Sugar-assisted glycopeptide ligation with complex oligosaccharides: Scope and limitations

Article abstract of DOI:10.1021/ja8010513

We have previously shown sugar-assisted ligation (SAL) to be a useful method for the convergent construction of glycopeptides. However to date SAL has only been carried out on systems where the thiol auxiliary is attached to a monosaccharide. For SAL to be truly applicable to the construction of fully elaborated glycopeptides and glycoproteins, it must be possible to carry out the reaction when the thiol auxiliary is attached to more elaborate sugars, as these are frequently what are observed in nature. Here we examine the effects of glycosylation at C-3, C-4, and C-6 of the C-2 auxiliary-containing glycan. Model glycopeptides where synthesized chemoenzymatically and reacted with peptide thioesters used in our previous work. These studies reveal that SAL is sensitive to extended glycosylation on the auxiliary-containing sugar. While it is possible to carry out SAL with extended glycosylation at C-4 and C-6, the presence of glycosylation at C-3 prevents the ligation from occurring. Additionally, with glycosylation at C-4 the ligation efficiency is affected by the identity of the N-terminal AA, while the nature of the C-terminal residue of the peptide thioester does not appear to affect ligation efficiency. These studies provide useful guidelines in deciding when it is appropriate to use SAL in the synthesis of complex glycopeptides and glycoproteins and how to choose ligation junctions for optimal yield.

Full text of DOI:10.1021/ja8010513

Published on Web 08/13/2008
Sugar-Assisted Glycopeptide Ligation with Complex
Oligosaccharides: Scope and Limitations
Clay S. Bennett,† Stephen M. Dean,† Richard J. Payne,†,‡ Simon Ficht,†
Ashraf Brik,†,§ and Chi-Huey Wong*,†,
Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road,
La Jolla, California 92037 and Genomics Research Center, Academia Sinica, 128 Section 2,
Academia Road, Nankang, Taipei 115, Taiwan
Received February 11, 2008; E-mail: wong@scripps.edu; chwong@gate.sinica.edu.tw
Abstract: We have previously shown sugar-assisted ligation (SAL) to be a useful method for the convergent
construction of glycopeptides. However to date SAL has only been carried out on systems where the thiol
auxiliary is attached to a monosaccharide. For SAL to be truly applicable to the construction of fully elaborated
glycopeptides and glycoproteins, it must be possible to carry out the reaction when the thiol auxiliary is
attached to more elaborate sugars, as these are frequently what are observed in nature. Here we examine
the effects of glycosylation at C-3, C-4, and C-6 of the C-2 auxiliary-containing glycan. Model glycopeptides
where synthesized chemoenzymatically and reacted with peptide thioesters used in our previous work.
These studies reveal that SAL is sensitive to extended glycosylation on the auxiliary-containing sugar.
While it is possible to carry out SAL with extended glycosylation at C-4 and C-6, the presence of glycosylation
at C-3 prevents the ligation from occurring. Additionally, with glycosylation at C-4 the ligation efficiency is
affected by the identity of the N-terminal AA, while the nature of the C-terminal residue of the peptide
thioester does not appear to affect ligation efficiency. These studies provide useful guidelines in deciding
when it is appropriate to use SAL in the synthesis of complex glycopeptides and glycoproteins and how to
choose ligation junctions for optimal yield.
Introduction
that in biological systems glycoproteins are produced as
heterogeneous mixtures of glycoforms and isoforms which are
Protein glycosylation is a complex co/post-translational
modification which has been estimated to be present in greater
than 50% of all human proteins.1 The attachment of oligosac-
charides to the protein backbone introduces a staggering array
of structural and functional diversity, far beyond that which is
available from the 20 canonical amino acids.2 Accordingly,
glycans have been shown to play important roles in a number
of biological processes, including protein folding, cellular
adhesion, cell targeting, and cell differentiation.3-7 In addition,
abnormal glycosylation of proteins is linked with several
disorders including autoimmune diseases and cancer.8,9 Despite
its importance, very little is understood about the consequences
of protein glycosylation at the molecular level, due to the fact
extremely difficult, if not impossible, to separate on a preparative
scale. As a result, there has been a significant amount of research
directed at the production of homogeneous glycoproteins through
chemical and chemoenzymatic methods.10-12
One of the most common approaches to the chemical
synthesis of proteins involves the union of smaller peptide
fragments through native chemical ligation (NCL).13,14 NCL
has proved to be extremely powerful for the convergent
synthesis of large glycopeptides and glycoproteins.15-21 Al-
(10) David, B. G. Chem. ReV. 2002, 102, 579–601.
