Cysteine-free peptide and glycopeptide ligation by direct aminolysis

Article abstract of DOI:10.1002/anie.200705298

(Chemical Equation Presented) Left to their own devices in a mixed-solvent system, peptides undergo efficient aminolysis with peptide thioesters (see scheme). This ligation method, which does not require coupling reagents, auxiliaries, or an N-terminal cysteine residue, is suitable for a variety of amino acids at the ligation junction. Its effectiveness was demonstrated by the synthesis of a 6.9-kDa section of the cancer-associated MUC1 tandem repeat.

Full text of DOI:10.1002/anie.200705298

Angewandte
Chemie
DOI: 10.1002/anie.200705298
Ligation Reactions
Cysteine-Free Peptide and Glycopeptide Ligation by Direct
Aminolysis**
Richard J. Payne, Simon Ficht, William A. Greenberg, and Chi-Huey Wong*
Native chemical ligation (NCL) is an extremely useful
technique for the synthesis of peptide and protein targets.[1,2]
The method, which relies on the chemoselective condensation
of a C-terminal peptide thioester with a peptide containing an
N-terminal cysteine residue to afford a native peptide bond,
has been implemented successfully in the total synthesis of
hundreds of proteins to date.[3] Furthermore, NCL has also
been applied effectively to the synthesis of glycoproteins.[4]
Although NCL has proved to be an extremely powerful
technique, certain limitations still exist. The most obvious is
the requirement for a cysteine residue at the ligation junction.
Alanine[5] and phenylalanine[6] disconnections were realized
recently by the use of desulfurization techniques and can
therefore be added to the NCL repertoire.[7] The combined
abundance of cysteine, alanine, and phenylalanine in protein
sequences is still relatively low, and as such, there is a high
probability that a particular target protein does not contain
one of these amino acids at a synthetically viable position. An
alternative strategy has been the development of cysteine-
free ligation techniques, whereby a thiol-containing auxiliary
is incorporated at the N terminus.[8,9] These methods have
proven useful for the synthesis of glycopeptides;[10,11] how-
ever, the use of such auxiliaries appears to be limited to
ligation sites with amino acid side chains of low steric bulk.
Recently, we reported an alternative peptide-ligation
strategy for the synthesis of glycopeptides: sugar-assisted
ligation (SAL). This method utilizes a glycopeptide in which
the carbohydrate is derivatized with a mercaptoacetate
auxiliary.[12,13] While investigating the mechanism of SAL,
we discovered that ligation reactions could proceed in an
intermolecular fashion between an N-terminal amine of a
glycopeptide and a C-terminal peptide thioester in the
absence of the thiol auxiliary, albeit at a lower rate and with
significant quantities of hydrolyzed thioester.[14] This obser-
vation suggests that the previously reported ligation reactions
may proceed by a direct aminolysis pathway in conjunction
with the intramolecular cyclization mechanism proposed
previously.
The direct coupling of a C-activated peptide with a
peptide containing a free amine was reported by Kemp and
co-workers in the 1970s.[15,16] In these studies, activated
C-terminal peptide esters (for example, N-ethylsalicylamide
and p-nitrophenyl esters) were used to couple peptides
efficiently in the absence of an N-terminal cysteine residue,
a thiol auxiliary, or exogenous activating reagents. Reactions
were conducted in dimethyl sulfoxide or N,N-dimethylform-
amide (DMF) and gave ligation products in high yields. The
limited solubility and potential racemization of peptides
under these conditions have prevented the exploitation of this
method. Clearly, a method that could overcome these draw-
backs would prove extremely useful for the synthesis of
peptides and glycopeptides that can not be otherwise
synthesized by NCL or SAL owing to the lack of a suitable
ligation junction.
