoate
(4j). White amorphous solid, yield: 68% (62 mg); [α]19D 47.1 (c 1.00,
CHCl3); 1H NMR (CDCl3, 400 MHz) δ = 1.36 (9H, s), 2.27 (1H, br
dd, J = 14.8 and 6.0 Hz), 2.33−2.54 (3H, m), 3.38 (1H, ddd, J = 14.0,
8.4, and 5.4 Hz), 3.52 (1H, ddd, J = 14.0, 9.2, and 4.8 Hz), 4.16 (1H, t,
J = 7.0 Hz), 4.21−4.35 (2H, m), 4.40−4.54 (1H, br m), 4.83 (2H, d,
J = 5.6 Hz), 5.30 (1H, d, J = 10.4 Hz), 5.41 (1H, d, J = 17.1 Hz), 5.52
(1H, d, J = 9.0 Hz), 6.02 (1H, ddt, J = 17.1, 10.4, and 5.6 Hz), 7.06−
7.36 (19H, m), 7.39 (2H, t, J = 7.4 Hz), 7.56 (2H, dd, J = 6.4 and 4.8
Hz), 7.76 (2H, d, J = 7.4 Hz), 8.02 (2H, d, J = 8.2 Hz); 13C NMR
(CDCl3, 75 MHz) δ = 27.9, 29.1, 38.2, 47.0, 49.0, 49.2, 65.8, 67.0,
67.0, 81.3, 118.5, 119.9, 125.1, 126.6, 127.0, 127.7, 127.8, 128.1, 129.5,
129.6, 129.9, 131.2, 131.9, 141.2, 143.7, 143.8, 144.5, 144.7, 155.2,
165.2, 169.0, 169.9; HRMS (ESI-TOF) m/z calcd for C54H52N2NaO7S
[M + Na]+ 895.3393, found 895.3373.
Allyl 4-[(Fmoc-L-Met-2-tritylsulfanylethyl)amino]benzoate (4o).
White amorphous solid, yield: 80% (690 mg); [α]18 68.3 (c 1.00,
D
1
CHCl3); H NMR (CDCl3, 400 MHz) δ = 1.56−1.78 (2H, m), 1.88
(3H, s), 2.14−2.25 (1H, m), 2.25−2.38 (2H, m), 2.42−2.60 (1H, m),
3.38 (1H, ddd, J = 13.0, 8.1, and 6.5 Hz), 3.55 (1H, ddd, J = 13.0, 8.2,
and 6.5 Hz), 4.18 (1H, t, J = 6.9 Hz), 4.25−4.39 (3H, m), 4.85 (2H, d,
J = 5.4 Hz), 5.32 (1H, d, J = 10.4 Hz), 5.43 (1H, d, J = 17.4 Hz), 5.48
(1H, d, J = 8.9 Hz), 6.05 (1H, ddt, J = 17.4, 10.4, and 5.4 Hz), 7.03−
7.36 (19H, m), 7.39 (2H, t, J = 7.4 Hz), 7.57 (2H, dd, J = 7.6 and 5.6
Hz), 7.76 (2H, d, J = 7.4 Hz), 8.06 (2H, d, J = 7.9 Hz); 13C NMR
(CDCl3, 75 MHz) δ = 15.4, 29.2, 29.7, 32.5, 47.1, 48.9, 51.0, 65.9,
66.9, 66.9, 118.6, 119.9, 125.1, 126.7, 127.0, 127.7, 127.8, 128.2, 129.5,
130.1, 131.3, 131.9, 141.3, 143.7, 143.8, 144.4, 144.6, 155.8, 165.2,
171.3; HRMS (ESI-TOF) m/z calcd for C51H49N2O5S2 [M + H]+
833.3083, found 833.3077.
Allyl 4-[(Fmoc-L-Thr(t-Bu)-2-tritylsulfanylethyl)amino]benzoate
(4k). White amorphous solid, yield: 84% (698 mg); [α]23 122.0
D
(c 1.00, CHCl3); 1H NMR (CDCl3, 400 MHz) δ = 0.89 (3H, d, J = 5.6
Hz), 1.03 (9H, s), 2.20−2.36 (1H, br m), 2.45−2.66 (1H, br m),
3.10−3.34 (1H, br m), 3.51−3.66 (1H, br m), 3.66−3.82 (1H, br m),
4.15−4.30 (1H, m), 4.22 (1H, t, J = 7.1 Hz), 4.35 (2H, d, J = 7.1 Hz),
4.86 (2H, d, J = 5.6 Hz), 5.33 (1H, d, J = 10.4 Hz), 5.44 (1H, d, J =
17.4 Hz), 5.54 (1H, d, J = 9.0 Hz), 6.06 (1H, ddt, J = 17.4, 10.4, and
5.6 Hz), 7.05−7.35 (19H, m), 7.40 (2H, t, J = 7.4 Hz), 7.61 (2H, dd,
J = 7.4 and 7.2 Hz), 7.77 (2H, d, J = 7.5 Hz), 8.06 (2H, d, J = 8.3 Hz);
13C NMR (CDCl3, 75 MHz) δ = 20.1, 28.1, 29.1, 47.2, 49.4, 56.5, 65.9,
Allyl 4-[(Fmoc-L-Tyr(t-Bu)-2-tritylsulfanylethyl)amino]benzoate
(4p). White amorphous solid, yield: 86% (773 mg); [α]22 25.9 (c
D
1
1.00, CHCl3); H NMR (CDCl3, 400 MHz) δ = 1.31 (9H, s), 2.23−
2.42 (2H, m), 2.67 (1H, dd, J = 13.0 and 5.9 Hz), 2.83 (1H, dd, J =
13.0 and 9.0 Hz), 3.24−3.43 (2H, m), 4.16 (1H, t, J = 7.0 Hz), 4.22−
4.43 (3H, m), 4.85 (2H, d, J = 5.5 Hz), 5.33 (1H, d, J = 10.5 Hz), 5.36
(1H, d, J = 8.9 Hz), 5.44 (1H, d, J = 17.2 Hz), 6.06 (1H, ddt, J = 17.2,
10.5, and 5.5 Hz), 6.79 (4H, s), 7.09−7.36 (19H, m), 7.40 (2H, t, J =
7.4 Hz), 7.56 (2H, dd, J = 6.8 and 2.4 Hz), 7.76 (2H, d, J = 7.5 Hz),
7.90 (2H, d, J = 8.0 Hz); 13C NMR (CDCl3, 75 MHz) δ = 28.8, 29.0,
39.3, 47.1, 48.9, 52.9, 65.8, 66.9, 67.0, 78.4, 118.6, 119.9, 124.1, 125.1,
125.1,126.7, 127.0, 127.7, 127.7, 127.8, 127.9, 128.1, 129.5, 129.7,
129.8, 130.0, 130.6, 130.8, 132.0, 141.3, 143.8, 143.8, 144.5, 144.5,
154.5, 155.2, 165.3, 171.0; HRMS (ESI-TOF) m/z calcd for
C59H57N2O6S [M + H]+ 921.3937, found 921.3939.