(11) Pratt, M. R.; Bertozzi, C. R. Chem. Soc. ReV. 2005, 34, 58–68.
(12) Bennett, C. S.; Wong, C. H. Chem. Soc. ReV. 2007, 36, 1227–1238.
(13) Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. H. Science
1994, 266, 776–779.
† The Scripps Research Institute.
(14) Dawson, P. E.; Kent, S. B. H. Annu. ReV. Biochem. 2000, 69, 923–
960.
‡ Present Address: School of Chemistry, University of Sydney, NSW
2006, Australia
(15) 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.
(16) Marcaurelle, L. A.; Mizoue, L. S.; Wilken, J.; Oldham, L.; Kent,
S. B. H.; Handel, T. M.; Bertozzi, C. R. Chem.sEur. J. 2001, 7, 1129–
1132.
§ Present Address: Department of Chemistry, Ben-Gurion University,
Beer Sheva, Israel
Academia Sinica.
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J. AM. CHEM. SOC. 2008, 130, 11945–11952 11945

A R T I C L E S
Bennett et al.
Scheme 1. Mechanism of Sugar-Assisted Ligation (SAL)
though useful, NCL is limited by the need for a N-terminal
cysteine, alanine, or phenylalanine residue, the latter two of
which are introduced following NCL through chemical
desulfurization of cysteine and ꢀ-mercaptophenylalanine,
respectively.22-24 One approach to circumvent the fact that many
glycoproteins do not possess these residues at strategically useful
positions along the protein backbone is the use of cysteine-free
ligation techniques. In this approach a thiol-containing auxiliary
is placed at the N-terminus of a peptide to act as a cysteine
surrogate.25,26 While the use of auxiliaries has expanded the
number of possible ligation junctions available for glycoprotein
synthesis, this approach is limited to sterically unencumbered
amino acids.27-30
Recently, our laboratory has introduced an alternative ap-
proach to glycopeptide ligation, termed sugar-assisted ligation
(SAL).31-35 This approach utilizes a glycopeptide in which the
thiol auxiliary is attached through a sugar (N-acetylglucosamine
or N-acetylgalactosamine) via either the C-2 acetamide or a
2-mercaptoacetate moiety on the C-3 hydroxyl group. Similar
to other auxiliaries, these glycopeptides undergo thioester
exchange with a peptide thioester, followed by an S f N acyl
shift to afford a native peptide linkage (Scheme 1). The reaction
shows high tolerance at the ligation junction and is orthogonal
to functional groups present in all proteinogenic amino acids.32
Following ligation, the thiol handle can be removed through
hydrogenolysis, in the case of the 2-mercaptoacetamido auxil-
iary, or deacetylation, in the case of the 2-mercaptoacetyl
auxiliary. While the use of chemical desulfurization is incom-
patible with unprotected cysteine residues, recent work from
the Kent group, the Danishefsky group, and our own laboratory
has shown that protection of cysteine residues with an aceta-
midomethyl(Acm)protectinggroupcircumventsthisproblem.23,36,37
The utility of this approach has been demonstrated through the
synthesis of the glycoprotein antibiotic diptericin,36 opening up
new avenues for the synthesis of complex glycopeptides and
possibly glycoproteins.38
approach is always not practical for glycoprotein synthesis as
the small differences in molecular weight between the starting
material and product make purification exceedingly difficult if
the reaction does not proceed to completion. Additionally,
because it is not possible to selectively elaborate one glycan in
the presence of others of the same type, the current approach is
not amenable to the selective synthesis of glycoproteins contain-
ing multiple glycans with differences in their composition.39 In
order to circumvent these problems, it is necessary to take a
more convergent approach, where the fully elaborated glycans
are attached to the peptide backbone using chemical or
enzymatic methods prior to ligation. We therefore sought to
examine if it was possible to carry out SAL in systems where
the thiol auxiliary was attached to more complex glycans.
In order to test the compatibility of SAL with complex
auxiliary-containing glycans we chose to examine model
glycopeptides 1a-d, 2a-d, 3a-d, and 4 in the reaction (Figure
1). These peptides would allow us to probe the effects of
glycosylation at C-3, C-4, and C-6 of the bridgehead sugar. By
using peptide backbones that have been examined in previous
studies, we could make direct comparison to the SAL reactions
of glycopeptides bearing monosaccharides. Importantly, com-
pounds 1a-d, 2a-d, and 3a-d could be prepared enzymati-
cally, providing us with an opportunity to examine the ability
of glycosyltransferases to effectively elaborate the auxiliary-
containing sugar. Here we report that extended glycosylation
of the auxiliary-containing sugar can have a profound effect on
Given our interest in the chemical synthesis of homogeneous
glycoproteins, our next goal was to apply SAL to the construc-
tion of glycopeptides containing complex glycans. Although we
had previously demonstrated that the auxiliary-containing glycan
can be enzymatically elaborated following ligation,32 this
(22) Yan, L. Z.; Dawson, P. E. J. Am. Chem. Soc. 2001, 123, 526–533.
