Enhanced epimerization of glycosylated amino acids during solid-phase peptide synthesis

Article abstract of DOI:10.1021/ja212188r

Glycopeptides are extremely useful for basic research and clinical applications, but access to structurally defined glycopeptides is limited by the difficulties in synthesizing this class of compounds. In this study, we demonstrate that many common peptide coupling conditions used to prepare O-linked glycopeptides result in substantial amounts of epimerization at the α position. In fact, epimerization resulted in up to 80% of the non-natural epimer, indicating that it can be the major product in some reactions. Through a series of mechanistic studies, we demonstrate that the enhanced epimerization relative to nonglycosylated amino acids is due to a combination of factors, including a faster rate of epimerization, an energetic preference for the unnatural epimer over the natural epimer, and a slower overall rate of peptide coupling. In addition, we demonstrate that use of 2,4,6-trimethylpyridine (TMP) as the base in peptide couplings produces glycopeptides with high efficiency and low epimerization. The information and improved reaction conditions will facilitate the preparation of glycopeptides as therapeutic compounds and vaccine antigens. This article not subject to U.S. Copyright. Published 2012 by the American Chemical Society.

Full text of DOI:10.1021/ja212188r

Article
pubs.acs.org/JACS
Enhanced Epimerization of Glycosylated Amino Acids During Solid-
Phase Peptide Synthesis
Yalong Zhang, Saddam M. Muthana, David Farnsworth, Olaf Ludek, Kristie Adams, Joseph J. Barchi, Jr.,
and Jeffrey C. Gildersleeve*
Chemical Biology Laboratory, National Cancer Institute, 376 Boyles Street, Building 376, Frederick, Maryland, 21702
S
* Supporting Information
ABSTRACT: Glycopeptides are extremely useful for basic
research and clinical applications, but access to structurally
defined glycopeptides is limited by the difficulties in
synthesizing this class of compounds. In this study, we
demonstrate that many common peptide coupling conditions
used to prepare O-linked glycopeptides result in substantial
amounts of epimerization at the α position. In fact,
epimerization resulted in up to 80% of the non-natural
epimer, indicating that it can be the major product in some
reactions. Through a series of mechanistic studies, we demonstrate that the enhanced epimerization relative to nonglycosylated
amino acids is due to a combination of factors, including a faster rate of epimerization, an energetic preference for the unnatural
epimer over the natural epimer, and a slower overall rate of peptide coupling. In addition, we demonstrate that use of 2,4,6-
trimethylpyridine (TMP) as the base in peptide couplings produces glycopeptides with high efficiency and low epimerization.
The information and improved reaction conditions will facilitate the preparation of glycopeptides as therapeutic compounds and
vaccine antigens.
clinical applications. Homogenous glycopeptides, however, are
difficult to obtain from natural sources and are often only
accessible through chemical synthesis.18 Synthesis typically
involves solid-phase peptide synthesis using amino acids that
contain the protected glycan (glyco-amino acids) as building
blocks. This approach allows precise control over the chemical
structure as well as access to larger quantities of material.
Moreover, one can obtain unnatural derivatives that are useful
for studying structure−activity relationships (SAR).
While the synthesis of standard peptides has become routine
and can be readily accomplished via automated methods, the
synthesis of glycopeptides/glycoproteins can be significantly
more challenging.1,19 Couplings of glyco-amino acids to a
growing peptide chain are often slow and inefficient. Moreover,
the outcome can be highly variable from one glycopeptide to
another. To complicate matters further, the building blocks
themselves typically need to be synthesized or are expensive
when commercially available. Therefore, the use of a large
excess of glyco-amino acid to increase the rate and drive the
reaction to completion is impractical. As a result of these
problems, scientists typically prepare the peptide precursor up
to the glycosylation site on an automated solid phase peptide
synthesizer and then carry out the coupling of the glyco-amino
acid manually.1,19 This allows one to follow the reaction
carefully, extend the coupling time as needed, repeat the
coupling if necessary, and examine alternative activating
INTRODUCTION
■
Glycopeptides are used extensively in basic research and a
number of them are under investigation for various clinical
applications.1 Glycopeptides are commonly used as structurally
defined models of complex glycoproteins for studying the roles
of glycosylation, for understanding relationships between
structure and function, and for modulating biological processes
involving the parent glycoprotein.2 Glycopeptide fragments of
glycoproteins also have significant potential as vaccine
antigens.3−8 For example, glycopeptides bearing the Tn antigen
(GalNAcα linked to a serine or threonine of a polypeptide
chain), the TF antigen (Galβ1−3GalNAcα linked to a serine or
threonine of a polypeptide chain), and the STn antigen
(NeuAcα2−6GalNAcα linked to a serine or threonine of a
polypeptide chain) have been investigated in a number of
clinical trials as antigens for cancer vaccines.4,5,9,10 Glycopep-
tides also have significant potential as therapeutic agents.
Glycopeptides found in human urine from patients with
interstitial cystitis have antiproliferative activity, and analogues
show promise as treatments for this debilitating disease.11,12
Glycopeptides found in venoms, such as contalukin-G of Conus
geographus13 and vespulakinins from yellow jacket venom,14 are
of interest as analgesics.15 Furthermore, prokaryotes are known
to produce a variety of antimicrobial glycopeptides, such as
glycocin F16 and sublancin 168.17 For these reasons,
glycopeptides have been the subject of intense research for
many years.
Access to structurally defined glycopeptides is crucial to
study their biological properties as well as to utilize them for
Received: December 30, 2011
Published: March 5, 2012
This article not subject to U.S. Copyright.