67.0, 67.7, 74.2, 118.6, 119.9, 125.2, 126.6, 127.0, 127.6, 127.7, 127.8,
127.9, 128.2, 129.5, 129.6, 131.2, 131.9, 141.3, 143.8, 144.0, 144.5,
145.4, 156.1, 165.3, 170.0; HRMS (ESI-TOF) m/z calcd for
C54H54N2NaO6S [M + Na]+ 881.3600, found 881.3580.
Allyl 4-[(Fmoc-L-Glu(Ot-Bu)-2-tritylsulfanylethyl)amino]benzoate
(4l). White amorphous solid, yield: 72% (665 mg); [α]19D 81.5 (c 1.00,
1
CHCl3); H NMR (CDCl3, 400 MHz) δ = 1.36 (9H, s), 1.65−1.83
(2H, m), 1.96−2.19 (2H, m), 2.23−2.37 (1H, m), 2.44−2.59 (1H, m),
3.33 (1H, ddd, J = 13.4, 8.8, and 5.9 Hz), 3.57 (1H, ddd, J = 13.4, 9.0,
and 6.1 Hz), 4.08−4.24 (1H, m), 4.18 (1H, t, J = 7.2 Hz), 4.32 (2H, d,
J = 7.2 Hz), 4.86 (2H, ddd, J = 5.6, 1.5, and 1.3 Hz), 5.33 (1H, dtd, J =
10.4, 1.3, and 1.2 Hz), 5.44 (1H, dtd, J = 17.2, 1.5, and 1.2 Hz), 5.53
(1H, d, J = 8.5 Hz), 6.06 (1H, ddt, J = 17.2, 10.4, and 5.6 Hz), 7.07−
7.36 (19H, m), 7.40 (2H, t, J = 7.4 Hz), 7.58 (2H, dd, J = 6.8 and 5.0
Hz), 7.76 (2H, d, J = 7.4 Hz), 8.05 (2H, d, J = 8.2 Hz); 13C NMR
(CDCl3, 75 MHz) δ = 27.5, 28.0, 29.1, 30.9, 47.1, 48.9, 51.1, 65.8,
66.9, 80.6, 118.6, 119.9, 125.1, 126.3, 127.0, 127.8, 128.3, 129.5, 129.6,
Allyl 4-[(Fmoc-L-His(Tr)-2-tritylsulfanylethyl)amino]benzoate
(4q). White amorphous solid, yield: 41% (745 mg); [α]20 32.9 (c
D
1
1.00, CHCl3); H NMR (CDCl3, 400 MHz) δ = 2.15−2.38 (1H, br
m), 2.52−2.71 (2H, br m), 2.71−2.87 (1H, br m), 3.17−3.37 (1H, br
m), 3.40−3.61 (1H, m), 4.18 (1H, t, J = 7.3 Hz), 4.20−4.35 (2H, m),
4.37−4.49 (1H, m), 4.87 (2H, d, J = 5.4 Hz), 5.34 (1H, d, J = 10.4
Hz), 5.45 (1H, d, J = 17.3 Hz), 6.07 (1H, ddt, J = 17.3, 10.4, and 5.4
Hz), 6.13 (1H, d, J = 8.0 Hz), 6.50 (1H, s), 7.02−7.45 (37H, m), 7.59
6954
dx.doi.org/10.1021/jo3011107 | J. Org. Chem. 2012, 77, 6948−6958

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Article
(2H, dd, J = 7.6 and 2.8 Hz), 7.76 (2H, d, J = 7.4 Hz), 8.02 (2H, d, J =
8.2 Hz); 13C NMR (CDCl3, 75 MHz) δ = 29.1, 30.4, 47.0, 49.2, 51.9,
65.7, 66.9, 67.0, 75.3, 118.5, 119.6, 119.8, 125.2, 125.3, 126.5, 127.0,
127.0, 127.1, 127.5, 127.7, 127.9, 128.0, 128.3, 129.4, 129.7, 131.0,
131.9, 135.6, 138.2, 141.1, 142.1, 143.8, 143.9, 144.5, 145.1, 155.7,
165.2, 170.6; HRMS (ESI-TOF) m/z calcd for C71H60N4NaO5S
[M + Na]+ 1103.4182, found 1103.4187.