In 1981, Blake reported a strategy based on silver-ion
mediation to facilitate peptide ligations via peptide thio-
acids.[17] The procedure was modified by Aimoto and co-
workers, who used a thioester as the acyl donor.[18,19] The
latter method exploits the reactivity of a peptide thioester,
which, in the presence of silver(I) and a suitable activating
agent, such as 1-hydroxy-1H-benzotriazole (HOBt) or
3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOOBt),
reacts with the N-terminal amine of a peptide to afford the
desired ligation product.[19] Recently, Danishefsky and co-
workers reported an extension of this method, whereby the
thioester was masked as a protected o-thiol-containing
phenolic ester.[20] Unfortunately, the reaction conditions
caused epimerization of the C-terminal thioester residue,
and therefore only non-epimerizable amino acids could be
incorporated at this position (glycine or proline). Addition-
ally, in contrast to NCL, the method lacked chemoselectivity
in the presence of cysteine and lysine residues, which required
protection with acetamidomethyl (Acm) and 1-(4,4-dimethyl-
2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (ivDde) groups,
respectively. We were therefore interested in pursuing a
general cysteine- and auxiliary-free ligation strategy for the
construction of peptides and glycopeptides without the use of
activating agents. The strategy should be applicable to a range
of peptide thioesters without causing epimerization of the
C-terminal residue. We believed that this goal was possible
through the use of suitable buffer conditions to facilitate the
direct aminolysis of a peptide and a C-terminal peptide
thioester (Scheme 1).
[*] Dr. R. J. Payne, Dr. S. Ficht, Prof. W. A. Greenberg, Prof. C.-H. Wong
Department of Chemistry, The Scripps Research Institute
10550 North Torrey Pines Road, La Jolla, CA 92037 (USA)
Fax: (+1)858-748-2409
E-mail: wong@scripps.edu
Prof. C.-H. Wong
The Genomics Research Center, Academia Sinica
128 Section 2, Academia Road, Nankang, Taipei 115 (Taiwan)
[**] R.J.P. and S.F. contributed equally to this research, which was
supported by the NIH and the Skaggs Institute for Chemical
Biology. R.J.P. is grateful for funding provided by the Lindemann
Trust Fellowship. S.F. is grateful for a postdoctoral fellowship
provided by the Deutsche Akademische Austauschdienst (DAAD).
In initial studies, we investigated the direct coupling of a
model peptide thioester 1 containing a C-terminal glycine
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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tion). We selected the following ligation buffer: 4:1 v/v NMP:
6m Gn·HCl, 1m HEPES, pH 8.5, as it displayed the most
suitable buffering range for the direct aminolysis reaction and
had been used successfully to suppress the hydrolysis of
peptide thioesters in previous ligation studies.[14] Under these
conditions, the ligation reaction between the peptide thioester
1 and peptide 2 reached completion within 48 h with minimal
thioester hydrolysis, and the desired product 3 was isolated in
89% yield (Scheme 2 and Table 1, entry 1).
Scheme 1. Proposed cysteine-free ligation reaction (R1 and R3 are
aminoacid side chain functionalities).
Table 1: Scope of the cysteine-free aminolysis reaction (R=
(CH2)2CONH2).
residue with a peptide 2 bearing an N-terminal glycine residue
by using the standard NCL buffer (conditions a,
Scheme 2).[2,3] Under these conditions, we observed the
formation of the desired product 3; however, the yield was
unacceptably low (40%) as a result of competing thioester
hydrolysis in the aqueous media.
Entry
Peptide
thioester
AA1
Peptide
AA2
(Glycosyl) amino
acid X
Yield [%][b]
1[c]
Gly
Ala
His
Gly
Ala
His
Gly
Ala
His
Gly
His
Gly
Ala
His
Gly
Ala
His
Gly
Gly
Gly
Gly
Gly
His
His
His
Ala
Ala
Ala
Asp
Asp
Glu
Glu
Glu
Tyr
Ser
Ser
Ser
Ser
Ser
Ser
Ser
Ser
Ser
Ser
Ser
Ser
Ser
Ser
Ser
Ser
Ser
89
quant.
quant.
57
40
41
66
54
37
quant.
89
quant.
quant.
90
39
34
12
98
65
2[c]
3[c]
4[d]
Scheme 2. Ligation of 1 and 2 under different conditions: a) 2
(14 mm), 1 (9 mm), Gn·HCl (6m), potassium phosphate (0.1m),
pH 8.5, PhSH (2% v/v), 378C, 48 h (40%); b) 2 (14 mm), 1 (9 mm),
4:1 v/v NMP/buffer: Gn·HCl (6m), HEPES (1m), pH 8.5, PhSH (2%
v/v), 378C, final pH value: 7.5, 48 h (89%). R=(CH2)2CONH2;
Gn·HCl=guanidine hydrochloride, HEPES=4-(2-hydroxyethyl)pipera-
zine-1-ethanesulfonic acid, NMP=N-methylpyrrolidinone.
