Efficient synthesis of complex glycopeptides based on unprotected oligosaccharides

Article abstract of DOI:10.1021/jo020570c

N-Glycopeptides containing 1 to 4 trisaccharide chains, with the carbohydrates vicinal to each other in the multivalent glycopeptides, were efficiently synthesized by using the glycosylated Fmoc-asparagine as a key building block. While the couplings of amino acids with glycopeptides could be achieved in the homogeneous solutions in N-methylpyrrolidinone (NMP) to give excellent yields, all products were conveniently isolated from the reaction mixtures through a precipitation method by using the free carbohydrate chains as phase tags. Commercially available pentafluorophenyl (Pfp) esters of amino acids were employed for the glycopeptide elongation. Longer glycopeptides were constructed by means of a highly convergent synthetic design that is based on the coupling of glycopeptide/peptide fragments. Hydrogen bond interactions between free oligosaccharides were proposed to explain the exceptionally high efficiency of the couplings between two glycosylated building blocks.

Full text of DOI:10.1021/jo020570c

Efficien t Syn th esis of Com p lex Glycop ep tid es Ba sed on
Un p r otected Oligosa cch a r id es
J ie Xue and Zhongwu Guo*
Department of Chemistry, Case Western Reserve University, Cleveland, Ohio 44106-7078
zxg5@po.cwru.edu
Received August 27, 2002
N-Glycopeptides containing 1 to 4 trisaccharide chains, with the carbohydrates vicinal to each other
in the multivalent glycopeptides, were efficiently synthesized by using the glycosylated Fmoc-
asparagine as a key building block. While the couplings of amino acids with glycopeptides could be
achieved in the homogeneous solutions in N-methylpyrrolidinone (NMP) to give excellent yields,
all products were conveniently isolated from the reaction mixtures through a precipitation method
by using the free carbohydrate chains as phase tags. Commercially available pentafluorophenyl
(Pfp) esters of amino acids were employed for the glycopeptide elongation. Longer glycopeptides
were constructed by means of a highly convergent synthetic design that is based on the coupling
of glycopeptide/peptide fragments. Hydrogen bond interactions between free oligosaccharides were
proposed to explain the exceptionally high efficiency of the couplings between two glycosylated
building blocks.
In tr od u ction
glycopeptides, both the enzymatic transglycosylation and
the enzymatic peptide and/or carbohydrate chain elonga-
tion have been exploited. Another interesting but inad-
equately addressed approach is the solid-phase synthesis
of glycopeptides with unprotected glycosyl amino acids
as building blocks.13 Nevertheless, owing to the remark-
able diversity and complexity of glycopeptide structures,
it is difficult for any individual method to meet the
demands of all kinds of synthetic endeavors, even though
the established methods have been successfully applied
to the preparation of numerous glycopeptides. Thus,
alternative methodologies are still desired.
Glycoproteins play an important role in various bio-
logical and pathological functions.1,2 To meet the growing
demand for synthetic glycoproteins and/or glycopeptides,
many useful synthetic methods have been developed in
the past two decades,3 such as solution-phase and solid-
phase syntheses with appropriately protected oligosac-
charides, as well as the elegant chemo-enzymatic synthe-
sis4-9 and chemoselective ligation method10-12 based on
free carbohydrates. For chemo-enzymatic syntheses of
(1) Varki, A. Glycobiology 1993, 3, 97-130.
A special property shared by all glycopeptide synthetic
targets, which may be utilized in organic synthesis, is
that they contain free carbohydrate chains possessing a
number of polar hydroxyl groups. The free hydroxyl
groups of carbohydrates did not affect the peptide cou-
pling reactions as indicated by the successful application
of glycosyl amino acids of unprotected monosaccharides
and disaccharides to conventional solid-phase synthesis
of glycopeptides.13 However, the high polarity of unpro-
tected carbohydrate conjugates makes them only soluble
in very polar organic solvents but not in less polar ones.