(23) Pentlute, B. L.; Kent, S. B. H. Org. Lett. 2007, 9, 687–690.
(24) Crich, D.; Banerjee, A. J. Am. Chem. Soc. 2007, 129, 10064–10065.
(25) Offer, J.; Boddy, C. N. C.; Dawson, P. E. J. Am. Chem. Soc. 2002,
124, 4642–4646.
(26) Low, D. W.; Hill, M. G.; Carrasco, M. R.; Kent, S. B. H.; Botti, P.
Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 6554–6559.
(27) Macmillan, D.; Anderson, D. W. Org. Lett. 2004, 6, 4659–4125.
(28) Wu, B.; Chen, J. H.; Warren, J. D.; Chen, G.; Hua, Z. H.; Danishefsky,
S. J. Angew. Chem., Int. Ed. 2006, 45, 4116–4125.
(29) Macmillan, D. Angew. Chem., Int. Ed. 2006, 45, 7668–7672.
(30) Chen, G.; Warren, J. D.; Chen, J.; Wu, B.; Wan, Q.; Danishefsky,
S. J. J. Am. Chem. Soc. 2006, 128, 7460–7462.
(31) Brik, A.; Yang, Y. Y.; Ficht, S.; Wong, C. H. J. Am. Chem. Soc.
2006, 128, 5626–5627.
(32) Brik, A.; Ficht, S.; Yang, Y. Y.; Bennett, C. S.; Wong, C. H. J. Am.
Chem. Soc. 2006, 128, 15026–15033.
(33) Ficht, S.; Payne, R. J.; Brik, A.; Wong, C. H. Angew. Chem., Int. Ed.
2007, 46, 5975–5979.
(34) Payne, R. J.; Ficht, S.; Tang, S.; Brik, A.; Yang, Y. Y.; Wong, C. H.
J. Am. Chem. Soc. 2007, 129, 13527–13536.
(35) Brik, A.; Wong, C. H. Chem.sEur. J. 2007, 13, 5670–5675.
(36) Yang, Y. Y.; Ficht, S.; Brik, A.; Wong, C. H. J. Am. Chem. Soc.
2007, 129, 7690–7701.
(39) For a recent example where enzymatic elaboration of a glycopeptide
containing two N-acetylglucosamine residues led to mixtures of
products, see: (a) Haneda, K.; Takeuchi, M.; Tagashira, M.; Inazu,
T.; Toma, K.; Isogai, Y.; Hori, M.; Kobayashi, K.; Takeuchi, M.;
Takegawa, K.; Yamamoto, K. Carbohydr. Res. 2004, 341, 181–190.
(37) Wan, Q.; Danishefsky, S. J. Angew. Chem., Int. Ed. 2007, 46, 9248–
9252.
(38) Hsieh-Wilson, L. Nature 2007, 445, 31–33.
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Sugar-Assisted Ligation with Complex Glycans
A R T I C L E S
Figure 1. Model auxiliary-containing glycopeptides to be examined in this study.
glycosidic linkage during SPPS. Hydrogenolysis of the benzyl
groups occurred with partial removal of the 2,2,2-trichloroethyl
carbamate (Troc), necessitating its reinstallation prior to global
acetylation. The resulting peracetylated disaccharide was reacted
with benzylamine in THF to effect selective cleavage of the
anomeric acetate, followed by treatment with 2,2,2-trichloro-
acetonitrile in the presence of potassium carbonate to afford
glycosyl trichloroacetimidate 10.44 This was coupled to protected
serine 1345 in the presence of TMSOTf and activated sieves to
afford glycosyl amino acid 12 in excellent yield. Removal of
the Troc protecting group was followed by HBTU/DIEA-
mediated coupling of the free amine to S-trityl-2-mercaptoacetic
acid46 and removal of the allyl protecting group47 to provide
glycosyl amino acid 6 in high yield.
