Site-Specific Incorporation of Multiple Thioamide Substitutions into a Peptide Backbone via Solid Phase Peptide Synthesis

Article abstract of DOI:10.1021/acs.joc.9b02486

Among various peptide modification strategies, thioamide substitution by replacing the carbonyl oxygen atom of an amide bond with a sulfur atom constitutes an invaluable tool for chemical biology, for use in peptide drug discovery and protein structure-fu

Full text of DOI:10.1021/acs.joc.9b02486

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Article
Site-Specific Incorporation of Multiple Thioamide Substitutions
into a Peptide Backbone via Solid Phase Peptide Synthesis
yang jinhua, Changliu Wang, Chaochao Yao, Chunqiu Chen, Yafang Hu, Guifeng He, and Junfeng Zhao
J. Org. Chem., Just Accepted Manuscript • Publication Date (Web): 02 Dec 2019
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Page 1 of 11
The Journal of Organic Chemistry
Site-Specific Incorporation of Multiple Thioamide Substitutions
into a Peptide Backbone via Solid Phase Peptide Synthesis
Jinhua Yang†, Changliu Wang†, Chaochao Yao†, Chunqiu Chen†, Yafang Hu†, Guifeng He† and Junfeng
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Zhao†‡*
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†College of Chemistry and Chemical Engineering, Jiangxi Normal University, Nanchang, 330022, Jiangxi, P. R. China
‡Key Laboratory of Chemical Biology of Jiangxi Province, Nanchang, 330022, Jiangxi, P. R. China
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Email: zhaojf@jxnu.edu.cn
Supporting Information
ABSTRACT: Among various peptide modification strategies, thioamide
substitution by replacing the carbonyl oxygen atom of an amide bond by a
sulfur atom, constitutes an invaluable tool for chemical biology including
peptide drug discovery and protein structure–function studies. However,
thioamide substitution effect has not been well studied because of the lack
of synthetic methods to site-specifically incorporating a thioamide bond
into a peptide backbone, particularly introducing multi thioamide
substitutions to peptide on a solid support. Herein, we reported a highly
efficient method to incorporate a thioamide bond to the peptide backbone
in a site-specific manner by employing α-thioacyloxyenamides, which are
formed from the addition of N-protected monothioamino acids and
ynamides, as a kind of novel thioacylating reagents in solid phase peptide
synthesis. This method is amenable for 19 of 20 proteinogenic amino acids
except for the His. Mono to multiple thioamide substitutions could be
incorporated into a growing peptide with no or low level of epimerization. By using this method, fully thioamide substituted hexapeptide
containing up to five continuous thioamide bonds could be synthesized smoothly. This synthetic methodology will spur the application of
the thioamide substitution tool for protein engineering and peptide drug discovery.
study of the physical and biological effect of thioamide
substitution is impeded mainly by the lack of efficient synthetic
strategies to site-specific incorporating thioamide bond into
peptide backbone.
■ INTRODUCTION
Site-specific modification and functionalization of peptides and
proteins can adjust their properties and functionalities and thus
evolves into an invaluable tool for peptide drug discovery and
protein structure–function studies.1 Compared to the
extensively studied post translation sidechain modifications,
little attention has been paid to the peptide backbone
modifications.2 Replacing the amide bond with other isosteric
functional groups is a straightforward strategy for peptide
backbone modification. Among various amide bond surrogates,
thioamide substitution, in which the amide bond oxygen atom
is replaced with a sulfur atom, is the simplest modification that
causes minimal perturbation.3 Although numerous studies have
revealed that the thioamide is isosteric to an amide bond with
the same or similar geometry conformations,3b both groups
retain many intrinsic different chemical and physical
properties.4 For example, the resistance of peptide to enzymatic
degradation and bioactive can be dramatically enhanced by
replacing the canonical peptide bond with a thioamide linkage.5
Thioamide exhibits higher nucleophilicity and electrophilicity,6
altered hydrogen-bonding propensity,7 unique spectroscopic
properties8 and greater metal binding affinity.9 Owing to these
features, thioamide has been employed as probes for studying
the dynamics of H-bond formation in β-sheets,10 α-helices,11, 10c
interaction of peptide bonds,12 cis/trans photoswitches,13
fluorescence quenchers,14 and metal-binding substrates.15
Although several intriguing features of thioamides have been
recruited for studying protein folding, stability, function, and
dynamics, the biological behavior of thioamide substitution on
peptides and proteins of interest still is unpredictable. Extensive
A broad range of synthetic methodologies for thioamide have
been developed over the past decades.16 However, only few of
them are amenable for incorporating a thioamide bond into a
growing peptide because of the difficult availability of chiral
starting material and the highly prone to racemization of the α-
chiral center of the thioamide residue. There are two strategies,
by employing thionating reagents and thioacylating reagents,
respectively, for synthesis of thioamide substituted peptide.
Thionating reagents such as P4S10,17 Lawesson’s reagent,18 and
other sulfur reagents19 have been used to selectively convert the
carbonyl oxygen atom of an amide bond to an sp2-hybridized
sulfur atom in the presence of ester functionality. However, this
method is limited to simple amides and dipeptides because there
is no selectivity for substrates containing more than one amide
bonds. In this regard, the thioacylating reagents are more
flexible because the modular nature enables them to incorporate
a thioamide bond in a site-specific manner. Alkyl dithioesters,20
thiobenzimidazolones,21 and thioacylnitrobenzotriazoles22 have
been employed as active thioacylating reagents for thioamide
substituted peptide synthesis. Thioacylnitrobenzotriazoles
prepared via three steps transformations from natural α-amino
acids (Figure 1, eq. 1) are the only one class of thioacylating
reagents that could be used to introduce a thioamide bond in
SPPS.4, 23 However, this method is limited to the 15 of the 20
common proteinogenic α-amino acids.2 In addition, the
preparation of thioacylnitrobenzotriazole involves the use of
toxic and malo-
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Page 2 of 11
Figure 1. Thioacylating Reagents Effective for Incorporating Thioamide Substitution in SPPS.
1
2
3
4
NH2
NH2
NO2
NH2
NO2
NO2
O
S
O
S
H
O2N
OH
H
H
H
NaNO2/AcOH
Lawesson's reagent
or P4S10
N
N
N
N
1)
Fmoc
Fmoc
N
N
Fmoc
N
Fmoc
N
H
H
R1
A
R1
R1
R1
NH2
N
D
5
B
C
6
7
8
9
R2
Amenable for 15 of 20 proteinogenic amino acids
Except Asn, Gln, Met, Thr, His
OR3
OR3
H2N
S
O
O
R2
Ts
OR3
N
O
S
H2N
R2
O
H
H
H
Me
(MYTsA)
H
N
NaSH
O
N
Ts
N
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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60
2)
N
Fmoc
OH
Fmoc
O
N
SH
Fmoc
N
H
Fmoc
R1
R1
R1
Me
R1
A
E
F
G
Amenable for 19 of 20 proteinogenic amino acids
Except His
dour thionating reagents. Meanwhile, monothioamino acids
have been used as thioacyl donor for thioamide incorporation
with the assistance of phosphorus coupling reagents.24
However, the co-formation of significant amount of
corresponding oxo-amide byproduct rendered it not feasible for
SPPS. Recently, we disclosed that ynamide could be used as a
racemization/epimerization-free coupling reagent for peptide
bond formation.25 Interestingly, thioamide substituted peptide
could be obtained in a racemization/epimerization-free manner
when monothioamino acid was employed as the acyl donor with
α-thioacyloxyenamide formed from the addition of
monothioamino acid to ynamide as an efficient thioacylating
reagent (Figure 1, eq. 2).26 Owing to the optimal balance
between the reactivity and stability of α-thioacyloxyenamides,
they can also be used to incorporate a thioamide bond site-
specifically in SPPS. Herein, we provided a systematic study
about the use of α-thioacyloxyenamide as thioacylating
reagents in SPPS.
chain reaction and Edman degradation during cleavage of the
thioamide substituted peptide from resin23d, 27a) as the model
reaction. The effect of solvent, amount of α-
thioacyloxyenamide, and coupling time on the reaction
efficiency have been studied systematically (Table 1).
Investigation on solvent disclosed that dimethylformamide
(DMF) is the optimal choice (Table 1, entry 6), which is
beneficial to suppress epimerization (Table 1, entries 2-4) and
the co-formation of oxoamide byproduct efficiently (Table 1,
entry 1). Interestingly, decreasing the amount of α-
thioacyloxyenamide and shortening reaction time from 1 h to
0.5 h have no significant influence on the conversion. On the
contrary, prolonged reaction time has deleterious effect on the
coupling and resulted more oxoamide byproduct formed. The
combination of 3 equiv of α-thioacyloxyenamide, DMF as the
solvent, and 1 h coupling time were identified to be the best
reaction conditions to incorporate a thioamide substitution to
the N-terminal of a growing peptide in SPPS.