6.93 (1H, t, J = 7.3 Hz), 7.09−7.37 (21H, m), 7.40 (2H, t, J = 7.4 Hz),
7.58 (2H, dd, J = 7.6 and 3.6 Hz), 7.66 (2H, d, J = 6.9 Hz), 7.71−7.82
(1H, m), 7.77 (3H, d, J = 7.6 Hz) ; 13C NMR (CDCl3, 75 MHz) δ =
29.1, 29.7, 47.1, 48.5, 52.0, 65.7, 66.7, 66.9, 110.0, 110.9, 118.4, 118.6,
119.6, 119.9, 122.2, 123.0, 125.1, 125.1, 126.6, 127.0, 127.4, 127.6,
127.7, 127.8, 129.3, 129.5, 129.6, 130.5, 132.0, 135.9, 141.2, 143.8,
144.3, 144.5, 155.4, 165.3, 171.7; HRMS (ESI-TOF) m/z calcd for
C57H50N3O5S [M + H]+ 888.3471, found 888.3444.
Allyl 4-[(Fmoc-L-His(MBom)-2-tritylsulfanylethyl)amino]benzoate
(4r). White amorphous solid, yield: 88% (91 mg); [α]20D 45.4 (c 1.00,
4-[(Fmoc-L-Val-2-tritylsulfanylethyl)amino]benzoic acid (10c).
1
White amorphous solid, yield: 80% (76 mg); [α]24 82.8 (c 0.40,
CHCl3); H NMR (CDCl3, 400 MHz) δ = 2.25 (1H, ddd, J = 12.8,
D
1
9.2, and 6.0 Hz), 2.44 (1H, ddd, J = 12.8, 8.8, and 6.0 Hz), 2.77 (1H,
dd, J = 14.7 and 6.7 Hz), 2.93 (1H, dd, J = 14.7 and 7.9 Hz), 3.20 (1H,
ddd, J = 14.0, 8.8, and 6.0 Hz), 3.60 (1H, ddd, J = 14.0, 9.2, and 6.0
Hz), 3.78 (3H, s), 4.07 (1H, d, J = 11.6 Hz), 4.12 (1H, d, J = 11.6 Hz),
4.15 (1H, t, J = 6.9 Hz), 4.24−4.41 (3H, m), 4.77 (1H, d, J = 11.2 Hz),
4.81 (1H, d, J = 11.2 Hz), 4.83 (2H, d, J = 5.5, 1.7, and 1.5 Hz), 5.30
(1H, dtd, J = 10.4, 1.5, and 1.3 Hz), 5.42 (1H, dtd, J = 17.2, 1.7, and
1.3 Hz), 5.50 (1H, d, J = 8.8 Hz), 6.03 (1H, ddt, J = 17.2, 10.4, and 5.5
Hz), 6.70 (1H, s), 6.72−6.81 (1H, br m), 6.84 (2H, d, J = 8.5 Hz),
7.05−7.35 (21H, m), 7.38 (2H, t, J = 7.3 Hz), 7.55 (2H, dd, J = 7.2
and 3.2 Hz), 7.74 (2H, d, J = 7.5 Hz), 7.94 (2H, d, J = 8.5 Hz); 13C
NMR (CDCl3, 75 MHz) δ = 27.5, 29.0, 47.0, 48.8, 51.2, 55.2, 65.9,
66.9, 69.0, 72.3, 114.0, 118.7, 120.0, 125.0, 126.1, 126.6, 127.0, 127.7,
127.8, 127.9, 128.1, 129.4, 129.6, 129.7, 129.9, 131.0, 131.9, 138.2,
141.3, 143.7, 144.3, 155.3, 159.6, 165.1, 170.6; HRMS (ESI-TOF) m/z
calcd for C61H56N4NaO7S [M + Na]+ 1011.3767, found 1011.3791.
Allyl 4-[(Fmoc-L-Lys(Boc)-2-tritylsulfanylethyl)amino]benzoate
CHCl3); H NMR (CDCl3, 400 MHz) δ = 0.75 (3H, d, J = 6.5 Hz),
0.81 (3H, d, J = 6.5 Hz), 1.75−1.94 (1H, m), 2.23−2.38 (1H, m),
2.48−2.65 (1H, m), 3.39 (1H, ddd, J = 13.9, 8.5, and 5.4 Hz), 3.60
(1H, ddd, J = 13.9, 8.8, and 5.2 Hz), 4.11 (1H, dd, J = 9.5 and 7.2 Hz),
4.22 (1H, t, J = 7.0 Hz), 4.34 (1H, dd, J = 10.4 and 7.0 Hz), 4.43 (1H,
dd, J = 10.4 and 7.0 Hz), 5.80 (1H, d, J = 9.5 Hz), 7.08 (2H, d, J = 8.1
Hz), 7.10−7.44 (19H, m), 7.63 (2H, dd, J = 7.6 and 2.8 Hz), 7.76 (2H,
d, J = 7.2 Hz), 8.09 (2H, d, J = 8.1 Hz); 13C NMR (CDCl3, 75 MHz)
δ = 17.3, 19.4, 29.1, 31.7, 47.2, 49.1, 56.5, 67.0, 67.0, 120.0, 125.1, 125.2,
126.7, 127.1, 127.3, 127.7, 127.8, 127.9, 128.4, 129.5, 131.7, 141.3, 143.8,
144.5, 145.3, 146.8, 156.1, 168.4, 171.7; HRMS (ESI-TOF) m/z calcd
for C48H45N2O5S [M + H]+ 761.3049, found 761.3057.
4-[(Fmoc-L-Phe-2-tritylsulfanylethyl)amino]benzoic acid (10f).