Solid-Phase Peptide and Glycopeptide Synthesis. Synthesis
of glycopeptides 14a-d (Scheme 3), which served as precursors
for enzymatic elaboration, was achieved using SPPS on Rink-
amide resin.48 Due to the complexity of 5, only 1 equiv of amino
acid was used and the coupling time was extended to 6 h. As
reported previously,32,34 these conditions were sufficient for
efficient coupling as determined by the UV absorption of the
Fmoc/piperidine adduct at 302 nm. Following coupling of 5,
the resin was split into four equal parts for the synthesis of Gly,
Asp, His, and Gly-Gly glycopeptides 14a-d. These targets were
chosen because the N-terminal Gly, Asp, and His glycopeptides
perform particularly well in SAL, while the Gly-Gly peptide
provides a simple extended glycopeptide model. Following the
final coupling, the acetate protecting groups on the sugar were
removed by hydrazinolysis prior to release from the resin using
Figure 2. Auxiliary-containing glycosyl amino acids required for glyco-
peptide synthesis.
the SAL reaction. The efficiency of the reactions appears to be
dictated by the position in which the bridgehead sugar is
modified.
Results and Discussion
Synthesis of Glycosyl Amino Acid Building Blocks. The initial
phase of this study involved the construction of the auxiliary-
containing glycosyl amino building blocks for solid-phase
peptide synthesis (SPPS) of the model glycopeptides (Figure
1). We envisioned that glycopeptides 1-3 could be constructed
through enzymatic elaboration of a monosaccharide-containing
glycopeptide following SPPS. As such most of the targets could
be synthesized from the previously reported amino acid 5
(Figure 2).34 For the construction of glycopeptide 4 it was
necessary to synthesize fucose-containing amino acid 6 (Figure
2), as there is no readily available enzyme for modification at
the C-6 position of the bridgehead GlcNAc.
Synthesis of 6 began with transformation of known glu-
cosamine derivative 740 into triol 8 through a three-step sequence
(Scheme 2). Triol 8 was coupled to fucosyl bromide 1241 under
halide ion conditions to afford disaccharide 9 in modest
yield.42,43 At this point it was necessary to replace the benzyl
protecting groups with acetates in order to stabilize the
(42) Lemieux, R. U.; Hendriks, K. B.; James; Stick, R. V.; James, K. J. Am.
Chem. Soc. 1975, 97, 4056–4062.
(43) For the use of halide-ion-mediated glycosylation on a similar system,
see: (a) Lee, H. H.; Baptista, J. A. B.; Krepinsky, J. J. Can. J. Chem.
1990, 68, 953–957.
(44) Schmidt, R. R.; Michel, J. Tetrahedron Lett. 1984, 25, 821–824.
(45) Vorheer, T.; Bannwarth, W. Bioorg. Med. Chem. Lett. 1995, 5, 2661–
2664.
(46) Kupihar, Z.; Schmel, Z.; Kele, Z.; Penke, B.; Kovacs, L. Bioorg. Med.
Chem. Lett. 2001, 9, 1241–1247.
(40) Paulsen, H.; Krogmann, C. Liebigs Ann. Chem. 1989, 1203–1213.
(41) Dejter-Juszynski, D.; Flowers, H. M. Carboydr. Res. 1971, 18, 215–
226.
(47) Ciommer, M.; Kunz, H. Synlett 1991, 8, 593–595.
(48) Albericio, F.; Carpino, L. A. Methods Enzymol. 1997, 289, 104–126.
9
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A R T I C L E S
Bennett et al.
Scheme 2. Synthesis of Glycosyl Amino Acid 6a
a Conditions: (a) (i) 30% HBr/AcOH, CH2Cl2; (ii) BnOH, Ag2CO3, AW300MS, CH2Cl2; (iii) 1:1 0.1 N NaOH (aq.)/MeOH, 50%, 3 steps; (b) TBAB, 12,
2:1 CH2Cl2/DMF, 4 Å MS, 37%; (c) (i) H2, 10% Pd/C, 3:1 5% formic acid in MeOH/ethyl acetate; (ii) TrocCl, NaHCO3, H2O; (iii) Ac2O, pyridine, 71%,
3 steps; (d) BnNH2, THF, 55%; (e) Cl3CCN, K2CO3, CH2Cl2, 59%; (f) 13, TMSOTf, AW300MS, CH2Cl2, -78 °C, 82%; (g) Zn, AcOH, 70%; (h)
TrtSCH2COOH, HBTU, DIEA, DMF, 95%; (i) Pd(PPh3)4, NMA, THF, 82%. AW300MS ) acid washed 3 Å molecular sieves, TBAB ) tetrabutylammonium
bromide, TrocCl ) 2,2,2-trichloroethyl chloroformate, HBTU ) 2-(1H-benzotriazole-1y-l)-1,1,3,3-tetramethylaminium hexafluorophosphate, DIEA ) N,N-
diisopropylethylamine, NMA ) N-methylaniline.
