Divergent behavior of glycosylated threonine and serine derivatives in solid phase peptide synthesis

Article abstract of DOI:10.1021/ol301723e

Solid phase peptide coupling of glycosylated threonine derivatives was systematically evaluated. In contrast to glycosylated serine derivatives which are highly prone to epimerization, glycosylated threonine derivatives produce only negligible amounts of epimerization. Under forcing conditions, glycosylated threonine analogs undergo β-elimination, rather than epimerization. Mechanistic studies and molecular modeling were used to understand the origin of the differences in reactivity. This article not subject to U.S. Copyright. Published 2012 by the American Chemical Society.

Full text of DOI:10.1021/ol301723e

ORGANIC
LETTERS
2012
Vol. 14, No. 15
3958–3961
Divergent Behavior of Glycosylated
Threonine and Serine Derivatives in Solid
Phase Peptide Synthesis
Yalong Zhang, Saddam M. Muthana, Joseph J. Barchi Jr., and Jeffrey C. Gildersleeve*
Chemical Biology Laboratory, National Cancer Institute, 376 Boyles Street,
Building 376, Frederick, Maryland 21702, United States
gildersj@mail.nih.gov
Received June 22, 2012
ABSTRACT
Solid phase peptide coupling of glycosylated threonine derivatives was systematically evaluated. In contrast to glycosylated serine derivatives
which are highly prone to epimerization, glycosylated threonine derivatives produce only negligible amounts of epimerization. Under forcing
conditions, glycosylated threonine analogs undergo β-elimination, rather than epimerization. Mechanistic studies and molecular modeling were
used to understand the origin of the differences in reactivity.
Glycopeptides have been used extensively in basic and
clinical research.1ꢀ5 Glycopeptides can serve as structu-
rally defined models of complex glycoproteins, and several
are in development as therapeutic agents. For example, a
glycopeptide isolated from urine has antiproliferative
activity,6 and glycosylation of an opioid peptide enhances
its in vivo performance.1 Glycopeptides are also being
developed as vaccines for cancer and HIV, and several
have progressed into clinical trials.2,7ꢀ9
Chemical synthesis allows precise control of the chemical
structure and access to large quantities of material. More-
over, one can obtain unnatural derivatives for studying
structureꢀactivity relationships.
The synthesis of glycopeptides can be significantly more
challenging than standard peptides. Glycopeptides are
commonly synthesized by coupling protected glyco-amino
acids to a growing peptide chain via solid phase glycopep-
tide synthesis.1 These couplings are often slow and ineffi-
cient. In addition, we recently demonstrated that many
commonly used peptide coupling conditions produce high
levels of epimerization for glyco-amino acids.11 In fact,
epimerization could produce as high as 80% of the un-
natural epimer. At present, however, the factors that
influence efficiency and epimerization are not well under-
stood, and additional studies are needed to develop effi-
cient and general peptide coupling conditions for
glycopeptides.
Chemical synthesis is an important tool for obtaining
structurally defined glycopeptides, since homogeneous
glycopeptides are difficult to obtain from natural sources.1,10
(1) Pratt, M. R.; Bertozzi, C. R. Chem. Soc. Rev. 2005, 34, 58.
(2) Westerlind, U.; Kunz, H. Carbohydr. Chem. 2010, 36, 1.
(3) Wilkinson, B. L.; Stone, R. S.; Capicciotti, C. J.; Thaysen-
Andersen, M.; Matthews, J. M.; Packer, N. H.; Ben, R. N.; Payne,
R. J. Angew. Chem., Int. Ed. 2012, 51, 3606.
(4) Gamblin, D. P.; Scanlan, E. M.; Davis, B. G. Chem. Rev. 2009,
109, 131.
(5) Payne, R. J.; Wong, C.-H. Chem. Commun. 2010, 46, 21.
(6) 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.
Our previous study focused on coupling glycosylated
serine derivatives to a growing peptide chain.11 In this
study, we evaluated efficiency and epimerization when
coupling glycosylated threonine derivatives. Nonglycosy-
lated threonine derivatives are known to react more slowly
(7) Dalgleish, A. G. Expert Rev. Vaccines 2004, 3, 665.
