High-pressure-promoted Fmoc-aminoacylation of N-ethylcysteine: preparation of key devices for the solid-phase synthesis of peptide thioesters

Article abstract of DOI:10.1016/j.tetlet.2009.11.034

In this study synthesis of Fmoc-aminoacyl-N-ethyl-S-triphenylmethylcysteine, a new N→S acyl migratory device for the preparation of peptide thioesters by Fmoc solid-phase peptide synthesis (SPPS) is described. Condensation of Fmoc-aminoacyl fluoride and N-ethyl-S-triphenylmethylcysteine allyl ester, readily prepared from known S-triphenylmethylcysteine allyl ester, was efficiently promoted in CH₂Cl₂ under high-pressure (800 MPa). When the reaction was performed with the additive DIEA, considerable epimerization at the chiral centers occurred, affording a mixture of diastereomers. When the preparation procedure for N-ethyl-S-triphenylmethylcysteine allyl ester was changed and the additive DIEA in the high-pressure reaction was excluded, Fmoc-aminoacyl-N-ethyl-S-triphenylmethylcysteines was obtained as a single stereoisomer without epimerization. The Fmoc-l-leucine adduct thus prepared was deallylated and used for the SPPS of a known decapeptide. A remarkable increase (44%) in the overall yield of the decapeptidethioester was achieved, compared to the 7% obtained by the stepwise on-resin Leu-Cys condensation method.

Full text of DOI:10.1016/j.tetlet.2009.11.034

Tetrahedron Letters 51 (2010) 407–410
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
High-pressure-promoted Fmoc-aminoacylation of N-ethylcysteine: preparation
of key devices for the solid-phase synthesis of peptide thioesters
a,
Yuko Nakahara a,b, Ichiro Matsuo b,c, Yukishige Ito b, Risa Ubagai a, Hironobu Hojo a, , Yoshiaki Nakahara
*
*
a Department of Applied Biochemistry, Institute of Glycoscience, Tokai University, Kitakaname 1117, Hiratsuka, Kanagawa 259-1292, Japan
b RIKEN, Institute of Physical and Chemical Research, Wako, Saitama, Japan
c Department of Chemistry and Chemical Biology, Gunma University, Tenjin-cho 1-5-1, Kiryu, Gunma 376-8515, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study synthesis of Fmoc-aminoacyl-N-ethyl-S-triphenylmethylcysteine, a new N?S acyl migratory
device for the preparation of peptide thioesters by Fmoc solid-phase peptide synthesis (SPPS) is
described. Condensation of Fmoc-aminoacyl fluoride and N-ethyl-S-triphenylmethylcysteine allyl ester,
readily prepared from known S-triphenylmethylcysteine allyl ester, was efficiently promoted in CH2Cl2
under high-pressure (800 MPa). When the reaction was performed with the additive DIEA, considerable
epimerization at the chiral centers occurred, affording a mixture of diastereomers. When the preparation
procedure for N-ethyl-S-triphenylmethylcysteine allyl ester was changed and the additive DIEA in the
high-pressure reaction was excluded, Fmoc-aminoacyl-N-ethyl-S-triphenylmethylcysteines was
Received 6 October 2009
Revised 6 November 2009
Accepted 10 November 2009
Available online 13 November 2009
obtained as a single stereoisomer without epimerization. The Fmoc-L-leucine adduct thus prepared
was deallylated and used for the SPPS of a known decapeptide. A remarkable increase (44%) in the overall
yield of the decapeptidethioester was achieved, compared to the 7% obtained by the stepwise on-resin
Leu-Cys condensation method.
Ó 2009 Elsevier Ltd. All rights reserved.
Peptide thioester is a crucial chemical motif in ligating peptide
segments by the thioester method1 and by native chemical liga-
tion.2 Therefore, concise solid-phase synthesis of peptide thioesters
especially by the Fmoc protocol is currently of interest to reseach-
ers.3 In a previous paper, we disclosed our new strategy for realiz-
ing post-SPPS thioesterification via an efficient N?S acyl migration
at the C-terminal N-alkylcysteine moiety (NAC-assisted thioesteri-
fication).4 Treatment with Reagent K detached the Fmoc peptide
assembled on the N-alkylcysteine-linked amide resin from the re-
sin,5 and the acyl migration followed by thiol exchange with the
additive 3-mercaptopropionic acid (see Scheme 1) exclusively con-
verted the released peptide to a peptide thioester.
tion of Fmoc-aminoacyl-N-ethylcysteines to eliminate this difficult
on-resin aminoacylation step.