5[d]
6[d]
7[d]
8[d]
9[d]
10[c]
11[c]
12[c]
13[c]
14[c]
15[d]
16[d]
17[d]
18[c]
19[c]
To suppress hydrolysis and increase the ligation yield, we
investigated a range of different solvent conditions. A general
strategy to prevent hydrolysis is the addition of cosolvents. We
examined various cosolvents in combination with a number of
suitable buffers. As the reaction relies on direct attack at the
thioester by a deprotonated amine, the buffer should operate
at pH 7.0–8.5. Buffers that contain nucleophilic amines (e.g.
tris(hydroxymethyl)aminomethane (Tris)) could not be used
owing to competing reactivity (see the Supporting Informa-
tion). Commonly used buffers that fulfill these criteria include
potassium phosphate, HEPES, and imidazole, each of which
was mixed with guanidine hydrochloride (Gn·HCl) as the
denaturant. N-Methylpyrrolidinone (NMP) was chosen as a
suitable cosolvent. The buffers were incubated in the
presence of Ac-LYRAA-S(CH2)2CONH2, which contains a
C-terminal alanine residue, so that any hydrolysis and
epimerization instigated by the buffer could be assessed
(see the Supporting Information). The use of potassium
phosphate, HEPES, and imidazole in combination with 6m
Gn·HCl in the absence of an organic cosolvent caused 80–
100% hydrolysis of the thioester in 48 h; thus, these
conditions were not suitable for the cysteine-free ligation
reaction (see Figure S1 in the Supporting Information). A 4:1
v/v mixture of NMP and HEPES or imidazole buffer
drastically suppressed hydrolysis of the thioester (< 15%
after 48 h) with no detectable epimerization of the C-terminal
alanine residue (see Figure S1 in the Supporting Informa-
Tyr
Tyr
Gly
Gly
Ser(b-GlcNAc)
Thr(a-GalNAc)
[a] Buffer: Gn·HCl (6m), HEPES (1m), pH 8.5. [b] Yield of the isolated
product. [c] Reaction time: 48 h. [d] Reaction time: 96 h.
To study the efficiency of this cysteine-free ligation
reaction in the presence of amino acids other than glycine,
peptides with a range of N-terminal residues (Gly, His, Ala,
Asp, Glu, and Tyr) and peptide thioesters with a range of
C-terminal residues (Gly, Ala, and His) were synthesized by
solid-phase peptide synthesis (SPPS; see the Supporting
Information). These peptides were ligated in the mixed-
solvent system (Table 1). The results show a general trend,
whereby the N-terminal amino acid of the peptide appears to
be more influential than the substitution of the C-terminal
thioester component on the yield of the aminolysis reaction.
When glycine was the N-terminal amino acid (Table 1,
entries 1–3), the ligation reactions were very efficient and
ligation products were obtained in quantitative yield for
peptide thioesters containing a C-terminal alanine or histidine
residue. The reaction yields were also excellent (89–100%)
for the ligation of peptides with N-terminal aspartic acid and
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glutamic acid residues (Table 1, entries 10–14). The incorpo-
ration of an N-terminal alanine residue resulted in diminished
ligation yields (Table 1, entries 7–9). Peptides containing
sterically demanding N-terminal residues were still able to
undergo direct aminolysis reactions in satisfactory yields.
Specifically, a peptide with an N-terminal histidine residue
reacted with a range of thioesters to give the desired products
in 40–57% yield (Table 1, entries 4-6), whereas lower yields
were observed when a peptide with an N-terminal tyrosine
residue was used (entries 15–17). All reactions were carried
out at a final pH value of 7.3–7.6 (as a result of the acidity of
the peptides, which were prepared as trifluoroacetate salts)
and appeared to be free of epimerization. The ligation
products were obtained with intact stereochemical integrity,
as deduced from the presence of a single peak in their HPLC
trace under analytical conditions suitable for the resolution of
epimerized peptides.
Having demonstrated the efficiency of the direct aminol-
ysis reaction for the synthesis of peptides, we next inves-
tigated the ligation of unprotected glycopeptides. Ligation
reactions between thioester 1 and glycopeptides containing
Ser-b-GlcNAc and Thr-a-GalNAc gave the desired ligation
products in good yields (Table 1, entries 18 and 19). Thus, the
direct aminolysis reactions also appear to be efficient when
glycans are present on the peptide backbone.