(8) Zhu, J.-L.; Warren, J. D.; Danishefsky, S. J. Expert Rev. Vaccines
2009, 8, 1399.
(9) Springer, G. F. Science 1984, 224, 1198.
(10) Buskas, T.; Ingale, S.; Boons, G.-J. Glycobiology 2006, 16, 113R.
(11) Zhang, Y.; Muthana, S. M.; Farnsworth, D.; Ludek, O.; Adams,
K.; Barchi, J. J.; Gildersleeve, J. C. J. Am. Chem. Soc. 2012, 134, 6316.
10.1021/ol301723e This article not subject to U.S. Copyright. Published 2012 by the American Chemical Society
Published on Web 07/20/2012

than the corresponding serine derivatives in solid phase
peptide coupling reactions.12 Since a slower coupling
rate would allow more time for epimerization, it was
possible that glycosylated threonine residues could be even
more prone to side reactions.
Two commonly used glyco-amino acids, Fmoc-Thr-
(Ac3GalNAcR)-OH and Fmoc-Thr(Ac3GlcNAcβ)-OH,
wereselectedfor thisstudy, along withthe nonglycosylated
counterpart as shown in Scheme 1. The desired glyco-
peptides, the unnatural epimers, and the truncated
(uncoupled) peptides were chemically synthesized and
used as standards for the development of HPLC assays
to evaluate efficiency and epimerization (see Supporting
Information (SI)). We selected seven coupling conditions
thathadpreviously beenusedtoprepare glycopeptides(see
Table 1 for references). They contain the most widely used
activation agents, bases, and solvents, as well as variations
in reaction times and equivalents of bases and reagents.
The results are summarized in Table 1. Little or no
truncated peptide was observed in any of the reactions.
Moreover, only negligible epimerization (e1.5%) was
detected for all of the coupling reactions. This result was
in stark contrast to our previous results with glycosylated
serine analogs. For example, coupling of Fmoc-Ser-
(Ac3GalNAcR)-OH using condition 2 produced ∼70%
epimerization, whereas <1% epimerization was observed
for glycosylated threonine derivatives. Under several
Scheme 1. Coupling Threonine and Glycosylated Threonine
Derivatives to ProGlyHex Resin
We carried out a systematic comparison by coupling
glyco-amino acids to a growing peptide chain, Pro-
Gly-Hex (Hex: 6-aminohexanoic acid), on solid phase.
Table 1. Summary of Yields, Epimerization, and β-Eliminaion for Various Peptide Coupling Conditions
Fmoc-Thr(R)-OH, where R =
no.
1
coupling conditionsa
Trt
Ac3GalNAcR
Ac3GlcNAcβ
AAs: 1.5 equiv
yields (%)b
98.3
<0.2
<0.2
98.2
0.4
98.8
0.3
HATU/HOAt: 1.2/1.2 equiv
NMM: 2.4 equiv
0/8 h in DMF13
epimerization (%)b
β-elimination (%)b
<0.2
0.5
2
3
4
5
6
7
AAs: 4.4 equiv
yields (%)b
epimerization (%)b
β-elimination (%)b
99.9
1.5
84.1
0.9
40.4
0.8
HATU: 4.4 equiv
NMM: 8.8 equiv
3/12 h in NMP14
AAs: 2 equiv
<0.2
<0.2
59.4
yields (%)b
epimerization (%)b
β-elimination (%)b
98.6
0.3
99.1
0.6
94.1
<0.2
5.0
HBTU/HOBt: 4.5/4.5 equiv
DIEA: 9 equiv
0/1 h in NMP15
<0.2
0.4
AAs: 3 equiv:
yields (%)b
epimerization (%)b
β-elimination (%)b
99.8
0.2
99.2
0. 7
0.7
97.9
0.3
DCC/HOBt: 18/18 equiv
1/64 h in NMP16
<0.2
1.9
AAs: 1.5 equiv
yields (%)b
epimerization (%)b
β-elimination (%)b
92.5
<0.2
<0.2
99.3
0.5
95.3
0.6
HBTU/HOBt: 1.5/1.5 equiv
DIEA: 1.5 equiv
0/0.33 h in DMF17
AAs: 2.5 equiv
0.3
2.2
yields (%)b
epimerization (%)b
β-elimination (%)b
98.3
0.2
99.0
0.4
98.6
0.3
BOP/HOBt: 2.5/2.5 equiv
DIEA: 2.5 equiv
0/4 h in DMF18
<0.2
0.3
1.1
