Fmoc synthesis of peptide thioesters without post-chain-assembly manipulation

Article abstract of DOI:10.1021/ja204088a

An operationally simple method for the synthesis of peptide thioesters is developed using standard Fmoc solid-phase peptide synthesis procedures. The method relies on the use of a premade enamide-containing amino acid which, in the final TFA cleavage step

Full text of DOI:10.1021/ja204088a

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Fmoc Synthesis of Peptide Thioesters without Post-Chain-Assembly
Manipulation
Ji-Shen Zheng,†,‡ Hao-Nan Chang,† Feng-Liang Wang,‡ and Lei Liu*,†,‡
†Department of Chemistry, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology (Ministry of Education),
Tsinghua University, Beijing 100084, China
‡Department of Chemistry, University of Science and Technology of China, Hefei 230026, China
S Supporting Information
b
yields.11,15 Thus, straightforward synthesis of peptide thioesters
ABSTRACT: An operationally simple method for the
synthesis of peptide thioesters is developed using standard
Fmoc solid-phase peptide synthesis procedures. The meth-
od relies on the use of a premade enamide-containing amino
acid which, in the final TFA cleavage step, renders the
desired thioester functionality through an irreversible in-
tramolecular N-to-S acyl transfer.
by using standard Fmoc-SPPS procedures without post-chain-
assembly manipulation remains an unsolved challenge.
Herein, we describe a practical method for direct synthesis of
peptide thioesters using just standard Fmoc SPPS procedures
(Scheme 1). A special amino acid is needed to achieve the goal,
but it can be readily available once made in large quantities. This
special amino acid carries an enamide moiety that is stable to both
amino acid coupling and Fmoc deprotection steps. The target
thioester is produced through an in situ N-to-S acyl transfer in the
TFA cleavage media, which generates an enamine intermediate
that is irreversibly hydrolyzed by water in the same cleavage cocktail.
The advantage of the new thioester synthesis method is twofold:
(1) the method is operationally simple because it uses standard
Fmoc SPPS protocols; (2) the crude peptide thioester cleaved
from the resin is fairly clean due to the irreversible nature of the
acyl transfer process.
Our study began with examining the enamides of amino acids
carrying mercaptoalkyl side chains. Compound 1 was tested first
(Scheme 2), which is completely converted by the TFA/TIPS/
H2O treatment. However, the desired thioester product is obtained
in only 17% yield, whereas detailed analysis of the reaction mixture
reveals the formation of a thiazole byproduct in 83% yield. We
hypothesized that the thiazole was produced through a favorable
aromatization process of the five-membered ring intermediate.
To prevent the formation of an aromatic ring, we next designed
compounds 2À4 that should undergo the acyl transfer through a
six-membered ring intermediate. Unfortunately, these compounds
fail to produce any thioester after many tests.
We then changed our strategy to put a methyl group on the
amide nitrogen, hoping that this would disfavor the formation of
a thiazole. To our delight, the treatment of compound 5 with
TFA/TIPS/H2O at room temperature for 12 h indeed generates
the desired thioesters in high yields (Scheme 3). For Gly the yield
is as high as 93%, whereas for amino acids with modest steric
hindrance (Phe, Ser, Leu) the yields are 70À86%. Even a sterically
demanding amino acid (i.e., Val) can be used in this method,
which gives the corresponding thioester in 55%yield. These results
verify the principle of the new thioester formation strategy.
Moreover, our test of compound 5 with the conditions for Fmoc
deprotection shows that 5 remains intact after treatment with
20% piperidine in DMF for 48 h.
-terminal peptide thioesters are key intermediates for the
C
chemical synthesis of proteins through native chemical
ligation.1 The Boc solid-phase peptide synthesis (SPPS) on a
thioester-linked resin has served as an effective protocol for the
preparation of peptide thioesters.2 In contrast, synthesis of a
peptide thioester through Fmoc SPPS is problematic because of
the requirement for repeated Fmoc removal under basic condi-
tions. However, many laboratories use Fmoc SPPS exclusively
due to safety and regulation concerns. Besides, Fmoc protocols
are favored for the synthesis of phosphorylated3 and glycosylated
peptides.4 In this context, many methods have been developed to
synthesize peptide thioesters through Fmoc SPPS.5 Some famous
examples include the safety-catch linker system,6 the C-terminal
peptide N-acylurea method,7 and the thiolysis of a backbone
pyroglutamyl imide.8 A common feature of these methods is the
requirement for a post-chain-assembly treatment (e.g., C-terminal
activation and thiolysis) that demands the expertise of a skilled
professional.
We reasoned that a more user-friendly Fmoc SPPS method for
peptides thioester synthesis may only use the standard Fmoc
procedures without any post-chain-assembly manipulation. The
earlier methods of optimizing Fmoc deprotection cocktails for
peptide thioester synthesis partially satisfy this criterion, but they
suffer from having low yields and racemization at the C-terminal
amino acid.9 Another related approach is the in situ peptide
thioester generation via an intramolecular O-to-S or N-to-S
acyl transfer.10,11 However, these acyl transfer methods do not
directly produce an isolable peptide thioester unless an alkylation
activation12 or thiolytic release13 step is added. For instance, the
recently developed N-alkylated Cys method14 needs to use excess
amounts of thiol to promote the thioester formation. Such a thiolytic
release step may induce fragmentation at normal Cys junctions.
On the other hand, direct ligation of the acyl transfer precursors
with an N-Cys peptide through in situ thioester generation often
suffers from significant hydrolysis leading to relatively low ligation
Received: May 3, 2011
Published: June 29, 2011
r
2011 American Chemical Society
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|

