A program for ligation at threonine sites: application to the controlled total synthesis of glycopeptides

Article abstract of DOI:10.1016/j.tet.2010.01.067

A method by which to accomplish formal threonine ligation has been developed. The method accomplishes ligations of two peptide domains. We have also demonstrated the ability to successfully ligate two independent glycopeptide domains.

Full text of DOI:10.1016/j.tet.2010.01.067

Tetrahedron 66 (2010) 2277–2283
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
A program for ligation at threonine sites: application to the controlled
total synthesis of glycopeptides
,
,b,
Jin Chen a y, Ping Wang a, Jianglong Zhu a, Qian Wan a, Samuel J. Danishefskya
*
a Laboratory for Bioorganic Chemistry, Sloan-Kettering Institute for Cancer Research, 1275 York Avenue, New York, NY 10065, USA
b Department of Chemistry, Columbia University, Havemeyer Hall, 3000 Broadway, New York, NY 10027, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A method by which to accomplish formal threonine ligation has been developed. The method accom-
plishes ligations of two peptide domains. We have also demonstrated the ability to successfully ligate
two independent glycopeptide domains.
Received 9 December 2009
Received in revised form 14 January 2010
Accepted 15 January 2010
Available online 22 January 2010
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
possessing targeted modifications in the carbohydrate or protein
domains. Of course, such targets could not necessarily be obtained
Glycoproteins are
a
class of naturally occurring bio-
through purely biologic means, since they would lack the viable
biosynthetic pathways for reaching the desired structural types. In
the context of our glycoprotein synthesis program, we have tar-
geted for total synthesis the clinically relevant glycoproteins, EPO
and hFSH.8 Several years ago, at the outset of our EPO total syn-
thesis endeavor, we identified a number of significant methodo-
logical shortcomings that would need to be addressed before
a viable total synthesis effort could be launched.
macromolecules, which are biosynthesized through post-trans-
lational protein glycosylation. A great deal of effort has been
directed toward the examination of the role that glycosylation plays
in various critical protein functions, such as protein folding, pro-
teolytic stability, and intercellular communication.1 Of particular
interest is the fact that many glycoproteins possess exploitable
therapeutic activity, and may serve as promising candidates in the
development of vaccines,2 diagnostic techniques,3 and therapeutic
agents.4 Prominent examples of therapeutically valuable glyco-
proteins include the erythropoietic agent, erythropoietin (EPO)4a,5
and the fertility agent, human follicle stimulating hormone
(hFSH).6 Despite considerable interest in this class of bio-
macromolecules, the field of glycobiology faces a significant ob-
stacle to the rigorous evaluation of glycoproteins: the isolation of
significant quantities of homogeneous glycoproteins from natural
sources is often prohibitively difficult, due to the fact that most
naturally occurring glycoproteins are biosynthesized as heteroge-
neous mixtures of glycoforms.
In particular, we were concerned with the dearth of general
techniques and protocols available for the merging of two glyco-
peptide fragments. To address this issue, we developed an efficient
method for the coupling of two differentially glycosylated peptide
fragments. This advance was predicated on the landmark break-
through of Kent and co-workers in the field of peptide synthesis,
termed native chemical ligation (NCL).9 NCL is a widely used
technique that allows for the merger of large peptide fragments,
one of which presents a C-terminal thioester, and the other an N-
terminal cysteine residue (Fig. 1a). Direct extension of the NCL
method to the realm of glycopeptide synthesis is complicated by
the difficulties associated with synthesizing pre-formed glyco-
peptide thioester. The solution developed in our laboratories
(outlined in Fig. 1b) involves a relatively stable C-terminal ortho-
thiophenolic ester, presented on one of the glycopeptides, and
a protected N-terminal cysteine residue incorporated on the other
fragment.10 Upon simultaneous reduction of the two disulfides, the
phenol moiety undergoes intramolecular O/S migration, pro-
viding an intermediate thioester, which undergoes thioester ex-
change with the free cysteine of the second glycopeptide. The
intermediate then suffers spontaneous intramolecular transfer to
yield the bidomainal glycopeptide adduct, incorporating two dif-
ferentiated sites of glycosylation. The general logic of this NCL
method has more recently been extended to a direct oxo-ester
Given the great difficulties associated with the isolation of ho-
mogeneous glycoproteins, we recognized that an opportunity for
chemical synthesis might lay in the challenge of using total
chemical synthesis to gain access to homogeneous glycoprotein
samples.7 The biology of such agents could then be studied in fur-
ther detail. Moreover, through chemical synthesis, it would be
possible to gain access to fully synthetic analog glycoproteins,
* Corresponding author. Tel.: þ1 212 639 5502; fax: þ1 212 772 8691.
E-mail address: s-danishefsky@ski.mskcc.org (S.J. Danishefsky).
Current Location: Department of Chemistry, Michigan Technological University,
y
1400 Townsend Dr., Houghton, MI 49931, USA
0040-4020/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2010.01.067

2278
J. Chen et al. / Tetrahedron 66 (2010) 2277–2283
(a)Native Chemical Ligation (Kent et. al.)
