Substrate-directed lewis-acid catalysis for peptide synthesis

Article abstract of DOI:10.1021/jacs.9b03850

A Lewis-acid-catalyzed method for the substrate-directed formation of peptide bonds has been developed, and this powerful approach is utilized for the new "remote" activation of carboxyl groups under solvent-free conditions. The presented method has the following advantages: (1) the high-yielding peptide synthesis uses a tantalum catalyst for any amino acids; (2) the reaction proceeds without any racemization; (3) the new substrate-directed chemical ligation using the titanium catalyst is applicable to convergent peptide synthesis. These advantages overcome some of the unresolved problems in classical peptide synthesis.

Full text of DOI:10.1021/jacs.9b03850

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Article
Substrate-Directed Lewis-Acid Catalysis for Peptide Synthesis
WATARU MURAMATSU, Tomohiro Hattori, and Hisashi Yamamoto
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b03850 • Publication Date (Web): 16 Jul 2019
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Journal of the American Chemical Society
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Substrate-Directed Lewis-Acid Catalysis for Peptide Synthesis
Wataru Muramatsu,* Tomohiro Hattori, and Hisashi Yamamoto*
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Molecular Catalyst Research Center, Chubu University, 1200 Matsumoto-cho, Kasugai, Aichi 487-8501, Japan.
Tantalum catalysis, Substrate-directed amidation, Solvent-free, Chemical ligation, Peptides, Protecting groups
ABSTRACT: A Lewis-acid-catalyzed method for the substrate-directed formation of peptide bonds has been developed, and this
powerful approach is utilized for the new “remote” activation of carboxyl groups under solvent-free conditions. The presented method
has the following advantages: 1) the high-yielding peptide synthesis uses a tantalum catalyst for any amino acids; 2) the reaction
proceeds without any racemization; 3) the new substrate-directed chemical ligation using the titanium catalyst is applicable to con-
vergent peptide synthesis. These advantages overcome some of unresolved problems in classical peptide synthesis.
has been stimulated. Indeed, since our initial report of a boron-
based catalyst for the amidation between carboxylic acids and
amines,10 a number of catalytic approaches have been re-
ported.11–13 All of these classical and modern amidation pro-
cesses are based on activation of the carboxylic acid moiety,
which unfortunately leads to issues of racemization of the pro-
duced peptides (Scheme 1A).5 Once racemization occurs, tedi-
ous purification becomes unavoidable. In particular, during
solid-phase peptide synthesis, the peptide must be cleaved from
the resin beads for purification and then linked again with beads
for the next reaction, which creates a major expense and other
troublesome issues for the synthesis. As an alternative, Kent’s
native chemical ligation (NCL) allows the coupling of two pep-
tides by the reaction of a-thioester with Cys-peptides.14,15 This
pioneering method facilitates preparation of large peptides by
chemical synthesis. Unfortunately, however, the Kent proce-
dure requires specific amino acid termini. If any amino acid ter-
minus could be used, chemical ligation would open an entirely
new entry to peptide synthesis. Not only large peptide synthesis,
but also smaller peptide synthesis could be simplified by gen-
eral chemical ligation using a convergent synthesis approach.
Thus, considerable room for improvement exists for avoiding
environmental degradation, purification involving racemization,
and non-convergent linear peptide synthesis. Herein we are
pleased to address some of these long-standing issues of peptide
synthesis using substrate-directed Lewis acid catalysis.
INTRODUCTION
In the current century, peptides are extensively exploited in
the pharmaceutical, agricultural, food, and cosmetic indus-
tries.1,2 Indeed, owing to their range of functionalities, low tox-
icities, and high specificities towards targeted proteins com-
pared to those of classical small molecules (i.e., <500 Da), the
global peptide therapeutics market is increasing in value annu-
ally by ~15%,3 and is currently expected to reach ~ USD 50
billion by 2025.4 To address the growing worldwide demand for
peptides, dramatic technological innovation to current synthetic
protocols is required in response to economic and environmen-
tal needs.