The first experiment was designed for direct aminoacylation of
N-ethyl-S-triphenylmethylcysteine74 in solution. When Fmoc-Gly-OH
1 (1.5 equiv) was activated with HATU (1-[bis(dimethylamino)methy-
lene]-1H-1,2,3-triazolo(4,5-b)pyridinium 3-oxide hexafluorophos-
hate) and DIEA (N,N-diisopropylethylamine) in DMF and then
reacted with 7 at room temperature, the coupling reaction took
place within 30 min to give dipeptide 9 in a 75% yield. However,
In this method, the decapeptide thioester carrying a C-terminal
Gly residue (EVTGHRWLKG-SCH2CH2COOH) was synthesized in a
good overall yield (34%). Synthesis of the other decapeptide thioes-
ters with a chiral amino acid at the C-terminus was also performed
with minimum epimerization, but the yields were lower (4.4–
7.0%). This remarkable decrease of the yield can be attributed to
insufficient loading of the C-terminal amino acid on the sterically
hindered N-ethylcysteine site. Even in the case of the least ste-
reo-demanding glycine,
a
large excess of Fmoc-glycine1
(10 equiv ꢀ 2) and a highly reactive agent were necessary to fulfil
the aminoacylation. Therefore, we set out to study the preforma-
* Corresponding authors.
E-mail address: yonak@keyaki.cc.u-tokai.ac.jp (Y. Nakahara).
Scheme 1. Post-SPPS thioesterification using N-ethylcysteine device.
0040-4039/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2009.11.034

408
Y. Nakahara et al. / Tetrahedron Letters 51 (2010) 407–410
Scheme 2. Preparation of Fmoc-aminoacyl N-ethylcysteines.
the same reactions using chiral Fmoc-amino acids, 2 and 3, pro-
duced a complex mixture from which the desired pure Fmoc-
aminoacylated N-ethyl-S-triphenylmethyl-cysteine was isolated
only in a low yield after cumbersome chromatographic purification
(see Scheme 2).
Definitive evidence on the epimerization was gained by a pep-
tide thioester synthesis with 12a and 12b.The known decapeptid-
ethioester (ATEVTGHRWL-SCH2CH2CO2H) 15 was synthesized as a
synthetic model since both the peptide and its C-terminally epi-
merized peptide had been synthesized and readily distinguished
from each other by HPLC.4 Dipeptide 12a (2 equiv) was used to at-
tach to the CLEAR-amide resin by activation with DCC–HOBt.10
Subsequently, the resin was subjected to chain assembly in an
automated peptide synthesizer under the FASTMOC program
(HBTU–HOBt, NMP).
The obtained decapeptide-bound resin had a reasonable in-
crease in weight. Then treatment with Reagent K released the pep-
tide, and ether precipitated it. The crude peptide was converted
into the thioester through N?S acyl migration and thiol exchange
with 3-mercaptopropionic acid (MPA) according to the reported
procedure. We next synthesized the decapeptide using 12b in
place of 12a. Figure 1a and b compares the HPLC data of peptide
thioesters produced from 12a and 12b. Peak 1 (M+H+: m/z
1257.92) and peak 2 (M+H+: m/z 1257.93) represent the decapep-
L
-
The second experiment was carried out with N-ethyl-S-triphen-
ylmethyl-cysteine allyl ester 8 to improve the preparation and iso-
lation of the dipeptidyl product. S-Triphenylmethyl-cysteine allyl
ester, prepared by the reported procedure,6 was N-ethylated with
acetaldehyde and NaBH3CN to give allyl ester 8 in a 75% yield. Cou-
pling of Fmoc-glycine 1 and 8 was readily promoted by HATU to
produce 10 in a 90% yield. However, using the same procedure, chi-
ral Fmoc-amino acids poorly produced the desired coupling prod-
ucts. Enhanced conditions are required to facilitate amide
formation with such poor nucleophiles as N-alkyl amino acid.7
After several experiments to screen conditions, we found that a
high-pressure reaction8 (800 MPa, 3 h) using Fmoc-aminoacyl flu-
orides (1.5 equiv) and 8 in CH2Cl2 in the presence of DIEA
(1.0 equiv) produced high yields of the coupling products. We
tested Fmoc-Leu-F 4, Fmoc-Asp(OBut)-F 5 and Fmoc-Gln(Trt)-F 6
in this reaction. Among the coupling products, 11 and 14 were ob-
tained as a separable mixture of major and minor components.