O
SH
HS
O
P1
S
O
O
H
H
N
H
N
N
Peptide 1
N
H
Peptide 2
+
SAr
Peptide 1
Peptide 1
S
R
H2N
Peptide 2
H2N
P2
O
O
+
O
+
HS-Ar
HS-Ar
(c)
(b)
→
Cysteine Alanine Reduction
NCL Phenolic Ester Variation
SH
HO
S
SH
O
O
O
O
Me
3. S
4. S
HS
S
N
H
1. Cleave S–S
H
H
N
TCEP, tBuSH
N
N
GP1
N
H
GP2
GP1
N
H
P2
GP1
O
GP1
N
H
P2
2. O
S
VA-044
O
O
SSEt
O
GP1
O
H
N
cys
ala
GP = glycopeptide
H2N
GP2
O
Threonine Ligation
(d)
SH
OH
(i)
Me
OH
OH
O
O
HS
O
H
desulfurization
H
H
+
N
N
N
Peptide 1
X
Peptide 1
N
H
Peptide 2
Peptide 1
N
H
Peptide 2
H2N
Peptide 2
O
(ii)
O
O
thr
γ
-thio-thr
Figure 1. (a) Native chemical ligation; (b) Glycopeptide NCL; (c) Free-radical-mediated reduction of Cys/Ala; (d) Proposed native chemical ligation at threonine.
variant, in which a phenolic ester equipped with para-NO2 or para-
CN substitution, is sufficiently activated to undergo direct cysteine
ligation without the need for the intermediacy of a thioester
species.11
Moreover, a dual native chemical ligation at lysine has also been
achieved recently. A -thiol group on the N-terminal lysine medi-
g
ates double chemical ligation at both
a and 3 amines, followed by
a free-radical desulfurization.20
These glycopeptide ligation protocols do suffer from an impor-
tant limitation in terms of generalizability, in that they require the
presence of a cysteine residue at the ligation site. In fact, there is
often a paucity of cysteine sites in naturally occurring proteins and
glycoproteins. The need for a menu of efficient cysteine-free ligation
methods thus remains quite high.
The development of a variety of methods by which to formally
accomplish NCL at a diverse range of amino acid residues could be
of substantial value. As noted above, due to its low frequency in
nature, direct ligation at cysteine itself is of limited practical value.
Furthermore, in developing a synthetic strategy toward a glyco-
protein or glycopeptide target, it is often desirable to merge two
pieces of relatively equal size. As the menu of amino acid ligation
options expands, greater flexibility may be brought to the design of
synthetic routes. Finally, the two-step ligation/reduction strategy
provides the opportunity to explore the consequences of protein
engineering with site-specifically modified glycoproteins.
In the hopes of broadening the range of options for amino acid
ligation, we sought to extend our two-step ligation/reduction
protocol to the development of a formal threonine ligation. Thre-
onine was selected for its relative abundance in nature, particularly
in comparison with cysteine. We describe herein the discovery and
formulation of a mild and efficient two-step formal threonine li-
gation protocol.
A number of useful solutions to the cysteine ligation problem
have been developed, many of which depend on the use of cysteine
or a thiol-containing amino acid surrogate in the ligation step.
Following thiol-mediated ligation, the cysteine or surrogate residue
is converted to the desired amino acid. In this way, methionine li-
gation has been achieved through homo-cysteine coupling, fol-
lowed by post-ligational methylation.12 Similarly, serine ligation
has been accomplished through NCL followed by conversion of
cysteine to serine.13 A number of cysteine-free ligation methods
make use of a two-step ligation–desulfurization sequence. For in-
stance, metal-based post-ligational thiol reduction has been used
to accomplish formal alanine14 and phenylalanine15 ligations. De-
spite the appeal of such a strategy, we noted that traditional de-
sulfurization methods suffer from a lack of substrate generality, due
to the susceptibility of many common functional groups (particu-
larly thiol moieties) to the standard harsh reduction conditions.
Based on the disclosure by Hoffmann and Walling in the 1950s,16
we recently developed a very mild and chemoselective free-
radical-mediated desulfurization strategy, which allows for the
post-translational conversion of cysteine to alanine in complex
glycopeptide and peptide settings (Fig. 1c).17 This method has
proven to be tolerant of a wide range of functionalities, including
sulfur-containing groupsdsuch as Thz, Cys(Acm), biotin, and thi-
oestersdas well as amino acids, including methionine, and even
complex carbohydrate moieties. This overall alanine ligation
strategy has now been successfully applied in the context of the
synthesis of a complex glycopeptide fragment of erythropoietin
(Ala1-Gly28).8c In addition, our mild sulfur reduction protocol has
been employed to accomplish a formal valine ligation, through the
coupling and post-ligational reduction of unnatural amino acid
The central idea is outlined in Figure 1d. Thus, as in the case of
the valine ligation,18 a thiol-containing threonine surrogate would
be incorporated at the N-terminus of Peptide 2. One could envision
two possible sites at which to install a thiol group onto the threo-
nine residue: at the
the -thiol threonine would serve as the more productive surrogate
in establishing the initial acylation event. Peptide 2, incorporating
the -thiol threonine, was expected to undergo trans-thioester-
b position or the g position. We expected that
g
g
ification with Peptide 1 (presented as either a thioester or activated
oxo-ester), to generate a thioester-linked intermediate, which
would then undergo spontaneous intramolecular acyl transfer,
generating the new amide bond. Subsequent radical-based de-
sulfurization would serve to remove the thiol, providing the target
peptide with threonine at the ligation site.
2. Results and discussion
Our first objective was to synthesize a
g-thiol threonine amino
surrogates (g b
-thiol valine18 or -thiol valine18,19) to valine residues.