AAs: 2.0 equiv
yields (%)b
epimerization (%)b
β-elimination (%)b
99.1
<0.2
<0.2
99.0
0.4
98.2
0.5
HATU/HOAt: 2.0/2.0 equiv
TMP: 2.0 equiv
0.3
0.3
0/2 h in DMF
a Glyco-amino acids were coupled to Pro-Gly-Hex resin. Preincubation time and coupling times are listed as x/y h (e.g., 3/12 h = 3 h preincubation
followed by 12 h reaction time). b All data have an error of less than 0.3%. The yield refers to the percentage of DþL products relative to the total peptide
(DþL products, β-elimination product, and truncated peptide). Abbreviations are as follows: 2-(7-Aza-1H-benzotriazol-1-yl)-N,N,N0,N0-tetramethyl
aminium hexafluorophosphate (HATU), 2-(1H-benzotriazol-1-yl)-N,N,N0,N0-tetramethylaminium hexafluorophosphate (HBTU), N,N0-dicyclohex-
ylcarbodiimide (DCC), benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (BOP), N,N-diisopropylethylamine (DIEA), N-methyl-
morpholine (NMM), 2,4,6-trimethylpyridine (TMP), dimethylformamide (DMF), and N-methyl-2-pyrrolidone (NMP).
Org. Lett., Vol. 14, No. 15, 2012
3959

conditions, however, an unknown peptide was produced
accounting for up to 60% of the mass. Further analysis
revealed the side product to be Fmoc-dehydroaminobuty-
rate-Pro-Gly-Hex-OH, which results from β-elimination
of the glycan moiety (Scheme 2, route b). Based on this
result, we assessed β-elimination for all three amino acids
and all seven conditions. β-Elimination primarily occurred
for Fmoc-Thr(Ac3GlcNAcβ)-OH.
The difference in behavior of glycosylated serine and
threonine derivatives in peptide coupling reactions could
be due to many factors. First, we anticipated that glyco-
sylated threonine derivatives would react more slowly than
serine derivatives; however, the relative rates had not been
previously measured. If glycosylated threonine derivatives
actually react faster, then there would be less time for
epimerization. To test this possibility, we measured the
relative coupling rates of other amino acids against threo-
nine in a competition assay. Fmoc-Thr(Trt)-OH was
mixed with the competing amino acid at an equivalent
amount. Themixture was activated and thencaptured with
the resin. The relative amounts of each peptide produced
after 5 min were measured via an HPLC assay. As sum-
marized in Table 2, the overall coupling rates of threonine-
based amino acids were slower than serine derivatives.
Therefore, a faster rate of peptide coupling does not
account for the large difference in epimerization levels.
for 3 h and then coupled them to ProGlyHex resin
(condition 2). The long preincubation step permits epimer-
ization. If the natural epimer is energetically favored and
the rate of epimerization is sufficient, extensive amounts of
the natural epimer would be formed in these reactions. In
actuality, very little epimerization was observed in these
reactions. Fmoc-D-Thr(Trt)-OH gave∼4% epimerization,
and Fmoc-D-Thr(Ac3GalNAcR)-OH gave <0.2% epimer-
ization. Since epimerization was not observed for either the
natural or unnatural epimer, we concluded that the rate of
epimerization was too slow to reach equilibrium. Interest-
ingly, Fmoc-D-Thr(Ac3GlcNAcβ)-OH produced 92.4% of
the β-elimination product under these conditions. Epimer-
ization may be occurring for this substrate, but rapid
β-elimination prevents measurement of the equilibrium.
