Effect of Copper Salts on Peptide Bond Formation Using Peptide Thioesters

Article abstract of DOI:10.1021/ol035742m

(Matrix presented) In the present paper, systematic studies revealed that Cu(I) salts in general and Cu(II) salts under certain circumstances promote effective reaction between peptide thiol esters and the N-terminal amino function of a second peptide seg

Full text of DOI:10.1021/ol035742m

ORGANIC
LETTERS
2003
Vol. 5, No. 24
4587-4590
Effect of Copper Salts on Peptide Bond
Formation Using Peptide Thioesters
,†
Raffaele Ingenito* and Holger Wenschuh
Jerini Peptide Technologies a DiVision of Jerini AG, InValidenstr. 130,
10115 Berlin, Germany
raffaele_ingenito@merck.com
Received September 10, 2003
ABSTRACT
In the present paper, systematic studies revealed that Cu(I) salts in general and Cu(II) salts under certain circumstances promote effective
reaction between peptide thiol esters and the N-terminal amino function of a second peptide segment to give the native amide bond for both
solution- and solid-phase syntheses. Chiral integrity was retained. Reaction conditions were optimized and applied to the synthesis of a small
protein, the identity of which was confirmed by NMR analysis.
Thiol esters of carboxylic acids were introduced as useful
acylating reagents for amines many years ago.1 However,
their application as protected amino acid thioesters for the
stepwise synthesis of peptides is only of limited use due to
their relatively low reactivity. In contrast, the use of peptide
thioesters as key intermediates for ligation and segment con-
densation processes has become increasingly important. Be-
sides the well-established process of native chemical ligation2
(NCL), a convergent peptide synthesis method3a,b (CPS) as
introduced by Blake3a and later improved by Aimoto4 applies
partially protected peptide thioesters as building blocks to
give native amide bonds, thus allowing the assembly of long
peptides and proteins not previously readily accessible by
conventional peptide synthesis methods.5 Whereas the NCL
method depends on the presence of Cys residues at certain
junction points, the latter method takes advantage of partially
protected peptide thioesters which can be reacted directly
with any primary amino group of a second partially protected
peptide segment. Since its invention, this method has been
continuously improved by the introduction of various addi-
tives to increase the reactivity of the peptide thioesters.
In particular, the combined use of silver ion in the presence
of HOOBt was demonstrated to be effective due to the in
situ transformation of the thioester into the more reactive
OOBt-ester.6
The present paper describes an extension of this method
to the use of copper salts as additives to accelerate the
reaction between peptide thiol esters and a second peptide
segment to give the native amide bond for both solution-
and solid-phase syntheses.
† Current address: IRBM/Merck Research Laboratories Rome Via
Pontina Km 30, 600-00040 Pomezia, Italy. Tel: +39 06 91093582. Fax:
+39 06 91093654.
(1) Wieland, T.; Bokelmann, E.; Bauer, L. Liebigs Ann. Chem. 1951,
573, 129.
(2) (a) Dawson, P. E.; Muir, T.; Clark-Lewis, I.; Kent, S. B. H. Science
1994, 266, 766. (c) Tam, J. P.; Lu, Y. A.; Chuan-Fa, L.; Shao, J. Proc.
Natl. Acad. Sci. U.S.A. 1995, 92, 12485.
(3) (a) Blake, J. Int. J. Peptide Protein Res. 1981, 17, 273. (b) Kimura,
T.; Takai, M.; Masui, Y.; Morikawa, T.; Sakakibara, S. Biopolymers 1981,
20, 1823.
(5) (a) Blake, J.; Yamashiro, D.; Ramasharma, K.; Li, C. H. Int. J. Peptide
Protein Res. 1986, 28, 468.
(6) (a) Kawakami, T.; Kogure, S.; Aimoto, S Bull. Chem. Soc. Jpn 1996,
69, 3331. (b) Kawakami, T.; Hasegwa, K.; Teruya, K.; Akaji, K.; Hotiuchi,
M.; Inagaki, F.; Kurihara, Y.; Uesugi, S.; Aimoto, S. J. Peptide Sci. 2001,
7, 474.
(4) Aimoto, S. Biopolymers 1999, 51, 247.
10.1021/ol035742m CCC: $25.00 © 2003 American Chemical Society
Published on Web 11/04/2003