Scheme 3. Fmoc-Based SPPS of Glycopeptides 14a-d
so that direct comparisons could be made with SAL reactions
bearing monosaccharides which were reported previously. The
N-termini of the peptide thioesters were capped with acetates
prior to removal from the resin in order to prevent any unwanted
aminolysis during the ligation reaction.50-52
Enzymatic Elaboration of Glycopeptides. Having synthesized
glycopeptides 14a-d, we next turned our attention to the
enzymatic synthesis of glycopeptides 1-3a-d. To achieve this,
we chose to focus on glycosyltransferase-mediated elaboration
of 14a-d, as many of these enzymes are known to have a broad
substrate tolerance with regard to the glycosyl acceptor.53
Importantly, from previous studies we already knew that the
presence of the sulfhydryl group at C-2 of the bridgehead sugar
did not interfere with at least one glycosyltransferase, ꢀ-1,4-
galactosyltransferase (GalT).32 As such, treatment of glycopep-
tides 14a-d with GalT and UDP-galactose (UDP-Gal) in the
presence of alkaline phosphatase and MnCl2 afforded the desired
disaccharide products 1a-d in 81-86% isolated yields (Scheme
5).54-56 These products were readily converted to sialic acid
containing trisaccharides 2a-d upon treatment with CMP-sialic
acid (CMP-sial) and R-2,3-(N)-sialyltransferase (SialT), under
conditions similar to those described for the GalT reaction.57
Conversion of 2a-d to glycopeptides 3a-d bearing the sialyl
trifluoroacetic acid/triisopropylsilane/thioanisole/water (17:1:1:
1). The resulting products were purified by HPLC to afford the
desired glycopeptides in 45-82% yield.
Synthesis of glycopeptide 4 was also achieved using Fmoc-
based SPPS (Scheme 4). Due to the challenges associated with
the construction of amino acid 6, we chose to only synthesize
a N-terminal Asp glycopeptide as glycopeptides possessing this
residue at their N-terminus provided some of the best yields
for SAL. Similar to the synthesis of 14a-d, we found that
acceptable yields could be achieved using 1 equiv of 6 and a
coupling time of 6 h. Because we were concerned about the
stability of the sensitive R-1,6-fucose linkage we chose to release
the glycopeptide from the resin prior to removal of the
carbohydrate protecting groups. Following removal from the
resin under standard conditions and HPLC purification the
glycopeptide, Zemple´n deacetylation at pH 9 (to prevent
ꢀ-elimination of the glycan)49 afforded glycopeptide 4.
Peptide thioesters bearing amino acids Gly, Tyr, His, and Ala
at the C-terminus were synthesized using the Boc-strategy, as
reported previously.32,34 These peptide thioesters were selected
(50) Kemp, D. S.; Bernstein, Z. W.; McNiel, G. N. J. Org. Chem. 1974,
39, 2831–2835.
(51) Kemp, D. S.; Choong, S. L. H.; Pekaar, J. J. Org. Chem. 1974, 39,
3841–3847.
(52) Payne, R. J.; Ficht, S.; Greenberg, W. A.; Wong, C. H. Angew. Chem.,
Int. Ed. 2008, 47, 4411–4415.
(53) Blixt, O.; Razi, N. Methods Enzymol. 2006, 415, 4411–4415.
(54) Schanbacher, F. L.; Ebner, K. E. J. Biol. Chem. 1970, 245, 5057–
5061.
(55) Beyer, T. A.; Sadler, J. E.; Rearicj, J. I.; Paulson, J. C.; Hill, R. L.
AdV. Enzymol. 1981, 52, 23–175.
(56) Wen, D. X.; Livingston, B. D.; Medzihradszky, K. F.; Kelm, S.;
Burlingame, A. L.; Paulson, J. C. J. Biol. Chem. 1992, 267, 21011–
21019.
(49) Brocke, C.; Kunz, H. Synthesis 2004, 525–542.
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Sugar-Assisted Ligation with Complex Glycans
A R T I C L E S
Scheme 4. Synthesis of Glycopeptide 4a
bearing a C-terminal glycine residue, under mixed solvent
system conditions (4:1 v/v N-methyl pyrrolidinone/6 M guani-
dine hydrochloride, 1 M HEPES, pH 8.5, 2% PhSH, 37 °C),33
in order to minimize peptide thioester hydrolysis. Under these
conditions glycopeptides 1a and 1b, possessing glycine and
aspartic acid at their N-terminus, underwent ligation with 15a
in 63% and 70% yields, respectively (Table 1, entries 1 and 2).