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Scheme 1. Peptide Thioester Synthesis through Standard
Fmoc SPPS Proceduresa
Scheme 4. Synthesis of Enamide-Containing Amino Acids
a Aa = amino acid.
Scheme 5. Preparation of Peptide Thioester through a
Standard Fmoc Procedure without Any Post-Chain-Assembly
Manipulation
Scheme 2. Thiazole Formation versus Acyl Transfer
Scheme 3. Thioester Formation from Compound 5a
group. In the final step, Alloc is converted to Fmoc to satisfy
the need of Fmoc SPPS, generating the desired special amino
acid 10.
The resulting enamide-containing amino acid 10 can be
coupled onto a standard resin (e.g., Rink amide AM resin) using
customary peptide coupling conditions (Scheme 5). Through
routine Fmoc SPPS protocols, the remaining amino acids are
assembled onto the resin in a stepwise manner according to the
predesigned sequence. At the end of peptide chain assembly,
standard TFA cleavage (12h, rt) is conducted to release the peptide
and remove the side-chain protection groups. During this step,
the in situ N-to-S acyl transfer takes place, which is followed by an
enamine hydrolysis to generate the desired peptide thioester.
Note that several previously established TFA cleavage condi-
tions have been examined and they do not show much difference
in the thioester conversion. As to the model 7-mer peptide
thioester 11, the isolated yields under the four cleavage condi-
tions are 81%, 76%, 79%, and 84%, respectively (Supporting
Information).
a Note that for Ser the starting material has a t-Bu protection group at
Ser’s OH, which is removed during the transformation.
To apply the above thioester formation method to SPPS, we
need to incorporate the enamide moiety into the C-terminal
amino acid. As shown in Scheme 4, this task can be accomplished
through several synthetic steps from a known compound 6.16
Hydroiodination of 6 by using iBu2AlH/I2 affords a vinyl iodide
7. The enamide 8 with Alloc protection is then obtained through
a key step of Cu-catalyzed CÀN cross-coupling.17 Next, trityl-
mercapto and N-methyl groups are installed to afford 9, which
upon reaction with succinic anhydride generates an acid linker
To examine the racemization at the C-terminal amino acid during
the thioester formation, we made the enantiomer of compound
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Table 1. Fmoc SPPS Synthesis of Peptide Thioesters
Scheme 7. Chemical Synthesis of Human Cox17 and the
Corresponding HPLC and MALDI-TOF Mass Dataa
Entry
target peptides
purity %a
yield %b
1
2
3
4
5
6
7
8
Fmoc-AKEAEKITTG-SRc
Fmoc-AKLGFSA-SR
91
88
85
82
91
75
85
56
70
81
64
68
78
60
70
32
Fmoc-IKEYFYTSGK-SR
H-AVRTTGIF-SR
Fmoc-GGAGSAQAMPL-SR
Fmoc-MFVFAVRTTGIF-SR
Fmoc-GDSKDVRKFI-SRd
Fmoc-AELVDALQFV-SRd
a Purity of crude peptides based on HPLC detection trace at 214 nm.
b Isolated yield based on the loading of the enamide-containing amino
acid. Please see the Supporting Information for more details. c R =
CH2COC3H6COC2H4ÀCONH2. d Cleavage for 24 h at 30 °C.
Scheme 6. HPLC Chromatogram of Crude Peptide Thioe-
sters with Different C-Terminal Amino Acid Residues after
the TFA Cleavage (12 h, rt)a
a MPAA = 4-mercaptophenylacetic acid, TCEP = tris(carboxyethyl)phos-
