Synthesis of methionine containing peptides related to native chemical ligation

Article abstract of DOI:10.1055/s-2003-38361

Methionine chemical ligation is based on a convenient direct thioester formation at the C-terminus of the ligation site, on homocysteine reduction with dithiothreitol in order to liberate the mercapto group of the homocysteine residue at the N-terminus, and on chemoselective S-methylation with methyl iodide.

Full text of DOI:10.1055/s-2003-38361

LETTER
659
Synthesis of Methionine Containing Peptides Related to Native Chemical
Ligation
S
ynthesis of Methi
a
onine Cont
n
aining Peptid
d
e
s
Related to
a
Native
C
h
s
emica
l
L
i
a
gation my Pachamuthu, Richard R. Schmidt*
Fachbereich Chemie, Universität Konstanz, Fach M 725, 78457 Konstanz, Germany
E-mail: Richard.Schmidt@uni-konstanz.de
Received 21 January 2003
Abstract: Methionine chemical ligation is based on a convenient
direct thioester formation at the C-terminus of the ligation site, on
homocysteine reduction with dithiothreitol in order to liberate the
mercapto group of the homocysteine residue at the N-terminus, and
on chemoselective S-methylation with methyl iodide.
Key words: chemical ligation, methionine ligation, peptides, S-me-
thylation
Solid phase peptide synthesis in combination with ‘native
chemical ligation’ of unprotected peptides has gained
enormous interest.1 The development of native chemical
ligation has opened the door to the synthesis of fully un-
protected peptides and even proteins. Although native
chemical ligation was already found by Wieland et al.2 in
1953, its potential in the synthesis of a variety of
proteins1a,3 including cyclic proteins4 was recognized only
in recent times.
The native chemical ligation involves a simple mixing of
two peptide segments under aqueous conditions in which
one peptide contains a thioester unit at the C-terminus and
the other one a cysteine residue at the N-terminus. The
first step involves formation of a thioester-linked interme-
diate and the second step rearrangement through a five-
membered transition state to form a native peptide bond.
Scheme 1 Peptide bond formation via a six-membered transition
state and chemoselective methylation of the homocysteine thiol group
This reaction could be successfully performed with long
peptides, however, in the ligation step three byproducts
were detected and in the methylation step long reaction
times led to overmethylation.9 In this paper, the methion-
ine chemical ligation is based on (i) convenient direct
thioester formation at the C-terminus of the ligation site,
(ii) in situ homocystine reduction with dithiothreitol
(DTT), thus avoiding the observed byproduct formation in
the ligation step, and (iii) chemoselective S-methylation
with methyl iodide which is less reactive than previously
employed methyl p-nitro-benzenesulfonate, thus restrict-
ing overmethylation.
Hence, a common required element is a cysteine residue
at the ligation site. However, this requirement is also a
limitation in terms of general applicability. Therefore, ef-
forts have been made to extend the applicability of native
chemical ligation to non-cysteine based ligations by em-
ploying Nα-oxyethanethiol5 or Nα-thiobenzyl linker6 as an
auxiliary at the ligation site. These auxiliaries have to be
removed after the ligation, in a separate step. Also other
approaches for the synthesis of non-cysteine containing
proteins were reported.7,8
Thioester 1, and for further studies, thioesters 3–7
(Table 1) were readily prepared by coupling of the amino
acids with ethanethiol in the presence of HOBt/DCC in
DMF at room temperature. Under these conditions
thioesters 1 and 3–7 were directly obtained from the cor-
responding amino acids in one step without protecting
other nucleophilic centres such as hydroxy, amide, or het-
erocyclic groups (isolated yields 70–82%). Thioester 2
was prepared by deprotecting N-Boc protected 1 using
20% TFA in dichloromethane.
Chemical ligation of a thioester at a peptide C-terminus
with a homocysteine (Hcy) residue at the N-terminus of a
second peptide is an interesting alternative; thus, after
S,N-acyl migration through a six-membered transition
state, a selective homocysteine S-methylation is required
in order to introduce a methionine residue at the ligation
site (Scheme 1).
Reaction of homocysteine, generated in situ by reduction
of homocystine using DTT as a reducing agent, with N-
Boc protected thioester 1 in tris buffer (pH ca. 8.0) pro-
duced the desired dipeptide bearing a free thiol group in
Synlett 2003, No. 5, Print: 10 04 2003.
Art Id.1437-2096,E;2003,0,05,0659,0662,ftx,en;G01203ST.pdf.
© Georg Thieme Verlag Stuttgart · New York
ISSN 0936-5214

