Efficient microwave-assisted tandem N- to S-acyl transfer and thioester exchange for the preparation of a glycosylated peptide thioester

Article abstract of DOI:10.1021/ol0616034

A peptide carrying a mercaptomethylated proline derivative at the C-terminus was prepared by solid-phase peptide synthesis (SPPS) and converted to the thioester of 3-mercaptopropionic acid (MPA) by aqueous MPA under microwave irradiation conditions. This

Full text of DOI:10.1021/ol0616034

ORGANIC
LETTERS
2006
Vol. 8, No. 20
4465-4468
Efficient Microwave-Assisted Tandem
N- to S-Acyl Transfer and Thioester
Exchange for the Preparation of a
Glycosylated Peptide Thioester
Fumihiro Nagaike, Yuko Onuma, Chie Kanazawa, Hironobu Hojo,* Akiharu Ueki,
Yuko Nakahara, and Yoshiaki Nakahara*
Department of Applied Biochemistry, Institute of Glycotechnology, Tokai UniVersity,
Hiratsuka, Kanagawa, 259-1292, Japan
hojo@keyaki.cc.u-tokai.ac.jp
Received June 29, 2006
ABSTRACT
A peptide carrying a mercaptomethylated proline derivative at the C-terminus was prepared by solid-phase peptide synthesis (SPPS) and
converted to the thioester of 3-mercaptopropionic acid (MPA) by aqueous MPA under microwave irradiation conditions. This post-SPPS
thioesterification reaction was successfully applied to the synthesis of a glycopeptide thioester composed of 25 amino acid (AA) residues,
which was then used for the preparation of a 61-AA glycopeptide by the thioester condensation method.
Segment condensation methods, such as the thioester method,1,2
and native chemical ligation3,4 have been highly optimized
for the chemical synthesis of proteins. The key intermediates
for these strategies are the peptide thioesters, which have
been mainly prepared by the tert-butoxycarbonyl (Boc) mode
solid-phase method. Because of the increasing interest in the
posttranslational modifications of protein, such as glycosy-
lation, the preparation of a peptide thioester by the 9-fluo-
renylmethoxycarbonyl (Fmoc) strategy, which does not use
harsh acidic conditions, has been reported.5-20 These methods
include the use of thioester-compatible Fmoc cleavage
cocktails with a preassembled thioester linkage on resin,6,10
post solid-phase peptide synthesis (post-SPPS) thioesterifi-
cation using sulfonamide linkers,7,9 aryl hydrazine support,15
protected peptide segments,5,8,11-13 and post-SPPS thioes-
terification by an O- to S- or N- to S-acyl transfer reaction.16-20
Some of these methods were actually applied to the prepara-
tion of glycosylated peptide thioesters, which led to the
successful synthesis of glycoproteins.7,16,21-27 However, the
(10) Clippingdale, A. B.; Barrow, C. J.; Wade, J. D. J. Pept. Sci. 2000,
6, 225-234.
(11) Swinnen, D.; Hilvert, D. Org. Lett. 2000, 2, 2439-2442.
(12) Mezo, A. R.; Cheng, R. P.; Imperiali, B. J. Am. Chem. Soc. 2001,
123, 3885-3891.
(1) Hojo, H.; Aimoto, S. Bull. Chem. Soc. Jpn. 1991, 64, 111-117.
(2) Aimoto, S. Biopolymer (Pept. Sci.) 1999, 51, 247-265.
(3) Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. H. Science
1994, 266, 776-779.
(4) Dawson, P. E.; Kent, S. B. H. Annu. ReV. Biochem. 2000, 69, 923-
960.
(13) Flavell, R. R.; Huse, M.; Goger, M.; Trester-Zedlitz, M.; Kuriyan,
J.; Muir, T. W. Org. Lett. 2002, 4, 165-168.
(14) Brask, J.; Albericio, F.; Jensen, K. Org. Lett. 2003, 5, 2951-2953.
(15) Camarero, J. A.; Hackel, B. J.; de Yoreo, J. J.; Mitchell, A. R. J.
Org. Chem. 2004, 69, 4145-4151.
(5) Futaki, S.; Sogawa, K.; Maruyama, J.; Asahara, T.; Niwa, M.; Hojo,
H. Tetrahedron Lett. 1997, 38, 6237-6240.
(16) Warren, J. D.; Miller, J. S.; Keding, S. J.; Danishefsky, S. J. J. Am.
Chem. Soc. 2004, 126, 6576-6578.
(6) Li, X.; Kawakami, T.; Aimoto, S. Tetrahedron Lett. 1998, 39, 8669-
(17) Botti, P.; Villain, M.; Manganiello, S.; Gaertner, H. Org. Lett. 2004,
6, 4861-4864.
8672.
(7) Shin, Y.; Winans, K. A.; Backes, B. J.; Kent, S. B. H.; Ellman, J.
A.; Bertozzi, C. R. J. Am. Chem. Soc. 1999, 121, 11684-11689.
(8) Alsina, J.; Yokumu, T. S.; Albericio, F.; Barany, G. J. Org. Chem.
1999, 64, 8761-8769.
(9) Ingenito, R.; Bianchi, E.; Fattori, D.; Pessi, A. J. Am. Chem. Soc.
1999, 121, 11369-11374.
(18) Kawakami, T.; Sumida, M.; Nakamura, K.; Vorherr, T.; Aimoto,
S. Tetrahedron Lett. 2005, 46, 8805-8807.
(19) Ollivier, N.; Behr, J.-B.; El-Mahdi, O.; Blanpain, A.; Melnyk, O.
Org. Lett. 2005, 7, 2647-2650.
(20) Ohta, Y.; Itoh, S.; Shigenaga, A.; Shintaku, S.; Fujii, N.; Otaka, A.
Org. Lett. 2006, 8, 467-470.
10.1021/ol0616034 CCC: $33.50
© 2006 American Chemical Society
Published on Web 09/07/2006