acid surrogate. To accomplish this, we drew on the earlier work of

J. Chen et al. / Tetrahedron 66 (2010) 2277–2283
2279
Table 1
Threonine extension by coupling and subsequent free-radical desulfurization
Me
OH
OMe
S
OH
OMe
O
Me
S
O
1) Couplinga
2) Desulfurizationb
+
Peptide
N
H
Peptide
H2N
X
O
O
γ -thiol-thr (5)
thr
Entry
1
Peptide
Coupling Product/Yield/Time
Desulfurization Product/Yield/Time
O
SH
FmocRNEDLSGAT
9 / 92 % / 2 h
OMe
FmocRNEDLSGAT
OMe
FmocRNEDLSGA
O
SSEt
SSEt
8 / 88% / 30 min
7
O
SH
FmocRLGDSTAGQT OMe
12 / 81% / 2 h
2
3
4
FmocRLGDSTAGQT
OMe
FmocRLGDSTAGQ
O
11 / 81% / 30 min
10
SH
O
OMe
FmocRLGDSTAGYT
15 / 89% / 2 h
OMe
FmocRLGDSTAGYT
FmocRLGDSTAGY
13
SPh
14 / 85% / 30 min
O
SH
FmocRLGDSTAGWT
OMe
FmocRLGDSTAGWT
OMe
FmocRLGDSTAGW
16
SPh
18 / 90% / 2 h
17 / 90% / 30 min
O
SH
FmocRTGDSAGTT
OMe
OMe
5
6
FmocRTGDSAGT
FmocRTGDSAGTT
OMe
SPh
21 / 84% / 2 h
20 / 77% / 1 h
19
SH
O
FmocRLGDSTAGLT
OMe
FmocRLGDSTAGLT
23 / 95% / 1 h
FmocRLGDSTAGL
22
SPh
NO2
24 / 80% / 2 h
SH
O
OMe
FmocKYDSRGFT
7
8
OMe
FmocKYDSRGFT
FmocKYDSRGF
25
O
27 / 80% / 2 h
26 / 95% / 30 min
NO2
SH
O
O
FmocRTGDSAGVT
OMe
FmocRTGDSAGVT
OMe
OMe
FmocRTGDSAGV
28
O
30 / 95% / 2 h
29 / 97% / 5 h
NO2
NO2
SH
FmocRTGDSAGIT
OMe
9
FmocRTGDSAGIT
32 / 90% / 5 h
FmocRTGDSAGI
O
33 / 82% / 2 h
31
SH
O
FmocVRSYTAGPT
OMe
10
FmocVRSYTAGPT
OMe
FmocVRSYTAGP
O
36 / 83% / 2 h
35 / 52% / 10 h
34
Key: (a) buffer at pH 6.5 (6.0 M Gn$HCl, 188.8 mM Na2HPO4), TCEP, rt (b) TCEP, VA-044, tBuSH, 37 ꢀC. VA-044¼2,2’-azobis-[2-(2-imidazolin-2-yl)propane] dihydrochloride.
TCEP¼tris(2-carboxyethyl)phosphine.
Rapoport and co-workers,21 in which allo-threonine was obtained
from -vinylglycine. Through modification of the Rapoport route,
we sought to accomplish the diastereoselective syntheses of the
-thiol threonine derivatives, 5 and 6 (Scheme 1). Compound 5 was
to the sodium salt of thioacetic acid, epoxide 2a was opened to
provide the acetylated thiol, 3. The latter was transformed, in
a straightforward fashion, to the target compounds, 5 and 6.
D
g
With g-thiol threonine derivative 5 in hand, we next sought to
to be used directly for single amino acid extension studies
(see Table 1), while compound 6 would be elaborated to peptide 37,
which would serve as the substrate in the threonine ligation studies
at the peptide level (see Table 2). Thus, as shown in Scheme 1,
vinylglycine 122 was epoxidized with an excess of mCPBA to afford
a 5:1 ratio of syn and anti epoxides, 2a and 2b, which could be
separated by chromatography. The major diastereomer, 2a, has the
desired syn configuration, perhaps as a consequence of hydrogen
bonding of mCPBA to the nitrogen functionality.23 Upon exposure
evaluate the feasibility of the proposed threonine ligation method.
We first examined the protocol in the context of a single amino acid
extension of a variety of peptide substrates. As shown in Table 1,
when a relatively sterically less demanding amino acid (Ala, Gln,
Tyr, Trp, Phe) was presented at the C-terminus of the peptide, the
amino acid extension generally proceeded very quickly and with
good yield (see entries 1–4 and 7). Even when the
b-branched
amino acid, threonine, was incorporated at the C-terminus, the
coupling was complete within 1 h (entry 5). As expected, as the C-

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J. Chen et al. / Tetrahedron 66 (2010) 2277–2283
NHFmoc
OMe
NHFmoc
OMe
NHFmoc
OMe
NHFmoc
OMe
mCPBA
AcSH, NaOAc
Toluene, DMF
RT, 2 h
AcS
S
+
→
O
O
S
CH2Cl2, 0 ºC RT,
OH
O
O
O
O
24 h
3
1
2a
2b
5
:
1
NH2
1) Boc2O,THF
MeOH, TEA
NHBoc
NHFmoc
1) 0.2 N NaOH, MeOH
0 ºC, 20 min
DEA
DMF
OMe
OH
S
OMe
S
S
S
2) 1 N NaOH,
H2O, THF
2) MMTS, DIEA
CH2Cl2, RT, 2 h
OH
5
O
RT, 2 h
OH
O
OH
O
4
6
Scheme 1. Synthesis of g-thiol threonine.
Table 2
NCL at threonine through ligation and subsequent free-radical desulfurization
Me
OH
Peptide B
S
OH
Peptide B
O
Me
S
O
1) NCLa
2) Desulfurizationb
+
Peptide A
N
H
H2N
Peptide A
X
O
O
37
thr
Peptide B = GAPRHSWG OMe
OH
O
HO
HO
HO
OH
O
HO
HO
HO
O
OH
OH
OH
O
O
O
O
O
HO
O
NH
HO
O
NHAc
NHAc
HO
HO
HO
O
OH
glycan (entry 4)
Entry
1
Peptide A
Ligation Product/Yield/Time
Desulfurization Product/Yield/Time
SH
O
FmocRLGDSTAGYTGAPRHSWG OMe
39 / 98% / 2 h
FmocRLGDSTAGYTGAPRHSWG
OMe
OMe
FmocRLGDSTAGY
13
SPh
SPh
38 / 70% / 1 h
O
SH
FmocRLGDSTAGWTGAPRHSWG
41 / 85% / 2 h
OMe
2
3
FmocRLGDSTAGWTGAPRHSWG
FmocRLGDSTAGW
16
40 / 73% / 1 h
NO2
SH
O
FmocRTGDSAGITGAPRHSWG
43 / 96% / 2 h
OMe
OMe
FmocRTGDSAGITGAPRHSWG
42 / 63% / 7 h
FmocRTGDSAGI
31
O
glycan
glycan
glycan
O
SH
4
OMe
FmocRLGNSTAGQTGAPRHSWG
FmocRLGNSTAGQ
OMe
O
FmocRLGNSTAGQTGAPRHSWG
45 / 82% / 2 h
SSEt
46 / 96% / 2 h
44
Key: (a) buffer at pH 6.5 (6.0 M Gn$HCl, 188.8 mM Na2HPO4), TCEP, RT; (b) TCEP, VA-044, tBuSH, 37 ꢀC.
