Chemical protein synthesis by kinetically controlled ligation of peptide O-esters

Article abstract of DOI:10.1002/cbic.200900789

(Chemical Equation Presented) Designer peptides: A large reactivity difference was observed between two peptide O-esters that can undergo peptide ligation through an situ O-to-S acyl shift. This observation allowed the designed of "one-pot" N-to-C sequential peptide fragment condensation through kinetically controlled ligation with more readily accessible peptide O-esters.

Full text of DOI:10.1002/cbic.200900789

DOI: 10.1002/cbic.200900789
Chemical Protein Synthesis by Kinetically Controlled Ligation of Peptide
O-Esters
Ji-Shen Zheng, Hong-Kui Cui, Ge-Min Fang, Wei-Xian Xi, and Lei Liu*[a]
Protein chemical synthesis can overcome potential limitations
of protein expression and produce proteins with predesigned
changes and modifications with atomic precision. The develop-
ment of increasingly efficient and general methods for peptide
ligation comprises a central objective.[1] One important chal-
lenge is to synthesize proteins by sequential ligation of three
or more unprotected peptide segments. Earlier strategies fo-
cused on sequential ligations toward the N terminus from a C-
terminal Cys peptide segment,[2] but the counterpart N-to-C se-
quential assembly of the peptide segments[3] was difficult, and
this prevented fully convergent protein synthesis. To solve the
problem Kent et al. recently invented kinetically controlled liga-
tion (KCL; Scheme 1).[4] The success of KCL relies on the large
pioneered peptide ligation through their use of the in situ O-
to-S acyl shift.
Our investigation began by examining model peptide O-
ester 1 (Table 1).[7] It was found that 1 (as an O-arylester) is not
suitable for Fmoc solid-phase peptide synthesis (SPPS) because
it cannot survive the conditions used for Fmoc cleavage. Sub-
sequently we focused on peptide O-alkylesters (2–12), which
have structures that differ by the number of carbon atoms be-
tween O and S and by the position and nature of substitution.
It was found that in cases in which the number of carbon
atoms between O and S equals to two, the reactants (4–9) can
be smoothly consumed. However, HPLC analysis indicated that
except for 5 and 6, all other peptide O-esters (4, 7–9) were
heavily hydrolyzed into peptideÀ
CO2H. Thus, the presence of a
side chain adjacent to the
oxygen atom is beneficial to the
O-to-S acyl shift (which possibly
proceeds through a five-mem-
bered ring intermediate), where-
as the presence of a side chain
adjacent to the sulfur atom does
the opposite. In cases in which
the number of carbon atoms
between O and S increases to
three, esters (10–12) show very
low conversions after 48 h; this
indicates that the corresponding
Scheme 1. Previous vs. new kinetically controlled ligation.
O-to-S acyl shift (through a six-
membered ring intermediate) is
reactivity difference between a peptide-a-thioarylester and a
peptide-a-thioalkylester. Despite this advance, difficulties still
exist for the preparation of peptide thioesters[5] and more trou-
blesome thioarylesters.[6] Here we describe a novel, practical
approach for chemical protein synthesis through kinetically
controlled ligation of peptide O-esters. The new method over-
comes the difficulties in the preparation of ligation segments.
The study also reveals interesting reactivity variations in the
intramolecular acyl shift reaction. This study was inspired by
the seminal work of Danishefsky,[7] Botti,[8] and others,[9] who
slow. Fusion of a phenyl ring into the linker (2) can dramatical-
ly accelerate the conversion, but in such cases only the hydro-
lyzed product was obtained. Finally, ester 3 (in which the
number of carbon atoms between O and S equals to four)
shows a modest conversion; however, one half of the product
is the hydrolyzed peptide.
The above systematic examinations showed that only 5 and
6 are useful for the peptide ligation through in situ O-to-S acyl
shift. Note that 6 (denoted as type I ester in the following dis-
cussion) was previously examined by Botti[8] and Muir.[9b,c]
Compared to 6, 5 (denoted as type II ester) exhibited an
improved ligation yield (93% vs. 84%) due to less hydrolysis.
More experiments showed that for the type II ester of Ala
(Table 2), the ligation yield was 73% at pH 6.75 (entry 1). This
value is also higher than the parallel value for the type I ester
of Ala under the same conditions (62%, entry 13). Replacement
of the ligation accelerator to MPAA[10] (entry 2) or change of
pH (entries 3–4) did not improve the ligation. On the other
hand, in cases in which the buffer was changed to imidazole
(entry 11) less hydrolysis and a higher ligation yield (89%) were
[a] J.-S. Zheng, H.-K. Cui, G.-M. Fang, W.-X. Xi, Prof. L. Liu
Department of Chemistry
Key Laboratory of Bioorganic Phosphorus Chemistry
and Chemical Biology (Ministry of Education), Tsinghua University
Beijing 100084 (China)
Fax: (+86)10-62771149
E-mail: lliu@mail.tsinghua.edu.cn
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.200900789.
ChemBioChem 2010, 11, 511 – 515
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511