Published 2012 by the American Chemical
Society
6316
dx.doi.org/10.1021/ja212188r | J. Am. Chem. Soc. 2012, 134, 6316−6325

Journal of the American Chemical Society
Article
Table 1. Summary of Yields and Epimerization Rates for Various Peptide Coupling Conditions
Fmoc-Ser(R)-OH, where R =
a
#
coupling conditions
AAs: 2.5 equiv;
Trt
Ac3GalNAcα
Ac4Galβ1−3Ac2GalNAcα
Ac3GlcNAcα
Ac3GlcNAcβ
b
1
yields (%)
75.1
0.8
69.1
5.1
83.9
4.3
100
7.7
69.0
8.1
b
b
b
b
b
b
b
b
b
b
b
b
b
HATU/HOAt: 1.1/1.1 equiv;
NMM 2.2 equiv; 0/3 h in DMF57
AAs: 1.5 equiv;
(D/(D+L) %)
b
2
yields (%)
81.0
2.2
74.6
11.4
96.7
21.2
89.9
15.0
92.0
20.0
HATU/HOAt: 1.2/1.2 equiv;
NMM: 4.0 equiv; 0/8 h in DMF58
AAs: 3.3 equiv;
(D/(D+L) %)
b
3
yields (%)
98.9
0.2
99.8
0.8
99.6
2.5
100
1.8
96.8
3.4
HATU/HOAt: 3.65/3.7 equiv;
DIPEA: 7.22 equiv; 0/12 h in DMF59
AAs: 4.4 equiv;
(D/(D+L) %)
b
4
yields (%)
63.4
37.6
99.4
69.8
83.0
65.6
100
1.9
HATU/HOAt: 4.4/0 equiv;
NMM: 8.8 equiv; 3/12 h in NMP59
AAs: 5 equiv;
(D/(D+L) %)
72.5
52.0
b
5
yields (%)
98.4
2.9
99.4
5.2
98.8
6.5
99.5
8.1
95.2
9.7
HATU/HOAt: 6.25/6.25 equiv;
NMM: 12.5 equiv; 0/6 h in NMP59
AAs: 2 equiv;
(D/(D+L) %)
b
6
yields (%)
99.5
0.4
96.7
7.9
99.4
7.4
100
94.9
7.3
HBTU/HOBt: 4.5/4.5 equiv;
DIPEA: 9 equiv; 0/1 h in NMP60
AAs: 3 equiv;
(D/(D+L) %)
11.5
b
7
yields (%)
99.5
0.1
99.7
1.9
97.8
2.4
90.5
1.7
98.6
2.8
DCC/HOBt: 18/18 equiv;
1/64 h in NMP61
(D/(D+L) %)
b
8
AAs: 2 equiv;
yields (%)
99.5
1.2
87.1
10.1
99.7
11.7
86.0
8.8
90.3
5.7
PyBOP/HOBt: 2/2 equiv;
DIPEA: 4 equiv; 0/24 h in DMF62
AAs: 1.5 equiv;
(D/(D+L) %)
b
9
yields (%)
95.8
0.7
88.1
2.1
98.0
3.7
85.6
4.4
86.0
3.9
HBTU/HOBt: 1.5/1.5 equiv;
DIPEA: 1.5 equiv; 0/0.33 h in DMF63
AAs: 5 equiv;
(D/(D+L) %)
b
10
11
12
13
yields (%)
99.6
0.8
99.9
5.2
99.5
7.8
100.0
13.3
96.7
8.6
HBTU/HOBt: 5/7.5 equiv;
NMM: 10 equiv; 0/5 h in DMF64
AAs: 5 equiv;
(D/(D+L) %)
b
yields (%)
99.6
0.5
99.7
2.9
99.0
2.9
100
5.5
98.2
8.0
DIC/HOBt: 10/10 equiv;
0.5/0.5 h in DMF65
(D/(D+L) %)
b
AAs: 2.5 equiv;
yields (%)
97.1
0.1
98.4
3.0
99.7
2.8
90.7
3.9
96.2
2.6
BOP/HOBt: 2.5/2.5 equiv;
DIPEA: 2.5 equiv; 0/4 h in DMF66
AAs: 2.0 equiv;
(D/(D+L) %)
b
yields (%)
98.7
0.2
99.1
1.1
99.5
0.3
100
1.6
98.9
0.6
HATU/HOAt: 2.0/2.0 equiv;
TMP: 2.0 equiv; 0/2 h in DMF
(D/(D+L) %)
a
Glyco-amino acids were coupled to Pro-Gly-Hex resin. For the coupling times, the first number refers to the preincubation time, and the second
b
number refers to the reaction time (e.g., 3/12 h = 3 h preincubation followed by 12 h reaction time). All yield and epimerization [D/(D+L)] data
have an error of less than 0.3%.
reagents and/or bases if necessary. Once the glyco-amino acid
has been successfully incorporated, the remaining peptide can
be completed via automated solid-phase peptide synthesis.
Although this approach can provide access to glycopeptides, it
is not ideal. This process is especially disadvantageous when
preparing large numbers of glycopeptides, such as in the
construction of a glycopeptide library.
Glycan microarrays, arrays containing numerous carbohy-
drates immobilized on a solid support in a spatially defined
arrangement, have become valuable tools for high-throughput
analysis of carbohydrate−macromolecule interactions.20−23
Given the importance of glycopeptides in biology, our
group24−28 and others29−37 have been interested in obtaining
large libraries of glycopeptides to enhance the diversity on
glycan microarrays. To facilitate these efforts, we have focused
on identifying efficient and general coupling conditions. In this
study, we carried out a systematic comparison of published
coupling conditions using four different Fmoc-protected glyco-
amino acids as well as the nonglycosylated counterpart. Both
efficiency and epimerization were evaluated using HPLC assays.
Interestingly, high rates of epimerization were observed under
many conditions, a fact that has important implications for
solid-phase synthesis of glycopeptides. Finally, we identify
conditions that provide high yields and low epimerization for a
range of glyco-amino acids.
MATERIALS AND METHODS
■
General Methods. Unless otherwise stated, reagents were
purchased from commercial suppliers and used without purification.
2-Chlorotrityl chloride resin, Fmoc-Ser(Trt)-OH, Fmoc-D-Ser(Trt)-
OH, and Fmoc-OSu were purchased from AnaSpec, Inc. (San Jose,
CA). Fmoc-6-aminohexanoic acid (Fmoc-Hex-OH), Fmoc-Gly-OH,
6317
dx.doi.org/10.1021/ja212188r | J. Am. Chem. Soc. 2012, 134, 6316−6325

Journal of the American Chemical Society
Article
Figure 1. Model coupling reaction and key synthetic materials. The “a” and “b” designations in the numbering refer to the L- and D-isomers of the
glycopeptides (or nonglycosylated peptides), respectively.
and Fmoc-Pro-OPfp were purchased from Novabiochem (Darmstadt,
Germany). 2-(7-Aza-1H-benzotriazol-1-yl)-N,N,N′,N′-tetramethyl uro-
nium hexafluorophosphate (HATU), 1-hydroxy-7-azabenzotriazole
(HOAt), 2-(1H-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hex-
afluorophosphate (HBTU), N-hydroxybenzotriazole (HOBt), N,N′-
dicyclohexylcarbodiimide (DCC), benzotriazol-1-yl-oxy-tris-pyrrolidi-
no-phosphonium hexafluorophosphate (PyBOP), dimethylformamide
(DMF), N-methyl-2-pyrrolidone (NMP), N,N-diisopropylethylamine
(DIEA), N-methylmorpholine (NMM), and 2,4,6-trimethylpyridine
(TMP) were from Sigma (St. Louis, MO).
Solid-Phase Peptide Coupling of Glyco-amino Acids and
Analysis of Efficiency and Epimerization. Synthesis, purification,
and characterization of the Fmoc-Pro-Gly-Hex-resin starting material
and the glycopeptide standards are described in the Supporting
Information. Glycopeptides with the natural configuration (L) contain
a number followed by an “a”, and those with the unnatural
configuration (D) are followed by a “b”. Prior to coupling the glyco-
amino acids, the Fmoc-Pro-Gly-Hex-resin (10.0 mg, 0.0037 mmol)
was swollen in DCM (2 mL) for 0.5 h at room temperature in a 3-mL
Baker disposable filtration column (Phillipsburg, NJ). The resin was
then treated with 20% piperidine in DMF (2× 0.5 mL) for 15 min to
remove the Fmoc protecting group, followed by washing with DCM
(3× 1.0 mL) and DMF (2× 1.0 mL).
Next, the appropriate amount of Fmoc-Ser(Trt)-OH or glyco-
amino acid, activating reagents, and base were combined in solvent
(150 μL total), mixed with a pipet 5 times, and then immediately
added to the resin. In certain cases, Fmoc-Ser(Trt)-OH or the glyco-
amino acids were preactivated for a period of time and then added to
the resin according to the specific conditions listed in Table 1. The
reaction was allowed to mix at room temperature with an Eppendorf
thermomixer 5436 (Hamburg, Germany) at a speed of 600 rpm, and
then the resin was washed with DCM (3 × 2 mL) then DMF (2 × 1
mL). Next, any unreacted Pro-Gly-Hex-resin was capped by treatment
with FmocOSu (12.3 mg, 0.037 mmol) and DIEA (6.4 μL, 0.037
mmol) in DMF (0.5 mL) for 2 h at 600 rpm at room temperature.
The resin was washed with DMF (2 × 1 mL) and DCM (3 × 2 mL),
and the glycopeptide was cleaved off the resin by incubating with a
mixture of TFA/DCM (90:10) (0.5 mL) at room temperature for 1.5
h at 500 rpm. After cleavage from the resin, the resulting peptides were
diluted with DCM (2 mL), concentrated under reduced pressure with
a rotary evaporator, dried under vacuum, and dissolved in acetonitrile
(1 mL). Solutions were filtered, and then product distributions were
evaluated using RP-HPLC (for specific HPLC conditions for each
substrate, see Supporting Information).