Next, the efficiency of incorporating one thioamide
substitution to the N-terminal of a tetrapeptide with the
tripeptide VGF bearing a bulky Val at the coupling site was
evaluated for α-thioacyloxyenamides derived from 19 Fmoc-
protected proteinogenic α-amino acids. As shown in Table 2, all
of them proceeded smoothly to offer the target thioamide
substituted tetrapeptide in quantitative conversion. It’s notable
that amino acids such as Met, Gln, Asn, and Thr, which can not
be used for thioacylnitrobenzotriazole method work well in our
case (Table 2, entries 3, 7, 9, 13 and 15).2 The bulky side-chain
protecting groups, such as Boc, tBu, and Trt, have no deleterious
effect on the conversion (Table 2, entries 4-11). The protected
hydrophilic and polar amino acids such as Arg, Asn, Ser, Lys,
and Tyr are also compatible with this method (Table 2, entries
20, 21 and Table 3, entries 13-15). Small amount of oxopeptide
was formed as the byproduct during the coupling of
thiocarbonyl esters of sterically hindered Val and Ile (Table 2,
entries 16-17). This might be attributed to the steric hindrance
between these bulky residues and bulky Val at the N terminal
(data not shown). This phenomenon could be avoided once the
bulky Val was replaced by Ala (Table 2, entries 16-17). Low
level of epimerization was observed for the thiocarbonyl esters
derived from Gln, Asp, Asn, and Ser, which are highly prone to
racemization upon activation, under the room temperature
conditions (Table 2, entries 7-9, and 11). As shown previously,
such slight epimerization could be suppressed by lowering the
■ RESULTS AND DISCUSSION
In our previous work, we have demonstrated that
thioacylating reagents α-thioacyloxyenamides could be
obtained as the major product when the addition reaction of
monothioamino acids and ynamide (MYTsA) were performed
under -40 oC with m-xylene as the solvent.26 To systematically
evaluate the potency of α-thioacyloxyenamides to incorporate
thioamide substitutions site-specifically to a growing peptide
backbone on a solid support, all of the α-thioacyloxyenamides
(3a-s) originated from 19 of 20 Fmoc-protected proteinogenic
α-amino acids were prepared under the optimzed reaction
conditions. As shown in Scheme 1, all of the target α-
thioacyloxyenamides except that of His could be obtained in
good yields under the optimized reaction conditions (Scheme
1).26 It should be noted that all of these α-thioacyloxyenamides
are stable and can be purified and characterized. No
deterioration can be detected after they have been kept in fridge
for two months.
With these thioacylating reagents in hand, the systematic and
comprehensive optimization of the reaction conditions to
incorporate one thioamide unit to the N-terminal of a growing
peptide on a solid support was conducted. Initial experiments
were carried out with the coupling of α-thioacyloxyenamide
derived from Fmoc-protected alanine with 0.03 mmol of
tripeptide VGF loaded on trityl chloride resin (2-CTC, which
can be cleaved under more milder condition to avoid the side
2
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The Journal of Organic Chemistry
Scheme 1. Synthesis of α-thioacyloxyenamides from N-
Fmoc Protected Monothioamino Acids
Table 1. Optimization of the Reaction Conditionsa
S
1
2
3
4
H
N
Fmoc
O
N
O
S
3a
(
)
H
H
Ts
m-xylene
Ts
N
N
Ts
+
N
Fmoc-AlaS-Val-Gly-Phe-OH
Fmoc
SH
Fmoc
O
N
NH2-Val-Gly-Phe-
-40 °C
R1
R1
Cleavage cocktailb
(MYTsA)
4
5a
1
2
3
5
6
7
8
9
S
S
H
N
H
S
Ts
N
Ts
H
Fmoc
O
N
Fmoc
O
N
N
Ts
Ts
Fmoc
Fmoc
O
N
N
SMe
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
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50
51
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53
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56
57
58
59
60
3a
3b
3c
, 67%
, 65%
, 68%
S
S
H
N
H
N
S
Ts
H
N
O
Fmoc
O
N
Ts
Fmoc
O
N
STrt
NH
Boc
O
OtBu
3d
, 68%
3e
3f
, 55%
, 63%
S
time
(h)
conversion
drd
entry
equiv
solvent
(4/5a)c
S
H
N
S
H
N
H
N
Ts
1e
2
3
3
3
3
3
3
1
2
3
3
1
1
NMP
MeOH
CH2Cl2
THF
1:99
69:31
3:97
> 99:1
98:2
Fmoc
O
N
Ts
Ts
Fmoc
O
O
N
Fmoc
O
H
N
Trt
N
Trt
OtBu
O
O
N
H
3
1
95:5
a
3g
3h
, 59%
, 50%
3i
, 40%
S
4
1
1:99
98:2
S
H
N
5f
6
1
DMF/CH2Cl2
DMF
1:99
> 99:1
> 99:1
> 99:1
> 99:1
> 99:1
> 99:1
H
N
S
Ts
Ts
H
Fmoc
O
N
Fmoc
O
N
N
Ts
Fmoc
O
N
1
< 1:99
2:98
tBuO
N
OtBu
Boc
7
1
DMF
3j
3k
3l
, 62%
, 65%
, 63%
8
1
DMF
1:99
S
S
9
0.5
2
DMF
1:99
S
H
N
H
N
Ts
Ts
Ts
N
O
N
Fmoc
Fmoc
O
N
N
10g
DMF
< 1:99
Fmoc
Fmoc
O
N
Ts
OMe
Fmoc
aReactions were carried out with 0.03 mmol of 4 at rt. bCleavage
cocktail: TFE:AcOH:CH2Cl2 = 1:3:6. cConversion (4/5a ratio) was
3m
3n
3o
, 68%
, 69%
, 66%
S
d
S
H
N
calculated based on HPLC at 254 nm. Product diastereomeric purity
S
H
H
N
N
Ts
O
was determined by HPLC. e8% (oxoamide: thioamide) oxoamide was
Ts
N
Fmoc
O
N
O
detected. DMF/CH2Cl2 (v/v) = 1:1. g2% (oxoamide:thioamide)
f
oxoamide was detected.
3p
, 66%
3q
3r
, 66%
, 66%
S
H
NH
S
N
Ts
conventional Fmoc deprotection conditions (piperidine pKa =
ca. 10 in DMF).4, 27 Petersson and co-works observed that a
more sterically hindered and non-nucleophilic base,1,8-
diazabicyclo[5.4.0]undec-7-ene (DBU), in DMF is beneficial to
suppress epimerization during the removal of Fmoc group of
thioamide-containing peptides.21a, 27a, 28 However, in our hand,
iterative exposure of thioamide-containing peptide to such
Fmoc deprotection conditions caused racemization during the
peptide elongation on a solid support.27a, 28a Fortunately, slight
modification of Petersson’s deprotection conditions by
employing a cocktail of DBU (1%)/n-C6CH13SH (25%) (v/v in
DMF) offered better results. To further illustrate the robustness
of our method, the synthesis of a series of dithioamide
substituted peptides, including i+1, i+2, and i+3, were studied
(Table 3).23d, 29 Interestingly, the excellent reaction efficiency
could be retained albeit a little longer reaction time was required
for the incorporation of the second thioamide substitution. This
might be at- tributed to the stronger electron-withdrawing effect
of the thio-amide bond than that of oxoamide and thus
decreasing the nucleophilicity of the N-terminal amino group of
the thioamide-containing peptide. It is found that this electron-
withdrawing
Fmoc
O
N
Boc
N
N
O
N
H
Boc
HN
Ts
N
Fmoc
N
Trt
3s
, 52%
3t
, NR
aThese reactions were carried out with 1 (0.15 mmol, 1.5 equiv), 2
(MYTsA, 0.1 mmol), m-xylene (1.0 mL), -40 oC, 8 h, Fmoc =
t
fluorenylmethoxy, Boc = tert-butoxycarbonyl, Trt = trityl, Bu = tert-
butyl, aIsolated yield. bCH2Cl2, -40 oC, 8 h. cNR = No reaction.
reaction temperature to 0 ℃.26 Scaling up the reaction to 0.4
mmol scale did not alter the coupling efficiency, which
showcase the viability of this method (Table 2, entry 1).
The success on the incorporation of monothioamide
substitution to the N-terminal of a growing peptide promoted us
to challenge the synthesis of multiple thioamide substituted
peptides, which involves iterative Fmoc deprotection of
endothiopeptide. It is noted that the α-proton of the thioamide
(pKa = ca. 13) residue is more acidic than that of the oxoamide
and thus it is highly prone to racemization for thioamide residue
under the
3
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Table 2. Synthesis of Mono-Thioamide Substituted
Table 3. Synthesis of Di- to Multi-Thioamide Bonds
Peptidesa
Substituted Peptide Sequencea
1
2
3
4
S
S
H
H
N
Ts
N
Ts
S
H
Fmoc
O
N
Fmoc
O
N
N
R2
3
(
)
3
)
(
R1
Xaa2
Xaa1
H2N Xaa3
N
Fmoc-XaaS-Val-Gly-Phe-OH
NH2-Val-Gly-Phe-
H
n
R1
Cleavage cocktailb
5
4
5
6
6
7
8
9
S
R2
entry
1
Xaa (5)
4/5c
<1:99
1:99
drd
H
N
H
Fmoc
N
Xaa3
Xaa2
Xaa1
N
H
OH
Ala (5a)e
Phe (5b)
>99:1
99:1
Cleavage cocktailb
N
R1
n
H
S
7
2
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
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3
Met (5c)
1:99
>99:1
>99:1
>99:1
>99:1
98:2
4
Lys(Boc) (5d)
Cys(Trt) (5e)
Glu(tBu) (5f)
Gln(Trt) (5g)
Asp(tBu) (5h)
Asn(Trt) (5i)
Tyr(tBu) (5j)
Ser(tBu) (5k)
Trp(Boc) (5l)
Gly (5m)
1:99
5
1:99
6
1:99
7
1:99
8
<1:99
<1:99
1:99
90:10
95:5
9
time
(h)
10
11
12
13
14
15
16
17
18
19
20
21
22
99:1
entry
sequence
ASASVGF (7a)
6/7c
drd
<1:99
1:99
97:3
1
2
3
4
5
1
3
1
4
4
1:99
3:97
1:99
4:96
7:93
>99:1
>99:1
92:8
>99:1
>99:1
>99:1
>99:1
>99:1
>99:1
>99:1
95:5
E(tBu)SASVGF (7b)
S(tBu)SASVGF (7c)
MSASVGF (7d)
<1:99
1:99
Pro (5n)
>99:1
>99:1
Thr(Me) (5o)
Ile (5p)f
2:98
K(Boc)SASVGF (7e)
2:98
<1:9
9
6
ASLASVGF (7f)
3
>99:1
Val (5q)f
2:98
7
8
FSLASVGF (7g)
K(Boc)SLASVGF (7h)
MSLASVGF (7i)
1
3
3
5
3
2:98
6:94
4:96
7:93
1:99
99:1
>99:1
>99:1
>99:1
99:1
Leu (5r)f
<1:99
2:98
Arg(Boc)2 (5s)
Ala (5t)g
9
2:98
>99:1
>99:1
>99:1
b
Glu (5u)h,i
<1:99
<1:99
10
11
E(tBu)SLASVGF (7j)
S(tBu)SLASVGF (7k)
Ala (5v)j,i
<1:9
9
aReactions were carried with 0.03 mmol of 4 at rt for 1 h. Cleavage
cocktail: TFE:AcOH:CH2Cl2 = 1:3:6. cConversion (4/5 ratio) was
12
ASLAASVF (7l)
2
>99:1
MSAMY(tBu)SAL (7m)
2
2
1:99
2:98
96:4
d
13
14
calculated based on HPLC at 254 nm. Product diastereomeric purity
was determined by HPLC. 4 (0.4 mmol) was used. fNH2-A-G-F-resin
e
K(Boc)SLK(Boc)ASAGF (7n)
E(tBu)SS(tBu)VE(tBu)SAPFN
>99:1
was used. gNH2-A-R(Pbf)-G-resin was used. hNH2-A-Q(Trt)- W(Boc)-
P-K(Boc)-resin was used. iThe product peptide was fully deprotected.