White amorphous solid, yield: 93% (176 mg); [α]19 53.5 (c 1.00,
D
1
CHCl3); H NMR (CDCl3, 400 MHz) δ = 2.23−2.43 (2H, m), 2.78
(1H, dd, J = 12.9 and 5.9 Hz), 2.91 (1H, dd, J = 12.9 and 9.2 Hz),
3.28−3.45 (2H, m), 4.18 (1H, t, J = 7.0 Hz), 4.25−4.45 (3H, m), 5.86
(1H, d, J = 8.9 Hz), 6.95 (2H, d, J = 7.2 Hz), 7.10−7.35 (22H, m),
7.39 (2H, t, J = 7.2 Hz), 7.59 (2H, dd, J = 7.2 and 1.2 Hz), 7.76 (2H,
d, J = 7.2 Hz), 7.93 (2H, d, J = 8.0 Hz); 13C NMR (CDCl3, 75 MHz)
δ = 28.9, 39.8, 47.1, 49.0, 53.0, 67.0, 67.1, 119.9, 125.1, 125.2, 126.7,
127.0, 127.7, 127.8, 127.9, 128.1, 128.5, 128.7, 129.5, 129.6, 129.6,
131.4, 132.1, 132.2, 132.2, 135.8, 141.3, 143.7, 143.8, 144.5, 144.6,
155.5, 168.8, 171.3; HRMS (ESI-TOF) m/z calcd for C52H45N2O5S
[M + H]+ 809.3049, found 809.3044.
(4s). White amorphous solid, yield: 80% (779 mg); [α]21 69.1 (c
D
1
1.00, CHCl3); H NMR (CDCl3, 400 MHz) δ = 1.01−1.30 (4H, br
m), 1.31−1.52 (2H, m) 1.41 (9H, s), 2.29 (1H, ddd, J = 12.8, 9.4, and
5.4 Hz), 2.53 (1H, ddd, J = 12.8, 9.2, and 5.1 Hz), 2.81−3.05 (2H,
br s), 3.31 (1H, ddd, J = 14.5, 9.2, and 5.4 Hz), 3.60 (1H, ddd, J =
14.5, 9.4, and 5.1 Hz), 4.10−4.26 (1H, m), 4.19 (1H, t, J = 7.2 Hz),
4.33 (2H, d, J = 7.2 Hz), 4.37 (1H, m), 4.86 (2H, ddd, J = 6.0, 2.0, and
1.6 Hz), 5.33 (1H, dtd, J = 11.2, 2.0, and 1.2 Hz), 5.38−5.48 (1H, m),
5.44 (1H, dtd, J = 16.8, 1.6, and 1.2 Hz), 6.06 (1H, ddt, J = 16.8,
11.2, and 6.0 Hz), 7.04−7.45 (19H, m), 7.40 (2H, t, J = 7.4 Hz), 7.59
(2H, dd, J = 7.2 and 4.8 Hz), 7.76 (2H, d, J = 7.5 Hz), 8.06 (2H, d, J =
8.2 Hz); 13C NMR (CDCl3, 75 MHz) δ = 22.1, 28.4, 29.2, 29.2, 32.5,
40.1, 47.1, 48.9, 51.3, 65.9, 66.9, 66.9, 79.1, 118.6, 119.9, 125.1, 126.6,
127.0, 127.6, 127.8, 128.3, 129.4, 129.6, 130.0, 131.2, 131.9, 141.2, 143.7,
143.8, 144.4, 144.7, 155.8, 155.9, 165.1, 171.7; HRMS (ESI-TOF) m/z
calcd for C57H60N3O7S [M + H]+ 930.4152, found 930.4143.
4-[(Fmoc-L-Ser(t-Bu)-2-tritylsulfanylethyl)amino]benzoic acid
(10h). White amorphous solid, yield: quant. (187 mg); [α]20 45.4
D
1
(c 1.00, CHCl3); H NMR (CDCl3, 400 MHz) δ = 1.08 (9H, s),
2.35−2.55 (2H, m), 3.20−3.34 (1H, m), 3.34−3.50 (2H, m), 3.50−
3.65 (1H, m), 4.19 (1H, t, J = 7.1 Hz), 4.32 (2H, d, J = 7.1 Hz), 4.35−
4.46 (1H, m), 5.68 (1H, d, J = 8.8 Hz), 7.10−7.35 (19H, m), 7.39
(2H, t, J = 7.3 Hz), 7.59 (2H, dd, J = 6.8 and 4.0 Hz), 7.75 (2H, d, J =
7.3 Hz), 8.06 (2H, d, J = 8.3 Hz); 13C NMR (CDCl3, 75 MHz) δ =
27.2, 29.1, 47.0, 49.3, 51.6, 62.5, 67.0, 67.1, 73.7, 119.9, 125.2, 126.7,
127.0, 127.7, 127.8, 127.9, 128.5, 129.1, 129.5, 131.5, 141.3, 143.7,
143.8, 144.5, 145.4, 155.8, 169.3, 169.3; HRMS (ESI-TOF) m/z calcd
for C50H49N2O6S [M + H]+ 805.3311, found 805.3332.