The classical approach to peptide synthesis constitutes the
coupling reaction between amines and carboxylic acids. In the
presence of a stoichiometric amount of a sophisticated coupling
reagent, peptides are prepared in a stepwise manner by reaction
with the amino functional group of the amino acid.5 Utilizing
well established solid-phase peptide synthesis,6 the peptide is
constructed by attaching a terminal amino acid to a resin bead,
and adding the subsequent amino acid residues in a sequential
manner, with the residual by-products being washed out at each
step. With these processes, producing peptide products requires
a huge volume of organic solvent and water in addition to sub-
stantial quantities of coupling reagent causing significant nega-
tive environmental impact.7–9 Giving these circumstances, in-
vestigation of various catalytic routes to peptide bond formation
Scheme 1. Formation of Peptide Bonds
A. Racemization via active ester during coupling-reagent-mediated reaction
B. Novel strategy for substrate-directed catalysis
Activated Esters
R2
Active Esters
O
WSC•HCl
Et3N, HOBt
Solvent
OR3
OR1
R1O
N
H
R2
Solvent-Free
cat. [M]
OR3
O
R2
O
O
R2
O
H
N
H
N
O
HN
R2
O
O
+
HN
R1O
R1O
N
H
OR4
R1O
N
H
OR3
O
O
N
N
O
R3
O
R4
O
[M]
H2N
O
OR3
N
R4
Directing Group
Racemization
OR1
O
R2
OR3
O
R2
O
O
R2
H
O
H
N
N
N
Oxazolone Intermediate
Seven-Membered Ring TS
HN
X
X
R1O
N
H
OR4
N
H
OR3
R1O
O
R2
R1O
O
R3
R4
O
O
[M]
O
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RESULTS AND DISCUSSION
potentially adsorb atmospheric moisture easily, unexpected hy-
1
drolysis of the catalyst by ambient moisture may impair the re-
producibility of the reaction. The use of microwave irradiation
dramatically shortened the time required to form the peptide
bond (92% yield with >99:1 er and >99:1 dr, entrie 5). Entries
1, 3, and 4 reveal that the minor diastereomer is not the expected
Boc-D-Ala-L-Ala-Ot-Bu,5 (from racemization at the N-terminal
L-Ala residue of 3a) but surprisingly Boc-L-Ala-D-Ala-Ot-Bu,
in which the C-terminal L-Ala residue of 3a was racemized. In
order to further examine this occurrence, 3a was employed un-
der the same reaction conditions, and racemization was not ob-
served by HPLC. In addition, GC revealed that 2 does not rac-
emize during the neutralization of L-Ala-Ot-Bu･HCl with the
basic anion exchange resins (99.8:0.2 er was obtained after tri-
fluoroacetylation of 2,23 see supporting information). In contrast,
we clarified by GC that 2 was only slightly racemized by heat-
ing (99.5:0.5 er was obtained after trifluoroacetylation of 2),
and the presence of Ta(OMe)5 slightly accelerated its racemiza-
tion (97.9:2.1 er was obtained after trifluoroacetylation of 2).
Whereas commonly used protecting groups for peptide synthe-
sis, such as Cbz, Troc, and Alloc, were tolerated in this ap-
proach to give excellent yields of 3b–d without any loss in ste-
reochemical integrity or additional side-product formation (en-
tries 6–8), replacement of these carbonate protecting groups
with Fmoc or Pht led to significantly reduced yields due to the
formation of side-products by the unexpected deprotection of
1e and 1g (entries 9 and 11). To reduce the reaction time, ele-
vated temperatures (i.e., 100 °C) were investigated, but signifi-
cantly lower yields of 3b–d were obtained, as compounds 1b–
d were converted into their corresponding dipeptides with the
concomitant release of phenoxy, trichloroethoxy, and allyloxy
anions from 1b–d, respectively; 1c was particularly sensitive to
the reaction temperature, it gave 3c in only 1% yield at 60 °C.
These results indicate that the carbonate protecting groups act
as directing groups, as shown in Scheme. 1B. The Bz and Ts
groups present in 1f and 1h also acted as good directing groups
to produce 3f and 3h in quantitative yields (entries 10 and 12).
Impressively, reactions with 1i and 1j provided 3i and 3j,24 re-
spectively, in moderate yields with no racemization under suit-
able conditions, despite the absence of the carbonyl oxygen at-
oms of the protecting groups (entries 13 and 14). These results
indicate that the nitrogen atoms of 1i and 1j can also act as di-
recting groups. However, side reactions were observed when
the reaction temperature was raised to 70 °C in an attempt to
improve the yields of 3i and 3j. In contrast, electrophilic Boc-
L-β-amino acid methyl esters, such as Boc-L-β-Ala-OMe and
Boc-L-β-HomoAla-OMe, failed to provide the desired peptides
in the presence of this catalyst because of the unstable eight-
membered ring transition states. As conditions for removing
these protecting groups are well-established,20,25 the Boc, Cbz,
Bz, and Bn moieties were smoothly deprotected to provide L-
Ala-L–Ala-Ot-Bu 4 in >90% yields using 4N HCl/1,4-dioxane
for the cleavage of the Boc group,26 Pd-catalyzed hydrogenation
for the deprotection of the Cbz and Bn groups, and the Meer-
wein reagent for the removal of the Bz group.27
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3
4
5
6
7
8
9
Novel Strategy for Substrate-Directed Lewis-Acid Catal-
ysis for Peptide bonds. Substrate-directed chemical reactions
are undoubtedly among the most powerful tools in modern or-
ganic chemistry.16,17 Our early efforts toward peptide bond for-
mation focused on the directing effects of hydroxy18 and oxime
groups19 in amino acid methyl esters, as such systems resolve
the issues of waste production and racemization; however, this
approach is unfortunately limited in substrate scope. Therefore,
we examined catalytic systems directed by the widely used car-
bonate protecting groups of simple amino acids.20 Our envis-
aged strategy is outlined in Scheme 1B. Initially, the Lewis
basic carbonyl oxygen atoms on Boc, Cbz, and Fmoc groups
preferentially interact with the Lewis acid catalyst. Subse-
quently, when a methyl ester moiety is available by proximity
to coordinate with the Lewis acid catalyst, the ester is selec-
tively activated. Most importantly, racemization via an oxazo-
lone intermediate is blocked by the formation of a seven-mem-
bered transition state. Finally, the amino group of the nucleo-
philic reaction partner site-selectively approaches the activated
methyl ester forming the desired peptide bond. We expected the
new process to rely on the directing effects of carbonate pro-
tecting groups to promote peptide bond formation between the
amino moieties and the inactive ester groups of the amino acids
while avoiding long-standing stereochemical problems.
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Tantalum-Catalyzed Peptide Bond-Forming Reactions.