It is thus possible to use the unprotected oligosaccharides
as “phase tags” to facilitate the product isolation in the
solution-phase synthesis of glycopeptides, i.e., the reac-
tions used to elongate the glycopeptide chains may be
performed in a polar organic solvent under homogeneous
conditions, while the product of each reaction can be
easily separated from the side products by adding a less
polar solvent to the reaction for precipitating the polar
glycopeptide after the reaction is accomplished. The
synthesis can take advantage of the benefits of both
(2) Dwek, R. A. Chem. Rev. 1996, 96, 683-720.
(3) Recent reviews about glycopeptide synthesis: (a) Herzner, H.;
Reipen, T.; Schultz, M.; Kunz, H. Chem. Rev. 2000, 100, 4495-4537.
(b) Meldal, M. In Neoglycoconjugates: Preparation and applications;
Lee, Y. C., Lee, R. T., Eds.; Academic Press: San Diego, CA, 1994; pp
145-198. (c) Arsequell, G.; Valecia, G. Tetrahedron: Asymmetry 1999,
10, 4045-3094. (d) Arsequell, G.; Valencia, G. Tetrahedron: Asymmetry
1997, 8, 2839-2876. (e) Kunz, H. Pure Appl. Chem., 1993, 65, 1223-
1232. (f) Osborn, H. M. I.; Khan, T. H. Tetrahedron Lett. 1999, 55,
1807-1850. (g) Pauslen, H. Angew. Chem., Int. Ed. Engl. 1990, 29,
823-838.
(4) Schuster, M.; Wang, P.; Paulson, J . C.; Wong, C.-H. J . Am. Chem.
Soc. 1994, 116, 1135-1136.
(5) Mizuno, M.; Haneda, K.; Iguchi, R.; Muramoto, I.; Kawakami,
T.; Aimoto, S.; Yamamoto, K.; Inazu, T. J . Am. Chem. Soc. 1999, 121,
284-290.
(6) Witte, K.; Sears, P.; Martin, R.; Wong, C.-H. J . Am. Chem. Soc.
1997, 119, 2114-2118.
(7) Wong, C.-H.; Schuster, M.; Wang, P.; Sears, P. J . Am. Chem.
Soc. 1993, 115, 5893-5901.
(8) Deras, I. L.; Takegawa, K.; Kondo, A.; Kato, I.; Lee, Y. C. Bioorg.
Med. Chem. Lett. 1998, 8, 1763-1766.
(9) Unverzagt, C. Angew. Chem., Int. Ed. Engl. 1996, 35, 2350-
2353.
(10) Roberge, J . Y.; Beebe, X.; Danishefsky, S. J . J . Am. Chem. Soc.
1998, 120, 3915-3927.
(11) Cohen-Anifeld, S.-T.; Lansbury, P. T. J . J . Am. Chem. Soc. 1993,
115, 10531-10537.
(12) Marcaurelle, L. A.; Mizoue, L. S.; Wilken, J .; Oldham, L.; Kent,
S. B. H.; Handel, T. M.; Bertozzi, C. R. Chem. Eur. J . 2001, 7, 1129-
1132.
(13) Otvos, L. J .; Urge, L.; Hollosi, M.; Wroblewski, K.; Graczyk,
G.; Fasman, G. D.; Thurin, J . Tetrahedron Lett. 1990, 41, 5889-5892.
10.1021/jo020570c CCC: $25.00 © 2003 American Chemical Society
Published on Web 02/21/2003
J . Org. Chem. 2003, 68, 2713-2719
2713

Xue and Guo
SCHEME 1a
solution-phase and solid-phase syntheses, namely the
faster and more efficient reactions in a homogeneous
solution and the convenient product isolation through the
precipitation method. The principle and the high ef-
ficiency of solution-phase organic synthesis with solid-
phase workups have been proved by numerous recently
designed phase tags.14-21 The uniqueness of using un-
protected carbohydrates as phase tags in glycopeptide
synthesis is that they are also an integral part of the
synthetic targets, so it is not necessary to cut these phase
tags off after the syntheses are finished. Furthermore,
the carbohydrate chains can be repeatedly used as phase
tags without limit until the final synthetic targets are
obtained. Another important advantage of this approach
is that it can avoid the final carbohydrate deprotection,
which is a necessary but sometimes problematic step in
a majority of other synthetic methods. Consequently, a
strategy for glycopeptide synthesis based on unprotected
oligosaccharides can be especially useful for the synthetic
targets containing acid-sensitive oligosaccharides and/
or reduction-liable peptides, as well as for those with
bulky side chains that may affect the coupling reactions.