terminus became more sterically hindered (Leu, Val, Ile, Pro), the
reaction rate suffered. However, coupling could still be accom-
plished within a reasonable time frame, and in moderate to good
yields (entries 6, 8–10). As shown in Table 1, a variety of different C-
terminal esters participated successfully in the extension protocol,
including thiophenyl ester, ortho-thiophenolic ester, and para-
nitrophenyl ester. The diversity of C-terminal esters amenable to
this protocol is of significance, as the ortho-thiophenolic ester is
compatible with glycopeptide ligation, while para-nitrophenyl es-
ter is particularly efficient at promoting coupling at sterically hin-
dered ligation sites. We also note that the threonine ligation
protocol is able to accommodate the presence of an unprotected
lysine in the substrate peptide (25, entry 7). All of the coupling
products obtained in Table 1 were subsequently subjected to our
standard radical-based desulfurization conditions to provide the
desired threonine extension products in very good yields.
We now sought to examine the two-step ligation/reduction
protocol in the context of a peptide-peptide coupling. Peptide 37,
possessing the g-thiol threonine surrogate at its N-terminus, was
prepared from compound 6 (see Supplementary data for details).
According to our general procedure, 37 and the peptide coupling
partner were dissolved in a guanidine buffer solution. Upon addi-
tion of TCEP, the disulfide moieties were cleaved to presumably give
rise to the free thiol functionalities, which then underwent the
anticipated ligation reaction. As shown in Table 2, we found the
ligation rate to be dependent on the nature of the C-terminal amino

J. Chen et al. / Tetrahedron 66 (2010) 2277–2283
2281
acid. Thus, when a less sterically demanding amino aciddsuch as
Tyr (13) or Trp (16)dwas present at the C-terminus, ligation was
complete within 1 h (entries 1 and 2). However, in the case of the
more hindered amino acid, Ile (31), the reaction took up to 7 h to
reach completion (entry 3). Once more, in each case, subsequent
free-radical-based desulfurization was readily achieved in high
yields through use of our mild reduction method. Finally, as shown
in Table 2, entry 4, our new protocol could be readily extended to
a glycopeptide ligation setting. Thus, peptide 44, presenting an N-
linked hexasaccharide domain, underwent ligation with peptide 37
to provide glycopeptide 45 in 82% yield. Upon exposure to our
previously described reduction conditions, glycopeptide 46, in-
corporating threonine at the ligation site, was obtained in 96%
yield.
suspension, which was centrifuged and the ether subsequently
decanted. The resulting solid was ready for HPLC purification.
4.2. Preparation and characterization of compounds 2a–6
4.2.1. (S)-Methyl 2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-2-
((S)-oxiran-2-yl) acetate (2a). To a stirred solution of 1 (200 mg,
0.593 mmol) in CH2Cl2 (6 mL) at 0 ꢀC was added m-chloroperbenzoic
acid (1.022 g, 5.93 mmol) and the reaction was warmed to rt. After
24 h, the mixture was filtered through a glass filter, the solids were
extracted with CH2Cl2, and the combined organic phase was washed
with 10% NaHCO3, water, dried, and evaporated. The two di-
astereomers were then separated by chromatography (CH2Cl2/
MeOH¼300:1) to give 2a (138 mg, 67% yield) as awhite solid.1H NMR
3. Conclusion
(500 MHz, CDCl3) d 2.59 (m,1H), 2.76 (m,1H), 3.46 (s,1H), 3.81 (s, 3H),
4.20 (t, J¼6.8 Hz,1H), 4.40 (d, J¼6.9 Hz, 2H), 4.70 (dd, J¼1.5 Hz, 8.9 Hz,
1H), 5.23 (d, J¼8.8 Hz, 1H), 7.29 (m, 2H), 7.32 (t, J¼6.3 Hz, 2H), 7.56 (t,
J¼6.0 Hz, 2H), 7.76 (d, J¼6.3 Hz, 2H); 13C NMR (125 MHz, CDCl3)
In conclusion, we have described the development of a useful
new entry in the field of native chemical ligation. Through an ef-
ficient two-step ligation/reduction protocol, it is now possible to
formally achieve NCL at threonine sites, in both peptide and gly-
copeptide settings. This methodological advance, taken in concert
with the previous entries of our group and others, has served to
significantly expand the NCL menu, which was originally restricted
to cysteine-based ligations. Further applications and extensions of
this method to the synthesis of important biologic level agents are
underway in our laboratory.
d
43.9, 47.1, 51.2, 53.0, 53.1, 67.2, 120.1, 125.1, 127.1, 127.1, 127.8, 141.3,
20
141.4, 143.6, 143.8, 156.2, 170.2; [
a
]
ꢂ5.21 (c 1.08, CHCl3); IR (liquid
D
film) (nmax/cmꢂ1): 3342, 3066, 3019, 2953, 2848, 1724; HRMS: m/e
calcd for C20H19NO5Naþ: 376.1161, found: 376.1153.