To verify the feasibility of our proposed strategy, we first tested
a series of solvents during the peptide-bond-forming reaction of
Boc-L-Ala-OMe 1a with 3.0 equivalents of L-Ala-Ot-Bu 2 in
the presence of 10 mol% Ta(OEt)5 at 50–100 °C for 24 h, ac-
cording to a previous report on a hydroxy-group-directed reac-
tion involving L-Ser and L-Thr;18 however no or very trace
amount of Boc-L-Ala-L-Ala-Ot-Bu 3a was obtained in common
solvents, such as toluene, DMF, or Et2O. When the reaction was
repeated in non-polar aliphatic solvents, such as n-pentane or c-
hexane, 3a was produced in slightly improved yields of ~10%.
Surprisingly, the absence of a solvent resulted in a notable im-
provement with 29% yield of 3a. We expected that the weak
C=O+–Ta bond formed between the Boc group and Ta(OEt)5
would be disrupted easily, compared to that of the O–Ta bond
formed between the OH group and Ta(OEt)5. After identifying
a metal catalyst and extensively optimizing the reaction condi-
tions (Tables T1 and 2, see supporting information), we found
that the solvent-free catalytic assembly of 1a and 2, which was
prepared by carefully neutralizing its HCl salt with a weakly
basic anion exchange resin, such as AmberlystTM A2121 or
DIAIONTM WA30, proceeded effectively at 45 °C in the pres-
ence of 10 mol% Ta(OMe)5 to furnish 3a in 98% isolated yield
with a >99:1 enantiomeric ratio (er) and >99:1 diastereomeric
ratio (dr) (entry 1 of Table 1). As expected, the reaction does
not proceed in the absence of Ta(OMe)5 (entry 2). In addition,
this catalysis facilitates the use of the HCl salts of amino acid
esters with Et3N in the presence of Ta(OMe)5, which is benefi-
cial, as these salts allow overwhelmingly longer storage without
racemization or polymerization; they are also significantly eas-
ier to handle than their free amines.22 Hence, the free amine pro-
vided by the in situ neutralization of L-Ala-Ot-Bu･HCl with
Et3N coupled smoothly with 1a to afford 3a in 92% yield with
>99:1 er and >99:1 dr (entry 3). A glove box is not indispensa-
ble for preparation of this reaction (96% yield with >99:1 er and
>99:1 dr, entry 4). However, since amino acids and Ta(OMe)5
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Journal of the American Chemical Society
Table 1. Examining Various Protecting Groups as Directing supports these reactions by facilitating the formation of a six-
Groups for Peptide Bond Formation.
1
or seven-membered transition state. In contrast, the presence of
bulky side chains at the α-positions of amino acids, especially
secondary alkyl groups, such as s-Bu in Ile, i-Pr in Val, and ter-
tiary groups such as t-Bu in Tle, detrimentally affected the
yields of these reactions (0–2% yields of 6d–f). It is reasonable
to assume that the inertness of 5d–f under the Ta(OMe)5-cata-
lyzed conditions is due to steric hindrance between the bulky
side chains of 5d–f and the bulky Boc group, which hinders for-
mation of the seven-membered transition state as outlined in
Scheme 1B. Hence, silyl esters, namely Boc-L-Ile-OTMS, Boc-
L-Val-OTMS, and Boc-L-Tle-OTMS, possessing potential leav-
ing groups with lower pKa values (TMSOH: 11, MeOH: 16),27
generated in situ from the corresponding carboxylic acids 7d–f
with 1-(trimethylsilyl)imidazole (TMSIM), at 50 °C were found
to be exquisitely effective in the Ta(OMe)5-catalyzed reaction,
to give 6d–f in yields of 37–97%, without loss in stereochemi-
cal integrity; 6d–f did not form in the absence of Ta(OMe)5 due
to the immobility of the substrate-directed activation of the silyl
esters. Silylating agents are known to activate amines29 or car-
boxylic acids30 in several amidation reactions; however, our
method operates in a completely different manner to these pro-
cesses. Our one-pot catalytic protocol with TMSIM was used to
further improve the yields of 6g, 6i, 6j, 6o, and 6u–x. In addi-
tion, this system satisfactorily enabled coordination with the
carbonyl oxygen atoms on ester groups located at the β- and γ-
positions, as well as those located at the α-positions of Asp and
Glu, respectively, to afford 6n and 6p in moderate to high yields.
As mentioned above, HCl salts of amino acids are preferable to
the corresponding free amines because they are easier to handle
and can be stored for long periods without polymerization
and/or racemization. This one-pot protocol involving silyl es-
ters is superior to the use of HCl salts of amino acids as nucle-
ophilic counterparts, giving the desired dipeptides 6q and 6r in
71 and 59% yields, respectively, because the HCl salts of amino
acids are neutralized in situ in the presence of imidazole. Fortu-
nately, this one-pot protocol involving TMSIM and the HCl
salts of amino acids dramatically improved the unresolved ami-
dation of amino acids bearing Fmoc groups; Fmoc is the most
used base-labile protecting group (85% yield and >99:1 dr, en-
try 9 of Table 1). The success of our novel method in LPPS
leads to its prospective application in SPPS. In addition to the
advantages of our method, most of the dipeptides obtained by
the one-pot protocol could be isolated in high purity with only
simple washing and extraction without column chromatography,
since the reactions via silyl esters do not provide any side-reac-
tion products. These advantages were used in the triply conver-
gent synthesis depicted in Scheme 4B.