a
Recently, we exploited the synthesis of glycopeptides
with unprotected oligosaccharides as building blocks and
as phase tags.22 The reactions to elongate the glycopep-
tide chains were carried out in the homogeneous solutions
of N-methylpyrrolidinone (NMP), and the expected gly-
copeptide products were easily isolated from the reaction
mixtures via the precipitation method by adding diethyl
ether to the reaction and isolating the precipitates via
filtration or centrifugation. In our preliminary studies,
N-hydroxybenzotriazole and N,N-dicyclohexylcarbodi-
imine (HOBt/DCC) were used as the condensation re-
agents. The new synthetic strategy was very successful
for several simple glycopeptides containing a monosac-
charide or a disaccharide chain by elongation of the
glycopeptides at both termini.
Reagents and conditions: (a) Ac2O, NaOAc, reflux, 94%. (b)
Et2O-BF3, TMSN3, DCM, rt, 80%. (c) NaOMe, MeOH, rt, quant
H2, Pd/C. (d) Fmoc-Asp(OBt)-OBut, NMP, rt, 2 h, 42%. (e) (i) 20%
piperidine, NMP, rt, 0.5 h; (ii) product precipitation by Et2O. (f)
(i) Fmoc-Gly-OPfp, NMP, rt, 2 h; (ii) product precipitation by Et2O.
(g) (i) Fmoc-Gln(Trt)-OPfp, NMP, rt, 2 h; (ii) product precipitation
by Et2O. (h) 25% TFA, DCM, 2 h.
a synthetic design that involves minimum C-terminal
elongations are desirable.
Resu lts a n d Discu ssion
Glycop ep tid e Ch a in Elon ga tion w ith th e P en ta -
flu or op h en yl (P fp ) Ester s of Am in o Acid s. Trying to
establish a more convenient protocol for glycopeptide
elongations, this research was focused on the one that
employs the Pfp esters of amino acids for the peptide
coupling,23,24 as it has been successfully used in the
preparation of glycopeptides by other methods.25-28 More
importantly, the relatively stable Pfp esters are com-
mercially available, and if this protocol is applicable to
the new strategy, the commercial active esters can be
directly used to construct glycopeptides. In this case the
only side product of the coupling reactions will be
pentafluorophenol that is soluble in the mixture of NMP
and ether and can be easily removed by diethyl ether. It
was therefore anticipated that the Pfp-protocol should
be especially helpful for the new synthetic strategy.
The elongation of glycopeptide chains by means of Pfp
esters was then investigated in the synthesis of glyco-
peptide 1 (Scheme 1), a simple model containing a
maltotriose. Though this is an artificial synthetic target,
Nevertheless, the applicability of this new strategy to
more complex structures remains to be proved. Moreover,
for wider uses its protocols may need optimization. For
example, though the above peptide coupling protocol gave
excellent results, it is necessary for the amino acids to
be activated just before the coupling reactions, and the
freshly prepared active esters in solution were directly
used without purification. Meanwhile, the acidic condi-
tions used to deprotect carboxyl groups in the process of
C-terminal elongation may affect the carbohydrate struc-
tures of the glycopeptides, especially if a synthesis
involves many steps of C-terminal elongation and the
synthetic target contains acid-sensitive glycosyl linkages.
Thus, a more convenient protocol for peptide coupling and
(14) Gravert, D.; J anda, K. D. Chem. Rev. 1997, 97, 489-510.
(15) Douglas, S. P.; Whitfield, D. M.; Krepinsky, D. M. J . Am. Chem.
Soc. 1991, 113, 5095-5097.
(16) Bosanac, T.; Yang, J .; Wilcox, C. S. Angew. Chem., Intl. Ed.
2001, 40, 1857-1879.
(23) Atherton, E.; Cameron, L. R.; Sheppard, R. C. Tetrahedron
1988, 44, 859-876.