Solid-Phase Peptide Coupling Using Condition 13. The
appropriate glyco-amino acid (2 equiv relative to the resin, 0.0073
mmol), HATU (2.78 mg, 0.0073 mmol), HOAt (1.0 mg, 0.0073
mmol), and TMP (0.98 μL, 0.0073 mmol) were mixed in DMF (150
μL) and immediately added to the resin (10 mg, 0.00366 mmol) in a
3-mL Baker disposable filtration column. The reaction was allowed to
mix at a speed of 600 rpm at room temperature for 2 h, followed by
washing with DCM (3 × 2 mL) then DMF (2 × 1 mL). Any
unreacted amino groups were capped by adding FmocOSu (12.3 mg,
0.037 mmol) and DIEA (6.4 μL, 0.037 mmol) in DMF (0.5 mL) and
shaking at room temperature for 2 h. The resin was washed with DMF
(2 × 1 mL) and DCM (3 × 2 mL), and the glycopeptide was cleaved
off the resin by treating the resin with a mixture of TFA/DCM (90:10)
(0.5 mL) at room temperature for 1.5 h.
Molecular Modeling. Calculation of conformational preferences
of the putative intermediates in amino acid racemizations (oxazolone,
see Figures 3 and 6) were performed using MacroModel within the
Maestro user interface (Schrodinger, LLC, New York, NY). Structures
were constructed within the Build facility in Maestro, and calculations
were performed in the Conformational Search utility in MacroModel.
Structures were first energy minimized using the OPLS-2005 force
field for 2000 steps using the Polak-Ribieri Conjugate Gradient
(PRCG) method with a convergence threshold of 0.001. Conforma-
tional searching was performed on a random minimized structure of
each oxazolone with no restraints. No explicit or implicit solvent was
used, but a distance dependent dielectric constant of 38 was used to
simulate the dielectric value of DMF. Thirty structures were calculated
around each rotatable bond and each conformation was minimized
6318
dx.doi.org/10.1021/ja212188r | J. Am. Chem. Soc. 2012, 134, 6316−6325

Journal of the American Chemical Society
Article
with either the PRCG or truncated newton conjugate gradient
(TNCG) method to a threshold of 0.005 in the OPLS-2005 force
field. Charges were read from the force field, and an extended bond
cutoff of 8.0, 20.0, and 4.0 Å were used for van der Waals, electrostatic,
and hydrogen bond distances, respectively. Mixed torsional/low-mode
sampling was performed for the searches, and structures were analyzed
for energy values and dihedral angles about the Hα−Cα−Cβ−Oβ
bond. Conformational clustering was performed using the con-
former_cluster.py script in the Chemoinformatics utility in Maestro.
Clusters were examined for energies, number-of-times-found for each
conformation/cluster, and appropriate bond angles.
containing the S- and R-epimers of the glyco-amino acids and
serine were synthesized, and HPLC assays were used to
measure yields and epimerization (for example, see Figure 2).
RESULTS AND DISCUSSION
■
Approach and Design. Many factors, such as coupling
reagents, reaction time, preincubation time, structure of the
glyco-amino acids, and the structure of the accepting peptides
on the solid support could affect the yields and epimerization
rates of glycopeptides during solid-phase synthesis. To find the
optimal coupling conditions for glyco-amino acids, we
investigated a variety of published conditions that had
previously been used for the solid-phase synthesis of
glycopeptides to determine which conditions would provide
the highest and most consistent yields and the lowest levels of
epimerization. On the basis of a survey of the literature, we
initially selected 12 conditions (see Table 1) that encompassed
the most widely used activating agents (HATU, HBTU, DCC,
PyBOP), bases (DIEA, NMM), and solvents (DMF, NMP). In
addition, the conditions were selected to include some variety
in terms of reaction times, equivalents of base, and equivalents
of reagents. A thirteenth condition was also evaluated, based on
a rationale that is discussed below.
Since the outcome of the coupling could be dependent on
the structure of the glyco-amino acid, we opted to evaluate four
different glyco-amino acids (see Figure 1) that are commonly
found in O-linked glycans. Two of the glyco-amino acids,
Fmoc-Ser(Ac3GalNAcα)-OH (7) and Fmoc-Ser(Ac4Galβ1-
3Ac2GalNAcα)-OH (9), are protected derivatives of the
tumor-associated carbohydrate antigens, Tn and TF. Synthesis
of Tn- and TF-containing glycopeptides is highly relevant to
cancer research and the development of Tn- and TF-based
cancer vaccines.5,38,39 We also looked at the coupling of other
glyco-amino acids, Fmoc-Ser(Ac3GlcNAcα)-OH (11) and its
β-linked analogue, Fmoc-Ser(Ac3GlcNAcβ)-OH (13). Attach-
ment of an O-β-GlcNAc to specific serine and threonine
residues of many cytosolic proteins is important for regulating
their biological activity,40,41 and the α-linked analogue is found
in prokaryotes.42 While not a complete evaluation of every
potential glyco-amino acid, this set would allow us to compare
effects of certain features, such as α- vs β-linkages, GalNAc vs
GlcNAc configuration, and mono- vs disaccharides, for a
biologically important group of glyco-amino acids. In each case,
the carbohydrate fragment was attached to a serine residue. For
comparison, we evaluated the coupling of Fmoc-Ser(Trt)-OH,
a protected form of serine that has been commonly used in
solid-phase peptide synthesis.
Figure 2. Representative RP-HPLC analysis of coupling reactions.
Traces of three potential products in the coupling reaction with Fmoc-
Ser(Ac3GalNAcα)-OH: Fmoc-Pro-Gly-Hex-OH (6) (starting material,
black); Fmoc-D-Ser(Ac3GalNAcα)-Pro-Gly-Hex-OH (2b, non-natural
epimer, red); Fmoc-L-Ser(Ac3GalNAcα)-Pro-Gly-Hex-OH (2a, de-
sired product, blue); mixture of three components at a ratio of 1:2:4
(purple).
Serine. Serine has previously been found to have a higher
rate of racemization than most other natural amino acids.43
Nevertheless, many existing coupling conditions produce high
efficiency couplings with low racemization. Of the 13
conditions that we tested, 10 produced the desired product
with greater than 95% yield, and all but one condition produced
the peptide with less than 3% racemization. Condition 4, which
involved preactivation of Fmoc-Ser(Trt)-OH with HATU/
HOAt/NMM/NMP for 3 h, led to both suboptimal yield
(63.4%) and substantial racemization (37.6%). Higher rates of
racemization were not entirely unexpected for this condition,
since long preactivation times are known to increase
racemization.44
Tn. Next, we evaluated the coupling efficiency of an Fmoc-
protected Tn-derivative, Fmoc-Ser(Ac3GalNAcα)-OH (7).
Compound 7 was synthesized following reported methods.45
The unnatural glyco-amino acid, Fmoc-D-Ser(Ac3GalNAcα)-
OH (8), was synthesized with similar procedures, and the
details can be found in the Supporting Information.
Results for the protected Tn antigen highlight the difficulties
of coupling glyco-amino acids. Although couplings of glyco-
amino acids can be less efficient than the nonglycosylated
counterparts, efficiency was not a significant problem for the
conditions we tested. The yields obtained for compound 7 were
similar to that of serine, where nine of thirteen conditions
provided over 95% yields. Epimerization, however, was a much
greater problem for Fmoc-Ser(Ac3GalNAcα)-OH (7) than for
nonglycosylated Fmoc-Ser(Trt)-OH. Eight of thirteen con-
ditions led to an epimerization of over 3%, and three of those
conditions led to over 10% epimerization. Moreover, condition
4 produced the unnatural epimer as the major product in the
reaction (70%). It is important to note that, unlike Fmoc-
Ser(Trt)-OH, epimerization of Fmoc-Ser(Ac3GalNAcα)-OH at
the α position of the amino acid produces a new diastereomer,
Since our goal was to identify conditions that would be
suitable for many different couplings, including difficult
linkages, we initially evaluated couplings of all glyco-amino
acids to a short peptide with an N-terminal proline residue,
which we anticipated would be challenging. In addition,
peptides with several other N-terminal residues were coupled
with Fmoc-Ser(Ac3GalNAcα)-OH (7) to test how the terminal
residue of peptides affected yields and epimerization. In each
case, standards of the truncated peptide and peptides
6319
dx.doi.org/10.1021/ja212188r | J. Am. Chem. Soc. 2012, 134, 6316−6325

Journal of the American Chemical Society
Article
Figure 3. General pathway for formation of the oxazolone and the mechanism of epimerization.