The fact that these yields were lower than those observed in
previous studies with the C-2 auxiliary-containing monosac-
charide (91% for glycine)34 suggests that the increased steric
bulk of the galactose moiety may effect the efficiency of the
reaction. This effect is even more pronounced with glycopeptide
1c, possessing a N-terminal histidine, which undergoes ligation
with 15a in 36% yield (entry 3). This result was surprising given
that in previous studies glycopeptides bearing an N-terminal
His gave good yields in SAL reactions. The lower yield observed
with the more complex glycan is presumably due to unfavorable
interactions between the larger His side chain and the auxiliary-
containing disaccharide during the S f N acyl shift. Finally,
extension of the N-terminus of the glycopeptide by an additional
glycine residue (1d) results in a drop in yield, presumably due
to the increased size of the proposed ring transition state (entry
4).
We next turned our attention to the effect of extended
glycosylation on the reaction with more sterically demanding
peptide thioesters. To this end, we examined the reaction of 1b
with peptide thioesters 15b-d, possessing Tyr, His, and Ala at
their C-terminus, respectively (table 1, entries 5-7). These
peptide thioesters had been used in our previous studies and
would therefore allow a better assessment of the effects of the
larger glycan on the reaction. Pleasingly, we found that the
presence of the sterically demanding Tyr residue in peptide
thioester 15b did not interfere with the reaction, providing the
ligation product in yields comparable to those observed with
peptide thioester 15a (entry 5). Similarly, glycopeptide thioester
15c, possessing a C-terminal His residue, also underwent ligation
in good yield (entry 6). Consistent with our previous studies,32-34
the ligation between 1b and peptide thioester 15d, possessing
an C-terminal Ala residue, gave a slightly lower yield (55%);
however this is still deemed to be synthetically useful (entry
7). These data suggest that the C-terminal residue of the peptide
thioester is less sensitive to the presence of larger glycans than
the N-terminus of the glycopeptide, in line with trends observed
in previous SAL studies.31-34
a PyBOP ) benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexaflu-
orophosphate; Pip ) piperidine; TFA ) trifluoroacetic acid; TIS )
triisopropylsilane; TA ) thioanisole; NMM ) N-methylmorpholine.
Lewis X motif using R-1,3-fucosyltransferase (FucT)58 proved
to be much more challenging, initially providing the desired
target glycopeptides in poor yields. This was not entirely
surprising given the proximity of the unnatural sulfhydryl group
to the C-3 glycosylation site on the bridgehead sugar. Pleasingly,
however, we found that it was possible to obtain target
glycopeptides 3a-d through the use of an excess of GDP-
fucose, FucT, and prolonged reaction times (see Supporting
Information).
Scope of Sugar-Assisted Ligation with Complex Glycans.
With the target glycopeptides in hand, we turned our attention
to investigating the efficiency of SAL in the presence of more
complex glycans. Our initial investigations focused on the
ligation between glycopeptides 1a-d with peptide thioester 15a,
In order to further probe the effects of glycosylation at C-4,
we examined the efficiency of SAL with trisaccharide glyco-
peptides 2a-d. Pleasingly, we found that the addition of a sialic
acid residue to C-3 of the terminal galactose had little effect on
the reaction between peptide thioester 15a and glycopeptides
2a and 2b (Table 2, entries 1 and 2). By comparison, the reaction
between glycopeptide 2c, possessing a N-terminal His, and 15a
proceeded in poor (14%) yield (entry 3), with no observable
increase in yield with prolonged reaction times. The large
decrease in yield upon moving to the larger glycan, relative to
that obtained with disaccharide-containing glycopeptide 1c
(Table 1, entry 3), further underscores our observation that larger
amino acids experience unfavorable steric interactions with the
extended glycans in the reaction. The increased bulk of the
glycan did not affect the reaction between 15a and extended
glycopeptide 2d, which gave the desired product in yields
comparable to those obtained with the disaccharide-containing
glycopeptide 1d. From these results, we conclude that for
sterically unencumbered amino acids extending glycosylation
(57) Wen, D. X.; Livingston, B. D.; Medzihradszky, K. F.; Kelm, S.;
Burlingame, A. L.; Paulson, J. C. J. Biol. Chem. 1992, 267, 21011–
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(58) Lin, S. W.; Yuan, T. M.; Li, J. R.; Lin, C. H. Biochemistry 2006, 45,
8108–8116.
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A R T I C L E S
Bennett et al.
