N-Alkyl cysteine-assisted thioesterification of peptides

Article abstract of DOI:10.1016/j.tetlet.2006.11.034

A new method for the preparation of peptide thioester by the post-solid phase peptide synthesis (SPPS) approach was developed. A series of N-alkyl cysteine derivatives were prepared and used as the C-terminus residue of the peptides prepared by the Fmoc S

Full text of DOI:10.1016/j.tetlet.2006.11.034

Tetrahedron Letters 48 (2007) 25–28
N-Alkyl cysteine-assisted thioesterification of peptides
Hironobu Hojo,* Yuko Onuma, Yuri Akimoto, Yuko Nakahara and Yoshiaki Nakahara*
Department of Applied Biochemistry, Institute of Glycotechnology, Tokai University, Hiratsuka, Kanagawa, 259-1292, Japan
Received 10 October 2006; revised 3 November 2006; accepted 7 November 2006
Abstract—A new method for the preparation of peptide thioester by the post-solid phase peptide synthesis (SPPS) approach was
developed. A series of N-alkyl cysteine derivatives were prepared and used as the C-terminus residue of the peptides prepared by
the Fmoc SPPS. The synthetic peptides released from resin by TFA were readily converted to the peptide thioester in aqueous
3-mercaptopropionic acid (MPA) without significant side reactions.
ꢀ 2006 Elsevier Ltd. All rights reserved.
A peptide thioester was first introduced in 1991 as a key
intermediate for the thioester method.1 Since then, its
usefulness was demonstrated by the chemical synthesis
of various proteins by the thioester method2 as well as
native chemical ligation.3,4 The preparation of peptide
thioesters has been mainly accomplished by the Boc
method. Recently, the Fmoc method has become more
prevalent than the Boc method, as the Fmoc method
does not use HF, which requires special precaution
and apparatus for its handling. In addition, the Fmoc
method is preferable for the preparation of peptides car-
rying an acid-sensitive moiety, such as carbohydrates.
However, application of the Fmoc method for peptide
thioester synthesis is difficult in that the thioester linkage
is labile to piperidine used for the Fmoc removal.
Recently, various Fmoc methods have been developed
to avoid this issue.5–21 Among them, post-SPPS thioesteri-
fication using a sulfonamide linker is widely used.7,9 In
this method, peptide thioester can be obtained by activa-
tion of the sulfonamide group by alkylation after the
peptide chain assembly, followed by thiolysis. However,
in one case the thioesterification reaction did not pro-
ceed well.22 Unverzagt and co-workers reported that
acetyl capping unexpectedly acetylates the nitrogen
atom in the sulfonamide linker, which results in cleavage
of the peptide chain during the solid-phase synthesis.23
Other Fmoc methods also have potential drawbacks.
Thus, further efforts are required to accomplish the
Fmoc compatibile peptide thioester preparation.
Recently, we reported that 5-mercaptomethyl proline
linked at the C-terminal of a peptide effectively pro-
motes thioesterification by 3-mercaptopropionic acid
(MPA) under microwave irradiation conditions.21 The
reaction seems to proceed via intramolecular N- to S-
acyl migration, followed by the intermolecular thioester
exchange reaction. This post-SPPS thioesterification is
fully compatible with the conventional Fmoc strategy
and gives peptide thioester in good yield. However, the
preparation of 5-mercaptomethyl proline is rather
tedious. In addition, without microwave irradiation,
the thioesterification is a slow reaction, which requires
about a week for completion. It seems that the driving
force of the first intramolecular N- to S-acyl shift reac-
tion is the lability of the amide bond with an imino acid,
proline.
Thus, we speculated that the same migration reaction
might proceed with other imino acids. Here, we exam-
ined a series of N-alkyl cysteine derivatives for use as
an N- to S-migratory device in the synthesis of peptide
thioesters modeled after the partial structure of the
emmprin intermediate24,25 as shown in Scheme 1.
The synthesis of Fmoc–N-alkylated cysteine derivatives
was carried out as shown in Scheme 2. Commercially
available Cys(Trt) was condensed with aldehydes to
form Schiff bases, which was reduced by NaBH4 and
purified by silica gel column chromatography. The puri-
fication of N-methyl–Cys(Trt) was achieved by reversed-
phase HPLC, since the separation from contaminated
Keywords: Fmoc solid-phase method; N–S Acyl transfer; Peptide
thioester.
*
Corresponding authors. Tel.: +81 463 58 1211; fax: +81 463 50 2075
(H.H.); e-mail addresses: hojo@keyaki.cc.u-tokai.ac.jp; yonak@
keyaki.cc.u-tokai.ac.jp
0040-4039/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2006.11.034

