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