Scheme 5. Enzymatic Elaboration of Glycopeptides 14a-da
a Abbreviations: UDP-Gal ) Uridine-5-diphosphate-R-D-galactose, disodium salt, ꢀ-1,4-GalT ) ꢀ-1,4-galactosyltransferase, HEPES ) 4-(2-hydroxyethyl)-
1-piperazine-1-ethanesulfonic acid, CMP-Sial ) cytosine monophosphate sialic acid, monosodium salt, R-2,3-SialT ) R-2,3-sialyltransferase, GDP-Fuc )
Guanosine-5-diphosphate-ꢀ-L-fucose, disodium salt, R-1,3-FucT ) R-1,3-fucosyltransferase, MES ) 2-(N-morpholino)-1-ethanesulfonic acid.
at C-4 beyond a disaccharide does not have a major impact on
the reaction. Ligation products could be desulfurized in an
efficient manner by treatment with 10% palladium on alumina
in the presence of tris(2-carboxyethyl)phosphine hydrochloride
(TCEP, see Supporting Information).32
we examined the reaction between glycopeptide 4 and peptide
thioesters 15a and 15d (Scheme 7). In both cases the SAL
reaction proceeded in moderate (44%) yield to afford glyco-
peptides 16a and 16b. While these yields are synthetically
useful, they are lower than those observed with auxiliary-
containing monosaccharides or C-4 substituted auxiliary-
containing glycans. This suggests that the glycan at C-6 may
be interacting with the peptide backbone. Importantly, given
the use of the generally more reactive glycopeptide possessing
a N-terminal Asp in the SAL reaction, these results indicate
that care must be used in selecting N-terminal residues in SAL
reactions when the auxiliary-containing glycan is substituted at
C-6.
N-Linked Ligation Study. Having examined the effects of
complex O-glycosylation on the efficiency of the SAL reaction,
we sought to determine if complex N-linked auxiliary-containing
glycopeptides were compatible with the reaction. To this end,
we envisioned enzymatic elaboration of glycopeptide 1732 using
endo-ꢀ-N-acetylglucosaminidase A or M (Endo A or M)
mediated transglycosylation.59,60 Unfortunately, all attempts to
transfer a complex oligosaccharide to the auxiliary-containing
glycan in 17 using Endo M failed. Rationalizing that the C-2
sulfhydryl group might be interfering with the reaction, we next
examined Endo A mediated transfer of sugar oxazoline 18,61
We next turned our attention to the more sterically congested
sialyl Lewis X glycopeptides 3a-d. Given the effects of steric
hindrance on the reaction observed with glycosylation at C-4,
we were concerned that the introduction of the fucose residue
onto the C-3 position of the bridgehead sugar would be
deleterious to the reaction. This indeed proved to be the case,
and we were unable to isolate any product from ligations
between 3a-d and peptide thioester 15a, even after prolonged
reaction times (Scheme 6). These results indicate that the SAL
reaction is not compatible with extended glycosylation at
positions adjacent to the thiol auxiliary. This observation
provides valuable information about which glycan structures
can be utilized for SAL reactions and should prove useful for
planning syntheses of complex glycopeptides and glycoproteins
in future studies.
We next examined the effect of glycosylation at C-6 of the
bridgehead sugar using glycopeptide 4. Given the distance from
the auxiliary, we were hopeful that this modification would not
have a significant negative effect on the reaction. To this end,
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11950 J. AM. CHEM. SOC. VOL. 130, NO. 36, 2008

Sugar-Assisted Ligation with Complex Glycans
A R T I C L E S
Table 1. Scope of SAL with Glycopeptides 1a-da
Table 2. Scope of SAL with Trisaccharide-Containing
Glycopeptides 2a-da
glycopeptide
(AA1)
thioester
(AA2)
ligation junction
-AA2-AA1-
isolated ligation
yieldb
entry
glycopeptide
(AA1)
ligation junction
-AA2-AA1-
isolated ligation
yieldb
entry
1
2
3
4
5
6
7
1a (Gly)
1b (Asp)
1c (His)
1d (Gly-Gly)
1b
15a (Gly)
15a
Gly-Gly
Gly-Asp
Gly-His
Gly-Gly
Tyr-Asp
His-Asp
Ala-Asp
63%
70%
36%
49%
70%
70%
55%
1
2
3
4
2a (Gly)
Gly-Gly
Gly-Asp
Gly-His
Gly-Gly
57%
70%
14%
44%
2b (Asp)
2c (His)
15a
15a
2d (Gly-Gly)
15b (Tyr)
15c (His)
15d (Ala)
a Conditions: 4:1 v/v NMP/6 M Gn ·HCl, 1 M HEPES, pH 8.5, 2%
PhSH, 37 °C, 96 h. b Isolated yield after HPLC purification.
1b
in lower, but still synthetically useful, ligation yields. In both
of these cases, the selection of the N-terminal amino acid in
the auxiliary-containing glycopeptide is critical, and sterically
unencumbered amino acids are necessary for optimal yields.