(17) Ley, S. V.; Massi, A.; Rodriguez, F.; Horwell, D. C.; Lewthwaite,
R. A.; Pritchard, M. C.; Reid, A. M. Angew. Chem., Int. Ed. 2001, 40,
1053-1055.
(24) Dryland, A.; Sheppard, R. C. Tetrahedron Lett. 1988, 44, 859-
876.
(25) Peters, S.; Bielfeldt, T.; Meldal, M.; Bock, K.; Paulsen, H.
Tetrahedron Lett. 1991, 32, 5067-5070.
(18) Perrier, H.; Labelle, M. J . Org. Chem. 1999, 64, 2110-2113.
(19) Hay, A. M.; Hobbs-Dewitt, S.; MacDonald, A. A.; Ramage, R.
Tetrahedron Lett. 1998, 39, 8721-8724.
(26) Paulsen, H.; Peters, S.; Bielfeldt, T.; Meldal, M.; Bock, K.
Carbohydr. Res. 1995, 268, 17-34.
(20) Studer, A.; Hadida, S.; Ferritto, R.; Kim, S.; J eger, P.; Wipf,
P.; Curran, D. P. Science 1997, 275, 823-826.
(27) Mathieux, N.; Paulsen, H.; Meldal, M.; Bock, K. J . Chem. Soc.,
Perkin Trans. 1 1997, 2359-2368.
(21) Curran, D. P. Angew. Chem., Int. Ed. 1998, 37, 1174-1196.
(22) Wen, S.; Guo, Z. Org. Lett. 2001, 3, 3773-3776.
(28) Ichiyanagi, T.; Takatani, M.; Sakamoto, K.; Nakahara, Y.; Ito,
Y.; Hojo, H.; Nakahara, Y. Tetrahedron Lett. 2002, 43, 3297-3300.
2714 J . Org. Chem., Vol. 68, No. 7, 2003

Efficient Synthesis of Complex Glycopeptides
the methods established herein will be applicable to
natural N-glycopeptides, and the results should be of
general significance. Actually, the protocols established
in the synthesis of 1 and other glycopeptides in this work
may also be adapted for O-linked glycopeptides. The key
building block 3, which has the unprotected oligosaccha-
ride linked to the side chain of Fmoc-Asn-OBut, was
prepared by reductive coupling between the azido deriva-
tive of maltotriose (2) and Fmoc-Asp-OBut as described
before.22 This product was purified by silica gel column
F IGURE 1. The convergent assembly of glycopeptides by
coupling peptide and/or glycopeptide fragments.
1
chromatography, and its H NMR spectrum proved the
complex glycopeptides, a convergent design for glycopep-
tide assembly (Figure 1) that is based on the coupling of
a glycopeptide fragment with another peptide or glyco-
peptide fragment was investigated. This is an approach
that has been commonly used in the synthesis of complex
peptides but not fully exploited in glycopeptide synthesis.
Glycopeptides built upon unprotected oligosaccharides
are especially suitable for this convergent design, as their
N- and C-termini can be easily and selectively exposed
under mild conditions. For instance, after a glycopeptide
is assembled by a procedure described above, it may be
treated by 25% TFA in DCM to deprotect the C-terminus
(Figure 1), and the resulting glycopeptide 7 is thus ready
to be activated and coupled to a peptide or glycopeptide
fragment 8 that bears a free N-terminus to give the
complex glycopeptide 9. Fragment 8 in turn can be
prepared either by solid-phase synthesis, in the case of
a simple peptide, or by this new synthetic strategy, in
the case of a glycopeptide. Finally, complete deprotection
of 9 will afford the synthetic target 10.
â-configuration of the newly formed glycosidic linkage (H-
1: δ 4.92, J 1,2 ) 9.2 Hz). Selective deprotection of the
3
Fmoc group in 3 by 20% piperidine in NMP and then
precipitation of the product by diethyl ether afforded 4
quantitatively. To the solution of 4 in NMP was added
the Pfp ester of Fmoc-Gly (4 equiv) with stirring at room
temperature. The reaction was monitored by TLC and
reversed-phase HPLC with a C18 column (Discovery 250
× 2.5 mm), which showed that the reaction finished
within 1 h and only one coupling product was observed,
whereupon diethyl ether (ca. 10 times the volume of
NMP) was added to precipitate the glycodipeptide prod-
uct, and the precipitate was isolated on centrifugation.