2
3
4
5
6
7
8
9
Ta(OMe)5 (10 mol%)
-Ala-Ot-Bu (2, 2.0 equiv)
L
R2
O
PG-
L
-Ala-OMe
PG-
L
-Ala-
L-Ala-Ot-Bu
45 °C, 72 h
(1a–j)
(3a–j)
R2
OMe
OMe
R'
or
HN
N
O
O
H
R
[Ta]
[Ta]
entry
product
dra
protecting group
(PG)
yield
(%)
10
11
12
13
14
15
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1
Boc
Boc
3a
3a
3a
3a
3a
3b
3c
3d
3e
98
0
>99:1
nd
2b
3c
4d
5e
6
7f
8g
9h
Boc
92
96
92
>99
93
98
>99:1
>99:1
>99:1
>99:1
>99:1
>99:1
nd
(>99:1)
>99:1
>99:1
>99:1
>99:1
>99:1
Boc
Boc
Cbz
Troc
Aloc
Fmoc
0
(85)
10
11g
12f
13g
14g
Bz
Pht
3f
3g
3h
3i
>99
10
Ts
>99
60
Bn
NCCH2
3j
45
aThe drs of 3a–j were determined by 1H NMR. bIn the absence of
c
Ta(OMe)5. 2·HCl and Et3N (2.0 equiv) were used instead of 2.
dWithout glove box. eThrough the use of microwave irradiation for
24 h. fThe reactions were carried out at room temperature. gThe re-
h
actions were carried out at 60 °C. The numbers in brackets are
yield and dr obtained under the following reaction conditions:
Fmoc-L-Ala-OH (2.0 equiv), 2·HCl (1.0 mmol), Ta(OMe)5 (10
mol%), TMSIM (2.0 equiv), 40 °C, 72 h.
We explored the versatility of our method under the opti-
mized conditions using a wide array of amino acids as electro-
philic coupling partners (Scheme 2). Most of the amino acids
were successfully coupled, including S-functionalized amino
acids 5s and 5t, which produced 6a–c, 6h–p, and 6s–u in mod-
erate to excellent yields. In particular, 6k–m were obtained
quantitatively at lower catalyst loadings (2.0 mol%) and/or over
a shorter reaction time (24 h) because the secondary directing
effect of the hydroxy group on Ser and Thr (5k and 5l),18 or the
tert-butyl ester group at the β-position of Asp (5m), strongly
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Scheme 2. Reaction Scope: Protected Amino Acid Methyl Esters (Highlighted in Blue)a
1
Ta(OMe)5 (10 mol%), TMSIM
2
3
4
5
L
-Ala-Ot-Bu (2)
or
Ta(OMe)5 (10 mol%)
R'
R'
R
O
OMe
OTMS
L
-Ala-Ot-Bu•HCl
L
-Ala-Ot-Bu (2)
H
PG
N
PG-AA-OMe
PG-AA-OH
HN
HN
O
N
H
Ot-Bu
O
40–70 °C, 72 h
30–50 °C, 72 h
R
R
O
[Ta]
[Ta]
(5a–x)
O
O
(7a–x)
Methods A and B
Methods C and D
(6a–x)
6
7
8
9
i-Bu
s-Bu
i-Pr
t-Bu
H
N
H
N
H
N
H
N
H
N
H
N
Boc
Boc
Boc
Boc
Boc
Boc
N
H
N
H
N
N
N
H
N
H
H
H
O
O
O
O
O
O
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
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40
41
42
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44
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46
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48
49
50
51
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53
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57
58
59
60
Boc-
L
-Gly–
L
-Ala (6a)
Boc-
D
-Ala–
L
-Ala (6b)
Boc-
L
-Leu–
L
-Ala (6c)
Boc-
L
-Ile–
L
-Ala (6d)
Boc-
L
-Val–
L
-Ala (6e)
Boc-
L
-Tle–L-Ala (6f)
>99% yield, >99:1 erb
95% yield, >99:1 drb
77% yield, >99:1 drb
97% yield, >99:1 drd
74% yield, >99:1 drd
97% yield, >99:1 drd
37% yield, >99:1 drd
(36 h)
(4-Ot-Bu)Ph
3-Ind
HO
HO
Ph
Bn
H
N
H
N
H
N
H
N
H
N
H
Boc
Boc
Boc
Boc
Boc
Boc
N
N
H
N
H
N
H
N
H
N
H
N
H
O
O
O
O
O
O
Boc-
L
-Phg–
L
-Ala (6g)
Boc-
L
-Phe–
L
-Ala (6h)
Boc-
L
-Tyr–
L
-Ala (6i)
Boc-
L
-Trp–
L
-Ala (6j)
Boc-
L
-Ser–
L
-Ala (6k)
Boc-
L
-Thr–L-Ala (6l)
75% yield, 70:30 drd,e
(50% yield, >99:1 dr)f
91% yield, >99:1 drb