Scheme 2. Potential Routes for Epimerization (a) and β-Elim-
ination (b)
Table 2. Relative Overall Coupling Rate vs Threonine
amino acida
relative reaction rate AA/Thr
To better evaluate the equilibria, we next attempted to
increase the rate of epimerization by varying the type of
base and increasing the number of equivalents of base. As
shown in Figure 1, we preincubated Fmoc-Thr-
(Ac3GlcNAcβ)-OH (a) or Fmoc-Thr(Ac3GalNAcR)-OH
(b), and HATU for 3 h with different equivalents of NMM
(a) or DIEA (b), respectively. Even under forcing condi-
tions, little D-epimer was detected. Instead the β-elimi-
nated peptide was observed as the major product for both
glyco-amino acids. For example, incubation of Fmoc-
Thr(Ac3GlcNAcβ)-OH with 12 equiv of NMM produced
∼90% β-eliminated product. Although we were unable to
measure the equilibrium ratio of epimers, it is clear that
glycosylated serine and threonine analogs produce differ-
ent side products in these coupling reactions.
In our previous study on peptide couplings of glycosy-
lated serine analogs, we found that the mild base, TMP,
provides high yields with little or no epimerization. To
further test the utility of this base, we evaluated the use of
higher equivalents of TMP, analogous to the studies above
with NMM and DIEA. Remarkably, epimerization and
β-elimination were <5% with up to 8 equiv of TMP for all
three amino acids. Therefore, TMP produces at least a
10-fold lower level of side products as compared to NMM
and DIEA. Based on these results and our previous results,
we consider condition 7 (2 equiv of glyco-amino acid, 2 equiv
of HATU and HOAt, and 2 equiv of TMP) to be the best
conditions we have examined for solid phase peptide
couplings involving glycosylated amino acids. Other mild
Fmoc-Thr(Trt)-OH
1
Fmoc-Ser(Trt)-OH
3.33 ( 0.53
0.70 ( 0.03
0.90 ( 0.02
2.62 ( 0.06
1.01 ( 0.04
Fmoc-Thr(Ac3GalNAcR)-OH
Fmoc-D-Thr(Ac3GalNAcR)-OH
Fmoc-Thr(Ac3GlcNAcβ)-OH
Fmoc-D-Thr(Ac3GlcNAcβ)-OH
a All reactions were carried out by mixing Fmoc-Thr(Trt)-OH
(2 equiv) and the listed amino acid (2 equiv) with HATU (4 equiv), HOAt
(4 equiv), and TMP (4 equiv) in DMF. The assays were conducted in triplicate.
A second possible explanation is that for glycosylated
threonine derivatives, the equilibrium between the natural
and unnatural epimer lies heavily in favor of the natural
epimer. If this was the case, epimerization could be occur-
ring in the reaction, but it would not produce significant
amounts of the unnatural epimer. To test this hypothesis,
we preincubated the unnatural epimers of Fmoc-D-Thr-
(Trt)-OH and D-glyco-amino acids with HATU/NMM
(12) Ragnarsson, U.; Karlsson, S.; Sandberg, B. Acta Chem. Scand.
1971, 25, 1487.
(13) Karch, F.; Hoffmann-Roder, A. Beilstein J. Org. Chem. 2010, 6, 47.
(14) Becker, T.; Kaiser, A.; Kunz, H. Synthesis 2009, 1113.
(15) Hojo, H.; Matsumoto, Y.; Nakahara, Y.; Ito, E.; Suzuki, Y.;
Suzuki, M.; Suzuki, A.; Nakahara, Y. J. Am. Chem. Soc. 2005, 127, 13720.
(16) Nakahara, Y.; Nakahara, Y.; Ogawa, T. Carbohydr. Res. 1996,
292, 71.
(17) Matsushita, T.; Hinou, H.; Kurogochi, M.; Shimizu, H.;
Nishimura, S. Org. Lett. 2005, 7, 877.
(18) Chen, L.; Jensen, K. J.; Tejbrant, J.; Taylor, J. E.; Morgan, B. A.;
Barany, G. J. Pept. Res. 2000, 55, 81.