Washing of the product with diethyl ether, which was
followed by drying under vacuum, gave 5 in a quantita-
tive yield. Both HPLC and NMR showed that the crude
product was homogeneous, and its NMR and MS spectra
agreed with the expected structure. These results proved
that the peptide coupling by using the Pfp-activated
esters of Fmoc-Gly was clean and effective.
This convergent method was examined in the prepara-
tion of a glycoheptapeptide 11 that could be achieved by
coupling the glycotetrapeptide 6 to a tripeptide 12
bearing a free N-terminus (Scheme 2). The fragment 12
was prepared from a commercial tripeptide 13 in three
conventional steps. On the other hand, the crude 6
obtained after condensation as described above was
directly used for the coupling reaction without further
purification. Thus, glycopeptide 6 was activated by reac-
tion with DCC and HOBt in NMP for 2 h to give 14,
whereupon 5 equiv of 13 was added to the reaction
mixture. After overnight stirring at room temperature,
ether was added to the reaction, and the precipitate was
isolated by centrifugation and analyzed by HPLC to show
only one peptide/glycopeptide peak that was identified
as the expected glycopeptide 15. This suggests that the
precipitation and washing process could efficiently re-
move the excess 13. Final deprotection of glycopeptide
15 by 20% piperidine in NMP afforded the synthetic
target 11 that was also isolated by the precipitation
method. The crude product (89% yield) was already very
pure as shown by both NMR and HPLC (g95%).
It is worth mentioning that the synthetic target 11
contains a typrosine residue that was previously shown
to be unstable to the reductive conditions used to remove
benzyl groups in the solid-phase synthesis of glycopep-
tides based on benzyl-protected glycosyl amino acids.29
Syn th esis of Mu ltiva len t Glycop ep tid es. Encour-
aged by the excellent results obtained above in regard
to the new peptide coupling protocol as well as the
fragment coupling method for glycopeptide synthesis, we
Following the same two-step protocol, i.e., (1) depro-
tection of the Fmoc group in a glycopeptide by 20%
piperidine in NMP and isolation of the product by the
precipitation method and (2) coupling of the glycopeptide
with the next amino acid in NMP (rt, 2 h) by using its
Pfp ester (3-4 equiv) and isolation of the product by the
precipitation method, the peptide chain of 5 was further
elongated with Fmoc-Gly-OPfp and Fmoc-Glu(Trt)-OPfp
sequentially to afford a glycotetrapeptide which, on
treatment with 25% trifluoroacetic acid (TFA) in dichlo-
romethane (DCM), was transformed to 6 bearing a free
C-terminus. The reactions were monitored and analyzed
by TLC and HPLC, while the reaction products were
characterized by means of NMR and MS. The final
synthetic target 1 was eventually obtained upon the
deprotection of 6 with 20% piperidine in NMP. The NMR
spectra and the HPLC diagram of 1 showed that its
purity was no less than 95%. These results suggested that
the Pfp ester protocol was very convenient and that the
coupling reactions were very efficient for glycopeptide
elongations. Nonetheless, further purification of the
product by gel filtration with a Bio Gel P4 column and
then by reversed-phase HPLC on a semipreparative C18-
column (25 × 1 cm) afforded 1 in an overall yield of 75%
(based on 3). The lower than perfect yield was mainly
due to the loss of products in the precipitation, analysis,
and material transfer processes in each step of the
glycopeptide synthesis, even though all the operations
were performed within the same reaction vessel to
minimize the product loss.
Glycop ep tid e Syn th esis by Cou p lin g of Glyco-
p ep tid e/P ep tid e F r a gm en ts. To minimize the number
of steps of C-terminal elongation in the preparation of
(29) Guo, Z.; Nakahara, Y.; Nakahara, Y.; Ogawa, T. Carbohydr.
Res. 1997, 303, 373-378.