jNH2-A-Y(tBu)-N(Trt)-F-resin was used.
15
2
4:96
96:4
(Trt)GK(Boc) (7o)
MSASASASA (7p)
16
17
10
5
1:99
2:98
>99:1
>99:1
ASASASASASA (7q)
induction effect could be interrupted by inserting of the normal
amide bond between the two thioamide bonds (Table 3, entries
1, 4, 6, 9, 12 and 13). The reaction time for the introduction of
the second thioamide moiety of the i+3 (2 h) was significantly
shorter than that of i+1 (4 h), i+2 (3 h). It is expected that
dithiopeptide would be highly prone to epimerization and to
cause other side reactions. Even though, our protocols gave the
required dithiopeptides in excellent conversion (93-99%), and
purity (different HPLC methods and HRMS were used to assess
the purity of final peptides 7a, 7f, and 7g, for details see SI). No
or low level of epimerization was observed for some special
amino acid residues. This promising result prompted us to
attempt the synthesis of multiple thioamide substituted
peptides, the properties of which have not been studied because
of their
aReactions were carried out with 0.03 mmol of 6 at rt for 1-10 h.
bCleavage cocktail: TFE:AcOH:CH2Cl2 = 1:3:6. cConversion (6/7
ratio) was calculated based on HPLC at 254 nm. dProduct
diastereomeric purity was determined by HPLC.
difficult availability. Multiple thioamide substituted pep tides
are frequently observed in natural products such as
thioviridamide family, which contains five continuous
thioamide bonds and possesses highly potent pro-apoptotic
activities and potent antiproliferative.30 However, chemical
synthesis of multiple thioamide substituted peptides on a solid
support have never been reported yet. To our delight, α-
thioacyloxyenamides proved to be effective thioacylating
4
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The Journal of Organic Chemistry
of CH2Cl2 repeatedly. The filtrate was concentrated about one-fifth
reagents to incorporate multiple thioamide substitutions into the
1
2
3
4
5
6
7
8
of its original volume and left to stand in the refrigerator for about
2 h. Then the mixture was filtered again and concentrated to give
the corresponding activated ester as a colorless foam, which was
immediately dissolved in CH2Cl2 (20.0 mL), followed by addition
of NaHS (15 mmol, 3.0 equiv, 70% purity) and 15-crown-5 (1.5
mmol, 0.3 equiv). Then the reaction mixture was stirred at rt for 3
h, diluted with CH2Cl2 and poured into ice water. The pH was
adjusted to ~3 by careful addition of 1 M HCl and extracted by
CH2Cl2 (3 × 20.0 mL). The combined organic layers were washed
with brine, dried over Na2SO4 and concentrated under reduced
pressure. The crude products 2 were used for the next reaction
without further purification.25a, 26a, 32
peptide backbone in SPPS. As shown in Table 3, tetra-
thioamide substituted peptide could be easily synthesized by
using our method (Table 3, entry 16). Even the fully thioamide
substituted homooligo-hexapeptides containing five continuous
thioamide bonds, the oxoamide congener of which is highly
hydrophobic and has a high tendency to aggregate could also be
synthesized on a solid support in an excellent yield by iterative
using of α-thioacyloxyenamide derived from Fmoc-protected
monothio-Alanine as the thioacylating reagent (Table 3, entry
17).28c, 31
9
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
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
A 5.0 mL round-bottomed flask was charged with MYTsA (0.1
mmol, 1.0 equiv), m-xylene (1.0 mL) and 2 (0.15 mmol, 1.5 equiv).
The reaction mixture was stirred at -40 C under air until starting
material MYTsA was fully consumed. The reaction mixture was
concentrated and purified by silica gel chromatography to afford
the α-thioacyloxyenamides 3. The products 3a, 3b, 3m, 3p, 3q and
3r have been synthesized and characterized in the previous work.25
The data given here are provided for convenience.
■ CONCLUSION
°
In conclusion, we have provided a highly efficient method
for site-specific incorporating thioamide substitutions to the
backbone of a growing peptide on a solid support. Thiocarbonyl
esters of 19 of 20 proteinogenic α-amino acids except His
worked well to offer the target thioamide substituted peptides
with no or low level of epimerization. Coupling conditions, a
modified Fmoc deprotection method and the feasibility of
incorporation of multiple thioamide substitutions have been
systematically studied. The thioamide substituted hexapeptide
containing up to five continuous thioamide bonds could also be
synthesized by using this method. This study paves the way for
the application of α-thioacyloxyenamides as a kind of
thioacylating reagents to incorporate a thioamide substitution to
a peptide backbone in a site-specific manner. No doubt, the
novel thioacylating strategy reported herein will spur the further
application of thioamide substitution tool in peptide and protein
chemical biology.
O-(1-((N,4-dimethylphenyl)sulfonamido)vinyl) (S)-2-((((9H-
fluoren-9-yl)methoxy)carbonyl)amino)-4-(methylthio)
butanethioate (3c). Yellow solid (41 mg, 68%); mp 46-50 ℃; Rf =
1
0.4 (PE/EA = 5:1); [α]25 D= + 40.8 (c = 0.87, CHCl3); H NMR
(400 MHz, CDCl3) δ 7.77 (d, J = 7.5 Hz, 2H), 7.72 (d, J = 8.2 Hz,
2H), 7.66 – 7.59 (m, 2H), 7.40 (t, J = 7.5 Hz, 2H), 7.35 – 7.28 (m,
4H), 5.67 (d, J = 9.0 Hz, 1H), 4.89 (d, J = 3.0 Hz, 1H), 4.83 – 4.71
(m, 1H), 4.61 (d, J = 3.0 Hz, 1H), 4.48 – 4.42 (m, 1H), 4.43 – 4.33
(m, 1H), 4.25 (t, J = 7.0 Hz, 1H), 3.01 (s, 3H), 2.67 – 2.50 (m, 2H),
2.43 (s, 3H), 2.38 – 2.23 (m, 1H), 2.13 (s, 3H), 2.03 – 1.89 (m, 1H);
13C{1H} NMR (100 MHz, CDCl3) δ 218.4, 155.8, 150.0, 144.5,
143.9, 143.8, 141.3, 132.7, 129.6, 128.2, 127.7, 127.1, 125.2,
120.0, 101.7, 67.1, 61.4, 47.2, 38.3, 34.1, 30.0, 21.6, 15.4 ppm; IR
(KBr) ν̃
3381, 2920, 1723, 1357, 1162, 743 cm−1; HRMS (ESI-
■ EXPERIMENTAL SECTION
TOF) calcd for C30H33N2O5S3 (M + H)+: 597.1546, found:
597.1540.
1H and 13C NMR spectra were recorded on a Bruker (400 and
100 MHz for 1H and 13C, respectively) instrument, and are
internally referenced to residual solvent signals, CDCl3 referenced
at δ 7.26 and 77.06 ppm. Data for 1H is reported as follows:
chemical shift (δ ppm), integration, multiplicity (s = singlet, d =
doublet, t = triplet, q = quartet, m = multiplet), broad peaks (br),
coupling constant (Hz) and assignment. Data for 13C NMR are
reported in terms of chemical shift, multiplicity (s = singlet, d =
doublet, t = triplet, q = quartet, m = multiplet), coupling constant
(Hz) and no special nomenclature is used for equivalent carbons.
