Native chemical ligation using removable Nα-(1-phenyl-2-mercaptoethyl) auxiliaries

Article abstract of DOI:10.1016/S0040-4039(01)00036-3

A new methodology to extend native chemical ligation is presented. This method makes use of a novel 1-phenyl-2-mercaptoethyl auxiliary moiety on the α-amino group of a first peptide segment, the auxiliary acts as a 1-amino-2-thiol-containing functional group to effect thioester-mediated, amide-forming ligation with a second thioester-containing peptide. Subsequent facile removal of the auxiliary from the newly formed amide gives products with only native peptide structures.

Full text of DOI:10.1016/S0040-4039(01)00036-3

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 1831–1833
Native chemical ligation using removable
a
N -(1-phenyl-2-mercaptoethyl) auxiliaries
Paolo Botti,* Michael R. Carrasco† and Stephen B. H. Kent*
Gryphon Sciences, 250 E Grand Ave, Suite 90, South San Francisco, CA 94080, USA
Received 12 June 2000; accepted 8 January 2001
Abstract—A new methodology to extend native chemical ligation is presented. This method makes use of a novel 1-phenyl-2-
mercaptoethyl auxiliary moiety on the a-amino group of a first peptide segment, the auxiliary acts as a 1-amino-2-thiol-containing
functional group to effect thioester-mediated, amide-forming ligation with a second thioester-containing peptide. Subsequent facile
removal of the auxiliary from the newly formed amide gives products with only native peptide structures. © 2001 Published by
Elsevier Science Ltd.
During the last few years thioester-mediated native
chemical ligation (NCL) of unprotected peptide
segments1 has been demonstrated to be uniquely effec-
tive for the rapid and efficient chemical synthesis of
numerous proteins.2 Although several other ligation
methods allow the chemoselective joining of two or
more unprotected peptide fragments in aqueous solu-
tion,3–5 only the NCL reaction leads to products with
an unmodified peptide bond at the ligation site. The
original NCL approach1 has been routinely used to join
two peptide segments at an Xxx-Cys sequence, where
Cys is the N-terminal amino acid of the first segment
and Xxx is an amino acid bearing an a-thioester at the
C-terminal end of the second segment. The 1-amino-2-
thiol arrangement of functionalities in the N-terminal
Cys is essential for thioester-mediated amide-forming
ligation.1 Thus, NCL does not allow the ligation of
unmodified polypeptide sequences devoid of suitably
located cysteine residues.
limited its practical utility. Canne et al. also tested an
Na-(2-mercaptoethyl) ligation auxiliary that showed
good rates of amide bond formation,6 but the auxiliary
was not designed for subsequent removal. A related
method based on the same principle made use of Na-(2-
mercaptobenzyl) as the auxiliary group, but this did not
solve the problem of the removal of the auxiliary
moiety after ligation.7
Here we present a novel, practical method that solves
these problems and extends NCL to the joining of
peptide segments at sites that do not contain cysteine
residues. Our method makes use of the Na-(2-mercapto-
ethyl) auxiliary group and takes advantage of its fast
ligation rates while allowing convenient and complete
removal of the auxiliary after ligation. 1-Phenyl-substi-
tution of the 2-mercaptoethyl auxiliary on the a-amino
group of the reacting peptide segment gave a benzyl-
amine derivative that is stable even under the strongly
acidic conditions normally used in the synthesis of a
peptide. The same group is readily cleaved under simi-
lar or even less acidic conditions when on the corre-
sponding amide formed in the ligation reaction. Thus, a
model peptide bearing either the Na-(1-phenyl-2-mer-
captoethyl) auxiliaries I and II was synthesized using
standard Boc chemistry solid phase peptide synthesis
(SPPS) methods,8 and reacted with a thioester-contain-
ing peptide. Ligation of the two segments gave the
corresponding phenyl-substituted N-(1-phenyl-2-mer-
captoethyl) amide. The auxiliary was then conveniently
removed under acidic conditions to provide the native
amide bond-linked polypeptide product. The general
ligation strategy and auxiliaries employed in this
approach are outlined in Scheme 1.
Several attempts have been made to extend the princi-
ples of NCL to join peptides at other than Xxx-Cys
ligation sites. Canne et al.6 presented the first extension
of the NCL reaction that made use of a Na-(thiol-con-
taining) auxiliary group in lieu of the cysteine side
chain thiol. The acid-stable, reductively cleavable Na-
(oxyethanothiol) auxiliary of necessity had a 1-amino-3-
thiol structure, and amide-forming ligation rates with
this auxiliary were much slower, which has severely
* Corresponding authors.
† Current address: Santa Clara University, Santa Clara, CA 95053,
USA.
0040-4039/01/$ - see front matter © 2001 Published by Elsevier Science Ltd.
PII: S0040-4039(01)00036-3