Table 2. Effects of the N-Terminal Amino Acid Residue on Efficiency and Epimerization
coupling of Fmoc-Ser(Ac3GalNAcα)OH to X-Gly-Hex-resin, where X =
Pro
Ser(Trt)
Leu
Ser(Ac3GalNAcα)
a
b
b
b
b
b
b
b
b
#
yields (%)
D/(D+L) (%)
yields (%)
D/(D+L) (%)
yields (%)
D/(D+L) (%)
yields (%)
D/(D+L) (%)
1
2
3
4
5
6
7
8
9
69.1
74.6
99.8
99.4
99.4
99.3
99.7
87.1
88.1
99.9
99.7
98.4
99.1
5.1
11.4
0.8
99.0
99.6
99.6
99.9
99.4
99.8
99.1
99.8
99.7
99.6
99.8
99.8
98.3
2.6
6.7
1.3
71.7
3.6
4.3
1.0
3.0
2.1
1.4
1.0
0.8
0.2
97.7
98.0
98.3
98.5
98.3
97.4
98.4
98.2
98.1
98.1
98.2
98.5
99.3
3.8
7.1
2.3
69.3
4.4
2.9
1.7
4.2
2.6
2.7
2.1
1.6
1.5
92.5
91.5
99.1
99.2
96.1
97.8
95.4
96.9
96.2
96.9
97.2
96.9
96.8
3.9
6.6
1.9
70.8
3.9
3.0
3.8
3.8
5.7
4.9
3.3
3.2
0.5
69.8
5.2
9.7
1.9
10.1
2.1
10
11
12
13
5.2
2.9
3.0
1.1
a
b
Entry number corresponds to the coupling conditions listed in Table 1. All yield and epimerization data have an error of less than 0.3%.
rather than an enantiomer. The production of 70% of the
unnatural epimer suggests that this diastereomer is energetically
favored or that L-proline selectively reacts with the unnatural
epimer. In either case, the major product of the reaction is the
unnatural epimer, demonstrating that one cannot assume that
the unnatural epimer will only be a minor contaminant.
of the glyco-amino acids and glycopeptides are in the
Supporting Information.
Like the protected Tn amino acid, Fmoc-Ser(Ac4Galβ1−
3Ac2GalNAcα)-OH produced substantially higher levels of
epimerization than the nonglycosylated Fmoc-Ser(Trt)-OH.
The larger glycan fragment, however, did not significantly affect
the yield or epimerization relative to the monoglycosylated
amino acid, indicating that the second monosaccharide residue
did not have a major effect on the outcome. As with the Fmoc-
Ser(Ac3GalNAcα)-OH, coupling with TMP as a base
(condition 13) produced excellent results.
α-GlcNAc. Next, we evaluated the coupling of Fmoc-
Ser(Ac3GlcNAcα)-OH. Fmoc-Ser(Ac3GlcNAcα)-OH (11) and
Fmoc-D-Ser(Ac3GlcNAcα)-OH (12) (see Figure 1) were each
synthesized from per-acetylated 2-azido-2-deoxy-D-glucopyra-
nosyl bromide according to a reported method (see Supporting
Information for details).45 The yields for peptide couplings with
Fmoc-Ser(Ac3GlcNAcα)-OH under the various published
conditions were similar to those of serine, Tn, and TF. The
extent of epimerization, however, was even higher for this
glyco-amino acid. For example, the percentage of epimerized
product observed under conditions 1 and 10 were 10 and 17
times higher for compound 11 than that of Fmoc-Ser(Trt)-OH.
Moreover, all but three conditions produced over 3%
epimerization. As previously observed for Tn and TF, condition
4 provided the highest epimerization (72.5%), and conditions
3, 7, and 13 provided the lowest epimerization (1.8%, 1.7% and
1.6%, respectively).
The epimerization process is thought to occur through
abstraction of the α-hydrogen of an oxazolone intermediate by
the base (see Figure 3).44 Therefore, one would expect that
lower amounts of base and use of a weaker base would lead to
less epimerization. Through a systematic comparison of bases,
Carpino and colleagues demonstrated that 2,4,6-trimethylpyr-
idine (TMP) is an excellent base for peptide couplings,
providing high yields with low epimerization.46 However, use of
TMP as a base for coupling glyco-amino acids has, to our
knowledge, only been used once previously, and the conditions
employed microwave irradiation to accelerate coupling.47
Therefore, it was not clear if TMP would be suitable for
coupling glyco-amino acids under nonmicrowave conditions.
To test this, we examined a thirteenth condition with 2 equiv of
glyco-amino acid, 2 equiv of HATU, 2 equiv of HOAt, and 2
equiv of TMP as the base. The coupling proceeded smoothly
and produced very little epimerization (see Table 1, entry 13).
TF. Glyco-amino acids with larger glycan chains could
potentially be more difficult to attach to a growing peptide
during solid-phase peptide synthesis. To address this issue, we
prepared Fmoc-Ser(Ac4Galβ1−3Ac2GalNAcα)-OH (9), and
Fmoc-D-Ser(Ac4Galβ1−3Ac2GalNAcα)-OH (10), the natural
and unnatural epimers of a protected TF antigen that contains a
disaccharide residue on the side chain of the amino acid. The
corresponding peptides were also prepared, and an HPLC assay
was developed to evaluate potential products from the peptide
coupling reactions. The detailed descriptions of the syntheses
β-GlcNAc. To study the effects of the stereochemistry of the
glycosidic-linkage on efficiency and epimerization, we evaluated
the coupling of Fmoc-Ser(Ac3GlcNAcβ)-OH (13). The
synthesis of Fmoc-Ser(Ac3GlcNAcβ)-OH (13) and Fmoc-D-
Ser(Ac3GlcNAcβ)-OH (14) were each carried out in one step
6320
dx.doi.org/10.1021/ja212188r | J. Am. Chem. Soc. 2012, 134, 6316−6325

Journal of the American Chemical Society
Article
according to a reported method48 starting from D-glucosamine
pentaacetate (see Supporting Information). As summarized in
Table 1, nine of the thirteen conditions provided good yields
(≥95%), and three of the conditions ranged from 69 to 92%
yield. Condition 4, however, provided an unexpectedly low
yield (2%). Repeating condition 4 without preactivation and
with 1-h preactivation provided 74% and 32% yield, with 8%
and 45% epimerization, respectively. This demonstrated that
extensive decomposition and epimerization occurs during the
preactivation step for condition 4. As previously described for
the other glyco-amino acids, the epimerization for Fmoc-
Ser(Ac3GlcNAcβ)-OH was much higher than for the protected
serine analogue. For example, the percentage of epimerized
product observed under conditions 3 and 11 were respectively
17 and 16 times higher for Fmoc-Ser(Ac3GlcNAcβ)-OH than
for Fmoc-Ser(Trt)-OH. In the case of Fmoc-Ser-
(Ac3GlcNAcβ)-OH, only conditions 7, 12, and 13 had under
3% epimerization.