J . Org. Chem, Vol. 68, No. 7, 2003 2715

Xue and Guo
SCHEME 2a
All these syntheses used glycosyl amino acid 3 as the
key building block. As shown above, the Fmoc in 3 could
be selectively removed by 20% piperidine in NMP to give
4 with the N-terminus exposed. On the other hand, its
C-terminus was selectively deprotected by treatment with
25% TFA in DCM to give 18 which, after condensation
to dryness under vacuum, was directly used in the next
step of the reaction.
The coupling of 18 to 4 was achieved via its Bt-ester
19 formed by reaction with HOBt and DCC (Scheme 3).
Thus, after 18 was stirred with DCC and HOBt (2.0
equiv) in NMP at room temperature for 2 h, 4 (1.0 equiv)
was added to the solution. This mixture was stirred at
room temperature for another 12 h. The glycopeptide
product was then precipitated out and washed with
diethyl ether to afford 20 that was characterized by NMR
and MS. The HPLC analysis (using a Waters Spherisorb
5 µm NH2 analytic column, 250 × 4.6 mm) of this product
indicated that it contained only one glycopeptide peak
and all the starting materials were consumed. It did not
need any purification for further transformations. This
coupling reaction was also studied by utilizing different
ratios of 18 to 4, and it was found that each experimental
entry produced a quantitative coupling in terms of the
minor substrate. Therefore, it is suggested that a 1:1 ratio
of the substrates should be employed for this coupling
reaction, as in such cases the intermediate purification
can be avoided.
a
Reagents and conditions: (a) Fmoc-OBt, DCM, rt, 95%. (b)
MeOH, SOCl2, rt, 82%. (c) 20% piperidine, NMP, rt, 0.5 h, quant.
(d) DCC, HOBt, NMP, rt, 2 h. (e) (i) rt, overnight; (ii) precipitation
by Et2O. (f) (i) 20% piperidine, NMP, rt, 0.5 h; (ii) precipitation by
Et2O, 89% (from 6).
Once the glycodipeptide 20 was obtained, its peptide
chain was elongated stepwise (Scheme 3) by the protocols
described in the synthesis of glycopeptide 1 to yield the
divalent glycopeptide 16. All the coupling reactions,
which were monitored by NMR and HPLC, gave excellent
results. In regard to the product isolation by the precipi-
tation method, it was proved to be more convenient to
work with these divalent structures than to work with
the monovalent glycopeptides, as the divalent glycopep-
tides could be more easily precipitated from the reaction
mixtures. Crude 16 (quantitative yield from 3) obtained
from the final precipitation showed only one glycopeptide
SCHEME 3a
1
peak by HPLC. Its H NMR spectrum also indicated that
this crude product was rather pure, except for being
contaminated by a small amount of piperidine (Figure
2a). The crude 16 was finally purified by a Bio Gel P4
column and then reversed-phase HPLC to afford 16 in
an overall yield of 93% (from 18). The structure of 16
was supported by its NMR (Figure 2b,c) and MS.
The tetravalent glycopeptide 17 (Scheme 4) was simi-
larly synthesized. First, the glycodipeptide 20 obtained
above was converted to 21 and 22 by selective deprotec-
tion of its But and Fmoc groups with 25% TFA/DCM and
20% piperidine/NMP, respectively. The workup proce-
dures for these reactions followed exactly that described
for 18 and 4. After 21 was activated by DCC/HOBt in
NMP, the two glycodipeptide fragments (21:22 1:1) were
coupled under the conditions described for 20 to give the
glycotetrapeptide containing four vicinal glycans, which
was precipitated and washed by diethyl ether. HPLC
analysis of this reaction indicated that the coupling
efficiency was about 85%. The product was then N-
deprotected by the standard protocol to afford 23. A part
of 23 was purified by gel filtration chromatography and
HPLC to get an isolated coupling yield of 75%, and the
structure of 23 was fully characterized. As shown, this
a
Reagents and conditions: (a) 25% TFA, DCM, rt, 2 h. (b) (i)
DCC, HOBt, NMP, rt, 2 h; (ii) precipitation by Et2O. (c) (i) 4, NMP,
rt, 12 h; (ii) precipitation by Et2O, quant. (d) (i) 20% piperidine,
NMP, rt, 0.5 h; (ii) precipitation by Et2O. (e) (i) Fmoc-Gly-OPfp,
NMP, rt, 2 h; (ii) precipitation by Et2O. (f) (i) Fmoc-Gln(Trt)-OPfp,
NMP, rt, 2 h; (ii) precipitation by Et2O, (g) 25% TFA, DCM, 2 h.