1832
P. Botti et al. / Tetrahedron Letters 42 (2001) 1831–1833
PEPTIDE 2
OCH3
CO-N-CH2-CO
PEPTIDE 1
HS
COS-alkyl
PEPTIDE 1
+
ligation
NH-CH2-CO
PEPTIDE 2
R
HS
HF (R = H),
or
R
OCH3
TFA (R = OCH3)
R = H, OCH3
PEPTIDE 1
CO-
PEPTIDE 2
NH-CH2-CO
Scheme 1. General ligation strategy with 1-phenyl-2-mercaptoethyl auxiliaries.
The general synthesis of N-terminal glycine peptides
containing the Na-(1-phenyl-2-mercaptoethyl) auxiliary
is illustrated in Scheme 2. Two different benzylamines
were prepared in three steps from the corresponding
substituted bromoacetophenones: 1-(4-methoxyphenyl)-
2-(4%-methylbenzylthio)-ethylamine (3a) and 1-(2,4-
dimethoxyphenyl)-2-(4%-methylbenzylthio)-ethylamine
(3b). Reaction of these amines with a resin-bound bro-
moacetylpeptide,9 followed by cleavage/deprotection,
gave peptides containing an N-terminal glycine residue
derivatized with either an Na-(1-(4-methoxyphenyl)-2-
mercaptoethyl) (I) or an Na-(1-(2,4-dimethoxyphenyl)-
2-mercaptoethyl) (II) auxiliary.
motrimethylsilane (TMSBr);10 while the dimethoxy-sub-
stituted auxiliary II was removed with 95% TFA, 2.5%
triisopropylsilane (TIS), and 2.5% water. The removal
of each auxiliary was accomplished in quantitative yield
as measured by high performance liquid chromatogra-
phy (HPLC).
The effectiveness of our new extended native chemical
ligation strategy is highlighted by analytical HPLC data
for model reaction 4 (Fig. 1). In this reaction, fragment
1-31 of Mouse Larc with a C-terminal Ala thioester was
ligated with the model peptide Na(Aux)GlySerTyrArg-
PheLeu bearing auxiliary I. After 40 h of ligation, the
reaction was virtually complete. The ligation product
was purified and then treated with {95% HF+5% p-
cresol} at 0°C for 1 h, resulting in quantitative removal
of the auxiliary I.
The feasibility of using Na-(1-phenyl-2-mercaptoethyl)
auxiliaries for native chemical ligation was tested with a
model peptide of sequence Na(Aux)GlySerTyrArg-
PheLeu. The N-terminus of the model peptide was
derivatized with either the Na-(1-(4-methoxyphenyl)-2-
mercaptoethyl) auxiliary (I), or the Na-(1-(2,4-di-
methoxy-phenyl)-2-mercaptoethyl) auxiliary (II). Each
of these peptides was successfully ligated with a variety
of thioester peptides (Table 1). After purification of the
ligation products, both auxiliary groups were efficiently
removed using acidic conditions: the methoxy-substi-
tuted auxiliary I was removed with 95% HF and 5%
p-cresol, or with trifluoroacetic acid (TFA)/bro-
In summary, we have demonstrated a novel method
that significantly extends the concept and practical util-
ity of native chemical ligation. Moreover, the rates of
ligation remained high, and the auxiliaries were easily
removed. Importantly, our strategy is compatible with
both Boc and Fmoc chemistries for peptide synthesis.
The ability to join peptide fragments at sites other than
Xxx-Cys sequences allows, inter alia, for convenient
O
R
O
R
Br
HS
S
1) H2N-O-CH2COOH
H3CO
NEt(i-Pr)2, DMF
2) Reduction
H3CO
1a,b
2a,b
1)
Br
O
NH2
H3CO
R
R
O
Peptide
Resin
NH
Peptide
SR'
H3CO
2) Acid cleavage/deprotection
HS
3a,b
4a,b
Scheme 2. General synthesis of derivatized peptides (a: R=H; b: R=OCH3; R%=p-Me benzyl).

P. Botti et al. / Tetrahedron Letters 42 (2001) 1831–1833
1833
Table 1. Results of ligation studies with Na(Aux)GlySerTyrArgPheLeu peptides
Model reaction
Peptide-aCO-SR (C-terminal thioester
residue)
Na-(Auxiliary)
Reaction time
(h)a
Ligation yield
(%)b
Auxiliary removal
conditions
1
2
3
4
Phe-Gly-Gly
Phe-Gly-Gly
TBP-A 1-67 (His)
Mouse Larc 1-31 (Ala)
I
16
16
16
16
40
16
40
16
40
\98
\98
87
81
92
73
85
69
76
HF
TFA
TFA
II
II
I
HF
5
6
Mouse Larc 1-31 (Ala)
MCP1 1-35 (Lys)
II
I
TFA
TFA/TMSBr
a Ligation conditions: 5 mM Na-(Aux) peptide in 6 M guanidinium phosphate buffer pH 7, 25°C, 2% thiophenol.
b Based on consumption of the Na-(Aux) peptide. Thioester peptides were used in 1.5-fold excess.
Figure 1. HPLC analyses of model ligation reaction 4 (Table 1). Peak 1=Na(Aux)Gly-SerTyrArgPheLeu; peak 2=Mouse
Larc(1-31)aCOSR; peak 3=ligation product with Na-auxiliary; peak 4=HF crude from the removal of the Na-auxiliary moiety.
synthetic access to native, non-cysteine containing pep-
tides and proteins. We will present data on the use of
our 1-phenyl-2-mercaptoethyl auxiliary ligation strategy
for the effective total synthesis of functional heme
proteins.11
5. Liu, C.-F.; Tam, J. P. Proc. Natl. Acad. Sci. USA 1994,
91, 6584–6588.
6. Canne, L. E.; Bark, S. J.; Kent, S. B. H. J. Am. Chem.
Soc. 1996, 118, 5891–5896.
7. Offer, J.; Dawson, P. E. Org. Lett. 2000, 2, 23–26.
8. Schno¨lzer, M.; Alewood, P.; Jones, A.; Alewood, D.;
Kent, S. B. H. Int. J. Peptide Protein Res. 1992, 40,
180–193.
References
9. Zuckermann, R. N.; Kerr, J. M.; Kent, S. B. H.; Moos,
W. H. J. Am. Chem. Soc. 1992, 114, 10646–10647.
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Pharm. Bull. 1988, 36, 4989–4992; (b) Yajima, H.; Fujii,
N.; Funakoshi, S.; Watanabe, T.; Murayama, E.; Otaka,
A. Tetrahedron 1988, 44, 805–819.
1. Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B.
H. Science 1994, 266, 776–779.
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