observed. The same behavior
was also observed for the type I
ester of Ala, in which the ligation
yield increased to 78% in the
imidazole buffer (entry 14). Final-
ly, the esters of amino acids car-
rying more complex side chains
(entry 6) could be successfully li-
gated, but the ligation yields
were low for the most sterically
hindered amino acids (entries 7–
8).
Table 1. Performance of different peptide O-esters.[a]
OÀR
Conversion
Hydrolysis
Ligation
The above results show that
the newly developed type II
ester is better than the type I
ester for peptide ligation. To
demonstrate the utility of the
type II ester in chemical protein
synthesis, we chose the S4 small
chain of napin (a plant antifun-
1
2
n.d.
n.d.
n.d.
100
100
31
0
gal defensive protein) as
a
model.[11] The synthesis started
with the Fmoc protected Rink
amide aminomethyl resin and
compound 13, which was pre-
pared readily from 3-mercapto-
propane-1,2-diol (Scheme 2). By
using the standard Fmoc proto-
col the desired peptide ester
(14) was successfully prepared.
Subsequently 14 was reacted
with the Cys-containing peptide
segment 15 [Eq. (1)] to afford
the desired product 16 smoothly
in 80% yield. Compared to the
standard native chemical liga-
tion, the major improvement of
the present approach is that all
the peptide segments used in
the ligation can be readily pre-
pared through the Fmoc meth-
od under mild conditions.
3
69
38
4
5
100
99
90
6
10
93
6
100
16
84
7
8
99
97
87
86
12
11
Interestingly and importantly,
it was found that the optimal
temperature for the ligation of
the type II ester of peptides is
about 408C. At 408C the ligation
between 5 and Cys is completed
in about 6 h, but at lower tem-
peratures the same reaction is
much slower (Figure 1). By com-
parison, the ligation between 6
and Cys is a much faster reaction
that is completed in about 2 h at
12–158C. Estimation of the initial
rates revealed that the reactivity
of 5 and 6 differs by about 30-
9
10
11
12
85
11
10
14
79
3
6
8
2
8
2
12
[a] All the values were measured by using HPLC. [b] n.d. not determined. [c] Diastereomers were difficult
to separate and therefore were used as a mixture.
512
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ChemBioChem 2010, 11, 511 – 515