NMR and mass spectral data indicated that the major and minor
products were diastereomers of each other. Table 1 (entries 1–3)
shows the yield and the specific rotation values of each coupling
product.
tides carrying C-terminal L-Leu (15) and D-Leu, respectively. Both
isomers were separated to determine their respective yields by
amino acid analysis. A distinct improvement in the yield of 15
(43%) was established over the previous one (7%).4 The C-terminal
isomer corresponding to peak 2 was isolated in a 2% derived from
the 12a experiment. In contrast, the product derived from 12b
demonstrated peaks 1 and 2 with an approximately equal inten-
sity. The ratio of the stereoisomers in the products appeared to re-
flect the enantiomeric purity of 12a and 12b, given that no
additional epimerization occurred in the acyl migration and thiol
exchange stages.
To determine the stereostructure by transformation into a
known oligopeptide, the Leu-derived major and minor products
11a and 11b were individually deallylated with Pd(Ph3P)4 and
dimedone in THF to form the carboxylic acids 12a and 12b. The
compound 12b was obtained as crystals. Recrystallization of 12b
from CH3CN made its specific rotation value almost null, and affor-
ded a sample suitable for X-ray analysis. The crystallographic data
Therefore, the high-pressure reaction of 4 and 8 in the absence
of the additive base was reinvestigated taking into account the po-
tential participation of DIEA or generated conjugate base Fꢁ in the
above epimerization. The reaction was promoted with no damage
to the acid-labile S-Trt group to again produce a mixture of two
stereoisomers in a high yield. However, the proportion shifted
more to the major isomer than the previous experiment. In addi-
tion, the separated minor isomer (11b0, see Table 1, entry 4) dis-
played a marked increase in its value of specific rotation (+36.8),
suggesting its high enantiomeric purity.11 Therefore, it is specu-
lated that excluding DIEA minimized the epimerization and that
showed that the sample was a racemate of Fmoc-
L-Leu-D-(Et)-
Cys(Trt)-OH and Fmoc- -Leu-
D
L
-(Et)Cys(Trt)-OH.9 Therefore, it be-
came clear that epimerization occurred in part at both the chiral
carbons of the Leu and Cys residues during this synthetic process,
and accordingly the major diastereomer 12a (11a) might have
been contaminated with its enantiomer to some extent.
11b0 arose from the
D-isomer contaminated in reactant 8. However,
Table 1
we were unable to determine whether or not the epimerization at
Cys had occurred before exposure to the high-pressure conditions.
An attempt to determine the enantiomeric purity of 8 by chro-
matographic analysis with chiral columns was unsuccessful.
Then S-triphenylmethyl-cysteine allyl ester was alternatively
synthesized by allylation of a cesium salt12 of Fmoc-S-triphenyl-
methyl-cysteine with allyl bromide and then by N-deprotection.