further studied their application to more complex syn-
thetic targets. The synthetic models were two N-linked
glycopeptides 16 and 17 (Schemes 3 and 4) that were
decorated by two and four vicinal maltotriose glycans,
respectively.
2716 J . Org. Chem., Vol. 68, No. 7, 2003

Efficient Synthesis of Complex Glycopeptides
SCHEME 4a
1
F IGURE 2. The H NMR (D2O, 600 MHz) spectra of 16: (a)
the crude product at 25 °C; (b) the purified product (after Bio
Gel P4 column chromatography and HPLC) at 25 °C; and (c)
the purified product at 50 °C.
coupling reaction turned out to be less efficient than that
between 18 and 4. However, the result was still quite
impressive if the extremely bulky structures of 21 and
22 are taken into consideration.
For further elongation of the peptide chain of 23, the
crude product was applied without purification, since the
expected final product 17, a tetravalent glycopeptide,
should be rather easily separated from the divalent side
products. Therefore, the peptide chain of 23 was elon-
gated by the established protocols. Final N- and C-
terminal deprotection of the resulting glycopeptide and
then purification by gel filtration chromatography and
RP HPLC afforded 17 in a 68% overall yield (from 20).
a
Reagents and conditions: (a) 25% TFA, DCM, 2 h, quant. (b)
(i) 25% piperidine, NMP, rt, 0.5 h; (ii) precipitation by Et2O. (c) (i)
DCC, HOBt, NMP, rt, overnight, quant.; (ii) precipitation by Et2O.
(d) (i) Fmoc-Gln(Trt)-OPfp, NMP, rt, 2 h; (ii) precipitation by Et2O.
(e) (i) Fmoc-Gly-OPfp, NMP, rt, 2 h; (ii) precipitation by Et2O.
1
The H NMR spectra of 17 at different temperatures are
shown in Figure 3.
As shown in Schemes 1-4, several glycopeptides were
synthesized by the new strategy in excellent yields. The
TLC, HPLC, and NMR analyses showed that the peptide
coupling reactions based on the Pfp esters of amino acids
(3-4 equiv) could complete within 1 h to give the ex-
pected products in perfect conversion, whereas most
coupling reactions were kept for at least 2 h to ensure
the complete coupling. Moreover, the purification of
intermediates by column chromatography or other so-
phisticated separation methods was not necessary in
these syntheses, except for the building block 3. The
crude final products, which were obtained after many
steps of deprotection, peptide coupling, and intermediate
precipitation, were already pure. These results also
supported the high efficiency of the peptide coupling
reactions and that of the precipitation method for product
isolation/purification. Thus, using commercial Pfp esters
of amino acids for the glycopeptide elongation was proved
to be a very convenient and efficient protocol, and the
convergent method for glycopeptide synthesis by coupling
glycopeptides with peptide or glycopeptide fragments was
also proved to be highly feasible.
F IGURE 3. The 1H NMR (D2O, 600 MHz) spectra of 17 at
different temperatures: (a) 25 and (b) 45 °C.
phase support,29 it was interesting to notice the extremely
high coupling efficiency of the bulky glycopeptides during
the synthesis of multivalent glycopeptides 16 and 17.
Several factors may be accountable for the outcome. For
instance, it is anticipated that achieving the coupling
reactions in a homogeneous NMP solution may signifi-
cantly improve the reaction efficiency over that performed
under the two-phase conditions. Moreover, the decreased
Compared to the coupling reaction between a benzyl-
protected glycosyl amino acid and a peptide on the solid-
J . Org. Chem, Vol. 68, No. 7, 2003 2717

Xue and Guo
of piperidine and NMP (1:4). After 1.5 h of stirring at room
temperature, 20 mL of diethyl ether was added to the reaction.