To better understand the origin of the differences, we first
measured the initial rates of epimerization. To accurately
measure the initial rates, we needed conditions wherein the
reactants would not be consumed to an appreciable degree
during the reaction. In addition, we needed conditions where
the epimerization would be slow enough to conveniently
measure using our assay. Since the base is consumed in the
reaction (through protonation), we needed to use a large excess
of base such that the concentration would not change
significantly during the reaction; however, a large excess of
DIEA or NMM would result in much faster rates of
epimerization, as well as undesired side reactions, making
analysis difficult. For these reasons, we chose to couple using 12
equiv of TMP as the base.
As shown in Figure 4, epimerization rates of the four glyco-
amino acids were significantly faster than the epimerization rate
Effects of N-Terminal Residue. The nature of the N-
terminal coupling partner can have a significant effect on the
rate of a peptide bond formation. We used a peptide with an N-
terminal proline residue for all of our initial studies.
Glycosylated serine residues in nature are often found next to
various other amino acids.49 To test the effects of N-terminal
amino acid structure on efficiency and epimerization, we
evaluated coupling of Fmoc-Ser(Ac3GalNAcα)-OH (7) to
peptides with a terminal serine and leucine, two amino acid
residues that are often found adjacent to the Tn antigen on
mucins. In general, the yields were high, but epimerization was
substantial for many of the coupling conditions (see Table 2).
The production of about 70% of the unnatural epimer under
condition 4 demonstrates that the energetic preference for this
diastereomer is not dependent on an N-terminal proline.
Synthesis of Clustered Tn Peptides. Mucins and other
glycoproteins often contain two or more glycosylated serine
and/or threonine residues linked consecutively on a peptide
chain. When these residues are all glycosylated, these
arrangements are referred to as “clustered” glycans. Clustered
Tn and STn antigens are more tumor-specific than single
antigens, and clustered Tn and STn glycopeptides are of high
interest as vaccine antigens.45,50,51 We anticipated that coupling
of Fmoc-Ser(Ac3GalNAcα)-OH (7) to a peptide chain
containing an N-terminal Ser(Ac3GalNAcα)-OH residue
could be even more difficult than coupling to a proline, serine,
or leucine. As before, we synthesized the appropriate standards
and developed an HPLC assay to detect the potential products.
While the yields were above 90% for all the conditions tested,
only two conditions, 3 and 13, had an epimerization of less than
3%. It is important to note that the preparation of clustered Tn
peptides requires at least two peptide couplings using glyco-
amino acids. Since the attachment of successive glyco-amino
acid residues is more difficult than coupling of the first glyco-
amino acid and the effects of epimerization are cumulative, the
preparation of clustered glycopeptides can be especially
difficult. Therefore, it is crucial to use optimal peptide coupling
conditions.
Figure 4. Initial rates of epimerization. Initial rates were measured
using the following coupling conditions: AAs/HATU/TMP = 1:1:12.
The initial rates were as follows: Fmoc-Ser(Ac3GlcNAcβ)-OH (13), k
= 0.37 0.01%/min; Fmoc-Ser(Ac3GlcNAcα)-OH (11), k = 0.27
0.01%/min; Fmoc-Ser(Ac3GalNAcα)-OH (7), k = 0.241 0.002%/
min; Fmoc-Ser(Ac4Galβ1−3Ac2GalNAcα)-OH (9), k = 0.197
0.005%/min; Fmoc-Ser(Trt)-OH, k = 0.066 0.003%/min.
of Fmoc-Ser(Trt)-OH. Fmoc-Ser(Ac3GlcNAcβ)-OH (13)
displayed the fastest initial rate of epimerization which was
about 6 times faster than that of Fmoc-Ser(Trt)-OH. Taken
together, these results show that a faster rate of epimerization is
one factor that contributes to the higher levels of epimerization
for glyco-amino acids.
Next, the extent of epimerization of glyco-amino acids was
measured. Condition 4, which included a 3-h preactivation step,
produced high levels of epimerization and, in some cases,
produced the unnatural epimer as the major product. We
hypothesized that the preincubation step allows for equilibra-
tion of the natural and unnatural epimer at the α position. If so,
extending the preincubation time would allow us to measure
the equilibrium ratios of natural and unnatural epimers. To test
this, we repeated this reaction using a variety of preincubation
times ranging from 0 to 24 h (see Figure 5). Since the activated
esters of the D- and L-glyco-amino acids are diastereomers and
could potentially have different coupling rates to an N-terminal
proline on the resin, an excess of resin was used to ensure that
all D- and L-isomers would be captured on the resin and
detected in the assay. For each of the glyco-amino acids tested,
much less epimerization was observed when no preincubation
was used, and the amount of epimerization increased as the
Mechanistic Studies. The differences in the levels of
epimerization observed for glyco-amino acids versus that for
serine could be due to a variety of reasons, such as differential
rates of epimerization, different rates of peptide coupling,
different energetic preferences for the natural versus unnatural
epimer, and enhanced coupling of the unnatural D-configured
activated esters relative to their L-counterparts.
6321
dx.doi.org/10.1021/ja212188r | J. Am. Chem. Soc. 2012, 134, 6316−6325

Journal of the American Chemical Society
Article
preincubation time increased, indicating that the epimerization
occurs during the preincubation period. The extent of
epimerization of Fmoc-Ser(Ac3GalNAcα)-OH (7) (protected
Tn, Figure 5a) reached a maximum of approximately 80%,
indicating that the equilibrium composition is 4:1. Similar
results were obtained with Fmoc-Ser(Ac4Galβ1−
3Ac2GalNAcα)-OH (9, Figure 5b) and Fmoc-Ser-
(Ac3GlcNAcα)-OH (11, Figure 5c). The extent of epimeriza-
tion could not be accurately determined for Fmoc-Ser-
(Ac3GlcNAcβ)-OH due to degradation of the starting material
during the preincubation period. These results indicate that the
unnatural epimer of these glyco-amino acids are favored over
the natural epimer and that this energetic preference is another
factor that contributes to the higher observed levels of
epimerization for glyco-amino acids.
stopped at 5 min to ensure the yield of each peptide was below
20% (total yield below 40%). Two equivalents of each amino
acid was used, which ensured that the consumption of each
amino acid was below 10%.
As summarized in Table 3, most glyco-amino acids coupled
more slowly than Fmoc-Ser(Trt)-OH. The slowest glyco-amino
a
Table 3. Relative Overall Rates of Peptide Coupling
amino acid
Fmoc-Ser(Trt)-OH
relative reaction rate GAA/Ser
1
Fmoc-D-Ser(Trt)-OH
1.06 0.05
0.69 0.02
0.84 0.03
0.46 0.01
0.64 0.02
1.19 0.03
0.99 0.02
1.20 0.09
Fmoc-Ser(Ac3GalNAcα)-OH
Fmoc-D-Ser(Ac3GalNAcα)-OH
Fmoc-Ser(Ac4Galβ1−3Ac2GalNAcα)-OH
Fmoc-Ser(Ac3GlcNAcα)-OH
Fmoc-D-Ser(Ac3GlcNAcα)-OH
Fmoc-Ser(Ac3GlcNAcβ)-OH
Fmoc-D-Ser(Ac3GlcNAcβ)-OH
a
All reactions were carried out by mixing Fmoc-Ser(Trt)-OH (2
equiv) and the listed glyco-amino acid (GAA, 2 equiv) with HATU (4
equiv) and TMP (4 equiv) in DMF. The total reaction time was 5 min,
and no preactivation was used.
acid, 9, showed an approximately 50% decrease in relative
coupling rate. The Fmoc-D-Ser(Trt)-OH gave the same
coupling rate as its enantiomer. Interestingly, the unnatural D-
isomer of Fmoc-Ser(Ac3GlcNAcα)-OH had a coupling rate
noticeably higher than that of the natural isomer.