The use of copper was primarily initiated by the goal to
use Cu(OBt)2 according to the “transesterification protocol”
described by Aimoto6 transferring the less reactive thioester
into a more reactive active ester and to take advantage of
the ability of Cu(OBt)2 to maintain the chiral integrity of
the activated C-terminal amino acid when used for active
ester based segment condensation either in solution7 or on
solid support.8
Initially, a simple model peptide thioester (T1: Msc-Leu-
Tyr-Arg-Ala-Gly-SR) was synthesized according to previous
Fmoc-based protocols,9 purified, and subsequently coupled
to another resin bound peptide (N1: H-Phe-Tyr(OtBu)-Gly-
Lys(Boc)-Ala-resin) in the presence of Cu(OBt)2/TMP (Table
1, entry g). In parallel, control experiments were performed
condensation reaction was repeated using CuCl2/TMP and
mixtures of CuCl2/TMP with thiophenate or HOBt. It was
found that addition of either CuCl2 or mixtures involving
thiophenate or HOBt gave no significant acceleration com-
pared to control experiments (Table 1, entries e and h).
Additional experiments were performed to examine the
influence of the oxidation state of copper. Thus, in contrast
to the use of CuCl2/TMP which gave none of the desired
product it was found that the addition of CuCl gave product
in the range of 35-45%.
These yields could be noticeably increased (>88% yield)
by prior addition of thiophenate to preactivate the alkyl
thioester (Table 1, entry j). On the other hand, use of the
commercially available CuI-thiophenolate salt as additive
gave significantly lower amounts of product, thus suggesting
that formation of a more reactive thioester prior to addition
of the CuI additive is advantageous with regard to the speed
of reaction. Furthermore, the combined use of CuCl and
HOBt (Table 1, entry k) showed a synergistic effect with
regard to yields of the target peptide when compared to the
use of CuCl/TMP (Table 1, entry I, 45%) or TMP/HOBt
(yield: 0%) alone.
Table 1. Comparison of Acylation Yields for Different
Additives on Solid Supporta
entry
additive
yield (%)
a
b
c
d
e
f
g
h
i
0
0
25
25
0
25
90
10
45
85
80
TMP
thiophenate
thiophenate/TMP
CuCl2/TMP
CuCl2/Thiophenate/TMP
Cu(OBt)2/TMP
CuCl2/HOBt/TMP
CuCl/TMP
thiophenate/CuCl/TMP
CuCl/HOBt/TMP
Since the first system examined involved a relatively
unhindered coupling (Gly/Phe; T1/N1), the most effective
additive combinations used [CuCl/thiophenate/TMP and Cu-
(OBt)2/TMP] and were also examined in the case of more
hindered couplings, e.g., Ala/Phe (T2/N1) and Leu/Phe (T3/
N1). It was found that in these cases high yields of coupling
products could also be obtained (Table 2). In the case of the
j
k
a Key: thioester peptide (T1), Msc-Leu-Tyr-Arg-Ala-Gly-S(CH2)2COOEt;
5 equiv of T1 in DMF, 0.2 M final concentration, 5 equiv of base, 5 equiv
of additives, 40 °C, 16 h.
Table 2. Comparison of Acylation Yields and Configurational
Loss for Different Ligation Points Using Different Methods on
Solid Supporta
in which the peptide thioester was reacted with the resin
bound C-terminal peptide without any additives or only in
the presence of a base (Table 1, entries a and b). In addition
the reaction was conducted with sodium thiophenate added
to the aliphatic thioester to increase reactivity via conversion
to the more reactive aromatic thioester. While the reactions
carried out without any additive or only with collidine (TMP)
as base gave no target material after 16 h, the addition of
sodium thiophenate yielded 25% of the target peptide (Table
1, entry c), which is obviously due to direct reaction of the
more reactive thioester with the resin bound peptide.
Interestingly, it was found that addition of Cu(OBt)2/TMP
significantly accelerated the speed of reaction resulting in a
reaction yield of >80% after 16 h (Table 1, entry g).
To elucidate whether the effect is related to a possible
transesterifcation process (thioester f active ester), the
T2 (T3)-N1
entry thioester
additive
yield (%) DL-isomer (%)
a
b
c
d
T2
T3
T2
T3
Cu(OBt)2/TMP
Cu(OBt)2/TMP
Thiophe/CuCl/TMP
Thiophe/CuCl/TMP
80
80
85
80
nd
2.6
nd
2
a Key: thioester peptides (T2), Msc-Leu-Tyr-Arg-Ala-Ala-S(CH2)2COOEt;
(T3), Msc-Leu-Tyr-Arg-Ala-LeuS(CH2)2COOEt; resin-bound peptide N1,
H-Phe-Tyr(tBu)-Gly-Lys(Boc)-Ala-resin; reaction time 16 h; nd, not
determined.
most hindered system (Leu/Phe), the time course for the
reaction (Figure 1) showed that reaction was slower at first
(ca. 10% after 4 h) but eventually reached a high level.
To extend these results to reactions carried out in solution,
peptide thioesters T1 and T3 were used for reaction with
the amino function of C-terminal peptide N2 in DMF. The
reaction conditions varied compared to the solid-phase
approach with regard to the reaction concentration (T1:10
mM; N2 3.3 mM) used.
It was found that after 24 h reaction yields were high in
all cases (Table 3). However, for the Leu-Phe ligation site,
when CuCl/thiophenate was used, the yield was only 50%,
most likely because of steric hindrance at the coupling site.
(7) Califano, J. C.; Devin, C.; Shao, J.; Blodgett, J. K.; Maki, R. A.;
Funk, K. W.; Tolle, J. C. Peptides 2000, Proceedings of 26th EPS, EDK,
Paris, France 2001.
(8) (a) Miyazawa, T.; Otomatsu, T.; Fukui, Y.; Yamadaq, T.; Kuwata,
S. Int. J. Peptide Protein Res. 1986, 28, 468. (b) Nishiyama, Y.; Tanaka,
M.; Saito, S.; Ishizuka, S.; Mori, T.; Kurita, K. Chem. Pharm. Bull. 1999,
47, 576. (c) Van Den Nest, W.; Yuval, S.; Albericio, F. J. Peptide Sci.
2001, 7, 115.
(9) Ingenito, R.; Bianchi, E.; Fattori, D.; Pessi, A. J Am. Chem. Soc.
1999, 121, 11369. (b) Ingenito, R.; Dreznjak, D.; Guffler, S.; Wenschuh,
H. Org. Lett. 2001, 4, 1187.
4588
Org. Lett., Vol. 5, No. 24, 2003