HRMS (ESI) spectra were obtained by the electrospray ionization
time-of-flight (ESI-TOF) mass spectrometry. The diastereomeric
ratio (dr) was determined by HPLC with C18 column with H2O and
acetonitrile (ACN) as eluent. Flash column chromatography
purification of compound was carried out by gradient elution using
ethyl acetate (EA) in light petroleum ether (PE). Unless otherwise
noted, materials obtained from commercial suppliers were used
without further purification. Analytical RP-HPLC was performed
on an UltiMate 3000 HPLC system using commercially available
columns (Phenomenex C18, 250 mm x 4.6 mm, 5 μm) at a flow
rate of 1.0 mL/min, detection at 220 or 254 nm. Mobile phase:
solvent A was 0.045% TFA (v/v) in H2O, solvent B was 10% H2O
and 0.039% TFA (v/v) in ACN. Gradient: a suitably adjusted
gradient of 20% B to 100% B was used.
O-(1-((N,4-dimethylphenyl)sulfonamido)vinyl) (S)-2-((((9H-
fluoren-9-yl)methoxy)carbonyl)amino)-6-((tert-butoxycarbonyl)
amino)hexanethioate (3d). Yellow solid (44 mg, 62%); mp 55-
58 ℃; Rf = 0.4 (PE/EA = 3:1); [α]25 D= + 29.0 (c = 0.7, CHCl3);
1H NMR (400 MHz, CDCl3) δ 7.75 (d, J = 7.5 Hz, 2H), 7.71 (d, J
= 8.0 Hz, 2H), 7.62 (t, J = 5.4 Hz, 2H), 7.39 (t, J = 7.5 Hz, 2H),
7.35 – 7.28 (m, 4H), 5.69 (d, J = 9.0 Hz, 1H), 4.88 (d, J = 3.1 Hz,
1H), 4.74 (s, 1H), 4.67 – 4.55 (m, 2H), 4.45 (dd, J = 10.4, 7.0 Hz,
1H), 4.33 (dd, J = 10.6, 7.2 Hz, 1H), 4.23 (t, J = 7.1 Hz, 1H), 3.17
– 3.06 (m, 2H), 3.00 (s, 3H), 2.41 (s, 3H), 1.97 – 1.88 (m, 1H), 1.82
– 1.68 (m, 1H), 1.57 – 1.37 (m, 13H); 13C{1H} NMR (100 MHz,
CDCl3) δ 219.2, 156.2, 155.8, 150.0, 144.5, 143.9, 143.8, 141.3,
132.6, 129.6, 128.2, 127.7, 127.1, 125.2, 120.0, 101.8, 79.0, 67.0,
61.9, 47.2, 40.1, 38.3, 34.2, 29.6, 28.5, 22.4, 21.6 ppm; IR (KBr) ν
3399, 2933, 1717, 1514, 1357, 1156, 737 cm−1; HRMS (ESI-TOF)
calcd for C36H43N3NaO7S2 (M + Na)+: 716.2435, found: 716.2429.
O-(1-((N,4-dimethylphenyl)sulfonamido)vinyl) (S)-2-((((9H-
̃
fluoren-9-yl)methoxy)carbonyl)amino)-3-(trityl
thio)
propanethioate (3e). Yellow solid (45 mg, 55%); mp 69-73 ℃; Rf
= 0.4 (PE/EA = 5:1); [α]25 D= + 40.2 (c = 0.60, CHCl3); 1H NMR
(400 MHz, CDCl3) δ 7.80 – 7.73 (m, 2H), 7.69 – 7.58 (m, 4H), 7.45
– 7.35 (m, 8H), 7.32 – 7.16 (m, 13H), 5.36 (d, J = 8.9 Hz, 1H), 4.85
(d, J = 2.9 Hz, 1H), 4.75 (d, J = 3.0 Hz, 1H), 4.53 – 4.39 (m, 2H),
4.38 – 4.28 (m, 1H), 4.23 (t, J = 7.0 Hz, 1H), 2.94 (s, 3H), 2.76 –
2.60 (m, 2H), 2.37 (s, 3H); 13C{1H} NMR (100 MHz, CDCl3) δ
215.8, 155.3, 149.3, 144.4, 144.3, 143.9, 143.8, 141.3, 129.6, 129.6,
128.1, 128.1, 127.7, 127.1, 126.9, 125.2, 120.0, 101.9, 67.3, 67.0,
General Procedure 1 for α-thioacyloxyenamides (3a-s).
To a stirred solution of the appropriate Fmoc protected amino
acid (5.0 mmol, 1.0 equiv) and N-hydroxysuccinimide (HOSu, 5.5
mmol, 1.1 equiv) in CH2Cl2 (20.0 mL), dicyclohexylcarbodiimide
(DCC, 5.5 mmol, 1.1 equiv) was added at 25 °C under N2. Then the
mixture was stirred at 25 °C for 2 h. The suspension was filtered to
remove the resulting white solid which was washed with 20.0 mL
60.7, 47.2, 37.4, 35.6, 21.6 ppm; IR (KBr) ν̃ 3405, 3051, 1728,
1498, 1357, 1163, 743 cm−1; MS (ESI-TOF) calcd for
C47H43N2O5S3 (M + H)+: 811.2329, found: 811.2318.
5
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The Journal of Organic Chemistry
Page 6 of 11
tert-butyl (S)-4-((((9H-fluoren-9-yl)methoxy)carbonyl) amino)-
7.40 (t, J = 7.4 Hz, 2H), 7.36 – 7.28 (m, 5H), 7.09 (d, J = 8.1 Hz,
1
2
3
4
5
6
7
8
5-((1-((N,4-dimethylphenyl)sulfonamido)vinyl)oxy) -5-thioxopen
tanoate (3f). Yellow solid (42 mg, 63%); mp 38-43 ℃; Rf = 0.4
(PE/EA = 5:1); [α]25 D= + 50.7 (c = 0.56, CHCl3); 1H NMR (400
MHz, CDCl3) δ 7.75 (d, J = 7.5 Hz, 2H), 7.71 (d, J = 8.2 Hz, 2H),
7.62 (t, J = 6.7 Hz, 2H), 7.38 (t, J = 7.5 Hz, 2H), 7.34 – 7.26 (m,
4H), 5.79 (d, J = 8.9 Hz, 1H), 4.88 (d, J = 3.0 Hz, 1H), 4.65 (d, J =
3.0 Hz, 1H), 4.64 – 4.57 (m, 1H), 4.45 (dd, J = 10.4, 7.0 Hz, 1H),
4.32 (dd, J = 10.5, 7.3 Hz, 1H), 4.22 (t, J = 7.1 Hz, 1H), 2.99 (s,
3H), 2.46 – 2.32 (m, 5H), 2.31 – 2.20 (m, 1H), 2.07 – 1.93 (m, 1H),
1.46 (s, 9H); 13C{1H} NMR (100 MHz, CDCl3) δ 218.2, 172.2,
155.8, 149.8, 144.5, 144.0, 143.8, 141.3, 132.9, 129.6, 128.2, 127.7,
127.1, 125.2, 120.0, 101.7, 80.8, 67.1, 61.8, 47.2, 38.0, 31.6, 29.4,
2H), 6.92 (d, J = 8.3 Hz, 2H), 5.52 (d, J = 8.9 Hz, 1H), 4.87 (q, J =
6.8 Hz, 1H), 4.82 (d, J = 2.6 Hz, 1H), 4.68 (d, J = 2.6 Hz, 1H), 4.43
– 4.32 (m, 2H), 4.21 (t, J = 7.1 Hz, 1H), 3.21 (dd, J = 13.9, 5.6 Hz,
1H), 3.07 – 2.98 (m, 4H), 2.41 (s, 3H), 1.32 (s, 9H); 13C{1H} NMR
(100 MHz, CDCl3) δ 217.7, 155.3, 154.4, 149.7, 144.5, 143.8,
141.3, 133.0, 130.5, 130.0, 129.6, 128.2, 127.7, 127.1, 125.2,
124.1, 120.0, 102.0, 78.4, 67.0, 62.7, 47.2, 39.9, 38.0, 28.9, 21.6
ppm; IR (KBr) ν̃
3375, 2986, 1728, 1363, 1168, 743 cm−1; MS
(ESI-TOF) calcd for C38H40N2NaO6S2 (M + Na)+: 707.2220, found:
707.2233.
9
O-(1-((N,4-dimethylphenyl)sulfonamido)vinyl) (S)-2-((((9H-
fluoren-9-yl)methoxy)carbonyl)amino)-3-(tert-butoxy)propane
thioate (3k). Yellow grass solid (40 mg, 63%); Rf = 0.4 (PE/EA =
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
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
28.1, 21.6 ppm; IR (KBr) ν̃
3375, 2915, 1717, 1363, 1079, 743 cm−1;
1
HRMS (ESI-TOF) calcd for C34H38N2NaO7S2 (M + Na)+: 673.2013,
found: 673.2009.