26
H. Hojo et al. / Tetrahedron Letters 48 (2007) 25–28
HS
(2 equiv to the amino groups in the resin) was intro-
duced by the DCC–HOBt method. Then, Fmoc–Gly
(10 equiv to the amino group) was reacted using HATU
(9.5 equiv) in the presence of DIEA (20 equiv) at 50 ꢁC
for 1 h. The reaction was repeated with the same
amount of Fmoc–Gly and reagents. Then the resin
was subjected to the automated synthesis using a peptide
synthesizer (ABI 433A) by the Fmoc method. After
completion of the chain assembly, the resin was treated
with Reagent K27 for 2 h at room temperature. Then the
crude peptide was analyzed by HPLC. The results are
shown in Figure 1 (0 h). The major peak (Peak A) had
the desired mass number of peptides 10 and 11.28 In
addition, Peak B also showed the desired mass number.
When Peak A collected by HPLC was redissolved in aq
NaHCO3 (pH 8) and immediately analyzed by HPLC, it
disappeared and was converted to Peak B. In contrast,
Peak B was slowly converted to Peak A under acidic
conditions (3% aq TFA, ꢀ12 h for completion). Peak
A was positive to Ellman’s reagent (5,50-dithiobis-(2-
nitrobenzoic acid)), whereas peak B was negative. These
results correspond well to the previous result that this
peptide is in an equilibrium between the thioester (Peak
B, 11) and the amide form (Peak A, 10).21 Then the
crude peptide was dissolved in 5% aq MPA (pH ꢀ 1)
and was made to stand at room temperature overnight.
As shown in Figure 1 (24 h), the peptide was cleanly
converted to the thioester of MPA 1228 without serious
side reactions. The reaction rate is almost comparable in
all N-alkyl cysteine, but becomes slightly higher accord-
ing to the increase of the alkyl chain from methyl to
isobutyl. These results are in marked contrast to the pre-
Peptide
Peptide
N
R
NH2
TrtS
O
O
deprotection
H
Peptide
N
R
H+
N
O
O
S
HN
R
O
NH2
O
HS
COOH
S
Peptide
COOH
O
Scheme 1. Post-SPPS thioesterification using N-alkyl cysteine as N- to
S-acyl transfer device.
R
O
O
H2N
1) R-CHO / aq KOH
2) NaBH4
HN
OH
STrt
OH
STrt
1
R= H; 2, CH3; 3,
CH(CH3)2; 4, C6H5; 5
R
O
Fmoc-OSu / aq Na2CO3
N
Fmoc
OH
STrt
R= H; 6, CH3; 7,
CH(CH3)2; 8, C6H5; 9
Scheme 2. Synthetic route for N-alkyl cysteine derivatives.
Cys(Trt) was incomplete using silica gel column chroma-
tography. The obtained N-alkyl cysteine derivatives
were protected by the Fmoc group using Fmoc–OSu
to obtain key devices 6–9.26 Then, these units were used
for the synthesis of emmprin (49–58), Glu-Val-Thr-Gly-
His-Arg-Trp-Leu-Lys-Gly by the Fmoc method. The
synthetic route for the N-ethyl derivative is exemplified
in Scheme 3. Fmoc–CLEAR amide resin was treated
with 20% piperidine NMP for 5 and 15 min. After wash-
ing with NMP, Fmoc–N-ethyl cysteine derivative 7
Fmoc CLEAR amide resin (NH2-resin)
O
Et
N
1) 20% piperidine/ NMP, 2)
Fmoc
OH
, DCC, HOBt
7
TrtS
1) 20% piperidine/ NMP, 2) Fmoc-Gly, HATU, DIEA (x2)
ABI 433A peptide synthesizer (FastMoc protocol)
Et
N
O
t
Glu(OBut)-Val-Thr(Bu )-Gly-His(Trt)-Arg(Pbf)-
Trp(Boc)-Leu-Lys(Boc)-Gly
NH-resin
TFA
TrtS
Et
O
H+
Et
N
O
HN
NH2
Glu-Val-Thr-Gly-His-
Glu-Val-Thr-Gly-His-
Arg-Trp-Leu-Lys-Gly
Arg-Trp-Leu-Lys-Gly
NH2
10
S
HS
11
COOH
HS
Figure 1. HPLC profile for thiol exchange reaction to obtain peptide
thioester 12 using MPA. HPLC elution conditions: Eluent; aqueous
acetonitrile containing 0.1% TFA, Column: Mightysil RP-18 GP
COOH
Glu-Val-Thr-Gly-His-Arg-Trp-Leu-Lys-Gly
12
S
Scheme 3. Synthetic route for model peptide thioester 12 using C-
terminal N-ethyl cysteine as N- to S-migratory device.
(4.6 · 150 mm) at a flow rate of 1 ml/min.
compounds derived from MPA.
denote non-peptidic
*