Finally, to determine whether the thioester/copper approach
would also be useful for longer peptide segments or even
small proteins the Z38-sequence11 of the B-domain of protein
A was synthesized. Segments 1-16 and 1-17 were chosen
as thioester components to be reacted with segments (17-
38) and (18-38) as C-terminal units, respectively. The
peptide thioester segments9 and the C-terminal segment were
prepared and purified according to known Fmoc-based
procedures and reprotected using Boc-OSu in solution as
reported.4,6b
Again the two methods [(thiophenate/CuCl/collidine and
Cu(OBt)2] shown to be most effective during the model
studies were applied in solution for the ligation. In these cases
yields were lower but still adequate12 (Table 4, entries 1-4).
Figure 1. Time course for reaction of peptide thioesters with resin-
bound peptide N1. Curve 1: T1 in the presence of Cu(OBt)2/TMP.
Curve 2: T1 in the presence of CuCl/thiophenate. Curve 3: T3 in
the presence of Cu(OBt)2/TMP. Curve 4: T3 in the presence of
CuCl/thiophenate/TMP.
Table 4. Z38 Domain Preparation by Copper-Mediated
Ligation in Solutiona
Z38
N-terminal C-terminal
yield
(%)
Time course experiments showed results similar to those
observed for the solid-phase reactions.
segment
segment
ligation point
additive
1
2
[1-17]
[1-17]
[18-39]
[18-39]
Ala/Leu
Ala/Leu
Cu(OBt)2
CuCl
Thioph/TMP
Cu(OBt)2
CuCl
40
40
It was also found that the use of copper salts accelerated
the hydrolysis of thioesters (1-5%) under the reaction
conditions used. This effect was more pronounced in cases
of more hindered junctions such as Leu-Phe or Glu-Ala (10-
30%) where the ligation times were prolonged.
3
4
[1-16]
[1-16]
[17-39]
[17-39]
Glu/Ala
Glu/Ala
65
45
Thioph/TMP
Since it is well-known that epimerization at the activated
C-terminal amino acid of the N-terminal segment is one of
the main drawbacks for segment condensation processes the
question of chiral integrity also needed to be addressed for
the new procedure.
To determine levels of epimerization during the condensa-
tion reaction, the thioester peptide T3 was synthesized having
either L-Leu or D-Leu at the C-terminal end so as to allow
preparation of the corresponding stereoisomeric standards
for HPLC comparison of the full length peptides after reac-
tion with N1 (Table 2, solid phase) and with N2 (Table 3,
solution). Relatively low values of configurational loss were
detected. Since the addition of copper salts during coupling
processes has been reported to suppress epimerization signi-
ficantly,8c,10 the extent of loss of configuration found in our
experiments might at least in part be related to this effect.
a Key: 10 mM final concentration in DMF; 60 h reaction time; Z38
sequence: AVAQSFNMQQQRRFYEALHDPNLNEEQRNAKIKSIRDD.
The product obtained from reaction with Cu(OBt)2 was
purified via GPC and preparative HPLC and analyzed using
MS and NMR. In addition to the expected MS (MS calcd
4563.1 [M + H]+, found 1141.4 [M + 4H]4+) it was found
that the Z38 mini-domain of protein A synthesized via
ligation using Cu(OBt)2 showed the same NMR structure as
a sample synthesized via stepwise approaches.11,12
In conclusion, it has been shown that amide bond
formation between a peptide thioester and a primary amine
in the form of a partially protected peptide can be accelerated
by addition of either CuCl or Cu(OBt)2. A systematic study
showed that the method was useful for different peptide
thioesters independent of their C-terminal nature. Also, it
was shown that the rate-enhancing effect of these additives
was evident for both solid- and solution-phase reactions. In
addition to the increased reaction rate, relatively low levels
Table 3. Comparison of Acylation Yields and Configurational
Loss for Gly-Phe and Leu-Phe Ligation Points for Different
Methods in Solutiona
(10) Akaji, K.; Kurijama, N.; Kiso, Y. J. Org. Chem. 1996, 61, 3350.
(b) Benoiton, N. L.; Lee, Y. C.; Steinaur, R.; Chen, F. M. Int. J. Pept.
Protein Res. 1992, 40, 559. (c) Blodgett, J. K.; Brammeier, N. M.; Califano,
J. C.; Devin, C.; Tolle, C. 16thAPS, June 26-July 1, 1999, Minneapolis,
MN, Poster 039.
(11) (a) Braisted, A. C.; Wells, J. A. Proc. Natl. Acad. Sci. U.S.A. 1996,
93, 5688. (b) Starovasnik, M. A.; Braisted, A. C.; Wells, J. A. Proc. Natl.
Acad. Sci. U.S.A. 1997, 94, 10080.
(12) Kochendoerfer, G. G.; Chen, S.; Mao, F.; Cressman, S.; Traviglia,
S.; Shao, H.; Hunter, C. L.; Low, D. W.; Cagle, E. N.; Carnevali, M.;
Gueriguian, V.; Keogh, P. J.; Porter, H.; Stratton, S. M.; Con Wiedeke,
M.; Wilken, J.; Tang, J.; Levy, J. J.; Miranda, L. P.; Crnogorac, M. M.;
Kalbag, S.; Botti, P.; Schindler-Horvat, J.; Savataski, L.; Adamson, J. W.;
Kung, A.; Kent, S. B. H.; Bradburne, J. A. Science 2003, 299, 884.
T1 (T3)-N2
entry thioester
additive
yield (%) DL-isomer (%)
a
b
c
d
T1
T3
T1
T3
thiophene/CuCl/TMP
thiophene/CuCl/TMP
Cu(OBt)2/TMP
75
50
75
75
4
7
Cu(OBt)2/TMP
a Key: (T1), Msc-Leu-Tyr-Arg-Ala-Gly-S(CH2)2COOEt; (T3), Msc-Leu-
Tyr-Arg-Ala-Leu-S(CH2)2COOEt; (N2), H-Phe-Tyr-Gly-Ser-Ala-NH2; reac-
tion time 24 h.
Org. Lett., Vol. 5, No. 24, 2003
4589