Steric, Electronic, and Conformational Factors Affect-
ing Epimerization. Although the experiments described
above demonstrated that initial rates of epimerization, the
amounts of D-isomer formed, and the coupling times of glyco-
amino acids are all quite distinct from those of their
nonglycosylated counterparts, a clearer understanding of the
structural features that may contribute to the higher
epimerization rates we desired. Therefore, we considered
several factors that could influence the relative rates of
epimerization.
One factor that could potentially contribute to enhanced
epimerization of glyco-amino acids relative to nonglycosylated
amino acids are inductive effects. Monosaccharide rings are
expected to exert a stronger electron-withdrawing inductive
effect than a trityl group. This could lead to enhanced acidity of
the α-hydrogen. To evaluate this possibility, we examined the
proton NMR chemical shifts of the α-hydrogens. Interestingly,
we observed no differences in chemical shifts of the α-
hydrogens for any of the serine analogues used in this study. In
contrast, the β-hydrogens of glycosylated serine residues were
shifted downfield approximately 0.5 ppm relative to the β-
hydrogens of trityl-protected serine. While this difference could
be due to inductive and/or anisotropic effects, we note that the
chemical shifts of the α- and β-hydrogens of tert-butyl-
protected serine were nearly identical with those of the trityl-
protected serine, indicating that anisotropic effects of the trityl
group did not have a significant effect on the chemical shifts of
the α- and β-hydrogens. Therefore, the NMR data support a
stronger electron-withdrawing effect exerted by the sugar ring;
however, this effect does not extend to the α-position of the
amino acids and does not appear to contribute to the increase
in epimerization.
Figure 5. Epimerization as a function of preactivation time. Fmoc-
Ser(Trt)-OH and glyco-amino acids were coupled using the following
coupling reagents and ratios: AAs/HATU/NMM = 1:1:2. Each amino
acid was preactivated for varying times and then the extent of
epimerization was measured as described in the Materials and
Methods: (a) Fmoc-Ser(Ac3GalNAcα)-OH (7); (b) Fmoc-Ser-
(Ac4Galβ1−3Ac2GalNAcα)-OH (9); (c) Fmoc-Ser(Ac3GlcNAcα)-
OH (11); and (d) Fmoc-Ser(Trt)-OH.
Finally, we measured the relative rates of coupling to the
resin using a competition assay. The epimerization process is
generally thought to occur via the activated intermediate(s)
(see Figure 3); once the glyco-amino acid has been coupled to
the growing peptide chain, the resulting amide would no longer
epimerize under the coupling conditions. A slower overall
coupling rate could allow more time for epimerization. We used
a competition assay to measure the relative coupling rates
wherein glyco-amino acids were mixed with an equivalent
amount of Fmoc-Ser(Trt)-OH, activated, and then captured
with resin as before. Using an HPLC assay, we measured
relative amounts of the glyco-peptides and serine-containing
peptides, which provide a measure of the relative rates of
coupling. Under standard conditions, the initial coupling rates
were too fast to measure in this assay. Therefore, the reaction
solution was diluted 10-fold, and the coupling reaction was
A second factor that could contribute to the enhanced
epimerization of glyco amino acids is conformational effects;
6322
dx.doi.org/10.1021/ja212188r | J. Am. Chem. Soc. 2012, 134, 6316−6325

Journal of the American Chemical Society
Article
Figure 6. Low-energy models of the oxazolone intermediates derived from (A) Fmoc-Ser(Trt)-OH and (B) Fmoc-Ser(Ac3GlcNAcβ)-OH (13)
generated from the Conformational Search utility in Macromodel 2011. Panels (C) and (D) are expansions of the oxazolone ring with the Hα−Cα−
Cβ−Oβ dihedral angle value labeled.
however, defining the preferred conformations of small, flexible
glyco-amino acids by NMR is difficult due to the limited
number of NMR-derived distance and angle restraints.
Moreover, peptide coupling and epimerization reactions can
proceed through intermediates that may have distinct
conformational preferences from the starting amino acids, but
these intermediates are not easily characterized due to their
instability. Therefore, we undertook a modeling study to
evaluate conformational preferences. Epimerization/racemiza-
tion is generally thought to proceed through an intermediate
oxazolone (see Figure 3).52,53 As a side note, β-elimination
could also occur through a similar mechanism, but we did not
observe β-elimination in any of our peptide-coupling
reactions.67 We reasoned that structural differences between
the various oxazolones could potentially contribute to differ-
ences in epimerization. Therefore, we compared conforma-
tional preferences of the putative oxazolone intermediates of
Fmoc-Ser(Trt)-OH and the glyco-amino acid that is most
prone to epimerization, viz. Fmoc-Ser(Ac3GlcNAcβ)-OH. The
oxazolones were constructed with the Build facility in Maestro
(Schrodinger, Inc.) and the program MacroModel 11.1 was
used to perform searches and analyze data. A series of
structures was calculated based on various criteria as the input
to the Conformational Search utility in Macromodel. To mimic
our reaction solvent, we used a distance-dependent dielectric
constant equal to the value for DMF (38). Full descriptions of
the search criteria (force fields, minimization settings, search
parameters) can be found in the Materials and Methods
section.
oxazolone, the Hα and Oβ atoms are nearly anti to each other
(∼165°), whereas these atoms adopt an angle of about 85° for
the trityl-protected oxazolone. The difference in preferred
conformations has two key effects. First, the α hydrogen of
Fmoc-Ser(Trt)-OH is more sterically shielded than the α
hydrogen of Fmoc-Ser(Ac3GlcNAcβ)-OH. The steric hin-
drance could make it more difficult for the base to abstract
the hydrogen from Fmoc-Ser(Trt)-OH. Second, the anti
configuration between the Hα and Oβ atoms of Fmoc-
Ser(Ac3GlcNAcβ)-OH maximizes orbital overlap between the
C−H σ-bond and the C−O σ*-antibonding orbital, which
would facilitate abstraction of the α-hydrogen from Fmoc-
Ser(Ac3GlcNAcβ)-OH. Although additional evidence will be
needed to verify this hypothesis, our initial analysis indicates
that a combination of steric and stereoelectronic effects
contribute to the enhanced rate of epimerization of glycosylated
amino acids relative to the nonglycosylated counterparts.
CONCLUSION
■
In this study, we demonstrate that glycosylated amino acids are
much more prone to epimerization than their corresponding
nonglycosylated counterparts. The enhanced epimerization
during solid-phase synthesis is due to a faster rate of
epimerization, an energetic preference for the unnatural epimer,
a slower overall rate of peptide coupling, and a conformational
preference in the putative oxazolone intermediate that can be
potentially stabilizing toward α-hydrogen abstraction. Of the 13
peptide coupling conditions that we systematically evaluated,
conditions 3 (HATU/HOAt/DIPEA/glyco-amino acid at a
ratio of about 1:1:2:1), 7 (DCC/HOBt/glyco-amino acid at a
ratio of 6:6:1), and 13 (HATU/HOAt/TMP/glyco-amino acid
at a ratio of about 1:1:1:1) provided the best overall yields
(>95%) and the lowest levels of epimerization (≤3%). These
conditions are compatible with automated solid-phase glyco-
The preferred conformations for the two oxazolones were
significantly different. Figure 6 shows the lowest-energy
conformers of the two oxazolones modeled (Figure 6, A and
B) along with the values of the Hα−Cα−Cβ−Oβ dihedral
angles in each (Figure 6, C and D). For the glycosylated
6323
dx.doi.org/10.1021/ja212188r | J. Am. Chem. Soc. 2012, 134, 6316−6325

Journal of the American Chemical Society
Article
peptide synthesis, but condition 7 is less desirable due to the
long reaction time and large excess of reagents used in the
reaction. Therefore, conditions 3 and 13 represent the best
current options for coupling glycosylated amino acids to a
growing peptide chain. It is important to note, however, that
additional studies are needed to more fully evaluate the scope
of the epimerization problem and the performance of these
peptide coupling conditions. For example, other factors, such as
the protecting groups on the glyco-amino acid and the nature
of the core amino acid (serine vs threonine), could also affect
the outcome. Moreover, longer sequences and/or more
aggregation prone sequences may be more difficult to
synthesize and more susceptible to side reactions. Nevertheless,
this study provides an important step toward general and
efficient peptide coupling conditions for synthesizing glyco-
peptides.