preparation of glycosylated peptide thioesters is still a major
problem in glycoprotein synthesis. The use of the sulfona-
mide linker requires alkylation at the final activation step,
but the reagent also alkylates the free hydroxyl groups at
the carbohydrate portion, unless they are protected.28 We
previously prepared a peptide thioester representing the
sequence of an extracellular matrix metalloproteinase inducer,
emmprin (34-58), carrying N-linked pentasaccharide by the
thioester-compatible Fmoc deblocking reagent, but the
overall yield was 1.8%. The low yield might be attributable
to the partial cleavage of the preformed thioester linkage
during Fmoc removal.22
To examine the reaction in Scheme 1, a proline derivative
carrying protected thiol group 1 was prepared according to
Scheme 2. Starting from the known compound 2,29 a vinyl
Scheme 2
Diketopiperazine formation is a well-known side reaction
during SPPS, especially when the C-terminal’s two residues
contain Pro residue(s). This reaction usually occurs during
the removal of the N-protecting group from the dipeptide.
We speculated that this reaction might occur on resin even
after the chain assembly has completed, if the peptide has
Pro-Pro at its C-terminus and if anchimeric assistance by a
thiol group promotes the cleavage of the dipeptide (Scheme
1, route A). Our initial attempt was to develop a post-SPPS
Scheme 1
group was introduced to the C5 position using vinylmagne-
sium bromide. The obtained compound 3 was converted to
glycol by osmium oxidation. The glycol moiety was oxida-
tively cleaved by KIO4 and then reduced by NaBH4 to obtain
alcohol 4. The hydroxyl group of compound 4 was tosylated
and converted to an STrt group to obtain compound 6. Then,
t
the Boc and Bu ester were removed by TFA. The amino
group of the obtained compound 7 was protected by the
Fmoc group to obtain key intermediate 1. The overall yield
of compound 1 was 19%. This unit was then used for the
synthesis of protected peptide resin, Fmoc-Leu-Lys(Boc)-
Gly-Pro(CH2STrt)-Pro-OCH2-Merrifield resin 9. However,
at the dipeptide stage, diketopiperazine derived from Pro-
(CH2STrt)-Pro was formed. Thus, the second and third amino
acids were introduced using Fmoc-Gly-Pro(CH2STrt) 8,
which was prepared by the coupling of compound 7 with
Fmoc-Gly-OSu. The obtained resin 9 was treated with
Reagent K30 for 1 h to generate free thiol groups. Thiol-
induced diketopiperazine formation (route A) was then
attempted using this resin under acidic to basic conditions.
However, we could not observe the progress of the reaction
by HPLC analysis.
thioesterification reaction based on route A. Unfortunately,
the reaction did not proceed as described below. However,
by the careful examination of the reaction, we found a novel
thioesterification reaction using 3-mercaptopropionic acid
(MPA) (route B). This procedure was further modified to
develop an efficient microwave-assisted post-SPPS thioes-
terification reaction, which was applied to the synthesis of a
glycosylated peptide thioester.
(21) Macmillan, D.; Bertozzi, C. R. Angew. Chem., Int. Ed. 2004, 43,
1355-1359.
(22) Hojo, H.; Haginoya, E.; Matsumoto, Y.; Nakahara, Y.; Nabeshima,
K.; Toole, B. P.; Watanabe, Y. Tetrahedron Lett. 2003, 44, 2961-2964.
(23) Hojo, H.; Matsumoto, Y.; Nakahara, Y.; Ito, E.; Suzuki, Y.; Suzuki,
M.; Suzuki, A.; Nakahara, Y. J. Am. Chem. Soc. 2005, 127, 13720-13725.
(24) Mezzato, S.; Schaffrath, M.; Unverzagt, C. Angew. Chem., Int. Ed.
2005, 44, 1650-1654.
To analyze the factors responsible for preventing the
progress of the reaction shown in route A, the peptide cleaved
under the forced conditions (Reagent K treatment at 60 °C
for 4 h) was examined by HPLC. The obtained peptide
showed two peaks on HPLC, as shown in Figure 1a (0 h).
(25) Wu, B.; Warren, J. D.; Chen, J.; Chen, G.; Hua, Z.; Danishefsky,
S. J. Tetrahedron Lett. 2006, 47, 5219-5223.
(26) Wu, B.; Chen, J.; Warren, J. D.; Chen, G.; Hua, Z.; Danishefsky,
S. J. Angew. Chem., Int. Ed. 2006, 45, 4116-4125.
(27) Yamamoto, N.; Takayanagi, A.; Yoshino, A.; Sakakibara, T.;
Kajihara, Y. Chem.-Eur. J. 2006, in press.
(29) Chiesa, M. V.; Manzoni, L.; Scolastico, C. Synlett 1996, 441-443.
(30) King, D. S.; Fields, C. G.; Fields, G. B. Int. J. Pept. Protein Res.
1990, 36, 255-266.
(28) Kajihara, Y.; Yoshihara, A.; Hirano, K.; Yamamato, N. Carbohydr.
Res. 2006, 341, 1333-1340.
4466
Org. Lett., Vol. 8, No. 20, 2006