Importantly, it appears that the peptide thioester is much less
sensitive to extended glycosylation at C-4 and C-6 on the
auxiliary-containing glycan, as even peptide thioesters possess-
ing bulky C-terminal residues smoothly undergo the reaction
with glycopeptides bearing appropriate N-terminal residues. SAL
is not tolerant of glycosylation at positions adjacent to the
auxiliary, however, as glycosylation at C-3 prevents the reaction
from taking place. Finally, both O-linked and N-linked auxiliary-
containing glycopeptides can be extended, although it appears
that the latter is more sensitive to larger glycans. Based on these
results, we urge caution in using SAL when a novel glycosy-
lation pattern is encountered in a glycoprotein, as structural
changes in more complex glycans can have major and unex-
pected effects on the reaction, as evidenced by the results
obtained with the C-6 substituted glycopeptide 4. Nevertheless,
1b
a Conditions: 4:1 v/v NMP/6 M Gn ·HCl, 1 M HEPES, pH 8.5, 2%
PhSH, 37 °C, 96 h. b Isolated yield after HPLC purification.
as this has been reported to be a much more efficient substrate
for Endo mediated transfer.62-64 Pleasingly, this strategy proved
to be more effective, delivering the target glycopeptide 19 in
38% yield (Scheme 8). This glycopeptide underwent SAL with
peptide thioester 15a to afford glycopeptide 20 in 40% yield
after HPLC purification.
Conclusion
The presence of extended glycosylation on the auxiliary-
containing glycan in SAL can have a significant impact on the
outcome of the reaction. Glycosylation at C-4 has the smallest
effect on the reaction, where in many cases the reaction proceeds
with high levels of efficiency. The effect of extended glycosy-
lation at C-6 on the SAL reaction is more pronounced, resulting
Scheme 6. Attempted Ligation of Glycopeptides 3a-d with Peptide Thioester 15a
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A R T I C L E S
Bennett et al.
Scheme 7. SAL Reactions between Glycopeptide 4 and Peptide Thioesters 15a and 15d
Scheme 8. Endo-ꢀ-N-acetylglucosaminidase A Mediated Elaboration of Auxiliary-Containing N-Linked Glycopeptide 17 and Use of the
Resulting Product in SALa,b
a Endo-mediated glycosylation conditions 20 mM phosphate buffer, pH 6.0. b SAL conditions 4:1 v/v NMP/6 M Gn ·HCl, 1 M HEPES, pH 8.5, 2%
PhSH, 37 °C, 96 h.
aqueous TCEP solution (10 mg/mL). The resulting solution was
purified by preparative HPLC (0-35% A/B, A ) 0.1% formic acid
in acetonitrile, B ) 0.1% formic acid in water) and analyzed by
MALDI-TOF/MS (matrix, 2,5-dihydroxybenzoic acid).
we feel that when appropriate SAL is a useful addition to the
glycopeptide, and potentially glycoprotein, synthesis toolbox,
as it permits access to ligation junctions that cannot be
constructed using other ligation strategies.
Acknowledgment. We thank Professor Chun-Hung Lin (Aca-
demica Sinica) for providing us with fucosyltransferases and
Professor Kaoru Takegawa (Kagawa University) for the gift of Endo
A-GST fusion protein plasmid. This work was supported by the
NIH and the Skaggs Institute of Chemical Biology. C.S.B. is
grateful for a NIH postdoctoral fellowship. R.J.P. is grateful for
funding provided by the Lindemann Trust Fellowship. S.F. thanks
the Deutsche Akademische Austauschdienst (DAAD) for a post-
doctoral fellowship. We thank Professor William A. Greenberg and
Dr. Sarah R. Hanson for help in preparation of the manuscript.
Experimental Section
General procedure for sugar-assisted ligation: A 20 mM solution
of the glycopeptide (1.5 equiv) in 4:1 v/v NMP/6 M Gn HCl, 1 M
HEPES, pH 8.5 was added to the peptide thioester (1 equiv). The
resulting solution was treated with thiophenol (2% v/v) and
incubated at 37 °C for 4 days, before quenching with 300 µL of
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Soc. 2005, 127, 9692–9693.
Supporting Information Available: Experimental procedures,
characterization of products, and other detailed results. This
material is available free of charge via the Internet at http://
pubs.acs.org.
(62) Li, H.; Li, B.; Song, H.; Breydo, L.; Baskakov, I. V.; Wag, L. X. J.
Org. Chem. 2005, 70, 9990–9996.
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Chem.sEur. J. 2006, 12, 3355–3364.
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JA8010513
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