O-(1-((N,4-dimethylphenyl)sulfonamido)vinyl) (S)-2-((((9H-
fluoren-9-yl)methoxy)carbonyl)amino)-5-oxo-5-(tritylamino)pen
5:1); [α]25 D= + 10.5 (c = 0.65, CHCl3); H NMR (400 MHz,
CDCl3) δ 7.77 (d, J = 7.5 Hz, 2H), 7.73 (d, J = 8.3 Hz, 2H), 7.66
(dd, J = 11.1, 7.5 Hz, 2H), 7.40 (t, J = 7.5 Hz, 2H), 7.37 – 7.27 (m,
4H), 5.83 (d, J = 9.1 Hz, 1H), 4.89 (d, J = 2.8 Hz, 1H), 4.82 (d, J =
2.8 Hz, 1H), 4.73 – 4.59 (m, 1H), 4.48 (dd, J = 10.6, 7.0 Hz, 1H),
4.36 (dd, J = 10.5, 7.3 Hz, 1H), 4.28 (t, J = 7.2 Hz, 1H), 3.83 (dd,
J = 9.1, 3.6 Hz, 1H), 3.66 (dd, J = 9.1, 4.0 Hz, 1H), 3.01 (s, 3H),
2.41 (s, 3H), 1.16 (s, 9H); 13C{1H} NMR (100 MHz, CDCl3) δ
216.9, 155.8, 149.4, 144.3, 144.0, 143.9, 141.3, 133.6, 129.6,
128.2, 127.7, 127.1, 125.2, 120.0, 102.2, 73.6, 67.2, 63.5, 62.3,
tanethioate (3g). Yellow solid (49 mg, 59%); mp 82-86 ℃; Rf
=
1
0.3 (PE/EA = 3:1); [α]25 D= + 58.6 (c = 0.6, CHCl3); H NMR
(400 MHz, CDCl3) δ 7.74 (d, J = 7.5 Hz, 2H), 7.65 – 7.55 (m, 4H),
7.40 – 7.33 (m, 2H), 7.30 – 7.26 (m, 4H), 7.25 – 7.21 (m, 11H),
7.20 – 7.15 (m, 4H), 5.91 (d, J = 8.4 Hz, 1H), 4.88 (d, J = 3.1 Hz,
1H), 4.67 (q, J = 6.9 Hz, 1H), 4.47 (d, J = 3.1 Hz, 1H), 4.42 (dd, J
= 10.4, 7.1 Hz, 1H), 4.29 (dd, J = 10.4, 7.3 Hz, 1H), 4.21 (t, J = 7.2
Hz, 1H), 3.00 (s, 3H), 2.52 – 2.35 (m, 5H), 2.25 (t, J = 7.4 Hz, 2H);
13C{1H} NMR (100 MHz, CDCl3) δ 218.2, 171.2, 155.9, 150.5,
144.7, 144.7, 144.0, 143.8, 141.3, 129.7, 128.8, 128.3, 127.9, 127.7,
127.2, 126.9, 125.3, 120.0, 101.5, 70.6, 67.2, 61.5, 47.2, 38.8, 33.0,
47.2, 37.3, 27.4, 21.6 ppm; IR (KBr) ν̃ 3381, 2969, 1728, 1363,
1162, 737 cm−1; HRMS (ESI-TOF) calcd for C32H36N2NaO6S2 (M
+ Na)+: 631.1907, found: 631.1908.
tert-butyl
(S)-2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)
amino)-3-((1-((N,4-dimethylphenyl)sulfonamido)vinyl)oxy)-3-
thioxo propyl)-1H-indole-1-carboxylate (3l). Yellow solid (48 mg,
62%); mp 55-59 ℃; Rf = 0.5 (PE/EA = 5:1); [α]25 D= + 40.9 (c =
30.1, 21.7 ppm; IR (KBr) ν̃ 3387, 3063, 1723, 1498, 1351, 1156,
737 cm−1; MS (ESI-TOF) calcd for C49H46N3O6S2 (M + H)+:
836.2823, found: 836.2836.
1
0.77, CHCl3); H NMR (400 MHz, CDCl3) δ 8.14 (d, J = 8.2 Hz,
1H), 7.78 – 7.67 (m, 4H), 7.61 (d, J = 7.8 Hz, 1H), 7.58 – 7.46 (m,
3H), 7.42 – 7.34 (m, 2H), 7.33 – 7.22 (m, 6H), 5.61 (d, J = 8.9 Hz,
1H), 4.99 (q, J = 7.0 Hz, 1H), 4.80 (d, J = 3.0 Hz, 1H), 4.63 (d, J =
3.0 Hz, 1H), 4.32 (q, J = 10.3, 8.9 Hz, 2H), 4.19 (t, J = 7.3 Hz, 1H),
3.41 (dd, J = 14.8, 5.7 Hz, 1H), 3.20 (dd, J = 14.6, 7.2 Hz, 1H),
3.01 (s, 3H), 2.39 (s, 3H), 1.65 (s, 9H); 13C{1H} NMR (100 MHz,
CDCl3) δ 217.6, 155.5, 149.9, 149.6, 144.5, 143.8, 141.3, 135.5,
132.8, 130.6, 129.6, 128.2, 127.7, 127.1, 125.2, 124.6, 124.5,
122.7, 119.9, 119.0, 115.3, 114.8, 101.7, 83.7, 67.2, 62.0, 47.1,
tert-butyl (S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-
4-((1-((N,4-dimethylphenyl)sulfonamido)vinyl)oxy)-4-thioxo
butanoate (3h). Yellow solid (32 mg, 50%); mp 40-45 ℃; Rf = 0.2
(PE/EA = 10:1); [α]25 D= + 26.8 (c = 1.65, CHCl3); 1H NMR (400
MHz, CDCl3) δ 7.77 (d, J = 7.6 Hz, 2H), 7.72 (d, J = 8.3 Hz, 2H),
7.64 (t, J = 8.3 Hz, 2H), 7.40 (t, J = 7.5 Hz, 2H), 7.35 – 7.28 (m,
4H), 5.99 (d, J = 9.3 Hz, 1H), 4.87 (d, J = 2.9 Hz, 1H), 4.85 – 4.77
(m, 1H), 4.68 (d, J = 3.1 Hz, 1H), 4.46 (dd, J = 10.2, 7.0 Hz, 1H),
4.33 (dd, J = 10.3, 7.4 Hz, 1H), 4.26 (t, J = 7.1 Hz, 1H), 3.01 (s,
3H), 2.89 (d, J = 5.6 Hz, 2H), 2.42 (s, 3H), 1.46 (s, 9H); 13C{1H}
NMR (100 MHz, CDCl3) δ 216.4, 169.2, 155.7, 149.8, 144.4,
143.9, 143.8, 141.3, 129.6, 128.2, 127.7, 127.1, 125.2, 120.0,
101.6, 81.9, 67.3, 58.6, 47.2, 39.6, 37.9, 28.1, 21.6 ppm; IR (KBr)
38.1, 30.1, 28.2, 21.6 ppm; IR (KBr) ν̃ 3387, 2920, 1734, 1357,
1168, 731 cm−1; HRMS (ESI-TOF) calcd for C41H41N3NaO7S2 (M
+ Na)+: 774.2278, found: 774.2274.
(9H-fluoren-9-yl)methyl
(S)-2-(((1-((N,4-dimethylphenyl)
ν̃
3381, 3063, 2974, 1734, 1357, 1162, 737 cm−1; MS (ESI-TOF)
sulfonamido)vinyl)oxy) carbonothioyl)pyrrolidine-1-carboxylate
(3n). Yellow grass solid (40 mg, 69%); Rf = 0.5 (PE/EA = 5:1);
[α]25 D= + 12.9 (c = 0.50, CHCl3); 1H NMR (400 MHz, CDCl3) δ
7.83 – 7.50 (m, 6H), 7.44 – 7.05 (m, 6H), 4.90 (d, J = 2.6 Hz, 0.5H),
4.82 – 4.74 (m, 1H), 4.72 (d, J = 2.7 Hz, 0.5H), 4.57 (d, J = 2.7 Hz,
0.5H), 4.54 – 4.39 (m, 1.5H), 4.36 – 4.07 (m, 2H), 3.80 – 3.63 (m,
1H), 3.63 – 3.48 (m, 1H), 2.98 (m, 3H), 2.38 (m, 3H), 2.24 (m, 2H),
2.07 (m, 1H), 1.98 – 1.82 (m, 1H); 13C{1H} NMR (100 MHz,
CDCl3) δ 219.2, 219.0, 154.7, 154.4, 150.4, 150.0, 144.5, 144.4,
144.3, 143.9, 143.8, 141.3, 141.3, 141.2, 132.7, 132.4, 129.6,
129.6, 128.3, 128.3, 127.7, 127.7, 127.1, 127.1, 125.3, 125.2,
125.2, 125.1, 120.0, 120.0, 101.7, 101.5, 68.2, 67.9, 67.5, 67.4,
47.4, 47.3, 47.2, 46.7, 38.5, 38.2, 33.0, 31.8, 23.8, 22.8, 21.6, 21.6
calcd for C33H36N2NaO7S2 (M +Na)+: 659.1856, found: 659.1865.
O-(1-((N,4-dimethylphenyl)sulfonamido)vinyl) (S)-2-((((9H-
fluoren-9-yl)methoxy)carbonyl)amino)-4-oxo-4-(tritylamino)
butanethioate (3i). Yellow solid (33 mg, 40%); mp 81-83 ℃; Rf =
1
0.2 (PE/EA = 3:1); [α]25 D= + 70.0 (c = 0.65, CHCl3); H NMR
(400 MHz, CDCl3) δ 7.73 (d, J = 7.5 Hz, 2H), 7.66 (d, J = 7.9 Hz,
2H), 7.61 (t, J = 8.0 Hz, 2H), 7.37 (t, J = 7.6 Hz, 2H), 7.24 (m,
20H), 6.98 (d, J = 8.7 Hz, 1H), 4.81 (m, 2H), 4.46 – 4.31 (m, 2H),
4.18 (m, 2H), 3.25 (dd, J = 15.1, 4.4 Hz, 1H), 3.06 – 2.91 (m, 3H),
2.87 (dd, J = 15.0, 5.0 Hz, 1H), 2.40 (s, 3H); 13C{1H} NMR (100
MHz, CDCl3) δ 216.2, 168.8, 156.1, 150.1, 144.6, 144.3, 144.0,
143.9, 141.2, 132.0, 129.6, 128.8, 128.2, 127.9, 127.6, 127.1,
127.0, 125.4, 119.9, 101.1, 71.0, 67.3, 59.1, 47.1, 39.0, 38.5, 21.6
ppm; IR (KBr) ν̃
3068, 2927, 1705, 1363, 1162, 743 cm−1; HRMS
(ESI-TOF) calcd for C30H30N2NaO5S2 (M + Na)+: 585.1488,
found: 585.1484.
ppm; IR (KBr) ν̃ 3381, 2927, 2850, 1663, 1462, 1376, 1161, 739
cm−1; MS (ESI-TOF) calcd for C48H44N3O6S2 (M + H)+: 822.2666,
found: 822.2660.