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K. Pachamuthu, R. R. Schmidt
LETTER
quantitative yield (Table 1). The thiol group was then Reaction of unprotected alanine thioester 2 with ho-
methylated using methyl iodide in ammonia in methanol mocysteine yielded quantitatively the Ala-Hcy ligation
affording the expected Boc-Ala-Met dipeptide 8 which product and chemoselective methylation of the thiol inter-
was isolated as free acid in 84% yield.10,11 Byproduct for- mediate produced S-methylated AlaMet dipeptide 9 in
mation was not observed. One-pot reaction of homocys- 76% isolated yield.10,11
tine, DTT, thioester 1 and methyl iodide in buffer solution
at room temperature also produced 8 albeit in lower yield.
Table 1 Reaction of Homocysteine with Aminoacids and Peptide Thioesters Followed by S-Methylation
Thioesters
Ligation reaction
Time (h)
Methylation reaction
Productse
Yield (%)
qub
Yield (%)
84b
5a
.5
6
1
8
9
quc
76c
2
96b
86b
3
10
11
12
24
qub
79b
4
5
88d
77d
5
Synlett 2003, No. 5, 659–662 ISSN 0936-5214 © Thieme Stuttgart · New York

LETTER
Synthesis of Methionine Containing Peptides Related to Native Chemical Ligation
661
Table 1 Reaction of Homocysteine with Aminoacids and Peptide Thioesters Followed by S-Methylation (continued)
Thioesters
Ligation reaction
Time (h)
Methylation reaction
Productse
Yield (%)
89b
Yield (%)
79b
30
6
13
6
91d
79d
7
14
a 1.25 equiv of thioester was employed.
b Obtained by recrystallization in petroleum ether–diethyl ether.
c Obtained by column chromatography (MeOH–CHCl3, 9:1).
d Obtained by recrystallization in ethyl acetate–petroleum ether.
e For physical data and comparison with literature data see ref.11
Also, reaction of thioesters 3–7 with homocysteine fol- In conclusion, the two-step synthesis of methionine con-
lowed by S-methylation yielded the corresponding me- taining peptides is convenient and very efficient. It is
thionine containing peptides 10–14 in very good yields noteworthy that in the ligation step no byproducts were
(Table 1).10,11 Further, as found in previous cysteine alky- observed and S-methylation of the thiol homocysteine
lation studies of unprotected peptides,12,13 there was no side chain with methyl iodide is a highly chemoselective
overmethylation of amines, alcohols, amides, and carbox- process which, besides cysteine, is compatible with all
ylate groups observed during methylation of the thiol side chains of the proteinogenic aminoacids. Hence, this
group. So both the ligation and the methylation step are method provides various possibilities for peptide and pro-
chemoselective and also racemisation was not observed. tein modification. Therefore, general application of this
Hence, this method seems to be an alternative or ideally approach to the synthesis of peptides and particularly of
complement the cysteine based native chemical ligation. glycopeptides is in progress in our laboratory.
This is also confirmed by the transformation of the N-ben-
zyl amide of homocysteine which is readily generated as
intermediate by reductive cleavage of homocysteine de-
Acknowledgment
This work was supported by the Fonds der Chemischen Industrie.
K. P. is grateful for an Alexander von Humboldt-Fellowship.
rivative 15 (Scheme 2); addition of the thioester of threo-
nine (3) led directly to the desired dipeptide amide