4.2.2. (2S,3S)-Methyl 2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-
4-(acetylthio)-3-hydroxybutanoate (3). Compound 2a was dissolved
in toluene (3.2 mL) and treated with a solution of sodium acetate
(88 mg, 1.077 mmol) and thioacetic acid (80 mL, 1.077 mmol) in
4. Experimental section
4.1. General
DMF (1.6 mL). The reaction mixture was stirred at rt for 2 h. The
mixture was concentrated under N2. The concentrate was dissolved
in EtOAc and washed with NH4Cl, water, and brine. The organic
layer was dried, concentrated, and purified by chromatography
(Hexane/EtOAc¼2:1) to give 3 (72 mg, 80%) as a light yellow foam.
Anhydrous THF, diethyl ether, CH2Cl2, toluene, and benzene were
obtained from a dry solvent system (passed through column of
alumina) and used without further drying. NMR spectra (1H and 13C)
were recorded on a Bruker Advance DRX-500 MHz, or a Bruker DRX-
600 MHz. Low-resolution mass spectral analyses were performed
with a JOEL JMS-DX-303-HF mass spectrometer or Waters Micro-
mass ZQ mass spectrometer.
1H NMR (500 MHz, CDCl3)
d 2.35 (s, 3H), 3.01 (m, 3H), 3.75 (s, 3H),
4.23 (m, 2H), 4.41 (d, J¼7.0 Hz, 2H), 4.51 (d, J¼9.3 Hz, 1H), 5.69 (d,
J¼9.4 Hz, 1H), 7.31 (m, 2H), 7.39 (m, 2H), 7.61 (m, 2H), 7.75 (m, 2H);
13C NMR (125 MHz, CDCl3)
d 30.6, 32.8, 47.2, 52.8, 57.3, 67.3, 71.5,
120.0, 120.0, 125.1, 125.2, 127.1, 127.8, 141.3, 141.4, 143.6, 143.8, 156.7,
170.9, 196.6; [
a
]
D
20 11.69 (c 0.68, CHCl3); IR (liquid film) (nmax/cmꢂ1):
HPLC: All separations of peptides and glycopeptides involved
a mobile phase of 0.05% TFA (v/v) in water (solvent A)/0.04% TFA in
acetonitrile (solvent B). Preparative and analytical HPLC separations
were performed using a Rainin HPXL solvent delivery system
equipped with a Rainin UV-1 detector. LC–MS chromatographic
separations were performed using a Waters 2695 Separations
Module and a Waters 996 Photodiode Array Detector equipped
3373, 3065, 3019, 2952, 2847, 1747, 1722, 1697; HRMS: m/e calcd for
C22H23NO6SNaþ: 452.1144, found: 452.1155.
4.2.3. (2S,3S)-Methyl 2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-
3-hydroxy-4-(methyldisulfanyl) butanoate (4). Compound 3 (47 mg,
0.11 mmol) was dissolved in MeOH (1.87 mL) and treated with
0.2 N NaOH solution (1.87 mL) at 0 ꢀC for 20 min. The reaction
mixture was carefully neutralized by the addition of 1 N HCl at 0 ꢀC,
diluted with EtOAc and washed with water and brine. The organic
layer was concentrated and dried in vacuo, generating 43 mg of
crude compound (2S,3S)-methyl 2-(((9H-fluoren-9-yl)methoxy)-
carbonylamino)-3-hydroxy-4-mercaptobutanoate, which was di-
rectly used in the next step.
with X-BridgeÔ C18 column (5.0
C18 column (3.5
m, 2.1ꢁ100.0 mm) or Varian Microsorb C18 col-
umn (2ꢁ150 mm) at a flow rate of 0.2 mL/min. HPLC separations
were performed using: X-BridgeÔ Prep C18 column OBDÔ (5.0 m,
mm, 2.1ꢁ150 mm), X-TerraÔ MS
m
m
19ꢁ150 mm), a flow rate of 16 mL/min. Microsorb 100-5 C18 col-
umn at a flow rate of 16.0 mL/min or Microsorb 300-5 C4 column at
a flow rate of 16.0 mL/min.
S-Methyl methanethiolsulfonate (36
mL, 0.38 mmol) and DIEA
Automated peptide synthesis was performed on an Applied
Biosystems Pioneer continuous flow peptide synthesizer. Peptides
were synthesized under standard automated Fmoc protocols. The
deblock mixture was a mixture of 100/5/5 of DMF/piperidine/DBU.
Upon completion of automated synthesis on a 0.05 mmol scale, the
peptide resin was washed into a peptide synthesis vessel with
CH2Cl2. The resin cleavage was effected by treatment with AcOH/
TFE/CH2Cl2 (2:2:6) for 2ꢁ1 h to yield peptidyl acids in good yield.