While we observed substantial amounts of epimerization in
many reactions, epimerization has been noted only infrequently
in previous publications. There are several factors that could
account for this discrepancy. First, glycopeptides containing an
unnatural epimer are often extremely difficult to separate from
the corresponding glycopeptides containing the natural epimer.
As the peptide sequence increases in length, the separation
becomes even more challenging. In many previous studies, the
glycopeptides of interest were over 10 amino acids long. In our
study, we used short sequences containing 3 amino acid
residues, which facilitated the separation of natural and
unnatural epimers. In addition, we chemically synthesized the
unnatural epimers as defined standards, which enabled the
development of suitable conditions for separating the natural
and unnatural epimers by HPLC. It should be noted, however,
that even for our short glycopeptides, separation was not trivial.
Second, it can be extremely difficult to detect the presence of
the unnatural epimer by standard characterization methods,
such as mass spectroscopy, elemental analysis, and NMR.
Therefore, these impurities could go undetected. Finally,
certain peptide linkages may be easier to form, leading to less
epimerization.
Epimerization of glycopeptides could have a major impact in
a number of situations. Many glycopeptides are in clinical
development as vaccine antigens. Previous studies have shown
that the immune system can mount a much stronger response
to peptides containing an unnatural epimer than to natural
peptides.54,55 Thus, even if the unnatural epimer is a minor
contaminant, it may be the dominant antigen recognized by the
immune system. Therefore, the presence or absence of the
unnatural epimer could have a major impact on the clinical
efficacy of a glycopeptide vaccine. For biologically active
glycopeptides, compounds containing an unnatural epimer can
have completely different activities. For example, the
antiproliferative factor (APF) present in urine of patients
with interstitial cystitis potently blocks proliferation of bladder
epithelial cells.11,56 In contrast, the corresponding glycopeptide
containing a D-proline at position 3 potently antagonizes the
activity of APF.11 Although the unnatural epimer is not at a
glycosylated position, this example demonstrates that variation
of stereochemistry at a single position can lead to a complete
switch in bioactivity. Our results demonstrate that formation of
the unnatural epimer can be a major problem and that judicious
choice of coupling conditions can minimize this undesired side
product.
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental details for the synthesis of glyco-amino acids and
glycopeptides, characterization data, and supplemental HPLC
analyses of glycopeptides. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
gildersj@mail.nih.gov
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported by the Intramural Research
Program of the NIH, NCI. We gratefully acknowledge James A.
Kelley (NIH/NCI) for obtaining mass spectrometry data for all
the compounds.
REFERENCES
■
(1) Buskas, T.; Ingale, S.; Boons, G. J. Glycobiology 2006, 16, 113R−
136R.
(2) Grogan, M. J.; Pratt, M. R.; Marcaurelle, L. A.; Bertozzi, C. R.
Annu. Rev. Biochem. 2002, 71, 593−634.
(3) Andersson, I. E.; Dzhambazov, B.; Holmdahl, R.; Linusson, A.;
Kihlberg, J. J. Med. Chem. 2007, 50, 5627−5643.
(4) Buskas, T.; Thompson, P.; Boons, G.-J. Chem. Commun. 2009,
5335−5349.
(5) Zhu, J.-L.; Warren, J. D.; Danishefsky, S. J. Expert Rev. Vaccines
2009, 8, 1399−1413.
(6) Batsalova, T.; Dzhambazov, B.; Klaczkowska, D.; Holmdahl, R. J.
Immunol. 2010, 185, 2701−2709.
(7) Andersson, I. E.; Andersson, C. D.; Batsalova, T.; Dzhambazov,
B.; Holmdahl, R.; Kihlberg, J.; Linusson, A. PLoS One 2011, 6, e17881.
(8) Hoffmann-Roeder, A.; Schoenhentz, J.; Wagner, S.; Schmitt, E.
Chem. Commun. 2011, 47, 382−384.
(9) Springer, G. F. Science 1984, 224, 1198−1206.
(10) Holmberg, L. A.; Sandmaier, B. M. Expert Rev. Vaccines 2004, 3,
655−663.
(11) Kaczmarek, P.; Keay, S. K.; Tocci, G. M.; Koch, K. R.; Zhang,
C.-O.; Barchi, J. J. Jr.; Grkovic, D.; Guo, L.; Michejda, C. J. J. Med.
Chem. 2008, 51, 5974−5983.
(12) Keay, S.; Kaczmarek, P.; Zhang, C.-O.; Koch, K.; Szekely, Z.;
Barchi, J. J. Jr.; Michejda, C. Chem. Biol. Drug Des. 2011, 77, 421−430.
(13) Kang, J.; Low, W.; Norberg, T.; Meisenhelder, J.; Hansson, K.;
Stenflo, J.; Zhou, G.-P.; Imperial, J.; Olivera Baldomero, M.; Rigby
Alan, C.; Craig, A. G. Eur. J. Biochem. 2004, 271, 4939−49.
(14) Yoshida, H.; Geller, R. G.; Pisano, J. J. Biochemistry 1976, 15,
61−4.
(15) Dhanasekaran, M.; Polt, R. Curr. Drug Delivery 2005, 2, 59−73.
(16) Venugopal, H.; Edwards, P. J. B.; Schwalbe, M.; Claridge, J. K.;
Libich, D. S.; Stepper, J.; Loo, T.; Patchett, M. L.; Norris, G. E.; Pascal,
S. M. Biochemistry 2011, 50, 2748−2755.
(17) Paik, S. H.; Chakicherla, A.; Hansen, J. N. J. Biol. Chem. 1998,
273, 23134−42.
(18) Wu, C.-Y.; Wong, C.-H. Chem. Commun. 2011, 47, 6201−6207.
(19) Hojo, H.; Nakahara, Y. Biopolymers 2007, 88, 308−324.
(20) Wu, C. Y.; Liang, P. H.; Wong, C. H. Org. Biomol. Chem. 2009,
7, 2247−2254.
(21) Oyelaran, O.; Gildersleeve, J. C. Curr. Opin. Chem. Biol. 2009,
13, 406−13.
(22) Lonardi, E.; Balog, C. I.; Deelder, A. M.; Wuhrer, M. Expert Rev.
Proteomics 2010, 7, 761−774.
(23) Rillahan, C. D.; Paulson, J. C. Annu. Rev. Biochem. 2011, 80,
797−823.
6324
dx.doi.org/10.1021/ja212188r | J. Am. Chem. Soc. 2012, 134, 6316−6325

Journal of the American Chemical Society
Article
(24) Manimala, J. C.; Li, Z.; Jain, A.; VedBrat, S.; Gildersleeve, J. C.
ChemBioChem 2005, 6, 2229−2241.
(52) Goodman, M.; McGahren, W. J. Tetrahedron 1967, 23, 2031−
50.
(53) Young, A. R.; Barcham, G. J.; Kemp, J. M.; Dunphy, J. L.; Nash,
A.; Meeusen, E. N. Glycoconjugate J. 2009, 26, 423−432.
(54) Stupp, Y.; Sela, M. Biochim. Biophys. Acta 1967, 140, 349−59.
(55) Pollack, S. J.; Hsiun, P.; Schultz, P. G. J. Am. Chem. Soc. 1989,
111, 5961−2.
(56) Kim, J.; Keay, S. K.; Dimitrakov, J. D.; Freeman, M. R. FEBS
Lett. 2007, 581, 3795−3799.