83% yield, >99:1 drb
97% yield, >99:1 drd
82% yield, >99:1 drc,e
97% yield, >99:1 drd,e
>99% yield, >99:1 drb
>99% yield, >99:1 drb
(2 mol% Ta, 24 h)
(2 mol% Ta, 24 h)
O
t-BuO
Boc
NH
O
O
t-BuO
Boc
NH
H
O
Trt
N
Trt
(
)
(
)
t-BuO
O
2
2
t-BuO
O
N
H
H
N
H
N
H
N
H
N
H
N
H
(
)
2
Boc
Boc
N
Boc
Boc
N
H
N
H
N
H
N
H
O
O
O
O
O
O
Boc-
L
-Asp–
L
-Ala (6m)
Boc-
L
-Asp–
L
-Ala (6n)
Boc-
L
-Glu–
L
-Ala (6o)
Boc-
L
-Glu–
L
-Ala (6p)
Boc-
L
-Asn–
L
-Ala (6q)
Boc-
L
-Gln–
L
-Ala (6r)
96% yield, >99:1 drb
45% yield, >99:1 drb
94% yield, >99:1 drd
64% yield, >99:1 drc
81% yield, >99:1 drd
42% yield, >99:1 drc,e
98% yield, >99:1 drd,e
71% yield, >99:1 dre,g
59% yield, >99:1 dre,g
(24 h)
H
N
NH
Boc
H
N
S
t-Bu
H
Boc
N
5-Imid(N-Bn)
(
)
(
)
4
3
MeS
Boc
Boc
Boc
Boc
H
N
H
N
H
N
H
N
H
N
Cbz
N
Boc
N
N
H
N
H
N
H
N
H
N
H
Boc
Boc- -Pro–
>99% yield, >99:1 drd
O
O
O
O
O
O
Cbz-
L
-Met–
L
-Ala (6s)
Boc-
L
-Cys–
L
-Ala (6t)
Boc-
L
-Lys–
L
-Ala (6u)
Boc-
L
-Arg–
L
-Ala (6v)
Boc-
L
-His–
L
-Ala (6w)
L
L-Ala (6x)
94% yield, >99:1 drb
97% yield, >99:1 drb
63% yield, >99:1 drb
90% yield, >99:1 drd
58% yield, >99:1 drd,e
45% yield, >99:1 drb
64% yield, >99:1 drd
aThe er of 6a was determined by HPLC. The ers and drs of 6g and 6h were determined by HPLC and 1H NMR. The drs of 6b–f and 6i–x
1
b
c
were determined by H NMR. Method A: 5 (1.0 mmol), 2 (2.0–3.0 equiv), and Ta(OMe)5 (10 mol%) were used. Method B: 5 (2.0–2.5
equiv), 2 (1.0 mmol), and Ta(OMe)5 (10 mol%) were used. dMethod C: 7 (2.0 equiv), 2 (1.0 mmol), TMSIM (2.0–2.2 equiv), and Ta(OMe)5
(10 mol%) were used. eThese reactions were carried out in solvent (CHCl3 or DMSO). fAfter separation of both diastereomers by normal-
phase silica-gel chromatography. gMethod D: 7 (2.0 equiv), 2·HCl (1.0 mmol), TMSIM (2.2 equiv), and Ta(OMe)5 (10 mol%) were used.
Similarly, these methods tolerated numerous functional
groups, such as ethers, esters, amides, and sulfides, in the elec-
trophilic amino acid species, as well as nucleophilic species,
during the preparation of dipeptides 9a–e and 9h–s; these reac-
tions proceeded without any racemization (Scheme 3). Unnatu-
ral amino acid, phenylglycine (Phg), is known to racemize more
easily than other amino acids,31 and this was unfortunately ob-
served using our substrate-directed method implemented for 6g
(>99:1 er and 70:30 dr) and 9f (>99:1 er and 93:7 dr). The rac-
emization of 6g and 9f is not due to oxazolone formation but
rather to enolization through direct deprotonation of the acidic
benzylic proton on 5g (or 6g) or 8f (or 9f), respectively.
However, 6g and its diastereomer could be separated by silica-
gel chromatography. In addition, 9f and its diastereomer can be
separated by recrystalization after cleavage of the Boc protect-
ing group.19 An abundance of side-products was observed when
this catalysis was applied to 8p at 70 °C, which is ascribable to
the instability of the S–C bond of the Trt group under the reac-
tion conditions. On the other hand, an excellent yield of 9p was
obtained using the TMSIM protocol at 50 °C. Although partial
racemization was observed for 9t (94:6 dr) and 9u (79:21 dr),
9t was fortunately isolated from the mixture of diastereomeric
products in 91% yield (92:8 ratio of rotational isomers) by sil-
ica-gel chromatography.