Allyl 4-[(Fmoc-L-Arg(Pbf)-2-tritylsulfanylethyl)amino]benzoate
(4t). White amorphous solid, yield: 95% (1101 mg); [α]22 46.3 (c
D
1
1.00, CHCl3); H NMR (CDCl3, 400 MHz) δ = 1.16−1.34 (2H, m),
1.41 (6H, s), 1.43−1.55 (2H, m), 2.06 (3H, s), 2.19−2.33 (1H, m),
2.43−2.51 (1H, m), 2.42- 2.51 (1H, m), 2.48 (3H, s), 2.54 (3H, s),
2.66−2.96 (2H, br m), 2.89 (2H, s), 3.23 (1H, ddd, J = 13.6, 9.0, and 5.4
Hz), 3.63 (1H, ddd, J = 13.6, 9.2, and 5.5 Hz), 4.06−4.20 (1H, m), 4.13
(1H, t, J = 6.4 Hz), 4.21−4.37 (2H, m), 4.85 (2H, d, J = 5.5 Hz), 5.32
(1H, d, J = 10.4 Hz), 5.43 (1H, d, J = 17.2 Hz), 5.92 (1H, d, J = 8.1 Hz),
5.98−6.16 (3H, m), 7.04−7.33 (19H, m), 7.37 (2H, t, J = 7.4 Hz), 7.54
(2H, t, J = 7.4 Hz), 7.74 (2H, d, J = 7.5 Hz), 8.04 (2H, d, J = 8.0 Hz);
13C NMR (CDCl3, 75 MHz) δ = 12.4, 17.9, 19.3, 24.6, 28.5, 29.1, 30.2,
40.5, 43.1, 47.0, 48.9, 50.9, 66.0, 66.9, 67.3, 86.4, 117.5, 118.7, 119.9,
124.7, 125.0, 125.1, 126.7, 127.0, 127.2, 127.7, 127.7, 127.8, 127.9, 128.3,
129.4, 130.2, 131.3, 132.5, 138.5, 141.2, 143.6, 144.4, 144.4, 144.4, 155.7,
156.3, 158.9, 165.1, 171.4; HRMS (ESI-TOF) m/z calcd for
C65H67KN5O8S2 [M + K]+ 1148.4068, found 1148.4044.
4-[(Fmoc-L-Asn(Tr)-2-tritylsulfanylethyl)amino]benzoic acid (10i).
White amorphous solid, yield: 83% (160 mg); [α]24 −17.0 (c 0.40,
D
1
CHCl3); H NMR (CDCl3, 400 MHz) δ = 2.28−2.47 (2H, t, J = 6.7
Hz), 2.63−2.86 (2H, m), 3.15−3.46 (2H, m), 4.11 (1H, t, J = 6.7 Hz),
4.23 (1H, dd, J = 10.3 and 6.7 Hz), 4.30 (1H, dd, J = 10.3 and 6.7 Hz),
4.39−4.69 (1H, m), 6.34 (1H, d, J = 7.6 Hz), 6.77 (1H, s), 6.92 (2H,
d, J = 7.4 Hz), 7.04−7.34 (32H, m), 7.36 (2H, t, J = 7.4 Hz), 7.55 (2H,
dd, J = 13.6 and 7.2 Hz), 7.74 (2H, d, J = 7.4 Hz), 7.82 (2H, d, J = 7.4
Hz); 13C NMR (CDCl3, 75 MHz) δ = 28.9, 39.3, 47.0, 49.5, 49.5, 67.0,
67.0, 70.7, 119.9, 125.2, 125.2, 126.6, 127.0, 127.0, 127.2, 127.7, 127.8,
127.9, 128.7, 129.4, 129.6, 131.5, 141.3, 143.5, 143.8, 144.2, 144.5,
144.7, 155.6, 168.2, 169.0, 170.3; HRMS (ESI-TOF) m/z calcd for
C66H56N3O6S [M + H]+ 1018.3890, found 1018.3877.
Allyl 4-[(Fmoc-L-Trp-2-tritylsulfanylethyl)amino]benzoate (4u).
4-[(Fmoc-L-Asp(Ot-Bu)-2-tritylsulfanylethyl)amino]benzoic acid
White amorphous solid, yield: 92% (852 mg); [α]23 82.1 (c 1.00,
(10j). White amorphous solid, yield: 98% (188 mg); [α]23 80.3 (c
D
D
1
1
CHCl3); H NMR (CDCl3, 400 MHz) δ = 2.16 (1H, ddd, J = 12.4,
1.00, CHCl3); H NMR (CDCl3, 400 MHz) δ = 1.37 (9H, s), 2.23−
8.4, and 7.2 Hz), 2.29 (1H, ddd, J = 12.4, 7.4, and 6.9 Hz), 2.93 (1H,
dd, J = 13.8 and 5.4 Hz), 3.06 (1H, dd, J = 13.8 and 9.2 Hz), 3.27−
3.47 (2H, m), 4.19 (1H, t, J = 7.0 Hz), 4.32 (1H, dd, J = 18.0 and 7.0
Hz), 4.35 (1H, dd, J = 18.0 and 7.0 Hz), 4.51 (1H, ddd, J = 9.2, 8.8,
and 5.4 Hz), 4.83 (2H, d, J = 5.5 Hz), 5.34 (1H, d, J = 10.4 Hz), 5.44
(1H, d, J = 17.3 Hz), 5.49 (1H, d, J = 8.8 Hz), 6.06 (1H, ddt, J = 17.3,
10.4, and 5.5 Hz), 6.18−6.52 (1H, br s), 6.76 (1H, d, J = 1.7 Hz),
2.44 (2H, m), 2.44−2.60 (2H, m), 3.29−3.46 (1H, m), 3.46−3.61
(1H, m), 4.17 (1H, t, J = 7.1 Hz), 4.29 (2H, d, J = 7.1 Hz), 4.45−4.61
(1H, br m), 5.77 (1H, d, J = 9.1 Hz), 7.07−7.35 (19H, m), 7.38 (2H, t,
J = 7.4 Hz), 7.58 (2H, dd, J = 7.6 and 2.0 Hz), 7.75 (2H, d, J = 7.4
Hz), 8.06 (2H, d, J = 8.2 Hz); 13C NMR (CDCl3, 75 MHz) δ = 28.0,
29.1, 38.3, 47.0, 49.0, 49.3, 67.0, 67.2, 81.5, 120.0, 125.2, 126.7, 127.0,
127.7, 127.8, 128.2, 128.5, 128.6, 129.5, 131.7, 132.1, 132.2, 141.3,
6955
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Article
143.7, 143.8, 144.5, 145.2, 155.4, 169.1, 169.4, 170.1; HRMS (ESI-TOF)
m/z calcd for C51H49N2O7S [M + H]+ 833.3260, found 833.3247.