fold. Such a large reactivity dif-
ference between 5 and 6 can be
attributed to their structures. In
5 the linker connecting the O
and S atoms carries an alkyl side
chain, whereas in 6 the side
chain is an acyl group. Because
an acyl group is electron with-
drawing whereas an alkyl group
is electron donating, the ester
group in 6 is expected to be
much more electrophilic than
that in 5. Thus, the intramolecu-
lar O-to-S acyl shift in 6 is more
favorable both kinetically and
thermodynamically than that in
5. The outcome is that 6 ligates
with Cys much faster than 5.
Table 2. Ligation of peptide O-esters with cysteine.[a]
Xaa
Medium
pH
Yield [%][b]
Ligation
Hydrolysis
1
2
3
4
5
6
7
8
9
Ala
Ala
Ala
Ala
Gly
Leu
Val
Pro
Ala
Ala
Ala
PBS
PBS
PBS
PBS
PBS
PBS
PBS
PBS
HEPES
Tris
6.75[c]
6.75[d]
6.50[c]
7.00[c]
6.75[c]
6.75[c]
6.75[c]
6.75[c]
6.75[c]
6.75[c]
6.75[c]
73
68
70
67
93
63
18
<2
76
75
89
25
30
24
24
6
35
25
16
24
19
9
10
11
Imi
An important application of
the large reactivity difference be-
tween the type I and II peptide
O-esters is the possibility of car-
rying out KCL,[4] for the first time
solely with peptide O-esters.
Three peptide segments (pep-
tide1-type I ester, Cys-peptide2-
type II ester, and Cys-peptide3)
can be sequentially ligated from
12
13
14
Gly
Ala
Ala
PBS
PBS
Imi
6.75[c]
6.75[c]
6.75[c]
84
62
78
16
37
20
PBS concentration was 0.2m, HEPES concentration was 1.0m, Tris concentration was 1.0m, Imi concentration
was 1.0m. [a] Peptide O-ester (1.6 mm) was reacted with Cys (2.0 mm) in 6m guanidine buffer containing 20%
NMP. [b] HPLC yields. [c] Accelerator 2% PhSH. [d] Accelerator 30 mm MPAA.
the
N
to the C terminus
(Scheme 1); this takes advantage
of the dual reactivity of the
middle segment (that is, a bi-
functional Cys-peptide2-type II
ester), which can be controlled
so that it can firstly react with a
peptide1-type I ester. To demon-
strate the utility of the KCL of
peptide O-esters, we report an
effective chemical synthesis of
[V15A]crambin. The same model
protein was previously synthe-
sized by Kent and coworkers.[2b,c]
Briefly, three peptide segments
were synthesized through Fmoc
SPPS (Scheme 3). The left seg-
ment (18) was firstly treated
with the middle segment (19)
under native chemical ligation
Scheme 2. Solid-phase synthesis of the type II ester of a peptide.
reaction conditions at 158C. After 1 h most of the reactants
were consumed. Mass analysis indicated that the major prod-
uct 21 is the ligated product of 18 and 19, whereas the main
byproducts (22 and 23) correspond to the hydrolyzed peptide
from 18 and cyclized peptide from 19. After 2 h the C-terminal
peptide segment 20 was added into the reaction mixture. The
ligation temperature was increased to 408C and the reaction
was completed overnight. The full-length 46-residue polypep-
ChemBioChem 2010, 11, 511 – 515
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www.chembiochem.org
513

There are three advantages of the above “one-pot” KCL
method: 1) First, starting from the three purified peptide seg-
ments, the one-pot synthesis can be completed to afford the
desired product with good yields and with only one final pu-
rification step in two days; 2) Second, the peptide segments in
the present KCL can be prepared through mild Fmoc methods,
which might be useful for the synthesis of more difficult tar-
gets such as glycoproteins; 3) Third, the N-terminal segment in
the present KCL can tolerate unprotected Cys residues (for ex-
ample, 18 contains two unprotected Cys residues), whereas in
the previous KCL an unprotected Cys residue in the left seg-
ment (as a peptide-athioarylester) might easily form a less re-
active thiolactone that will impede the KCL.[4b]
In summary, through systematic examinations we have con-
firmed one and identified a new peptide O-ester that can be
used for peptide ligation through in situ O-to-S acyl shift.
These peptide O-esters can be readily prepared through mild
Fmoc solid-phase synthesis and thus we have extended the
practical utility of native chemical ligation. Most importantly, a
large reactivity difference was observed between the two pep-
tide O-esters. This allows, for the first time, a “one-pot” N-to-C
sequential condensation of peptide segments through kineti-
cally controlled ligation with more readily accessible peptide O-
esters instead of peptide thioesters.
Acknowledgements
This work was supported by NSFC (No. 20802040 and No.
20932006), and the Specialized Research Fund for the Doctoral
Program of Higher Education of China (Grant No.
200800030074).
Figure 1. Rates for the ligation of peptide O-esters. A) The ligation between
5 (1.6 mm) and Cys (2.0 mm) at different temperatures. B) The ligation be-
tween 5 (1.6 mm) and Cys (2.0 mm) versus the ligation between 6 (1.6 mm)
and Cys (2.0 mm) at 12–158C.
tide chain of [V15A]crambin was successfully obtained in 59%
yield (HPLC yield).
Keywords: ligation · peptide O-esters · peptide thioesters ·
peptides · solid-phase synthesis
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Received: December 29, 2009
Published online on February 15, 2010
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