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Scheme 3. Reaction Scope: Amino Acid tert-Butyl Esters (Highlighted in Pink)a
1
2
3
4
Ta(OMe)5 (10 mol%), TMSIM
L
Ta(OMe)5 (10 mol%)
L
R'
O
OMe
OTMS
-AA-Ot-Bu (8a–u)
-AA-Ot-Bu (8a–u)
H
N
Boc
Boc-
L
-Ala-OMe
Boc-
L
-Ala-OH
HN
HN
O
O
N
H
Ot-Bu
40–70 °C, 72 h
30–50 °C, 72 h
t-BuO
t-BuO
[Ta]
R
O
O
[Ta]
(1a)
(10)
O
Methods A and B
Method C
(9a–u)
5
6
7
8
9
O
O
O
O
O
O
H
N
H
H
N
H
N
H
H
N
N
N
Ot-Bu
Ot-Bu
Ot-Bu
Ot-Bu
Ot-Bu
Ot-Bu
O
Ph
O
O
O
i-Bu
-Ala–
O
s-Bu
-Ala–
O
i-Pr
-Ala–
Boc-
L
-Ala–Gly (9a)
Boc-
L
-Ala–
D
-Ala (9b)
Boc-
L
L
-Leu (9c)
Boc-
L
L
-Ile (9d)
Boc-
L
L
-Val (9e)
Boc-
L
-Ala–L-Phg (9f)
65% yield, >99:1 erb
96% yield, >99:1 erd
97% yield, >99:1 drb
90% yield, >99:1 drb
93% yield, >99:1 drb
91% yield, >99:1 drb
>99% yield, 83:17 drb
65% yield, 93:7 drc
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
O
H
O
H
O
H
O
H
O
H
O
H
N
N
N
N
N
N
Ot-Bu
Ot-Bu
Ot-Bu
Ot-Bu
Ot-Bu
Ot-Bu
O
Bn
O
O
O
O
t-BuO
O
O
Ph(4-Ot-Bu)
3-Ind(N-Boc)
Ot-Bu
Ot-Bu
Boc-
L
-Ala–
L
-Phe (9g)
Boc-
L
-Ala–
L
-Tyr (9h)
Boc-
L
-Ala–
L
-Trp (9i)
Boc-
L
-Ala–
L
-Ser (9j)
Boc-
L
-Ala–
L
-Thr (9k)
Boc-L-Ala–L-Asp (9l)
95% yield, 98:2 drb
95% yield, >99:1 drb
51% yield, >99:1 drb
91% yield, >99:1 drd
78% yield, >99:1 drc
96% yield, >99:1 drd
71% yield, >99:1 drc
98% yield, >99:1 drd
61% yield, >99:1 drb
>99% yield, >99:1 drd
O
H
N
O
H
N
O
H
N
O
H
N
O
H
N
O
H
N
Ot-Bu
Ot-Bu
Ot-Bu
Ot-Bu
Ot-Bu
Ot-Bu
(
)
(
)
3
(
)
2
4
O
O
O
O
Trt
O
Boc
O
O
SMe
O
N
H
NH
S
Ot-Bu
NH2
Mtr
HN
N
H
Boc-
L
-Ala–L-Glu (9m)
Boc-
L
-Ala–
L
-Asn (9n)
Boc-
L
-Ala–
L
-Met (9o)
Boc-
L
-Ala–
L
-Cys (9p)
Boc-
L
-Ala–
L
-Lys (9q)
Boc-L-Ala–L-Arg (9r)
86% yield, >99:1 drd
34% yield, >99:1 drc
71% yield, >99:1 drd
96% yield, >99:1 drd,e
>99% yield, >99:1 drb
>99% yield, >99:1 drd
26% yield, >99:1 drc
99% yield, >99:1 drd
43% yield, >99:1 drb
98% yield, >99:1 drd
O
H
O
N
N
N
Ot-Bu
Ot-Bu
O
O
O
Ot-Bu
5-Imid(N-Trt)
O
Boc-
L
-Ala–
L
-His (9s)
Bz-
L
-Ala–
L
-Pro (9t)
Boc-
L-Ala–L-N-MeAla (9u)
66% yield, 79:21 drd
98% yield, >99:1 drd
71% yield, 94:6 drb,f
(65% yield, >99:1 dr)g
97% yield, 96:4 drd,f
(91% yield, >99:1 dr)g
aThe er of 9a was determined by HPLC. The ers and drs of 9f and 9g were determined by HPLC and 1H NMR. The drs of 9b–e and 9h–u
were determined by 1H NMR. bMethod A: 1a (1.0 mmol), 8 (2.0–3.0 equiv), and Ta(OMe)5 (10 mol%) were used. cMethod B: 1a (2.0–2.5
equiv), 8 (1.0 mmol), and Ta(OMe)5 (10 mol%) were used. dMethod C: Boc-L-Ala-OH 10 (2.0 equiv), 8 (1.0 mmol), TMSIM (2.0–2.2 equiv),
and Ta(OMe)5 (10 mol%) were used. eThe reaction was carried out in CHCl3. f1f or Bz-L-Ala-OH 11 was used instead of 1a or 10, respec-
tively. gAfter separation of both diastereomers by normal-phase silica-gel chromatography.
Applications to the Substrate-Directed “Remote” Peptide
Bond-Forming Reactions. Tantalum catalysts are known to
activate the carbonyl oxygen of an amino acid ester at least six
atoms away from directing groups as electrophilic coupling
partners.18,19 Hence, we envisaged that our catalytic system
might be suitable for substrate-directed ‘remote’ amidations
with dipeptide methyl esters as electrophilic coupling partners,
facilitated by ligands (Scheme 4A). To explore this concept, we
examined the solvent-free reaction of Boc-L-Ala–L-Ala-OMe
12 and 2.0 equivalents of L-Ala-Ot-Bu 2 with a 10 mol%
Ta(OMe)5, which gave the desired tripeptide 13a in 41% yield
with >99:1 dr. Upon screening several bidentate and tetraden-
tate ligands for the tantalum catalysis, the use of L1–3 gave im-
proved yields (additional screening data were listed in Scheme
S1 and Table T3, see supporting information). Since the methyl
ester group is located about 5–6Å away from the Boc directing
group, we designed the bitantalum catalyst using ligands L432
and L5.33 Gratifyingly, when L5 was employed with Ta(OMe)5,
13a was obtained in 92% yield. Thus, one tantalum coordinates
to the Boc group, and the other tantalum activates the terminal
methyl ester.