O-(1-((N,4-dimethylphenyl)sulfonamido)vinyl)
(2S,3R)-2-
((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methoxybutane
thioate (3o). Yellow solid (40 mg, 66%); mp 48-50 ℃; Rf = 0.4
(PE/EA = 8:1); [α]25 D= + 47.8 (c = 0.88, CHCl3); 1H NMR (400
MHz, CDCl3) δ 7.77 (d, J = 7.6 Hz, 2H), 7.74 (d, J = 8.3 Hz, 2H),
7.69 (d, J = 7.4 Hz, 1H), 7.65 (d, J = 7.4 Hz, 1H), 7.45 – 7.37 (m,
O-(1-((N,4-dimethylphenyl)sulfonamido)vinyl) (S)-2-((((9H-
fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-(tert-butoxy)phenyl)
propanethioate (3j). Yellow solid (36 mg, 65%); mp 54-57 ℃; Rf =
1
0.4 (PE/EA = 5:1); [α]25 D= + 40.4 (c = 0.67, CHCl3); H NMR
(400 MHz, CDCl3) δ 7.83 – 7.66 (m, 5H), 7.59 (t, J = 7.4 Hz, 2H),
6
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2H), 7.36 – 7.29 (m, 4H), 5.76 (d, J = 9.9 Hz, 1H), 4.92 (d, J = 2.9
elution with 50-90% B with a flow rate of 1.0 mL/min over 30 min
1
2
3
4
5
6
7
8
Hz, 1H), 4.67 (d, J = 2.9 Hz, 1H), 4.54 – 4.43 (m, 2H), 4.37 – 4.24
(m, 2H), 4.13 (qd, J = 6.2, 2.1 Hz, 1H), 3.33 (s, 3H), 3.01 (s, 3H),
2.43 (s, 3H), 1.25 (d, J = 6.3 Hz, 3H); 13C{1H} NMR (100 MHz,
CDCl3) δ 217.2, 156.6, 150.0, 144.4, 144.0, 143.8, 141.3, 132.9,
129.6, 128.3, 127.7, 127.1, 125.3, 120.0, 102.5, 76.9, 67.2, 66.2,
gave the target peptide. 98.85% conversion of crude HPLC traces;
tR = 12.513 min; MS (ESI-TOF) calcd for C34H39N4O6S (M + H)+:
631.26, found 630.81.
Synthesis of Fmoc-PheS-Val-Gly-Phe-OH (5b). Peptide 5b was
obtained using the procedure 2. HPLC analysis was performed
using C18 analytical column, 254 nm UV detection. Gradient
elution with 60-90% B with a flow rate of 1.0 mL/min over 30 min
gave the target peptide. 98.60% conversion of crude HPLC traces;
tR = 12.091 min (major); MS (ESI-TOF) calcd for C40H43N4O6S(M
+ H)+: 707.29, found 706.97.
57.2, 47.2, 38.1, 21.6, 16.6 ppm; IR (KBr) ν̃ 3428, 3057, 2933,
1723, 1357, 1085, 731 cm−1; HRMS (ESI-TOF) calcd for
C30H32N2NaO6S2 (M + Na)+: 603.1594, found: 603.1591.
O-(1-((N,4-dimethylphenyl)sulfonamido)vinyl) (S)-2-((((9H-
fluoren-9-yl)methoxy)
carbonyl)amino)-5-(1,3-bis(tert-butoxy
9
carbonyl)guanidino)pentanethioate (3s). Yellow solid (45 mg,
55%); mp 66-69 ℃; Rf = 0.4 (PE/EA = 3:1); [α]25 D= + 41.5 (c =
0.48, CHCl3); 1H NMR (400 MHz, CDCl3) δ 11.52 (s, 1H), 8.36 (t,
J = 5.6 Hz, 1H), 7.75 (d, J = 7.6 Hz, 2H), 7.71 (d, J = 7.9 Hz, 2H),
7.64 (t, J = 9.0 Hz, 2H), 7.39 (t, J = 7.4 Hz, 2H), 7.35 – 7.27 (m,
4H), 5.92 (d, J = 9.0 Hz, 1H), 4.87 (d, J = 3.1 Hz, 1H), 4.68 (d, J =
3.0 Hz, 1H), 4.64 – 4.53 (m, 1H), 4.50 – 4.33 (m, 2H), 4.23 (t, J =
6.9 Hz, 1H), 3.51 (dd, J = 12.8, 6.2 Hz, 1H), 3.39 (dd, J = 12.7, 6.1
Hz, 1H), 2.99 (s, 3H), 2.41 (s, 3H), 2.03 – 1.91 (m, 1H), 1.79 – 1.59
(m, 3H), 1.50 (s, 9H), 1.48 (s, 9H); 13C{1H} NMR (100 MHz,
CDCl3) δ 218.6, 163.6, 156.3, 155.8, 153.3, 149.7, 144.4, 144.0,
143.8, 141.3, 133.0, 129.6, 128.2, 127.7, 127.1, 125.2, 119.9,
101.8, 83.1, 79.2, 67.0, 62.1, 47.2, 40.3, 37.9, 31.4, 28.3, 28.1, 25.6,
Synthesis of Fmoc-MetS-Val-Gly-Phe-OH (5c). Peptide 5c was
obtained using the procedure 2. HPLC analysis was performed
using C18 analytical column, 254 nm UV detection. Gradient
elution with 60-90% B with a flow rate of 1.0 mL/min over 30 min
gave the target peptide. 99.00% conversion of crude HPLC traces;
tR = 10.260 min (major); MS (ESI-TOF) calcd for C36H43N4O6S2
(M + H)+: 691.26, found 690.96.
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
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Synthesis of Fmoc-Lys(Boc)S-Val-Gly-Phe-OH (5d). Peptide 5d
was obtained using the procedure 2. HPLC analysis was performed
using C18 analytical column, 254 nm UV detection. Gradient
elution with 60-90% B with a flow rate of 1.0 mL/min over 30 min
gave the target peptide. 99.03% conversion of crude HPLC traces;
tR = 11.918 min; MS (ESI-TOF) calcd for C42H54N5O8S (M + H)+:
788.37, found 788.18.
21.6 ppm; IR (KBr) ν̃ 3328, 2974, 1728, 1640, 1357, 1126, 743
Synthesis of Fmoc-Cys(Trt)S-Val-Gly-Phe-OH (5e). Peptide 5e
was obtained using the procedure 2. HPLC analysis was performed
using C18 analytical column, 254 nm UV detection. Gradient
elution with 60-90% B with a flow rate of 1.0 mL/min over 30 min
gave the target peptide. 99.02% conversion of crude HPLC traces;
tR = 24.000 min; MS (ESI-TOF) calcd for C53H51N4O6S2 (M - H)-:
903.33, found 903.14.
cm−1; MS (ESI-TOF) calcd for C41H52N5O9S2 (M + H)+: 822.3201,
found: 822.3195.
General Procedure
2
for Incorporation of the
Monothioamide on a Solid Support.
Normal peptide assembly by Fmoc based SPPS (4). 2-CTC
resin (39 mg, 0.03 mmol) with a loading of 0.77 mmol/g, was
placed into 5.0 mL fritted syringes, the resin was swelled in CH2Cl2
(3.0 mL, 30 min). The syringe was then drained, Fmoc-Phe-OH (35
mg, 3.0 equiv), N,N-Diisopropyl-ethylamin (DIPEA, 10.0 μL, 2.0
equiv) and 3.0 mL DMF/CH2Cl2 (v/v 1:1) were added into the
fritted syringe. The loading reaction was left to react 2 h at rt before
being drained. Then, the resin was washed with DMF (3.0 mL x 4)
and CH2Cl2 (3.0 mL x 4). The solution of 20% piperidine in DMF
(3.0 mL) was added to the fritted syringes to react 2 min before
being drained. A further 3.0 mL of 20% piperidine was added to
react 18 min. Then, the resin was again carefully washed with DMF
(3.0 mL x 4) and CH2Cl2 (3.0 mL x 4). The peptide elongation was
carried out by adding a solution of Fmoc protected amino acid (3.0
equiv), HBTU (35 mg, 3.0 equiv) and DIPEA (30.0 μL, 6.0 equiv)
in 3.0 mL DMF/CH2Cl2 (1:1). After 1 h, the resin was washed with
DMF (3.0 mL x 4) and CH2Cl2 (3.0 mL x 4). The resin-bound
peptide 4 was obtained by using standard SPPS protocols as
described above.23d, 26a, 28a
Synthesis of Fmoc-Glu(tBu)S-Val-Gly-Phe-OH (5f). Peptide 5f
was obtained using the procedure 2. HPLC analysis was performed
using C18 analytical column, 254 nm UV detection. Gradient
elution with 60-90% B with a flow rate of 1.0 mL/min over 30 min
gave the target peptide. 98.80% conversion of crude HPLC traces;
tR = 13.185 min; MS (ESI-TOF) calcd for C40H47N4O8S (M - H)-:
743.31, found 743.45.