of epimerization were observed. The methodology could be
successfully extended to the reaction of longer segments as
demonstrated by application to a 38-mer protein domain
which was assembled in a reasonable yield.12
formation, mechanistic details on the reaction must await
further studies.
Acknowledgment. This work was supported by the FiTE
program of the European EFRE fund and the Senate of
Berlin. We thank S. Guffler for preparation of the hydroxy-
benzotriazole salt used and for helpful discussion throughout
the work. D. Dreznjak is thanked for excellent technical
assistance. M. Schade and J. Kahman are acknowledged for
NMR characterization of the Z domain of protein A. Prof.
L. A. Carpino is thanked for critical reading of the
manuscript.
Whereas for the copper(I) salt the cuprous ion itself seems
to be the key component for the accelerating effect for the
copper(II) salt [Cu(OBt)2] transesterification (thioester f
active ester) must be considered as a key step since the rate-
enhancing effect was much less pronounced in the case of
CuCl2 vs CuCl. Although various copper species in general
were recently shown to have rate-accelerating effects for a
variety of reactions7,8,13 such as those of amines with aryl
halides14 their influence on amide bond formation by means
of peptide thioesters has not yet been described. While it
seems to be evident that cuprous ion is involved in the
transition complexes which facilitate the final amide bond
Supporting Information Available: Complete protocols
for the synthesis, purification, and characterization of all
peptides used in this work, including ESI, HPLC, and NMR
characterization of the Z domain. This material is available
free of charge via the Internet at http://pubs.acs.org.
(13) (a) Zhang, L.; Tam, J. P. Tetrahedron Lett 1997, 38, 4375.
(14) (a) Kwong, F. Y.; Buchwald, S. L. Org. Lett. 2003, 5, 793. (b)
Zanon, J.; Klapars, A.; Buchwald, S. L. J. Am. Chem. Soc. 2003, 125, 2890.
OL035742M
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Org. Lett., Vol. 5, No. 24, 2003