solvent system. With the exception of peptide 7, the ligation
reactions proceeded in good yields (69–93%) which were
comparable with the yield observed in the NCL reaction of 4
(94%; Table 2, entry 1). We performed kinetic studies to gain
insight into the relative rates of these ligation reactions in the
presence and absence of internal cysteine residues (see
Figure S2 in the Supporting Information). These studies
showed that although NCL is faster than direct aminolysis,
reactions still proceeded at synthetically useful rates. Fur-
thermore, these rates suggest that ligation reactions proposed
previously to occur via large-ring (> 21-membered-ring) SAL
transition states may actually proceed through direct inter-
molecular aminolysis.[14]
Having established that the ligation tolerates the incor-
poration of unprotected cysteine residues, we synthesized a
series of peptides based on 8 with N-terminal glycine,
histidine, alanine, aspartic acid, and glutamic acid residues
(see the Supporting Information). These peptides were
ligated under the mixed-solvent conditions with peptide
thioesters containing C-terminal glycine, alanine, and histi-
dine residues (see Table S1 in the Supporting Information for
ligation reactions and yields). These reactions followed
similar general trends and afforded the products in compa-
rable yields to those described in Table 1. Thus, the presence
of unprotected cysteine residues does not have a marked
effect on ligation efficiency.
The presence of internal cysteine residues is known to be
problematic in peptide ligation reactions as a result of the
reversible formation of unproductive thioesters and thiolac-
tones and consequent lowering of the reaction rate.[21] The
current strategy employed to overcome this issue relies on the
protection of internal cysteine residues with an Acm
group.[5,20–22] We were interested in assessing the efficiency
of our ligation reaction in the presence of unprotected
internal cysteine residues to streamline synthetic efforts and
prevent additional protection and deprotection steps in the
synthetic sequence. To this end, the six hexameric peptides
4–9 were synthesized by SPPS (see the Supporting Informa-
tion). These constructs contain cysteine residues at all
possible positions along the backbone. Peptide 4 has an N-
terminal cysteine residue, which was expected to undergo an
NCL reaction with a peptide thioester, whereas peptide 9
contains a cysteine residue at the C terminus (Table 2).
Peptides 4–9 were treated with the peptide thioester 1,
which contains a C-terminal glycine residue, in the mixed-
As a result of the lower reaction rate displayed by the
cysteine-free ligation relative to that of NCL, we anticipated
chemoselectivity issues in the presence of the free e-amino
side chain of lysine. To establish whether this concern was
founded, we synthesized peptide 10 with an internal lysine
residue (see the Supporting Information). The treatment of
this peptide with peptide thioester 1, which contains a
C-terminal glycine residue, gave a 2:1 regioisomeric mixture
of the desired ligation product 12 (66%) and the undesired
product with an acylated lysine side chain (34%; Scheme 3).
Attempts to bias the formation of the desired ligation product
by modifying the pH value of the buffer and the concen-
tration of the reaction mixture were unsuccessful. In a similar
manner to that described by Danishefsky and co-workers,[20]
we used the ivDde group for orthogonal protection of the
e-amino functionality. The lysine(ivDde)-containing peptide
11 was ligated to the peptide thioester 1 in 86% yield, and the
ivDde protecting group was subsequently removed in high
yield by hydrazinolysis.
Owing to our interest in the synthesis of glycopeptides and
glycoproteins, our vision for this method extended to the
Table 2: Ligation of peptides containing cysteine residues (R=
(CH2)2CONH2).[a]
Entry
Peptide
Yield [%][b]
1[c]
2[c]
3[c]
4[d]
5[c]
6[c]
H-CSPGYS-NH2 (4)
H-GCPGYS-NH2 (5)
H-GSCGYS-NH2 (6)
H-GSPCYS-NH2 (7)
H-GSPGCY-NH2 (8)
H-GSPGYC-NH2 (9)
94
69
93
44
84
87
[a] Reaction conditions: 4:1 v/v NMP/buffer: Gn·HCl (6m), HEPES
(1m), pH 8.5), PhSH (2% v/v), 378C, final pH value: 7.3–7.6. [b] Yield of
the isolated product. [c] Reaction time: 48 h. [d] Reaction time: 96 h.
Scheme 3. Chemoselectivity of the cysteine-free aminolysis reaction in
the presence of an internal lysine residue. [a] Buffer: Gn·HCl (6m),
HEPES (1m), pH 8.5. R=(CH2)2CONH2.