H. Hojo et al. / Tetrahedron Letters 48 (2007) 25–28
27
Table 2. Preparation of peptide thioesters having chiral amino acids at
the C-terminus
vious result using mercaptomethylated Pro, which re-
quired one week for the reaction to complete even in
40% aq MPA.21 The yields of peptide thioesters are sum-
marized in Table 1. The decreased yield in the iBu and
Bn derivatives compared to the Et derivative was de-
rived from the incomplete introduction of the C-termi-
nal Gly residues due to steric hindrance. It is known
that the introduction of amino acid after N-alkylated
amino acid is difficult.29,30 Thus, in point of the ease
of the preparation and the high yield of the peptide
thioester, the N-ethyl derivative seems to be the most
practical device for peptide thioester preparation. By
the semi-preparative scale synthesis using the N-ethyl
derivative, preparation of product 12 in 10 mg scale
was easily accomplished.
Sequence
Yieldb (%)
D/Lc
ATEVTGHRWL-SRa
TEVTGHRWLK-SRa
WLKGGVVLKE-SRa
7.0
4.4
5.3
<0.068
<0.025
N.D.
a SR denotes SCH2CH2COOH.
b Isolated yields based on the amino groups in the initial resin.
c Calculated by the comparison of peak areas of the peptide thioester
composed of all L-amino acids with C-terminally epimerized peptide
thioester.
prepared from a commercially available cysteine deriva-
tive. The thioesterification after peptide chain assembly
proceeds without serious side reactions in good yields.
This method is fully compatible with the conventional
Fmoc strategy. Thus, the novel method eliminates prob-
lems inherent in peptide thioester preparation by the
Fmoc strategy. In addition, due to the mild reaction
conditions for the thioesterification, this method would
be easily extended to the preparation of peptide thio-
esters carrying acid-sensitive moieties, such as carbo-
hydrates. Further optimization of the introduction of
the amino acid next to C-terminal N-alkyl cysteine as
well as suppression of the epimerization during thioesteri-
fication are now under study. The preparation of glyco-
protein using the novel strategy is also in progress.
We next examined the synthesis of peptide thioesters
having chiral amino acids at their C-terminus to exam-
ine the general applicability of this method. Based on
the results in Table 1, the N-ethyl Cys derivative was
used in this synthesis. To the N-ethyl–Cys(Trt)–CLEAR
amide resin, Fmoc–Leu, Lys(Boc), or Glu(OBut) was
introduced by HATU-DIEA. The introduction was
repeated using the same amount of amino acid and
reagents. However, the coupling yield of these chiral
amino acids was less than 50%, which decreased the
total yield of the peptide thioester. Further optimization
of the coupling conditions of chiral amino acids is re-
quired. Chain elongation on this resin was then achieved
using the peptide synthesizer. After completion of the
chain assembly, the resin was treated with Reagent K.
Then the crude peptide was dissolved in 5% MPA and
the thioesterification reaction was carried out. To ana-
lyze the epimerization ratio during thioesterification, a
peptide thioester carrying D-amino acids was also pre-
pared in the same manner. As in the case of Gly series
in Table 1, the conversion proceeded without significant
side reactions, but at slower rates. The complete conver-
sion required 2–3 days. The result of the synthesis is
shown in Table 2. The epimerization ratio of C-terminal
Glu was not determined, since the peptide thioesters
having C-terminal L-Glu and D-Glu were not separated
on HPLC. These data show that the novel method is
applicable to the preparation of peptide thioesters carry-
ing chiral amino acids at their C-termini in acceptable
yields and epimerization ratio.
Acknowledgements
This work was partly supported by a Grant-Aid for
Scientific Research from the Ministry of Education,
Culture, Sport, Sciences and Technology of Japan.
We thank Tokai University for a grant-in-aid for high-
technology research. We also thank the Japan Society
for the promotion of Science for a Grant-in-Aid for
Creative Scientific Research (No. 17GS0420).
References and notes
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Table 1. Result of the N-alkyl cysteine assisted thioesterification
HS
5% aq MPA
EVTGHRWLKG
NH2
EVTGHRWLKG-SCH2CH2COOH
N
R
O
R
Yielda (%)
Methyl
Ethyl
iButyl
Bn
33
34
28
17
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a Isolated yields based on the amino groups in the initial resin.