4-[(Fmoc-L-Glu(Ot-Bu)-2-tritylsulfanylethyl)amino]benzoic acid
for 90−120 min. The reaction was filtered to give filtrate to which was
added cold Et2O to afford a precipitate. The formed precipitate was
collected by centrifugation and thoroughly washed with Et2O to give a
crude model SEAlide peptide 7 (H-VQGSX-NAr-L-NH2: X = G (7a),
A (7b), V (7c), F (7f), S (7h), N (7i), D (7j), E (7l), and H (7r)).
The crude materials, except for 7j, were purified by reversed-phase
HPLC on preparative HPLC column using linear gradient using solvent
system consisting of 0.1% TFA in H2O and 0.1% TFA in CH3CN.
For Asp-containing peptide 7j, linear gradient of CH3CN in 10 mM
NH4OAc buffer was used.
7a (X = G). Analytical HPLC conditions: linear gradient of 0.1%
TFA/CH3CN in 0.1% TFA/H2O, 5−45% over 30 min, retention
time = 18.4 min; LRMS (ESI-Ion Trap) m/z calcd for C32H52N9O9S
[M + H]+ 738.4, found 738.1.
7b (X = A). Analytical HPLC conditions: linear gradient of 0.1%
TFA/CH3CN in 0.1% TFA/H2O, 5−45% over 30 min, retention
time = 18.7 min; LRMS (ESI-Ion Trap) m/z calcd for C33H54N9O9S
[M + H]+ 752.4, found 752.2.
(10l). White amorphous solid, yield: 86% (164 mg); [α]24 57.0
D
1
(c 1.00, CHCl3); H NMR (CDCl3, 400 MHz) δ = 1.38 (9H, s),
1.66−1.87 (2H, m), 2.00−2.24 (2H, m), 2.25−2.40 (1H, m), 2.45−
2.60 (1H, m), 3.30−3.45 (1H, m), 3.58 (1H, ddd, J = 13.4, 8.8, and 5.9
Hz), 4.20 (1H, t, J = 7.2 Hz), 4.16−4.28 (1H, m), 4.35 (2H, d, J = 7.2
Hz), 5.92 (1H, d, J = 8.3 Hz), 7.06−7.37 (19H, m), 7.39 (2H, t, J = 7.4
Hz), 7.61 (2H, dd, J = 7.6 and 2.4 Hz), 7.76 (2H, d, J = 7.4 Hz), 8.10
(2H, d, J = 8.0 Hz); 13C NMR (CDCl3, 75 MHz) δ = 25.6, 28.0, 29.1,
30.8, 47.1, 49.0, 51.1, 67.1, 68.0, 80.7, 119.9, 125.2, 126.7, 127.0, 127.2,
127.7, 127.8, 127.9, 128.3, 128.6, 129.5, 131.8, 132.1, 141.3, 143.7,
143.8, 144.5, 144.9, 155.9, 168.6, 171.3, 172.0; HRMS (ESI-TOF) m/z
calcd for C52H51N2O7S [M + H]+ 847.3417, found 847.3391.
4-[(Fmoc-L-His(MBom)-2-tritylsulfanylethyl)amino]benzoic acid
(10r). White amorphous solid, yield: 90% (52 mg); [α]20 27.8 (c
D
1
1.00, CHCl3); H NMR (CDCl3, 400 MHz) δ = 2.16−2.30 (1H, m),
2.38−2.54 (1H, m), 2.81 (1H, dd, J = 14.8 and 6.6 Hz), 2.97 (1H, d,
J = 14.8 and 7.7 Hz), 3.24−3.38 (1H, m), 3.52−3.67 (1H, m), 3.73
(3H, s), 4.08−4.26 (1H, m), 4.13 (1H, d, J = 11.6 Hz), 4.20 (1H, d,
J = 11.6 Hz), 4.28−4.48 (3H, m), 4.85 (1H, d, J = 10.7 Hz), 4.96 (1H,
d, J = 10.7 Hz), 5.63 (1H, d, J = 8.5 Hz), 6.65−6.92 (1H, m), 6.71 (1H,
s), 6.83 (2H, d, J = 8.4 Hz), 7.06−7.44 (22H, m), 7.50−7.64 (3H, m),
7.66−7.79 (2H, m), 8.07 (2H, d, J = 8.2 Hz); 13C NMR (CDCl3, 75
MHz) δ = 27.4, 29.1, 47.1, 48.7, 51.6, 55.2, 66.9, 69.5, 72.9, 114.1, 120.0,
125.1, 126.7, 126.8, 127.1, 127.6, 127.7, 127.8, 127.9, 129.5, 129.7, 131.4,
131.9, 137.7, 141.3, 143.5, 143.7, 144.4, 155.4, 159.6, 168.3, 170.7;
HRMS (ESI-TOF) m/z calcd for C58H53N4O7S [M + H]+ 949.3635,
found 949.3661.
7c (X = V). Analytical HPLC conditions: linear gradient of 0.1%
TFA/CH3CN in 0.1% TFA/H2O, 5−45% over 30 min, retention
time = 20.8 min; LRMS (ESI-Ion Trap) m/z calcd for C35H58N9O9S
[M + H]+ 780.4, found 780.5.
7f (X = F). Analytical HPLC conditions: linear gradient of 0.1%
TFA/CH3CN in 0.1% TFA/H2O, 5−45% over 30 min, retention
time = 23.4 min; LRMS (ESI-Ion Trap) m/z calcd for C39H58N9O9S
[M + H]+ 828.4, found 828.6.