Based on this observation, we were encouraged to apply the
Ta(OMe)5/L5 complex system in triply convergent syntheses of
tri- and tetrapeptides (Scheme 4B). Although Boc-L-Ala-OMe
1a was initially selected as the electrophilic coupling partner,
the reaction of 1a with L-Ala-OMe 16a in the presence of cata-
lytic Ta(OMe)5 unexpectedly furnished cyclic L-Ala–L-Ala as
the major product, rather than the desired Boc-L-Ala–L-Ala-
OMe 12. To make matters worse, the one-pot process for the
three-component peptide-forming reaction of Boc-L-Ala-
OTMS generated in situ from Boc-L-Ala-OH 10 and TMSIM,
with 16a as the second component and 2 as the third component,
gave only trace amounts of 13a due to the unexpected effect of
the imidazole generated in the second peptide-forming reaction.
Accordingly, after the first peptide-forming reaction using 10
and 16a was carried out in the presence of Ta(OMe)5 and
TMSIM, the imidazole was removed by simply washing with
water prior to treatment of the crude 12 with 2 in the presence
of the Ta(OMe)5/L5 complex to give 13a in 83% yield without
any stereochemical problems. The protocol was also extended
to the preparation of tri- and tetrapeptides 13a–n that contain a
variety of amino acid residues as well as other functional groups
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Page 6 of 10
Scheme 4. Substrate-Directed “Remote” Peptide Bond-Forming Reactionsa
1
2
3
4
A. Ligand scope
Ta(OMe)5 (10 mol%)
Ligand (10 mol%)
-Ala-Ot-Bu (2, 2.0 equiv)
L
Boc-
L-Ala–L-Ala–L-Ala-Ot-Bu
Boc-
L
-Ala–
L-Ala-OMe
5
70 °C, 48 h
(13a, >99:1 dr for all ligands)
(12)
6
7
Ligand:
8
9
N
t-Bu
t-Bu
H
O
O
H
O
[Ta]
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
[Ta]
N
N
N
N
5~6 Å
N
N
N
[Ta]
5~6 Å
N
N
[Ta]
N
O
OH
OH
HO
L5, 82% yield (92% yield)b
L1, 66% yield
L2, 68% yield
L3, 74% yield
L4, 71% yield
B. Triply convergent synthesis base on the Ta(OMe)5/L5 complex system
Ta(OMe)5 (10 mol%), TMSIM (2.0 equiv)
PG-
(7a, 7c, 7h, 7o, 7u, 10, 14, 15, 2.0 equiv)
Ta(OMe)5 (10 mol%), L5 (10 mol%)
-CC-Ot-Bu
(2, 4, 8a, 8c, 8e, 8o, 17, 18, 2.0 equiv)
L
-AA-OH
L
L-BB-OMe
PG-
L
-AA–
L-BB–L-CC-Ot-Bu
60 °C, 24 h
without purification
70 °C, 48 h
(16a–d)
(13a–n, >99:1 dr)
Boc-
L
-Ala–
L
-Ala–
L
-Ala-Ot-Bu
Cbz-
L
-Ala–
L
-Ala–
L
-Ala-Ot-Bu
Cbz-Gly–
L
-Ala–
L
-Ala-Ot-Bu
Boc-
Boc-
L
L
-Leu–
L-Ala–L-Ala-Ot-Bu
83% yield (13a)
81% yield (13b)
91% yield (13c)
91% yield (13d)
Boc-
L
-Phe–
L
-Ala–
L
-Ala-Ot-Bu
Boc-
L
-Ala–
L
-Leu–Gly-Ot-Bu
Boc-
L
-Ala–
L
-Met–
L
-Ala-Ot-Bu
-Ala–
L-Ala–L-Leu-Ot-Bu
83% yield (13e)
88% yield (13f)
91% yield (13g)
65% yield (13h)
Boc-
L
-Ala–
81% yield (13i)c
L
-Ala–
L
-Val-Ot-Bu
Boc-
L
-Ala–
L
-Ala–
L
-Met-Ot-Bu
Boc-Gly–
L
-Phe–
L
-Ala-Ot-Bu
Boc-
L
-Ala–
L-Ala–L-Ala–L-Ala-Ot-Bu
78% yield (13l)c,d
95% yield (13j)
70% yield (13k)
Boc-
L
-Glu(t-Bu)–
L
-Ala–Gly–
L
-Ala-Ot-Bu
Boc-L-Lys(Boc)–L-Ala–L-Leu–Gly-Ot-Bu
87% yield (13m)
81% yield (13n)
aThe drs of 13a–n were determined by H NMR. The reaction was carried out for 72 h. L-Val-Ot-Bu 8e and L-Ala-L-Ala-Ot-Bu 4 (3.0
1
b
c
equiv) were used. dThe reaction was carried out at 80 °C.
Applications to the Substrate-Directed Catalytic Chemi-
cal Ligation Using Amino Acid Terminus without Any Pre-
functionalization.34 The chemical ligation between two differ-
ent peptides is one of the most important and unsolved pro-
cesses for the generation of longer peptides.35–40 NCL was pio-
neered by Kent,14,15 and more recently, Bode invented a novel
α-ketoacid/hydroxylamine ligation system.41 The great contri-
bution of these chemical ligations by Kent and Bode is note-
worthy as the milestone of modern peptide chemistry. Although
these reactions proceed efficiently in diluted aqueous condi-
tions in the presence of many functional groups, the former pro-
cess is still problematic with respect to generality, as thioester
groups and unprotected-Cys residues are required at the C-ter-
minal and N-termini of the corresponding peptides, respectively.