28
H. Hojo et al. / Tetrahedron Letters 48 (2007) 25–28
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26. Selected data. Compound 7: [a]D ꢁ39.6 (c 1.1 in CHCl3).
Rf 0.31 (2:1 Toluene–ethyl acetate containing 1% AcOH).
1H NMR (CDCl3): d 4.51–4.33 (m, 2H, Fmoc–CH2–), 4.19
(brt, J = 6.6 Hz, 0.6H, Fmoc–CH–), 4.09 (m, 0.4H,
Fmoc–CH–), 3.39 (m, 0.4H, –CH2–CH3), 3.28 (m, 0.6H,
–CH2–CH3), 3.11 (dd, J = 5.8, 8.8 Hz, 0.6H, aH), 2.98–
2.85 (m, 0.4H; aH, 1.2H; bH ·2), 2.74–2.59 (m, 1H; –
CH2CH3, 0.4H; bH), 2.39 (dd, J = 9.3, 13.7 Hz, 0.4H,
bH), 0.91–0.85 (m, 3H, –CH3). HRFABMS: found, m/z
636.21236: Calcd for C39H35NNaO4S, m/z 636.21845.
27. King, D. S.; Fields, C. G.; Fields, G. B. Int. J. Pept.
Protein Res. 1990, 36, 255–266.
28. MALDI-TOF MS of peptide 10: found, m/z 1312.8
(M+H)+, calcd for (M+H)+ 1312.7. Amino acid analysis
of peptide 10: Thr0.95Glu0.78Gly2Val0.97Leu1.00Lys1.06
-
21. Nagaike, F.; Onuma, Y.; Kanazawa, C.; Hojo, H.; Ueki,
A.; Nakahara, Y.; Nakahara, Y. Org. Lett. 2006, 8, 4465–
4468.
His1.03Arg0.93. MALDI-TOF MS of peptide 11: Found,
m/z 1312.7 (M+H)+: Calcd for (M+H)+, 1312.7. Amino
acid analysis of peptide 11: Thr0.90Glu0.90Gly2Val0.95
-
22. Marcaurelle, L. A.; Mizoue, L. S.; Wilken, J.; Oldham, L.;
Kent, S. B. H.; Handel, T. M.; Bertozzi, C. R. Chem. Eur.
J. 2001, 7, 1129–1132.
Leu0.94Lys0.99His0.98Arg0.96. MALDI-TOF MS of peptide
12: Found, m/z 1270.8 (M+H)+: Calcd for (M+H)+,
1270.6. Amino acid analysis of peptide 12:
23. Mezzato, S.; Schaffrath, M.; Unverzagt, C. Angew. Chem.,
Int. Ed. 2005, 44, 1650–1654.
Thr0.95Glu0.87Gly2Val0.94Leu1.03Lys1.02His0.98Arg0.92.
29. Teixido, M.; Albericio, F.; Giralt, E. J. Pept. Res. 2005,
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