7h (X = S). Analytical HPLC conditions: linear gradient of 0.1%
TFA/CH3CN in 0.1% TFA/H2O, 5−45% over 30 min, retention
time = 17.5 min; LRMS (ESI-Ion Trap) m/z calcd for C33H54N9O10S
[M + H]+ 768.4, found 768.1.
Representative Experiment for the Check of Racemization
during Coupling Reaction of Fmoc Amino Acid to the Aniline
Linker. Fmoc-Ser(t-Bu)-incorporated aniline linker 4h (50 mg,
0.059 mmol) was treated with 20% piperidine in DMF (0.5 mL) at
room temperature. After 10 min, the solvents were completely
evaporated off using oil pump. To the residue was added preactivated
Boc-L-Leu or Boc-D-Leu derivative, prepared from reaction of Boc-L/D-
Leu-OH (16 mg each, 0.065 mmol) with HBTU (22 mg, 0.059 mmol)
and DIPEA (10 mL, 0.059 mmol) in DMF (0.5 mL) for 10 min at
ambient temperature. After 30 min reaction, a mixture of EtOAc and
saturated NaHCO3 aq was added to quench each reaction. The
organic layer was washed with saturated NaHCO3 aq, 10% citric acid
aq, and brine and dried over MgSO4. After removal of the solvent, each
sample obtained from the coupling with Boc-L-Leu-OH or Boc-D-Leu-
OH was analyzed by reversed-phase HPLC on analytical column.
Because major coupling product (D-Leu-L-Ser dipeptide) resulting from
the Boc-D-Leu-coupling corresponds to the epimer of the reacemiza-
tion product (L-Leu-D-Leu diepetide), extent of the racemization was
determined as integration of minor peak correspond to D-Leu-L-Ser
dipeptide as fraction of the sum of major and minor dipeptides by
HPLC analysis of Boc-L-Leu-coupling sample.
General Experimental Procedure for the Synthesis of SEAlide
Peptides 7. To NovaSyn TGR resin (Rink amide type: 0.25 mmol
amine/g, 0.40 g, 0.10 mmol) was coupled Fmoc-Leu-OH (100 mg,
0.30 mmol) with the aid of diisopropylcarbodiimide (DIPCDI) (48 mL,
0.30 mmol) and HOBt·H2O (48 mg, 0.30 mol) in DMF at room
temperature for 2 h, followed by Fmoc removal with 20% piperidine
in DMF, to give Leu-incorporated resin (H-Leu-NovaSyn TGR
resin). The resulting resin was treated with an equimolar mixture of
an acid-type N-Fmoc amino acyl-N-sulfanylethylaniline linker (Fmoc-
X-NAr-OH 10: 0.20 mmol), HATU (76 mg, 0.20 mmol), and DIPEA
(36 mL, 0.20 mmol) at room temperature for 3 h to yield an Fmoc-X-
NAr-incorporated resin. On the resulting resin, standard Fmoc SPPS
(coupling with an Fmoc amino acids (3 equiv) using DIPCDI/
HOBt·H2O (3.0 equiv each) (2 h) and Fmoc removal with 20%
piperidine in DMF (10 min)) was performed for peptide chain
elongation to give a protected peptide resin for a model SEAlide
peptide 7. The completed resin was exposed to a TFA-based reagents
cocktail (TFA−thioanisole−m-cresol−EDT−H2O, 80:5:5:5:5 or
90:2.5:2.5:2.5:2.5, (v/v), 50 μL/1 mg of resin) at room temperature
7i (X = N). Analytical HPLC conditions: linear gradient of 0.1%
TFA/CH3CN in 0.1% TFA/H2O, 5−45% over 30 min, retention
time = 17.1 min; LRMS (ESI-Ion Trap) m/z calcd for C34H55N10O10S
[M + H]+ 795.4, found 795.2.
7l (X = E). Analytical HPLC conditions: linear gradient of 0.1%
TFA/CH3CN in 0.1% TFA/H2O, 5−45% over 30 min, retention
time = 18.9 min; LRMS (ESI-Ion Trap) m/z calcd for C35H56N9O11S
[M + H]+ 810.4, found 810.2.
7r (X = H). Analytical HPLC conditions: linear gradient of 0.1%
TFA/CH3CN in 0.1% TFA/H2O, 10 to 40% over 30 min, retention
time = 15.0 min; LRMS (ESI-Ion Trap) m/z calcd for C36H56N11O9S
[M + H]+ 818.4, found 818.4.
7j (X = D). Analytical HPLC conditions: linear gradient of 0.1%
TFA/CH3CN in 0.1% TFA/H2O, 10 to 40% over 30 min, retention
time = 16.6 min; LRMS (ESI-Ion Trap) m/z calcd for C34H54N9O10S
[M + H]+ 796.4, found 796.2.
7j-thioester Analytical HPLC conditions: linear gradient of 0.1%
TFA/CH3CN in 0.1% TFA/H2O, 10 to 40% over 30 min, retention
time = 17.0 min; LRMS (ESI-Ion Trap) m/z calcd for C34H54N9O10S
[M + H]+ 796.4, found 796.2.
Synthesis of N-Terminal Cysteinyl Peptide 8. N-Terminal
cysteinyl peptide 8 was prepared by standard Fmoc SPPS protocol on
NovaSyn TGR resin (Rink amide type: 0.25 mmol amine/g, 0.40 g,
0.10 mmol) by procedures identical to those reported.12a
General Experimental Procedure for NCL of SEAlide
Peptides 7 with N-Terminal Cysteinyl Peptide 8. Model NCLs
of SEAlide peptides 7 (1.0 mM) with N-terminal cysteinyl peptide 8
(1.0 mM) were performed in 3 M Gn·HCl−0.5 M Na phosphate
buffer in the presence of 30 mM MPAA and 40 mM TCEP (pH 7.3)
at 37 °C. For each reaction, at each time point 5 μL aliquots were
withdrawn and analyzed by analytical reversed-phase HPLC on
Cosmosil 5C18-AR-II analytical column (4.6 × 250 mm; detection at
220 nm) with a concave gradient (curve 7 of the Waters 600E) of 0.1%
TFA/CH3CN in 0.1% TFA/H2O, 5−45% over 30 min. The extent of
ligation was quantified by integration of the ligated products 9 as a
fraction of the sum of the unreacted N-terminal cysteinyl peptide and
the ligated products.