intermediate which on S-methylation afforded
BocThrMetNHBn dipeptide 16 in good yield. Thus, also
amide bonds of the homocysteine intermediate are stable
under the reaction conditions.
References
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Scheme 2 Synthesis of BocThrMetNHBn (16)
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662
K. Pachamuthu, R. R. Schmidt
LETTER
which was purified by recrystallization or column
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chromatography. All products were characterized by NMR
data.
(11) Compounds 8–14, 16 were characterized by [α]D, 1H NMR
20
and 13C NMR data. 8: Mp 56–60 °C (lit.14: 59–62 °C); [α]D
–27.7 (c 1, MeOH). 9: Mp 82–88 °C; [α]D20 +27.8 (c 1,
MeOH). 10: Mp 50–54 °C; [α]D20 –15.3 °C (c 1, MeOH). 11:
Mp 70–80 °C; [α]D20 –2.2 (c 1, MeOH). 12: Mp 145–149 °C
(lit.15: 149–154 °C); [α]D20 –19.0 (c 1, DMF) (lit.15 [α]D –20
(c 1, DMF). 13: Mp 83–88 °C (lit.16: 90 °C); [α]D –3.5 (c 1,
MeOH). 14: Mp 65 °C (petroleum ether/ethyl acetate);
[α]D20 –62.0 (c 1, MeOH). 1H NMR (250 MHz, CDCl3): δ =
1.36 (d, J = 6.5 Hz, 3 H), 1.43 (s, 9 H), 1.99–2.30 (m, 6 H),
2.05 (s, 3 H), 2.46 (t, 6.4 Hz, 2 H), 2.90–3.10 (m, 2 H), 3.50–
4.60 (m, 4 H), 6.20–6.40 (br s, 1 H), 7.40–7.60 (br s, 1 H).
13C NMR (62.9 MHz, CDCl3): δ = 15.4, 18.1, 25.0, 28.4,
30.8, 40.8, 47.5, 48.0, 54.4, 60.8, 77.2, 79.6, 155.5, 171.5,
173.8, 177.8; MALDI: m/z = 456 [M + K+]. 16: Mp 52–
56 °C; [α]D20 –39.5 (c 1, MeOH). 1H NMR (250 MHz,
CDCl3): δ = 1.12 (d, J = 6.4 Hz, 3 H), 1.40 (s, 9 H), 1.90–2.2
(m, 2 H), 2.03 (s, 3 H), 2.50 (t, J = 7.4 Hz, 2 H), 3.33 (d,
J = 3.0 Hz, 1 H), 4.06–4.09 (m, 1 H), 4.23–4.26 (m, 1 H),
4.39 (t, J = 5.4 Hz, 2 H), 4.56–4.65 (m, 1 H), 5.50 (d, J = 7.6
Hz, 1 H), 6.9 (br s, 1 H), 7.2–7.4 (m, 6 H). 13C NMR (62.9
MHz, CDCl3): δ = 15.2, 18.5, 28.2, 30.1, 31.2, 43.5, 52.6,
58.7, 67.2, 80.4, 127.4, 127.6, 128.6, 137.8, 156.2, 170.9.
171.2. MALDI: m/z = 462 [M + Na+].
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(10) General Procedure for the Synthesis of Peptides 8–14,
16: To a stirred solution of homocystine (200 mg, 0.75
mmol) in 0.1 M Tris buffer (10 mL, pH ca. 8.0) containing 6
M guanidinium hydrochloride was added DTT (230 mg, 1.5
mmol) and the reaction mixture was allowed to stir at r.t.
After 1 h, thioester (1.5 mmol) was added to the reaction
mixture. After completion of the reaction (TLC monitoring),
the reaction mixture was washed with CHCl3 and the aq
layer was neutralized with dilute HCl; the aq layer was then
extracted with EtOAc followed by washing the EtOAc layer
with brine and the organic layer was dried over anhyd
Na2SO4. Evaporation of the solvent yielded the
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recrystallization. This thiol was immediately taken up in
NH3 in MeOH (3 mL) and MeI (3 equiv) was added into the
reaction mixture at 0 °C. This reaction mixture was allowed
to stir at 0 °C for 2.0 h and at r.t. for 30 min. Then the
reaction mixture was neutralized with dilute HCl followed
by extraction with EtOAc, washing the EtOAc layer with
H2O and the organic layer was dried over anhyd Na2SO4.
Evaporation of the organic solvent yielded the crude product
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Synlett 2003, No. 5, 659–662 ISSN 0936-5214 © Thieme Stuttgart · New York