thioester of MPA 12 within 12 h without serious side
reactions. By elongation of the reaction time (168 h, 7 days),
the amide 11 was also converted to the desired thioester. It
seems that under these conditions (pH ) 1.3), the amide 11
is gradually converted to the thioester 10, which is then
transthioesterified by MPA. The yield, calculated by the area
ratio of the product to all peptidic components eluted by
HPLC, was 70%. Under these conditions, the hydrolyzed
peptide was less than 15%. To enhance the reaction rate,
the thioesterification reaction was performed under micro-
wave irradiation. This technique is becoming popular even
in peptide synthesis.31,32 In 40% MPA, the reaction was
almost completed within 30 min under microwave irradiation
conditions (150 W, 80 °C) (Figure 1b). The reaction was
slower in 20% and 10% MPA; nevertheless, it took only
about 1 h for completion. Considering the easiness in the
purification step, the reaction in these decreased concentra-
tions of MPA might be favorable. On the basis of the
successful clean and quick conversion to the thioester, we
thought this reaction could become a novel method for
obtaining peptide thioesters despite the failure in diketopip-
erazine formation.
To examine the general applicability of this method for
peptide thioester preparation, an N-acetylglucosaminylated
peptide thioester representing the sequence of emmprin (34-
58) was prepared as shown in Figure 2a. In this synthesis,
we intended to obtain a peptide thioester directly from the
solid support. Thus, amino PEGA resin, which retains high-
swelling ability in aqueous media, was used to achieve an
efficient thioesterification reaction in aq MPA. The C-
terminal proline that was unnecessary in route B was omitted.
Thus, Fmoc-Pro(CH2STrt) 1 was directly introduced to the
amino groups in the resin using the 1,3-dicyclohexylcarbo-
diimide (DCC)-1-hydroxybenzotriazole (HOBt) method.
Then, the sequence of emmprin (34-58) was synthesized
using the ABI433A peptide synthesizer by the 0.1 mmol
scale FastMoc protocol (0.1 Ω MonPrevPk), which uses
O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium hexafluo-
rophosphate (HBTU)-HOBt-N,N-diisopropylethylamine
(DIEA) as a coupling reagent (the details of the protocol
are described in the Supporting Information). Asn44 was
introduced using Fmoc-Asn(GlcNAcBn3) by the DCC-
HOBt method. After the completion of the peptide chain
assembly, the resin was treated by Reagent K, followed by
the “low-acidity TfOH” mixture (TfOH, Me2S, m-cresol, 1,2-
ethanedithiol, TFA)22,23,33,34 at -10 °C for 2 h to remove
the benzyl groups of the carbohydrate moiety. Then, the resin
was treated with aq MPA under microwave irradiation
conditions. In contrast to the model experiment shown in
Figure 1, the reaction in 40% MPA was too drastic to obtain
the desired thioester in high yield. In MPA concentrations
Figure 1. (a) HPLC profile of the thioesterification of a peptide
mixture of 10 and 11. HPLC elution conditions: eluent, aqueous
acetonitrile containing 0.1% TFA; column, Mightysil RP-18 GP
(4.6 × 150 mm) at a flow rate of 1 mL/min. * denotes the
hydrolyzed product of 12. (b) Time course of the yield of the peptide
thioester 12 under microwave irradiation conditions in aq MPA.
Both peaks had the same mass number. When peak A
collected by HPLC was redissolved in aq NaHCO3 (pH 8)
and immediately analyzed by HPLC, it was completely
converted to peak B. In contrast, the conversion of peak B
to peak A occurred slowly under acidic conditions (3% aq
TFA, 2 days). Peak A was negative to Ellman’s reagent (5,5′-
dithiobis-(2-nitrobenzoic acid)), whereas peak B was positive.
These data supported the possible structures of 10 and 11
shown in Figure 1a, which means that the thioesterification
occurs under acidic conditions. However, the formation of
diketopiperazine on resin might be slower than the reverse
S- to N-acyl migration in a weakly acidic or basic condition,
which prevents the reaction shown in route A.
On the basis of these results, we attempted the intermo-
lecular thioester exchange reaction in the presence of an
excess amount of MPA (Scheme 1, route B). The peptide
mixture of 10 and 11 was dissolved in 40% aq MPA at room
temperature, and the formation of Fmoc-Leu-Lys-Gly-
SCH2CH2COOH 12 was monitored by HPLC. As shown in
Figure 1a, the thioester 10 was converted to the tripeptide
(31) Matsushita, T.; Hinou, H.; Kurogochi, M.; Shimizu, H.; Nishimura,
S.-I. Org. Lett. 2005, 7, 877-880.
(32) Murray, J. K.; Farooqi, B.; Sadowsky, J. D.; Scalf, M.; Freund, W.
A.; Smith, L. M.; Chen, J.; Gellman, S. H. J. Am. Chem. Soc. 2005, 127,
13271-13280.
(33) Tam, J. P.; Heath, W. F.; Merrifield, B. R. J. Am. Chem. Soc. 1986,
108, 5242-5251.
(34) Takano, Y.; Habiro, M.; Someya, M.; Hojo, H.; Nakahara, Y.
Tetrahedron Lett. 2002, 43, 8395-8399.
Org. Lett., Vol. 8, No. 20, 2006
4467