Though difficult to discuss its precise enantiomeric purity with
such a small value range, a higher enantiomeric purity of the newly
synthesized sample (80) was expected, owing to the slight increase
in its specific rotation value (+7.0, cf. 8: +5.5). Thus, high-pressure
Dipeptides prepared by high-pressure reaction
a
a
Entry Fluoride Cys DIEA Major
product (%)
[
a
]
D
Minor
product (%)
[a]
D
1
2
3
4
5
6
7
4
5
6
4
4
5
6
8
8
8
8
80
80
80
+
+
+
ꢁ
ꢁ
ꢁ
ꢁ
11a (70)
13ab (74)
14a (67)
11a0 (68)
11c (94)
13c (77)
14c (76)
ꢁ28.7 11b (21)
ꢁ33.9
+10.1
ꢁ17.7 14b (25)
ꢁ32.2 11b0 (14)
ꢁ33.5
+8.9
+36.8
ꢁ56.1
ꢁ20.2
a
Specific rotation values were measured at 20 2 °C for solutions in CHCI3.

Y. Nakahara et al. / Tetrahedron Letters 51 (2010) 407–410
409
Figure 1. HPLC profiles of the synthetic peptide thioesters synthesized using Fmoc-Leu-(Et)Cys(Trt)-OH, (a) 12a and (b) 12b: Peaks 1 and 2 correspond to the peptide
thioesters carrying C-terminal
B, acetonitrile containing 0.1% TFA, flow rate, 1 ml/min. Non-peptidyl contaminants.
L
-Leu and
D
-Leu, respectively. Conditions: column, Mightysil RP-18, 4.6 ꢀ 150 mm (5
lm); eluent A, distilled water containing 0.1% TFA, eluent
*
reactions with 80 were performed using the fluorides 4, 5 and 6 by
excluding DIEA. Every reaction produced a single coupling product
as shown in Table 1 (entries 5–7: 11c, 13c, and 14c). These results
supported the high enantiomeric purity of 80 and the lack of epi-
merization in the high-pressure reactions.
Finally, peptide thioester 15 using 12c, derived by deallylation
of 11c, was synthesized. The result was in accord with our expec-
tations as shown in Figure 2, in which 15 appeared as a single ste-
reoisomer. The production of the C-terminal isomer was not
observed. Therefore, it was concluded that no epimerization oc-
curred during the post-SPPS thioester synthesis. The yield of iso-
lated 15 was 44%.
derivative was attached to resin with DCC–HOBt, and then nine
amino acids were condensed on the resin by the Fmoc protocol
with an automated peptide synthesizer. Cleavage of the peptide
from the resin with Reagent K followed by thioester exchange with
the additive 3-mercaptopropionic acid produced a stereochemi-
cally pure decapeptide thioester in 44% yield. Thus, the thioester
synthesis including N?S acyl migration and thiol exchange steps
exclusively proceeds with configurational retention of the C-termi-
nal amino acid. As a consequence, the method demonstrated here
extends the scope of NAC-assisted synthesis of peptide thioesters,
which should allow us to design the peptide segments usable in
chemical ligation more freely.
In summary, solid-phase synthesis of peptide thioester was
remarkably improved by the use of Fmoc-
triphenylmethyl- -cysteine, which we efficiently prepared through
a high-pressure reaction. The enantiomerically pure -leucine
L-aminoacyl-N-ethyl-S-
Acknowledgments
L
L
This work was supported by a grant-in-aid for creative scientific
research (17GS0420) from the Japan Society for the Promotion of
Science, and in part by a grant-in-aid for scientific research from
the Ministry of Education, Sports, Science and Technology of Japan
(20380069).We thank Dr. T. Chihara for operating the high-pres-
sure equipment and Dr. D. Hashizume for X-ray crystallography
at Riken. The authors are indebted to Daicel Chemical Industries,
LTD for the analysis of compound 9 with a variety of chiral col-
umns. We also thank Tokai University for their support with a
grant-in-aid for high technology research.
Supplementary data
Supplementary data (experimental procedures, NMR and MS of
8, 80, 9, 10, 11a, 11b, 11a0, 11b0, 11c, 12a, 12b, 12c, 13ab, 13c, 14a,
14b, 14c and 15) associated with this article can be found, in the
online version, at doi:10.1016/j.tetlet.2009.11.034.
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a
11. The [
a] value was in fair agreement with that (+35.2) of the major dipeptide
D
separately synthesized by high-pressure coupling of 4 and
D
-(Et)Cys(Trt)-OAll
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with HOBt, HBTU, and DIEA (see Ref. 6).
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