Synthesis of Fmoc-Gln(Trt)S-Val-Gly-Phe-OH (5g). Peptide 5g
was obtained using the procedure 2. HPLC analysis was performed
using C18 analytical column, 254 nm UV detection. Gradient
elution with 60-90% B with a flow rate of 1.0 mL/min over 30 min
gave the target peptide. 98.70% conversion of crude HPLC traces;
tR = 14.013 min (major); MS (ESI-TOF) calcd for C55H56N5O7S(M
+ H)+: 930.39, found 930.23.
Synthesis of Fmoc-Asp(tBu)S-Val-Gly-Phe-OH (5h). Peptide 5h
was obtained using the procedure 2. HPLC analysis was performed
using C18 analytical column, 254 nm UV detection. Gradient
elution with 50-90% B with a flow rate of 1.0 mL/min over 30 min
gave the target peptide. 99.23% conversion of crude HPLC traces;
tR = 18.360 min (major); MS (ESI-TOF) calcd for C39H47N4O8S(M
+ H)+: 731.31, found 730.91.
Synthesis of the Thioamide Substituted Tetrapeptide (5).
Prior to the thioamide coupling, the 4 (0.03 mmol) was washed with
DMF. The solution of 3a (3.0 equiv) in DMF (3.0 mL) was taken
into the fritted syringes. The reaction was shaken at rt for 1 h. Then,
the solvent was drained off and the resin was thoroughly washed
with DMF (3.0 mL x 4) and CH2Cl2 (3.0 mL x 4). The peptide 5a
was obtained after treat 10 mg resin with 100.0 μL of cleavage
cocktail (2,2,2-Trifluoroethanol (TFE)/AcOH/CH2Cl2 - 1:3:6) for
30 min. The CH2Cl2 was removed from the cocktail. Then the
solution of water/acetonitrile (v/v 1:1, 200.0 μL) was added to the
residue. The resin was filtered off and the filtrates was analysed by
HPLC and MS. Peptide 5u and 5v were obtained after treat 30 mg
resin with 100.0 μL of cleavage cocktail (TFA/TIPS/Thioanisole/
H2O = 95:2.5:2.5:2.5) for 1 h. The cleavage mixture was
evaporated to an oil and then cold ether was added. The precipitate
was collected and analysed by HPLC and MS.
Synthesis of Fmoc-Asn(Trt)S-Val-Gly-Phe-OH (5i). Peptide 5i
was obtained using the procedure 2. HPLC analysis was performed
using C18 analytical column, 254 nm UV detection. Gradient
elution with 60-90% B with a flow rate of 1.0 mL/min over 30 min
gave the target peptide. 99.19% conversion of crude HPLC traces;
tR = 18.318 min (major); MS (ESI-TOF) calcd for C54H53N5O7S(M
+ H)+: 916.37, found 916.01.
Synthesis of Fmoc-Tyr(tBu)S-Val-Gly-Phe-OH (5j). Peptide 5j
was obtained using the procedure 2. HPLC analysis was performed
using C18 analytical column, 254 nm UV detection. Gradient
elution with 40-90% B with a flow rate of 1.0 mL/min over 30 min
gave the target peptide. 99.78% conversion of crude HPLC traces;
tR = 24.581 min (major); MS (ESI-TOF) calcd for C44H49N4O7S(M
- H)-: 777.33, found 777.45.
Synthesis of Fmoc-AlaS-Val-Gly-Phe-OH (5a). Peptide 5a was
obtained using the procedure 2. HPLC analysis was performed
using C18 analytical column, 254 nm UV detection. Gradient
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Synthesis of Fmoc-Ser(tBu)S-Val-Gly-Phe-OH (5k). Peptide 5k
gave the target peptide. 98.01% conversion of crude HPLC traces;
1
2
3
4
5
6
7
8
was obtained using the procedure 2. HPLC analysis was performed
using C18 analytical column, 254 nm UV detection. Gradient
elution with 60-90% B with a flow rate of 1.0 mL/min over 30 min
gave the target peptide. 99.65% conversion of crude HPLC traces;
tR = 13.370 min (major); MS (ESI-TOF) calcd for C38H47N4O7S(M
+ H)+: 703.32, found 703.12.
tR = 17.550 min. MS (ESI-TOF) calcd for C44H58N7O9S2+ (M + H)+:
892.37, found 892.26.
Synthesis of NH2-GluS-Ala-Gln-Trp-Pro-Lys-OH (5u). HPLC
analysis was performed using C18 analytical column, 254 nm UV
detection. Gradient elution with 5-50% B with a flow rate of 1.0
mL/min over 30 min gave the target peptide. 100% conversion of
crude HPLC traces; tR = 14.357 min. MS (ESI-TOF) calcd for
C35H52N9O9S+ (M + H)+: 774.36, found 774.30.
Synthesis of Fmoc-Trp(Boc)S-Val-Gly-Phe-OH (5l). Peptide 5l
was obtained using the procedure 2. HPLC analysis was performed
using C18 analytical column, 254 nm UV detection. Gradient
elution with 70-90% B with a flow rate of 1.0 mL/min over 30 min
gave the target peptide. 99.17% conversion of crude HPLC traces;
tR = 14.834 min; MS (ESI-TOF) calcd for C47H52N5O8S (M + H)+:
846.35, found 846.33.
Synthesis of NH2-AalS-Ala-Tyr-Asn-Phe-OH (5v). HPLC
analysis was performed using C18 analytical column, 254 nm UV
detection. Gradient elution with 5-50% B with a flow rate of 1.0
mL/min over 30 min gave the target peptide. 100% conversion of
crude HPLC traces; tR = 18.433 min. MS (ESI-TOF) calcd for
C28H37N6O7S+ (M + H)+: 601.24, found 601.15.
9
10
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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
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Synthesis of Fmoc-GlyS-Val-Gly-Phe-OH (5m). Peptide 5m was
obtained using the procedure 2. HPLC analysis was performed
using C18 analytical column, 254 nm UV detection. Gradient
elution with 40-90% B with a flow rate of 1.0 mL/min over 30 min
gave the target peptide. 99.58% conversion of crude HPLC traces;
tR = 15.706 min; MS (ESI-TOF) calcd for C33H37N4O6S (M - H)-:
615.23, found 615.67.
General Procedure 3 for the Incorporation of Multi-
thioamide on a Solid Support.
Fmoc Removal. After the first thioamide incorporation, the 6
(0.03 mmol) was washed with DMF. A solution of DBU/n-
C6CH13SH (1%/25%, v/v in DMF, 3.0 mL) was added to the fritted
syringes to react 2 min before being drained. A further 3.0 mL of
DBU/n-C6CH13SH was added to react 2 min. Then, the resin was
carefully washed with DMF (3.0 mL x 4) and CH2Cl2 (3.0 mL x 4).
Subsequent Thioamide Bonds Incorporation. After the first
thioamide was incorporated into the peptide sequence, the
subsequent Fmoc deprotections and peptide elongation were
treated with DBU/n-C6CH13SH and HBTU/DIPEA conditions. The
second thioamide incorporation proceeded as described above
general procedure 1 to get target 7 on the resin. The peptide 7 was
obtained after treat 10 mg resin with 100.0 μL of cleavage cocktail
(TFE/AcOH/CH2Cl2 - 1:3:6) for 30 min. The CH2Cl2 was removed
from the cocktail. Then the solution of water/acetonitrile (v/v 1:1,
200.0 μL) was added to the residue. The resin was filtered off and
the filtrates was analysed by HPLC and MS.
Synthesis of Fmoc-ProS-Val-Gly-Phe-OH (5n). Peptide 5n was
obtained using the procedure 2. HPLC analysis was performed
using C18 analytical column, 254 nm UV detection. Gradient
elution with 60-90% B with a flow rate of 1.0 mL/min over 30 min
gave the target peptide. 98.85% conversion of crude HPLC traces;
tR = 18.054 min; MS (ESI-TOF) calcd for C35H39N4O6S (M - H)-:
655.26, found 655.24.
Synthesis of Fmoc-Thr(Me)S-Val-Gly-Phe-OH (5o). Peptide 5o
was obtained using the procedure 2. HPLC analysis was performed
using C18 analytical column, 254 nm UV detection. Gradient
elution with 40-90% B with a flow rate of 1.0 mL/min over 30 min
gave the target peptide. 98.72% conversion of crude HPLC traces;
tR = 18.833 min; MS (ESI-TOF) calcd for C36H43N4O7S (M + H)+:
673.27, found 673.20.
Synthesis of Fmoc-ASASVGF-OH (7a). Peptide 7a was obtained
using the procedure 3 after 1 h. HPLC analysis was performed
using C18 analytical column, 254 nm UV detection. Gradient
elution with 50-90% B with a flow rate of 1.0 mL/min over 30 min
gave the target peptide. 99.42% conversion of crude HPLC traces;
tR = 14.520 min; HRMS (ESI-TOF) calcd for C37H42N5O6S2 (M -
H)-: 716.2582, found 716.2593.
Synthesis of Fmoc-IleS-Ala-Gly-Phe-OH (5p). Peptide 5p was
obtained using the procedure 2. HPLC analysis was performed
using C18 analytical column, 254 nm UV detection. Gradient
elution with 50-90% B with a flow rate of 1.0 mL/min over 30 min
gave the target peptide. 97.22% conversion of crude HPLC traces;
tR = 14.701 min; MS (ESI-TOF) calcd for C35H39N4O6S (M - H)-:
643.26, found 643.36.