were protected by the Boc groups using Boc-OSu. The
obtained peptide 14 was condensed with C-terminal peptide
emmprin (59-94) 15 by the thioester method following the
previous synthesis (Scheme 3). The segment coupling
Scheme 3
proceeded well as in the previous synthesis.22 The removal
of Boc and Acm groups, followed by air oxidation, gave
the final product, the extracellular first Ig domain of emmprin
(34-94) carrying GlcNAc 16 (25% based on peptide 15).
In conclusion, we have developed a novel post-SPPS
thioesterification reaction under microwave irradiation condi-
tions. This method is fully compatible with the conventional
Fmoc solid-phase method and gives a peptide thioester in
good yield. The obtained peptide thioester was successfully
applied to the synthesis of the N-acetylglucosaminylated
peptide composed of 61 amino acid residues by the thioester
method, demonstrating the usefulness of the thioester ob-
tained by this novel method. A series of potent N to S
migratory devices for thioesterification are currently being
investigated.
Figure 2. Synthesis of peptide thioester 13: (a) synthetic route,
(b) RPHPLC profile. Elution conditions: same as those in Figure
1a. Peaks A (eluted as a right-side shoulder of a large peak derived
from MPA) and B: see text. C: Defective peptide thioester, which
lacks C-terminal HRWLKG. D: Hydrolyzed product of 13. E:
Peptide 13.
of 10% and 20%, the desired thioester 13 was obtained in
good purity within 1 h as shown in Figure 2b. The isolated
yield of the product was 4.2% and 3.7% in the case of 10%
and 20% MPA, respectively, based on the amino group in
the initial resin. These yields are over 2 times higher than
that of the previous synthesis,22 demonstrating the efficiency
of the new method. The amino acid analysis of the residual
resin showed that the peptide that remained on the resin was
as low as 5%. However, it has to be noted that the peptide
bond between Asp45 and Ser46 showed marked lability to
10-20% MPA treatment. The peptide bond was cleaved in
about a 25% ratio to the desired product (peak A in Figure
2b, emmprin (48-58)-SCH2CH2COOH; peak B, [Asn-
(GlcNac),44 Cys(Acm)41]-emmprin (34-45)). At present, we
have not searched for conditions to avoid this issue, but a
further increase in the yield might be expected by solving
this problem.
Acknowledgment. This work was partly supported by a
Grant-in-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).
Supporting Information Available: Experimental pro-
cedures for 1 and 3-16. This material is available free of
charge via the Internet at http://pubs.acs.org.
Then, the use of this thioester for segment coupling was
demonstrated. The amino groups of the peptide thioester 13
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