The peptidyl acids were modified on C-terminus and/or N-termi-
nus. The resulting peptides were subjected to a deprotection
cocktail (60.0 mg of phenol, 0.2 ml of water, 0.15 ml of triisopro-
pylsilane, and 3.0 ml TFA) for 2.0 h. TFA was removed by N2. The oily
(12 L, 0.11 mmol) were added to CH2Cl2 (0.5 mL). The crude resi-
m
due of (2S,3S)-methyl 2-(((9H-fluoren-9-yl)methoxy)carbonyl-
amino)-3-hydroxy-4-mercaptobutanoate in CH2Cl2 (0.8 mL) was
added dropwise to the above solution and stirred at rt for 2 h. The
reaction mixture was concentrated and purified by chromatogra-
phy (Hexane/EtOAc¼2:1), to provide 4 (33 mg, 70% in two steps) as
a light yellow foam. 1H NMR (500 MHz, CDCl3)
d 2.42 (s, 3H), 2.65
(dd, J¼9.2 Hz 14.2 Hz, 1H), 2.71 (d, J¼2.5 Hz, 1H), 2.85 (dd, J¼3.8 Hz,
14.0 Hz,1H), 3.79 (s, 3H), 4.22 (t, J¼6.8 Hz,1H), 4.43 (m, 3H), 4.51 (d,
J¼9.4 Hz, 1H), 5.58 (d, J¼9.4 Hz, 1H), 7.29 (m, 2H), 7.37 (t, J¼7.1 Hz,
2H), 7.60 (t, J¼7.1 Hz, 2H), 7.75 (m, 2H); 13C NMR (125 MHz, CDCl3)
d
22.9, 41.4, 47.2, 52.9, 57.1, 67.2, 69.9, 120.0, 120.1, 125.1, 127.1, 127.8,
residue was triturated with diethyl ether to give
a
white
141.3, 141.4, 143.6, 143.8, 156.7, 170.9; [a]
20 48.90 (c 0.27, CHCl3); IR
D

2282
J. Chen et al. / Tetrahedron 66 (2010) 2277–2283
(liquid film) (nmax/cmꢂ1): 3406, 3019, 2952, 2918, 2850, 1724;
HRMS: m/e calcd for C21H23NO5S2Naþ: 456.0915, found: 456.0909.
mixture was stirred at 37 ꢀC. The reactions were monitored by LC–
MS and purified directly by HPLC upon consumption of the starting
material.
4.2.4. (2S,3S)-Methyl 2-amino-3-hydroxy-4-(methyldisulfanyl)butanoate
(5). To a stirred solution of 4 (57 mg, 0.131 mmol) in DMF (4.3 mL)
was added diethylamine (1.4 mL). The reaction mixture was stirred
at rt for 2 h and the solvent was evaporated. The residue was pu-
rified by chromatography (CH2Cl2/MeOH¼40:1) to give 5 (25 mg,
4.4.1. MS characterization of peptides 7–46. Compound 7. ESIMS
calcd for C56H74N12O17S2 [MþH]þ m/z¼1251.48, found: 1251.60.
Compound 8. ESIMS calcd for C53H75N13O19
m/z¼1230.51, found: 1231.12.
S
[MþH]þ
89% yield) as a clear oil. 1H NMR (500 MHz, CDCl3)
d
2.43 (s, 3H),
Compound 9. ESIMS calcd for C53H75N13O19 [MþH]þ m/
z¼1198.54, found: 1198.88.
2.84 (dd, J¼6.0 Hz,13.9 Hz,1H), 2.92 (dd, J¼7.0 Hz,13.9 Hz,1H), 3.66
(d, J¼3.1 Hz, 1H), 3.76 (s, 3H), 4.17 (m, 1H); 13C NMR (125 MHz,
Compound 10. ESIMS calcd for C58H79N13O17S2 [MþH]þ m/
z¼1294.53, found: 1294.79.
CDCl3) d 23.1, 41.7, 52.5, 56.2, 70.6, 174.3; [a]
22 76.7 (c 0.3, CHCl3); IR
D
(liquid film) (nmax/cmꢂ1): 3366, 3303, 2952, 2918, 1738, 1437, 1245,
1174, 1022; HRMS: m/e calcd for C6H13NO3S2Hþ: 212.0415, found:
212.0413.
Compound 11. ESIMS calcd for C55H79N14O19S [MþH]þ m/
z¼1273.55, found: 1274.08.
Compound 12. ESIMS calcd for C55H80N14O19 [MþH]þ m/
z¼1241.58, found: 1241.91.
4.2.5. (2S,3S)-2-(tert-Butoxycarbonylamino)-3-hydroxy-4-(methyl-
disulfanyl) butanoic acid (6). To a solution of 5 (28 mg, 0.133 mmol)
and Boc2O (57.8 mg, 0.265 mmol) in THF (0.7 mL) and MeOH
(0.5 mL) was added TEA (0.056 mL, 0.399 mmol). The mixture was
stirred for 2 h at room temperature. The reaction mixture was
extracted with EtOAc three times. Combined organic layers were
dried and purified by chromatography (Hex/EtOAc¼2:1) to give
(2S,3S)-methyl 2-(tert-butoxycarbonylamino)-3-hydroxy-4-(methyl-
disulfanyl) butanoate (30 mg, 75% yield) as a clear oil. 1H NMR
Compound 13. ESIMS calcd for C60H76N12O16S [MþH]þ m/
z¼1253.53, found: 1254.20.
Compound 14. ESIMS calcd for C59H81N13O19S [MþH]þ m/
z¼1308.56, found: 1308.92.
Compound 15. ESIMS calcd for C59H81N13O19 [MþH]þ m/
z¼1276.59, found: 1277.07.
Compound 16. ESIMS calcd for C62H77N13O15S [MþH]þ m/
z¼1276.55, found: 1276.81.
Compound 17. ESIMS calcd for C61H82N14O18S [MþH]þ m/
z¼1331.58, found: 1331.93.
(500 MHz, CDCl3)
d 1.43 (s, 9H), 2.42 (s, 3H), 2.71 (m, 1H), 2.88 (m,
1H), 3.78 (s, 3H), 4.42 (d, J¼8.8 Hz, 2H), 5.32 (d, J¼8.4 Hz, 1H); 13C
Compound 18. ESIMS calcd for C61H82N14O18 [MþH]þ m/
z¼1299.60, found: 1300.02.
NMR (125 MHz, CDCl3) d 22.9, 28.3, 41.6, 52.8, 56.7, 70.0, 80.4, 156.1,
171.2; [
a
]
20 59.84 (c 0.37, CHCl3); IR (liquid film) (nmax/cmꢂ1): 3407,
Compound 19. ESIMS calcd for C49H63N11O15S [MþH]þ m/
z¼1078.43 [Mþ2H]2þ m/z¼539.72, found: 1078.58, 540.12.