For each reaction yielding the ligated peptides 9, a whole reaction
mixture was fractionated by preparative reversed-phase HPLC on
Cosmosil 5C18-AR-II preparative column (20 × 250 mm) after 24−72 h
6956
dx.doi.org/10.1021/jo3011107 | J. Org. Chem. 2012, 77, 6948−6958

The Journal of Organic Chemistry
Article
reactions to yield the desired ligated peptide. Epimerization during
couplings of 9b, 9h, and 9r was examined by comparative HPLC
analyses of crude ligation products with D-amino acid-incorporated
reference peptides, which were synthesized by standard Fmoc SPPS.
9a (X = G). Analytical HPLC conditions: linear gradient of 0.1%
TFA/CH3CN in 0.1% TFA/H2O, 5−45% over 30 min, retention
time = 12.0 min; LRMS (ESI-Ion Trap) m/z calcd for C43H73N16O12S
[M + H]+ 1037.5, found 1037.4.
9b (X = A). Analytical HPLC conditions: linear gradient of 0.1%
TFA/CH3CN in 0.1% TFA/H2O, 5−45% over 30 min, retention
time = 12.3 min; LRMS (ESI-Ion Trap) m/z calcd for C44H75N16O12S
[M + H]+ 1051.5, found 1051.6.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported in part by a Grant-in Aid for
Scientific Research (KAKENHI). A.O. and A.S. are grateful for
research grants from the Takeda Science Foundation. K.S.
thanks the Yoshida Scholarship Foundation for a scholarship.
K.T. thanks Japan Society for the Promotion of Science for a
fellowship.
9c (X = V). Analytical HPLC conditions: linear gradient of 0.1%
TFA/CH3CN in 0.1% TFA/H2O, 10 to 40% over 30 min, retention
time = 12.3 min; LRMS (ESI-Ion Trap) m/z calcd for C46H79N16O12S
[M + H]+ 1079.6, found 1079.8.
9f (X = F). Analytical HPLC conditions: linear gradient of 0.1%
TFA/CH3CN in 0.1% TFA/H2O, 5 to 35% over 30 min, retention
time = 18.7 min; LRMS (ESI-Ion Trap) m/z calcd for C50H79N16O12S
[M + H]+ 1127.6, found 1128.0.
9h (X = S). Analytical HPLC conditions: linear gradient of 0.1%
TFA/CH3CN in 0.1% TFA/H2O, 5−45% over 30 min, retention
time = 12.0 min; LRMS (ESI-Ion Trap) m/z calcd for C44H75N16O13S
[M + H]+ 1067.5, found 1067.8.
9i (X = N). Analytical HPLC conditions: linear gradient of 0.1%
TFA/CH3CN in 0.1% TFA/H2O, 5−45% over 30 min, retention
time = 12.2 min; LRMS (ESI-Ion Trap) m/z calcd for C45H76N17O13S
[M + H]+ 1094.6, found 1094.2.
9l (X = E). Analytical HPLC conditions: linear gradient of 0.1%
TFA/CH3CN in 0.1% TFA/H2O, 10 to 25% over 30 min, retention
time = 11.9 min; LRMS (ESI-Ion Trap) m/z calcd for C46H77N16O14S
[M + H]+ 1109.6, found 1109.2.
9r (X = H). Analytical HPLC conditions: linear gradient of 0.1%
TFA/CH3CN in 0.1% TFA/H2O, 5−45% over 30 min, retention
time = 16.7 min; LRMS (ESI-Ion Trap) m/z calcd for C47H77N18O12S
[M + H]+ 1117.6, found 1117.6.
NCL of pure 7j with 8 was also conducted under reaction condi-
tions identical to those employed for other SEAlide peptides. After
24 h reaction, HPLC analysis of the reaction showed the formation of
two ligated peptides, which had the same molecular weight. Further
analysis for determining which peptide is α- or β-peptide was not
attempted.
9j (early eluted) Analytical HPLC conditions: linear gradient of
0.1% TFA/CH3CN in 0.1% TFA/H2O, 5−45% over 30 min, retention
time = 19.0 min; LRMS (ESI-Ion Trap) m/z calcd for C45H75N16O14S
[M + H]+ 1095.5, found 1095.5.
REFERENCES
■
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9j (later eluted) Analytical HPLC conditions: linear gradient of
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ASSOCIATED CONTENT
■
S
* Supporting Information
HPLC charts of crude model SEAlide Peptides 7, of
representative NCL of SEAlide peptides 7 with N-terminal
cysteinyl peptide 8, of check of racemization during coupling
reaction of Fmoc amino acids to the aniline linker, and of check
of epimerization of C-terminal amino acids during NCL.
1
Copies of H and 13C NMR spectra of compounds 4c, 4d, 4e,
4f, 4g, 4h, 4i, 4j, 4k, 4l, 4m, 4n, 4o, 4p, 4q, 4r, 4s, 4t, 4u, 10c,
10f, 10h, 10i, 10j, 10l, and 10r. This material is available free of
charge via the Internet at http://pubs.acs.org
AUTHOR INFORMATION
■
Corresponding Author
*Tel: +81-88-633-7283. Fax: +81-633-9505. E-mail: aotaka@
ph.tokushima-u.ac.jp.
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Article
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