Similarly, in the latter case, the advance preparations of α-ke-
toacid residues at the C-terminal and hydroxylamine residues at
the N-terminal are essential. Clearly, a more general chemical
ligation system for direct peptide bond formation between the
amino group of one peptide and the carboxyl group of another
peptide is much valuable. Herein, we investigated Lewis acid-
catalyzed chemical ligation between peptides to demonstrate
the robustness of our concept in terms of generality, utility, sig-
nificance, and originality (Scheme 5). Our approach eliminates
the inconvenience of laborious prefunctionalization at the mu-
tually ligation junctions of peptides. This success of our simple
approach in catalytic chemical ligation leads to its application
to the “all catalytic convergent peptide synthesis” which solves
the problems of multistep processes of linier SPPS and LPPS.
Additionally, it will be helpful for prospective development of
chemical ligation between polypeptides and/or proteins without
any limitation and the use of prefunctionalized polypeptides as
well as proteins. After a series of optimization studies (Table
T4, see supporting information), we found that Cbz-Gly–Gly–
L-Phe-OH 19a underwent peptide bond formation with 8c and
TMSIM in the presence of a Ti(Oi-Pr)4 catalyst to give 21a in
72% yield and with 94:6 dr. The tetrapeptide 21a is a common
building block for the synthesis of Met- and Leu-enkephalins,42
and the physical and spectroscopic data for 21a are in good
agreement with those for the known data.43 Gratifyingly, we
achieved catalytic coupling reaction with 2–10 mol% Ti(Oi-Pr)4
as the catalyst to give oligopeptides 21a–g bearing a series of
functional groups in yields of up to >99% and with high dia-
stereomeric ratios. We believe that this is an elegant solution for
the long-standing problem of general chemical ligation of pep-
tides.
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Scheme 5. Lewis Acidic Metal-Catalyzed Peptide Coupling Reactiona
1
2
3
4
Ti(Oi-Pr)4 (10 mol%)
TMSIM (2.2 equiv)
peptide-Ot-Bu (4, 8c–e, 8l, 8o, 20)
OTMS
PG-peptide–peptide-Ot-Bu
(21a–g)
PG-peptide-OH
HN
O
40–50 °C, 36–72 h
[Ti]
O
(19a–g, 2.0 equiv)
5
6
7
8
Cbz-Gly–Gly–
L
-Phe–
L
-Leu-Ot-Bu (21a)
Cbz-Gly–Gly–
L
-Leu–
L
-Met-Ot-Bu (21b)
Cbz-L-Pro–L-Leu–Gly–L-Val-Ot-Bu (21c)
72% yield, 94:6 dr
(in DMSO)
99% yield, 97:3 dr
98% yield, >99:1 dr
(2 mol% Ti)
9
Boc-Gly–Gly–Gly–
L
-Ala–
L
-Ala-Ot-Bu (21d)
Cbz-Gly–Gly–Gly–Gly–
L
-Ile-Ot-Bu (21e)
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
>99% yield, >99:1 dr
(2 mol% Ti, in DMSO)
>99% yield, >99:1 dr
(5 mol% Ti, in CHCl3)
Cbz-Gly–
L
-Pro–
L
-Leu–Gly–
L
-Asp(t-Bu)-Ot-Bu (21f)
Boc-L-Ala–L-Met–L-Ala–Gly–L-Ala–L-Ala-Ot-Bu (21g)
>99% yield, >99:1 dr
(2 mol% Ti, in CHCl3)
98% yield, >99:1 dr
(in CHCl3)
aThe drs of 21a–g were determined by 1H NMR.
*E-mail: muramatsu@isc.chubu.ac.jp (W.M.)
*E-mail: hyamamoto@isc.chubu.ac.jp (H.Y.)
CONCLUSION
ORCID
Wataru Muramatsu: 0000-0001-7183-7981
Tomohiro Hattori: 0000-0003-1720-0191
Hisashi Yamamoto: 0000-0001-5384-9698
Over the past century, a number of technological advances in
peptide synthesis have been made. However, despite remarka-
ble progress in catalytic and stoichiometric peptide bond-form-
ing reactions, the amide bond formation for peptides still re-
mains one of the top challenges.44 In this article, we introduced
a substrate-directed strategy that makes a seminal contribution
to the field of peptide synthesis and demonstrated the dramatic
potential of this novel Lewis acid-catalyzed approach. The new
method addresses some of the critical issues associated with
classical SPPS and LPPS routes, including racemization, waste
production, complicated operation, tedious purification
steps,45,46 limited applicability, and generality of peptide chem-
ical ligation. In particular, our tantalum-catalyzed method com-
pletely blocks the generation of an oxazolone intermediate,
which causes numerous purification steps for racemization. In
addition, this approach can be exploited in the triply convergent
synthesis of tri- and tetrapeptides. Thus, since our method does
not cause any racemization, it will be able to significantly sim-
plify the process without some of the tedious purification steps
in classical peptide synthesis. Furthermore, our new chemical
ligation method using non-prefunctionalized peptides opens ef-
ficient and straightforward all catalytic convergent synthetic ap-
proach for oligopeptide synthesis, and it will be able to generate
a variety of (3 x 2n)-residue peptides (n = 1, 2, 3 ･･･) in very
few steps. We therefore expect that the presented Lewis acid
method constitutes an important new protocol for future peptide
synthesis.
Notes
The authors declare no competing interests.
ACKNOWLEDGMENT
We thank the Japan Science and Technology Agency (JST), Adapt-
able and Seamless Technology Transfer Program through Target-
driven R&D (A-STEP).
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website.
Experimental procedures, characterization data, 1H- and 13C NMR
spectra, HPLC data, and GC data (PDF)
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AUTHOR INFORMATION
Corresponding Author
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