Compound 20. ESIMS calcd for C48H68N12O18S [MþH]þ m/
z¼1133.46, found: 1134.01.
D
2979, 2918, 1752, 1716; HRMS: m/e calcd for C11H21NO5S2Naþ:
334.0759, found: 334.0749.
(2S,3S)-methyl
2-(tert-butoxycarbonylamino)-3-hydroxy-4-
(methyldisulfanyl) butanoate (20 mg, 0.064 mmol) was dissolved
in THF (1 mL) and treated with 1 N NaOH solution (1 mL). The
mixture was stirred for 2 h at rt. The reaction mixture was washed
with Et2O and the aqueous layer was adjusted to pH 3 using 1 N
HCl, and extracted with Et2O three times. The combined organic
layer was dried and purified by chromatography (CH2Cl2/
MeOH¼20:1 to 15:1) to give 6 (14 mg, 74% yield). 1H NMR
Compound 21. ESIMS calcd for C48H68N12O18 [MþH]þ m/
z¼1101.49, found: 1101.83.
Compound 22. ESIMS calcd for C57H78N12O15S [MþH]þ m/
z¼1203.55, found: 1204.02.
Compound 23. ESIMS calcd for C56H83N13O18S [MþH]þ m/
z¼1258.58, found: 1259.00.
Compound 24. ESIMS calcd for C56H83N13O18 [MþH]þ m/
z¼1226.61, found: 1227.09.
(500 MHz, CDCl3)
d 1.39 (s, 9H), 2.38 (s, 3H), 2,70 (m, 1H), 2.84 (m,
1H), 4.44 (m, 2H), 5.45 (d, J¼8.8 Hz, 1H); 13C NMR (125 MHz, CDCl3)
Compound 25. ESIMS calcd for C60H70N12O16 [MþH]þ m/
z¼1215.51, [Mþ2H]2þ m/z¼608.26; found: 1216.04, 608.71.
Compound 26. ESIMS calcd for C59H76N12O16S [MþH]þ m/
z¼1241.52, [Mþ2H]2þ m/z¼621.27; found: 1241.94, 621.63.
Compound 27. ESIMS calcd for C59H76N12O16 [MþH]þ m/
z¼1209.56, [Mþ2H]2þ m/z¼605.29; found: 1210.14, 605.69.
Compound 28. ESIMS calcd for C50H64N12O17 [MþH]þ m/
z¼1105.46, found: 1105.80.
20
d
23.0, 28.3, 41.4, 56.6, 70.0, 80.7, 156.4, 174.7; [
a
]
37.56 (c 0.28,
D
CHCl3); IR (liquid film) (nmax/cmꢂ1): 3393, 2979, 2929, 2853, 1710,
1691; HRMS: m/e calcd for C10H19NO5S2Naþ: 320.0602, found:
320.0602.
4.3. General procedure for native chemical ligation
at threonine site
Compound 29. ESIMS calcd for C49H70N12O17S [MþH]þ m/
z¼1131.48, [Mþ2H]2þ m/z¼566.25; found: 1131.87, 566.81.
Compound 30. ESIMS calcd for C49H70N12O17 [MþH]þ m/
z¼1099.51, found: 1099.86.
To a solution of Peptide A (1.0 equiv, 4
(1.5 equiv, 6
mol) in 0.5 mL of Guanidine bufferz was added 0.5 M
bond-breakerÒ TCEP solution (Pierce) (5.0 equiv, 20
mol). The
mmol) and Peptide B
m
m
Compound 31. ESIMS calcd for C51H66N12O17 [MþH]þ m/
z¼1119.48, found: 1120.10.
reaction mixture was stirred at room temperature. The reactions
were monitored by LC–MS and purified directly by HPLC upon
consumption of the starting material.
Compound 32. ESIMS calcd for C50H72N12O17S [MþH]þ m/
z¼1145.50, [Mþ2H]2þ m/z¼573.26; found: 1145.93, 573.68.
Compound 33. ESIMS calcd for C50H72N12O17 [MþH]þ m/
z¼1113.52, found: 1113.87.
4.4. General procedure for desulfurization
Compound 34. ESIMS calcd for C58H72N12O16 [MþH]þ m/
z¼1193.53, found: 1193.59.
To a solution of the peptide (0.6 mM) in 200.0
buffer) and 100.0 L of CH3CN were added 200.0 L of 0.5 M bond-
breakerÒ TCEP solution (Pierce), 20.0
L of 2-methyl-2-propane-
L of radical initiator (0.1 M in water). The reaction
mL of water (or
m
m
Compound 35. ESIMS calcd for C57H78N12O16S [MþH]þ m/
z¼1219.55, found: 1220.01.
m
thiol and 10.0
m
Compound 36. ESIMS calcd for C57H78N12O16 [MþH]þ m/
z¼1187.57, found: 1188.02.
Compound 37. ESIMS calcd for C44H65N15O12S2 [MþH]þ m/
z¼1060.45, found: 1060.44.
z
Guanidine buffer: To 1.0 mL of 6.0 M guanidine buffer, were added 26.8 mg of
Na2HPO4. The pH value of resulting solution was nearly 6.5.

J. Chen et al. / Tetrahedron 66 (2010) 2277–2283
2283
Compound 38. ESIMS calcd for C97H133N27O28S [Mþ2H]2þ m/
z¼1078.99, [Mþ3H]3þ m/z¼719.66, found: 1079.34, 719.96.
Compound 39. ESIMS calcd for C97H133N27O28 [Mþ2H]2þ m/
z¼1063.00, [Mþ3H]3þ m/z¼709.00, found: 1063.35, 709.23.