phine, Gn = guanidine.
in good yields and purities (Table 1 and Scheme 6). Note that the
thioester formation step requires more time (24 h, 30 °C) for Ile
and Val.
The method was further applied to the synthesis of a 67-mer
protein, namely, human cytochrome oxidase 17 (human Cox17),18
as shown in Scheme 7. This protein recruits copper ions to mito-
chondria for incorporation into the Cox apoenzyme. The 29-mer
peptide thioester fragment 15 was prepared using the Fmoc
method described in Scheme 7, which was successfully isolated in
42% yield. The other fragment 16 which is a 38-mer peptide was
also synthesized using the standard Fmoc SPPS method. The liga-
tion was performed under the standard conditions. Within 6 h at
room temperature, the desired full-length peptide was obtained
in about 95% ligation yield according to the HPLC and MALDI-
TOF analysis.
In summary, we have introduced a new method for the synthe-
sis of peptide thioesters using just routine Fmoc SPPS procedures.
The method relies on the use of a premade enamide-containing
amino acid which, in the final TFA cleavage step, renders the
desired thioester functionality through an irreversible intramo-
lecular N-to-S acyl transfer. An important feature of the new
method is its operational simplicity and potential to be auto-
matized with conventional procedures. Straightforward access to
peptide thioesters may widen the application of peptide ligation
methods for the synthesis of complex post-translationally mod-
ified proteins. The current limitation of the method is that the
synthesis of enamide-containing amino acids remains laborious.
We expect that more efficient amino acid loading may ultimately
be achievable using analogous linker principles.
a Detailed yields can be found in Table 1. S* denotes Ser[β-D-Glc-
(OAc)4]. R = CH2COC3H6COC2H4ÀCONH2.
10 corresponding to a D-alanine. With this compound, we then
used the above Fmoc SPPS method to prepare another 7-mer
peptide thioester 12 (i.e., Fmoc-Ala-Lys-Leu-Gly-Phe-Ser-[D]Ala-
COSR, where R = CH2COC3H6COC2H4ÀCONH2). HPLC
analysis of peptides 11 and 12 (Supporting Information) reveals
that the epimerization at the C-terminal chiral center is less than
1%. To test the utility of the new method for the synthesis of peptide
thioesters with post-translational modifications, we applied it to
the preparation of two glycopeptide thioesters, i.e. Fmoc-Gly-
Phe-Ser[β-D-Glc(OAc)4]-Ala-Lys-Leu-Gly-Phe-Ser-Ala-COSR
(13, a 10-mer) and Fmoc-Glu-Arg-Met-Lys-Tyr-Val-Cys-Gly-
Phe-Ser[β-D-Glc(OAc)4]-Ala-Lys-Leu-Gly-Phe-Ser-Ala-COSR
(14, a 17-mer). The two model glycopeptides were obtained in 56%
and 49% isolated yields. Moreover, we have made the enamide-
containing amino acids for Gly, Lys, Phe, Leu, Val, and Ile
(Supporting Information). With these special amino acids in
hand we can readily prepare the corresponding peptide thioesters
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’ AUTHOR INFORMATION
Corresponding Author
lliu@mail.tsinghua.edu.cn
’ ACKNOWLEDGMENT
This study was supported by NSFC (Nos. 20932006, 91013007)
and the national “973” grants from the Ministry of Science and
Technology (No. 2011CB965300).
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