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synthesis of a homogeneous glycopeptide of biological
poration of cancer-associated glycans into the MUC1 repeat-
ing unit is recognized as a promising target for production of
immunostimulating antigens and the development of cancer
vaccines.[24,25]
As an initial demonstration, the TN antigen (a-GalNAc)
was attached to two threonine residues within the 20-mer
repeat unit. The key glycopeptide thioester fragment 14 was
synthesized on sulfamylbutyryl resin by using an activation
and thiol-release strategy (see the Supporting Information).[4]
Trifluoroacetamide-protected glycine was coupled as the
relevance. We chose to synthesize a native trimeric 60-mer
section of the cancer-associated MUC1 tandem-repeat glyco-
protein. MUC1 is a heavily O-glycosylated glycoprotein that
is present at the interface between epithelial cells and their
extracellular matrix.[23] The extracellular domain consists of
tandem repeating units comprising twenty amino acid resi-
dues. In cancer, MUC1 is overexpressed and aberrantly
glycosylated with truncated glycans, resulting from down-
regulation of glycosyltransferases. Therefore, specific incor-
Scheme 4. Synthesis of a 60-mer MUC1 repeat glycopeptide by application of the cysteine-free aminolysis. [a] Buffer: Gn·HCl (6m), HEPES (1m),
pH 8.5. R=(CH2)2CO2Et; Fmoc=9-fluorenylmethoxycarbonyl, TFA=trifluoroacetic acid, TMS=trimethylsilyl.
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N-terminal amino acid to facilitate sequential fragment
condensations under the direct aminolysis conditions without
generating cyclization and polymerization by-products. After
complete assembly on the resin, activation of the sulfonamide
linkage with trimethylsilyldiazomethane[26] and release from
the solid support upon treatment with ethyl-3-mercaptopro-
pionate gave the desired 20-mer diglycopeptide thioester 14
in 27% yield (see the Supporting Information). The treat-
ment of 14 with dilute aqueous NaOH for 48 h followed by
brief hydrazinolysis gave the fully deprotected diglycopeptide
15 (92% yield), which was ligated with the protected
glycopeptide thioester 14 under the conditions for direct
aminolysis (Scheme 4). After 60 h, the reaction had reached
completion (as determined by LC–MS), and the ligation
mixture was treated with 5% hydrazine to deacetylate the
glycans and remove the N-terminal trifluoroacetamide group.
After purification by HPLC, the desired dimeric 4.6-kDa
tetraglycopeptide of MUC1 was isolated in 77% yield. The
MUC1 dimer 16 was submitted to a subsequent ligation with
the 20-mer glycopeptide thioester 14 to afford, after hydra-
zinolysis and HPLC purification, the trimeric 6.9-kDa MUC1
repeat hexaglycopeptide 17 (1.2 mg) in 80% yield, as verified
by analytical HPLC, LC–MS, and MALDI-TOF mass spec-
trometry. The efficiency with which these large glycopeptide
fragments can be constructed by the direct aminolysis method
demonstrates clearly its potential for the production of
complex peptides and even proteins, with or without post-
translational modifications.
larger oligomeric MUC1 constructs by extending the iterative
ligation strategy reported here, with the intention of produc-
ing new immunogenic glycopeptides for the production of
immunostimulating antigens. It is hoped that these constructs
may ultimately aid in the development of cancer vaccines.
Received: November 18, 2007
Revised: January 15, 2008
Published online: April 29, 2008
Keywords: glycopeptides · glycoproteins · ligation · peptides ·
.
vaccines
[1]T. Wieland, E. Bokelmann, L. Bauer, H. U. Lang, H. Lau, Justus
Liebigs Ann. Chem. 1953, 583, 129 – 149.
[2]P. E. Dawson, T. W. Muir, I. Clarklewis, S. B. H. Kent, Science
1994, 266, 776 – 779.
[3]P. E. Dawson, S. B. H. Kent, Annu. Rev. Biochem. 2000, 69, 923 –
960.
[4]Y. Shin, K. A. Winans, B. J. Backes, S. B. H. Kent, J. A. Ellman,
C. R. Bertozzi, J. Am. Chem. Soc. 1999, 121, 11684 – 11689.
[5]B. L. Pentelute, S. B. H. Kent, Org. Lett. 2007, 9, 687 – 690.
[6]D. Crich, A. Banerjee, J. Am. Chem. Soc. 2007, 129, 10064 –
10065.
[7]Other cysteine residues in the peptide or protein must be
protected when these desulfurization methods are used.
[8]J. Offer, C. N. C. Boddy, P. E. Dawson, J. Am. Chem. Soc. 2002,
124, 4642 – 4646.