(57) Brocke, C.; Kunz, H. Synthesis 2004, 525−542.
(58) Karch, F.; Hoffmann-Roeder, A. Beilstein. J. Org. Chem. 2010, 6,
47.
(25) Li, Q.; Anver, M. R.; Butcher, D. O.; Gildersleeve, J. C. Mol.
Cancer Ther. 2009, 8, 971−979.
(26) Zhang, Y.; Li, Q.; Rodriguez, L. G.; Gildersleeve, J. C. J. Am.
Chem. Soc. 2010, 132, 9653−9662.
(27) Li, Q.; Anver, M. R.; Li, Z.; Butcher, D. O.; Gildersleeve, J. C.
Int. J. Cancer 2010, 126, 459−68.
(28) Li, Q.; Rodriguez, L. G.; Farnsworth, D. F.; Gildersleeve, J. C.
ChemBioChem 2010, 11, 1686−91.
(29) Laurent, N.; Voglmeir, J.; Wright, A.; Blackburn, J.; Pham, N. T.;
Wong, S. C. C.; Gaskell, S. J.; Flitsch, S. L. ChemBioChem 2008, 9,
883−887.
(59) Becker, T.; Kaiser, A.; Kunz, H. Synthesis 2009, 1113−1122.
(60) Hojo, H.; Matsumoto, Y.; Nakahara, Y.; Ito, E.; Suzuki, Y.;
Suzuki, M.; Suzuki, A.; Nakahara, Y. J. Am. Chem. Soc. 2005, 127,
13720−13725.
(30) Westerlind, U.; Schroeder, H.; Hobel, A.; Gaidzik, N.; Kaiser,
A.; Niemeyer, C. M.; Schmitt, E.; Waldmann, H.; Kunz, H. Angew.
Chem., Int. Ed. 2009, 48, 8263−8267.
(31) Ohyabu, N.; Hinou, H.; Matsushita, T.; Izumi, R.; Shimizu, H.;
Kawamoto, K.; Numata, Y.; Togame, H.; Takemoto, H.; Kondo, H.;
Nishimura, S.-I. J. Am. Chem. Soc. 2009, 131, 17102−17109.
(32) Song, X.; Lasanajak, Y.; Rivera-Marrero, C.; Luyai, A.; Willard,
M.; Smith, D. F.; Cummings, R. D. Anal. Biochem. 2009, 395, 151−
160.
(61) Nakahara, Y.; Nakahara, Y.; Ogawa, T. Carbohydr. Res. 1996,
292, 71−81.
(62) Campo, V. L.; Carvalho, I.; Allman, S.; Davis, B. G.; Field, R. A.
Org. Biomol. Chem. 2007, 5, 2645−2657.
(63) Matsushita, T.; Hinou, H.; Kurogochi, M.; Shimizu, H.;
Nishimura, S. Org. Lett. 2005, 7, 877−880.
(64) Seitz, O.; Wong, C.-H. J. Am. Chem. Soc. 1997, 119, 8766−8776.
(65) Shin, Y.; Winans, K. A.; Backes, B. J.; Kent, S. B. H.; Ellman, J.
A.; Bertozzi, C. R. J. Am. Chem. Soc. 1999, 121, 11684−11689.
(66) Chen, L.; Jensen, K. J.; Tejbrant, J.; Taylor, J. E.; Morgan, B. A.;
Barany, G. J. Pept. Res. 2000, 55, 81−91.
(67) The reaction of 13 under condition 4 produced a complex
mixture of degradation products. We cannot rule out the formation of
β-elimination products in this case.
(33) Kracun, S. K.; Clo, E.; Clausen, H.; Levery, S. B.; Jensen, K. J.;
Blixt, O. J. Proteome Res. 2010, 9, 6705−6714.
(34) Pedersen, J. W.; Blixt, O.; Bennett, E. P.; Tarp, M. A.; Dar, I.;
Mandel, U.; Poulsen, S. S.; Pedersen, A. E.; Rasmussen, S.; Jess, P.;
Clausen, H.; Wandall, H. H. Int. J. Cancer 2010, 128, 1860−1871.
(35) Steentoft, C.; Schjoldager, K. T.; Clo, E.; Mandel, U.; Levery, S.
B.; Pedersen, J. W.; Jensen, K.; Blixt, O.; Clausen, H. Glycoconjugate J.
2010, 27, 571−582.
(36) Wandall, H. H.; Blixt, O.; Tarp, M. A.; Pedersen, J. W.; Bennett,
E. P.; Mandel, U.; Ragupathi, G.; Livingston, P. O.; Hollingsworth, M.
A.; Taylor-Papadimitriou, J.; Burchell, J.; Clausen, H. Cancer Res. 2010,
70, 1306−1313.
(37) Weinrich, D.; Koehn, M.; Jonkheijm, P.; Westerlind, U.;
Dehmelt, L.; Engelkamp, H.; Christianen, P. C. M.; Kuhlmann, J.;
Maan, J. C.; Nuesse, D.; Schroeder, H.; Wacker, R.; Voges, E.;
Breinbauer, R.; Kunz, H.; Niemeyer, C. M.; Waldmann, H.
ChemBioChem 2010, 11, 235−247.
(38) Dalgleish, A. G. Expert Rev. Vaccines 2004, 3, 665−668.
(39) Ouerfelli, O.; Warren, J. D.; Wilson, R. M.; Danishefsky, S. J.
Expert Rev. Vaccines 2005, 4, 677−685.
(40) Vosseller, K.; Sakabe, K.; Wells, L.; Hart, G. W. Curr. Opin.
Chem. Biol. 2002, 6, 851−857.
(41) Wells, L.; Hart, G. W. FEBS Lett. 2003, 546, 154−158.
(42) Olszewski, N. E.; West, C. M.; Sassi, S. O.; Hartweck, L. M.
Biochim. Biophys. Acta 2010, 1800, 49−56.
(43) Di Fenza, A.; Tancredi, M.; Galoppini, C.; Rovero, P.
Tetrahedron Lett. 1998, 39, 8529−8532.
(44) Albericio, F.; Carpino, L. A. Methods Enzymol. 1997, 289, 104−
126.
(45) Kuduk, S. D.; Schwarz, J. B.; Chen, X.-T.; Glunz, P. W.; Sames,
D.; Ragupathi, G.; Livingston, P. O.; Danishefsky, S. J. J. Am. Chem.
Soc. 1998, 120, 12474−12485.
(46) Carpino, L. A.; Ionescu, D.; El-Faham, A. J. Org. Chem. 1996, 61,
2460−5.
(47) Lee, D. J.; Harris, P. W. R.; Kowalczyk, R.; Dunbar, P. R.;
Brimble, M. A. Synthesis 2010, 763−769.
(48) Arsequell, G.; Krippner, L.; Dwek, R. A.; Wong, S. Y. C. J. Chem.
Soc., Chem. Commun. 1994, 2383−4.
(49) Kim, Y. S.; Gum, J. Jr.; Brockhausen, I. Glycoconjugate J. 1996,
13, 693−707.
(50) Coltart, D. M.; Royyuru, A. K.; Williams, L. J.; Glunz, P. W.;
Sames, D.; Kuduk, S. D.; Schwarz, J. B.; Chen, X.-T.; Danishefsky, S. J.;
Live, D. H. J. Am. Chem. Soc. 2002, 124, 9833−9844.
(51) Sakai, K.; Yuasa, N.; Tsukamoto, K.; Takasaki-Matsumoto, A.;
Yajima, Y.; Sato, R.; Kawakami, H.; Mizuno, M.; Takayanagi, A.;
Shimizu, N.; Nakata, M.; Fujita-Yamaguchi, Y. J. Biochem. 2010, 147,
809−817.
6325
dx.doi.org/10.1021/ja212188r | J. Am. Chem. Soc. 2012, 134, 6316−6325