3960
Org. Lett., Vol. 14, No. 15, 2012

peptide coupling conditions may also be useful for making
glycopeptides.19
While the above experiments provided interesting in-
sight into the reaction pathways, it was still not clear why
glyco-threonine analogs have much lower levels of epimer-
ization than glyco-serine analogs. Previous NMR studies
haveshown thatglyco-serine and glyco-threonine can have
significantly different preferred conformations in water,20
but little information was available for protected deriva-
tives in organic solvents. Therefore, we carried out mole-
cular modeling to determine if differences in confor-
mational preferences might contribute to differences in
reactivity. To mimic the reaction solvent, a distance de-
pendent dielectric constant of 38 was used. Since epimer-
ization and β-elimination are thought to proceed through
an oxazolone intermediate21 (see Scheme 2), the correspond-
ing oxazolone intermediates of Fmoc-Thr(Ac3GalNAcR)-
OH and Fmoc-Ser(Ac3GalNAcR)-OH were modeled using
methods reported previously.11
Figure 2. Low-energy models of the oxazolone intermediates
derived from (A, C) Fmoc-Ser(GalNAcR)-OH and (B, D)
Fmoc-Thr(GalNAcR)-OH generated from the Conformational
Search utility in Macromodel 2011. Panels (C) and (D) focus on
the oxazolone ring and the HRꢀCRꢀCβꢀOβ dihedral angle.
hydrogen. Therefore, the combination of steric and stereo-
electronic effects may contribute to the low epimerization of
glyco-threonine analogs compared with the glyco-serine
ones. When abstraction of the R-hydrogen is possible in a
threonine derivative, formation of a more highly substituted
alkene relative to serine derivatives likely contributes to the
preference for β-elimination over epimerization.
In summary, we demonstrate that serine and threonine
based glyco-amino acids display significantly different beha-
viors in solid phase peptide coupling reactions. Glyco-threo-
nine derivatives produce little or no epimerization but,
occasionally, give rise to β-elimination. In contrast, glyco-
serine derivatives are highly prone to epimerization. The
difference in reactivity is likely due to distinct differences in
conformational preferences of the reactive intermediates. For-
tunately, use of TMP as a base in the peptide coupling
reactions significantly reduces both epimerization and β-elim-
ination side reactions. Finally, our studies show that even small
structural changes to glyco-amino acids, such as the presence
or absence of a single methyl group, can have a dramatic effect
on side reactions. Therefore, a more detailed understanding of
the factors that contribute to epimerization and β-elimination
will significantly improve our ability to synthesize glycopep-
tides with high efficiency and predictability.
Figure 1. Epimerization and β-elimination as a function of base.
(a) Fmoc-Thr(Ac3GlcNAcβ)-OH; (b) Fmoc-Thr(Ac3GalNAcR)-
OH. Glyco-amino acids were coupled using the following cou-
pling agents and ratios: AAs/HATU/base = 1/1/x. Each amino
acid was preincubated for 3 h, and then the extent of epimerization
and β-elimination were measured as described in the SI.
The preferred conformations of the glyco-serine and
glyco-threonine oxazolones showed distinct differences
(see Figure 2). In particular, the HRꢀCRꢀCβꢀOβ di-
hedral angle for the threonine derivative was ∼60°
(Figure 2D), placing the β-methyl and the O-GalNAc
groups gauche to the R-hydrogen. In contrast, the
HRꢀCRꢀCβꢀOβ dihedral angle for the serine derivative
was ∼180° (Figure 2, C), placing the O-GalNAc group
anti to the R-hydrogen. The difference has two key effects.
First, the R-hydrogen of glyco-threonine analogs is more
sterically shielded than that of glyco-serine derivatives, which
could significantly hinder abstraction of the R-hydrogen.
Second, overlap of the CβꢀO σ*-antibonding orbital with
the CRꢀH σ-bond in the glyco-serine derivative, but not the
threonine derivative, could facilitate abstraction of this
Acknowledgment. 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.
Supporting Information Available. Experimental de-
tails for the syntheses of glyco-amino acids and supple-
mental HPLC data. This material is available free of
charge via the Internet at http://pubs.acs.org.
(19) Mitchell, S. A.; Pratt, M. R.; Hruby, V. J.; Polt, R. J. Org. Chem.
2001, 66, 2327.
(20) Corzana, F.; Busto, J. H.; Jimenez-Oses, G.; Garcia de Luis, M.;
Asensio, J. L.; Jimenez-Barbero, J.; Peregrina, J. M.; Avenoza, A. J. Am.
Chem. Soc. 2007, 129, 9458.
(21) Albericio, F.; Carpino, L. A. Methods Enzymol. 1997, 289, 104.
The authors declare no competing financial interest.
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