[9]D. W. Low, M. G. Hill, M. R. Carrasco, S. B. H. Kent, P. Botti,
Proc. Natl. Acad. Sci. USA 2001, 98, 6554 – 6559.
[10]D. Macmillan, D. W. Anderson, Org. Lett. 2004, 6, 4659 – 4662.
[11]B. Wu, J. H. Chen, J. D. Warren, G. Chen, Z. H. Hua, S. J.
Danishefsky, Angew. Chem. 2006, 118, 4222 – 4231; Angew.
Chem. Int. Ed. 2006, 45, 4116 – 4125.
In summary, we have described the discovery of an
effective new method for the assembly of peptides and
glycopeptides by direct aminolysis in the absence of an
N-terminal auxiliary or cysteine residue. Ligation reactions
[12]A. Brik, Y. Y. Yang, S. Ficht, C. H. Wong, J. Am. Chem. Soc.
2006, 128, 5626 – 5627.
are performed in
a mixed-solvent system containing
N-methylpyrrolidinone and guanidine hydrochloride/
HEPES buffer. This medium allows reactions to proceed at
reasonable rates with minimal hydrolysis of the thioester
component. The ligation products were isolated in good
yields, and the reactions proceeded without epimerization.
The method proved to be general, as demonstrated by the use
of a wide range of amino acid residues at the ligation junction.
Furthermore, the method is chemoselective in the presence of
all naturally occurring side chains, with the exception of
lysine, which must be protected.
[13]A. Brik, S. Ficht, Y. Y. Yang, C. S. Bennett, C. H. Wong, J. Am.
Chem. Soc. 2006, 128, 15026 – 15033.
[14]S. Ficht, R. J. Payne, A. Brik, C. H. Wong, Angew. Chem. 2007,
119, 6079 – 6083; Angew. Chem. Int. Ed. 2007, 46, 5975 – 5979.
[15]D. S. Kemp, Z. W. Bernstein, G. N. McNeil, J. Org. Chem. 1974,
39, 2831 – 2835.
[16]D. S. Kemp, S. L. H. Choong, J. Pekaar, J. Org. Chem. 1974, 39,
3841 – 3847.
[17]J. Blake, Int. J. Pept. Protein Res. 1981, 17, 273 – 274.
[18]S. Aimoto, N. Mizoguchi, H. Hojo, S. Yoshimura, Bull. Chem.
Soc. Jpn. 1989, 62, 524 – 531.
[19]S. Aimoto, Biopolymers 1999, 51, 247 – 265.
[20]G. Chen, Q. Wan, Z. Tan, C. Kan, Z. Hua, K. Ranganathan, S. J.
Danishefsky, Angew. Chem. 2007, 119, 7527 – 7531; Angew.
Chem. Int. Ed. 2007, 46, 7383 – 7387.
[21]T. Durek, V. Y. Torbeev, S. B. H. Kent, Proc. Natl. Acad. Sci.
USA 2007, 104, 4846 – 4851.
[22]Y. Y. Yang, S. Ficht, A. Brik, C. H. Wong, J. Am. Chem. Soc.
2007, 129, 7690 – 7701.
[23]S. Dziadek, H. Kunz, Synlett 2003, 1623 – 1626.
[24]M. A. Tarp, H. Clausen, Biochim. Biophys. Acta 2008, 1780,
546 – 563.
This method clearly shows potential for application in the
synthesis of a host of native (glyco)peptide and (glyco)protein
targets that can not be constructed by other currently
available ligation techniques. Additionally, the buffer system
may prove useful in preventing thioester hydrolysis in other
ligation reactions (e.g. NCL). To demonstrate its value for the
production of more-complex targets, the method was imple-
mented successfully in the synthesis of a 6.9-kDa section of
the cancer-associated MUC1 glycoprotein, which contains six
glycosylation sites. Current efforts in our laboratory are
focused on the use of the direct aminolysis reaction for the
construction of more-complex MUC1 glycopeptides by
incorporating other cancer-associated glycans, such as the
Thomsen–Friedenreich antigen (T antigen), sialyl T, and
GloboH. Furthermore, we are attempting to synthesize
[25]S. Dziadek, D. Kowalczyk, H. Kunz, Angew. Chem. 2005, 117,
7798 – 7803; Angew. Chem. Int. Ed. 2005, 44, 7624 – 7630.
[26]S. Mezzato, M. Schaffrath, C. Unverzagt, Angew. Chem. 2005,
117, 1677 – 1681; Angew. Chem. Int. Ed. 2005, 44, 1650 – 1654.
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