Synthesis of Fmoc-E(tBu)SASVGF-OH (7b). Peptide 7b was
obtained using the procedure 3 after 3 h. HPLC analysis was
performed using C18 analytical column, 254 nm UV detection.
Gradient elution with 60-90% B with a flow rate of 1.0 mL/min
over 30 min gave the target peptide. 96.66% conversion of crude
HPLC traces; tR = 14.316 min; MS (ESI-TOF) calcd for
C43H54N5O8S2 (M + H)+: 832.34, found 831.97.
Synthesis of Fmoc-ValS-Ala-Gly-Phe-OH (5q). Peptide 5q was
obtained using the procedure 2. HPLC analysis was performed
using C18 analytical column, 254 nm UV detection. Gradient
elution with 50-90% B with a flow rate of 1.0 mL/min over 30 min
gave the target peptide. 97.94% conversion of crude HPLC traces;
tR = 13.036 min; MS (ESI-TOF) calcd for C34H36N4O6S (M - H)-:
629.24, found 629.20.
Synthesis of Fmoc-S(tBu)SASVGF-OH (7c). Peptide 7c was
obtained using the procedure 3 after 1 h. HPLC analysis was
performed using C18 analytical column, 254 nm UV detection.
Gradient elution with 60-90% B with a flow rate of 1.0 mL/min
over 30 min gave the target peptide. 99.25% conversion of crude
HPLC traces; tR = 13.095 min (major); MS (ESI-TOF) calcd for
C41H52N5O7S2 (M + H)+: 790.33, found 790.01.
Synthesis of Fmoc-LeuS-Ala-Gly-Phe-OH (5r). Peptide 5r was
obtained using the procedure 2. HPLC analysis was performed
using C18 analytical column, 254 nm UV detection. Gradient
elution with 50-90% B with a flow rate of 1.0 mL/min over 30 min
gave the target peptide. 99.88% conversion of crude HPLC traces;
tR = 15.873 min; MS (ESI-TOF) calcd for C35H39N4O6S (M - H)-:
643.26, found 643.38.
Synthesis of Fmoc-MSASVGF-OH (7d). Peptide 7d was
obtained using the procedure 3 after 4 h. HPLC analysis was
performed using C18 analytical column, 254 nm UV detection.
Gradient elution with 60-90% B with a flow rate of 1.0 mL/min
over 30 min gave the target peptide. 95.51% conversion of crude
HPLC traces; tR = 11.121 min; MS (ESI-TOF) calcd for
C39H48N5O6S3 (M + H)+: 778.28, found 777.92.
S
Synthesis of Fmoc-Arg(Boc)2 -Val-Gly-Phe-OH (5s). Peptide
5s was obtained using the procedure 2. HPLC analysis was
performed using C18 analytical column, 254 nm UV detection.
Gradient elution with 50-90% B with a flow rate of 1.0 mL/min
over 30 min gave the target peptide. 98.43% conversion of crude
HPLC traces; tR = 19.433 min (major); MS (ESI-TOF) calcd for
C47H60N7O10S (M - H)-: 914.41, found 914.47.
Synthesis of Fmoc-K(Boc)SASVGF-OH (7e). Peptide 7e was
obtained using the procedure 3 after 4 h. HPLC analysis was
performed using C18 analytical column, 254 nm UV detection.
Gradient elution with 60-90% B with a flow rate of 1.0 mL/min
over 30 min gave the target peptide. 92.92% conversion of crude
Synthesis of Fmoc-AlaS-Val-Arg(pbf)-Gly-OH (5t). Peptide 5t
was obtained using the procedure 2. HPLC analysis was performed
using C18 analytical column, 254 nm UV detection. Gradient
elution with 50-80% B with a flow rate of 1.0 mL/min over 30 min
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HPLC traces; tR = 12.843 min; MS (ESI-TOF) calcd for
HPLC analysis was performed using C18 analytical column, 254
1
2
3
4
5
6
7
8
C45H59N6O8S2 (M + H)+: 875.38, found 875.25.
nm UV detection. Gradient elution with 80-100% B with a flow
rate of 1.0 mL/min over 30 min gave the target peptide. tR = 24.133
Synthesis of Fmoc-ASLASVGF-OH (7f). Peptide 7f was
obtained using the procedure 3 after 3 h. HPLC analysis was
performed using C18 analytical column, 254 nm UV detection.
Gradient elution with 60-90% B with a flow rate of 1.0 mL/min
over 30 min gave the target peptide. 99.63% conversion of crude
HPLC traces; tR = 11.883 min; HRMS (ESI-TOF) calcd for
C43H53N6O7S2 (M - H)-: 829.3423, found 829.3425.
-
min (major). MS (ESI-TOF) calcd for C98H127N12O19S2 (M - H)-:
1839.88, found 1839.75.
Synthesis of Fmoc-MSASASASA-OH (7p). Peptide 7p was
obtained using the procedure 3 after 10 h. HPLC analysis was
performed using C18 analytical column, 254 nm UV detection.
Gradient elution with 70-90% B with a flow rate of 1.0 mL/min
over 30 min gave the target peptide. 99.24% conversion of crude
HPLC traces; tR = 8.151 min; MS (ESI-TOF) calcd for
C32H40N5O4S5 (M - H)-: 718.17, found 717.94.
Synthesis of Fmoc-FSLASVGF-OH (7g). Peptide 7g was
obtained using the procedure 3 after 1 h. HPLC analysis was
performed using C18 analytical column, 254 nm UV detection.
Gradient elution with 60-90% B with a flow rate of 1.0 mL/min
over 30 min gave the target peptide. 98.10% conversion of crude
HPLC traces; tR = 15.975 min (major); HRMS (ESI-TOF) calcd for
C49H55N6O7S2 (M -H)-: 905.3736, found 905.3750.
9
10
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13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
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Synthesis of Fmoc-ASASASASASA-OH (7q). Peptide 7q was
obtained using the procedure 3 after 5 h. HPLC analysis was
performed using C18 analytical column, 254 nm UV detection.
Gradient elution with 50-90% B with a flow rate of 1.0 mL/min
over 30 min gave the target peptide. 97.75% conversion of crude
HPLC traces; tR = 16.620 min; MS (ESI-TOF) calcd for
C33H41N6O4S5 (M - H)-: 745.18, found 745.25.
Synthesis of Fmoc-K(Boc)SLASVGF-OH (7h). Peptide 7h was
obtained using the procedure 3 after 3 h. HPLC analysis was
performed using C18 analytical column, 254 nm UV detection.
Gradient elution with 60-90% B with a flow rate of 1.0 mL/min
over 30 min gave the target peptide. 93.64% conversion of crude
HPLC traces; tR = 16.006 min; MS (ESI-TOF) calcd for
C51H70N7O9S2 (M + H)+: 988.47, found 988.39.
■ ASSOCIATED CONTENT
Supporting Information
Synthesis of Fmoc-MSLASVGF-OH (7i). Peptide 7i was
obtained using the procedure 3 after 3 h. HPLC analysis was
performed using C18 analytical column, 254 nm UV detection.
Gradient elution with 60-90% B with a flow rate of 1.0 mL/min
over 30 min gave the target peptide. 95.62% conversion of crude
HPLC traces; tR = 13.833 min; MS (ESI-TOF) calcd for
C45H57N6O7S3 (M - H)-: 889.26, found 889.35.
The Supporting Information is available free of charge on the ACS
Publications website at http://pubs.acs.org.
1
Optimization of reaction conditions; H- and 13C NMR spectra;
HPLC data; ESI-MS spectra; additional experimental procedures
(PDF)
■ AUTHOR INFORMATION
Synthesis of Fmoc-E(tBu)SLASVGF-OH (7j). Peptide 7j was
obtained using the procedure 3 after 5 h. HPLC analysis was
performed using C18 analytical column, 254 nm UV detection.
Gradient elution with 60-90% B with a flow rate of 1.0 mL/min
over 30 min gave the target peptide. 93.34% conversion of crude
HPLC traces; tR = 17.450 min; MS (ESI-TOF) calcd for
C49H65N6O9S2 (M + H)+: 945.42, found 945.04.
Corresponding Author
*E-mail: zhaojf@jxnu.edu.cn
ORCID
Junfeng Zhao: 0000-0003-4843-4871
Synthesis of Fmoc-S(tBu)SLASVGF-OH (7k). Peptide 7k was
obtained using the procedure 3 after 3 h. HPLC analysis was
performed using C18 analytical column, 254 nm UV detection.
Gradient elution with 60-90% B with a flow rate of 1.0 mL/min
over 30 min gave the target peptide. 98.54% conversion of crude
HPLC traces; tR = 17.791 min (major); MS (ESI-TOF) calcd for
C47H63N6O8S2 (M + H)+: 903.41, found 902.99.
Notes
The authors declare no competing financial interests.
■ ACKNOWLEDGMENT
This work was supported by the National Natural Science
Foundation of China (21762021, 91853114), and the Science and
Technology Project of Jiangxi Provincial Education Department
(GJJ150297).
Synthesis of Fmoc-ASLAASVF-OH (7l). Peptide 7l was obtained
using the procedure 3 after 2 h. HPLC analysis was performed
using C18 analytical column, 254 nm UV detection. Gradient
elution with 60-90% B with a flow rate of 1.0 mL/min over 30 min
gave the target peptide. 100% conversion of crude HPLC traces; tR
= 13.330 min; MS (ESI-TOF) calcd for C44H55N6O7S2 (M - H)-:
843.36, found 843.44.
■ REFERENCES
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