Compound 40. ESIMS calcd for C99H134N28O27S [Mþ2H]2þ m/
z¼1090.50, [Mþ3H]3þ m/z¼727.33, found: 1090.91, 727.57.
Compound 41. ESIMS calcd for C99H134N28O27 [Mþ2H]2þ m/
z¼1074.50, [Mþ3H]3þ m/z¼716.68, found: 1074.79, 716.97.
Compound 42. ESIMS calcd for C88H124N26O26S [Mþ2H]2þ m/
z¼997.46, [Mþ3H]3þ m/z¼665.31, found: 997.82, 665.60.
Compound 43. ESIMS calcd for C88H124N26O26 [Mþ2H]2þ m/
z¼981.47, [Mþ3H]3þ m/z¼654.65, found: 981.72, 654.97.
Compound 44. ESIMS calcd for C98H146N16O46S2 [Mþ2H]2þ m/
z¼1174.46, found: 1175.42.
Harvey, D. J.; Dwek, R. A.; Rudd, P. M.; de Llorens, R. Glycobiology 2003, 13,
457–470.
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Assoc. 1994, 86, 129–135; (b) Durand, G.; Seta, N. Clin. Chem. 2000, 46, 795–805;
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Hackenberger, C. P. R.; Schwarzer, D. Angew. Chem., Int. Ed. 2008, 47, 10030–
10074; (b) Gamblin, D. P.; Scanlan, E. M.; Davis, B. G. Chem. Rev. 2009, 109, 131–
163; (c) Pratt, M. R.; Bertozzi, C. R. Chem. Soc. Rev. 2005, 34, 58–68; (d) Brik, A.;
Wong, C.-H. Chem.d Eur. J. 2007, 13, 5670–5675 For selected examples of recent
syntheses or semisyntheses of homogeneous protein and glycoproteins, see: (e)
Piontek, C.; Silva, D. V.; Heinlein, C.; Pohner, C.; Mezzato, S.; Ring, P.; Martin, A.;
Schmid, F. X.; Unverzagt, C. Angew. Chem., Int. Ed. 2009, 48, 1941–1945; (f)
Becker, C. F. W.; Liu, X.; Olschewski, D.; Castelli, R.; Seidel, R.; Seeberger, P. H.
Angew. Chem., Int. Ed. 2008, 47, 8215–8219; (g) Pentelute, B. L.; Gates, Z. P.;
Dashnau, J. L.; Vanderkooi, J. M.; Kent, S. B. H. J. Am. Chem. Soc. 2008, 130,
9702–9707; (h) Yamamoto, N.; Tanabe, Y.; Okamoto, R.; Dawson, P. E.; Kajihara, Y.
J. Am. Chem. Soc. 2008, 130, 501–510; (i) Kochendoerfer, G. G.; Chen, S.-Y.; Mao, F.;
Cressman, S.; Traviglia, S.; Shao, H.; Hunter, C. L.; Low, D. W.; Cagle, E. N.; Car-
nevali, M.; Gueriguian, V.; Keogh, P. J.; Porter, H.; Stratton, S. M.; Wiedeke, M. C.;
Wilken, J.; Tang, J.; Levy, J. J.; Miranda, L. P.; Crnogorac, M. M.; Kalbag, S.; Botti, P.;
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burne, J. A. Science 2003, 299, 884–887.
8. Significant progress has been made toward the syntheses of both of these
complex glycoproteins.For recent progress toward EPO, see: (a) Tan, Z.; Shang,
S.; Halkina, T.; Yuan, Y.; Danishefsky, S. J. J. Am. Chem. Soc. 2009, 131, 5424–
5431; (b) Yuan, Y.; Chen, J.; Wan, Q.; Tan, Z.; Chen, G.; Kan, C.; Danishefsky, S. J.
J. Am. Chem. Soc. 2009, 131, 5432–5437; (c) Kan, C.; Trzupek, J. D.; Wu, B.; Wan,
Q.; Chen, G.; Tan, Z.; Yuan, Y.; Danishefsky, S. J. J. Am. Chem. Soc. 2009, 131,
5438–5443 For recent progress toward hFSH, see: (d) Nagorny, P.; Fasching, B.;
Li, X.; Chen, G.; Fasching, B.; Aussedat, B.; Danishefsky, S. J. J. Am. Chem. Soc.
2009, 131, 5792–5799.
Compound 45. ESIMS calcd for C133H199N31O57S [Mþ2H]2þ m/
z¼1588.18, [Mþ3H]3þ m/z¼1059.12, found: 1588.27, 1059.25.
Compound 46. ESIMS calcd for C133H199N31O57 [Mþ2H]2þ m/
z¼1572.19, [Mþ3H]3þ m/z¼1048.46, found: 1572.66, 1048.74.
Acknowledgements
Support for this research was provided by the National Institutes
of Health (CA28824 to SJD). We thank Prof. W.F. Berkowitz for
helpful discussions. Special thanks go to Ms. Rebecca Wilson for
editorial advice and consultation and to Ms. Dana Ryan for assis-
tance with the preparation of the manuscript. We thank Dr. George
Sukenick, Ms. Hui Fang, and Ms. Sylvi Rusli of the Sloan-Kettering
Institute’s NMR core facility for mass spectral and NMR spectro-
scopic analysis.
9. Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. H. Science 1994, 266, 776–779.
10. Warren, J. D.; Miller, J. S.; Keding, S. J.; Danishefsky, S. J. J. Am. Chem. Soc. 2004,
126, 6576–6578.
Supplementary data
11. Wan, Q.; Chen, J.; Yuan, Y.; Danishefsky, S. J. J. Am. Chem. Soc. 2008, 130,
15814–15816.
Experimental procedures, NMR spectra, LC–MS spectra, com-
pound characterization. Supplementary data associated with this
article can be found, in the online version, at doi:10.1016/
j.tet.2010.01.067.
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