Following centrifugation at 3000 rpm for 5 min, the solvents
were poured off carefully, while the precipitate remained in
the reaction flask. This crude product was then suspended in
20 mL of diethyl ether with vigorous shaking on a vortex
mixer, which was followed by centrifugation. The ether solu-
tion was removed from the vessel, and the remaining solid was
washed by ether 2 to 3 more times. The precipitate was finally
dried under vacuum to afford the product that was directly
used for the next step of the reaction.
A Typ ica l P r oced u r e for th e P ep tid e Cou p lin g w ith
P fp Ester s of Am in o Acid s. A glycosyl amino acid or
glycopeptide with an exposed N-terminus (56 µmmol) was
dissolved in NMP (2 mL), and to the solution was added the
Pfp ester of an Fmoc-amino acid (3-4 equiv) with stirring at
room temperature. The reaction process was monitored by TLC
and HPLC (on a C18 or an NH2 column). The reaction usually
took about 1 h to finish. Then, 20 mL of diethyl ether was
added to precipitate the product, and the precipitate was
collected following centrifugation at 3000 rpm for 5 min. This
crude product, after being washed 4 times with diethyl ether
(as described above) and dried under vacuum, was directly
applied to the next step of the reaction.
A Typ ica l P r oced u r e for th e C-Ter m in a l Dep r otection
(of Bu t). After the tert-butyl ester of a glycosyl amino acid or
a glycopeptide (33 µmol) was treated with a mixture of TFA
and DCM (1:3, 2 mL) at room temperature for 2 h, the reaction
mixture was condensed to dryness. The residue was washed
by diethyl ether 3 to 4 times as described above to afford the
desired product that was used for the next step of the reaction
or to afford the final product that was then thoroughly purified
by HPLC.
L-Glu t a m in yl-glycyl-glycyl-{N-[r-D-glu cop yr a n osyl-
(1f4)-r-D-glu cop yr a n osyl-(1f4)-â-D-glu cop yr a n osyl]}-L-
a sp a r a gin e (1). After N-terminal deprotection of 3 (50 mg,
56 µmol) following the typical procedure shown above, the
resulting glycosyl amino acid 4 (38 mg, quantitative) was
dissolved in NMP (2 mL) and treated with Fmoc-Gly-OPfp (107
mg, 230 µmol) at room temperature for 2 h to accomplish the
coupling reaction. Precipitation followed by washing and
drying of the product as described above eventually afforded
the glycodipeptide 5 (53 mg) in quantitative yield.
size of the unprotected oligosaccharide chains, in com-
parison to that of the protected ones, may also contribute
to the high coupling efficiency, as it may reduce the steric
interactions between the glycans of two glycosylated
substrates. Meanwhile, the results may also suggest
potential hydrogen bonding between the free carbohy-
drate chains, as this can help to bring together the two
substrates involved and thus facilitate their coupling. To
prove this hypothesis, more detailed studies are neces-
sary. Nevertheless, it is generally accepted, as well as
observed,30 that hydrogen bonds between free oligosac-
charides are feasible, especially in organic solvents that
interact with carbohydrates less effectively than water.
This research has demonstrated that the new strategy
can be efficient and useful for the synthesis of a variety
of complex N-glycopeptides, including ones that contain
multiple vicinal oligosaccharide chains. To the best of our
knowledge, this is the first report of the chemical
synthesis of a glycopeptide with 4 vicinal complex oli-
gosaccharides. Additionally, the successful preparation
of 11 also suggests that the synthetic method based on
unprotected glycosyl amino acids can be especially suit-
able for glycopeptides containing vulnerable peptide
sequences.
Exp er im en ta l Section
Gen er a l Meth od s. Thin-layer chromatography (TLC) was
performed on silica gel GF254 with an eluent of MeOH, DCM,
and AcOH (3:6:1 to 6:3:1), and the detections were achieved
by charring with 2% H2SO4/EtOH. Commercial anhydrous
solvents were used without further purification.
N-(9-F lu or en ylm eth oxyca r bon yl)-N-[r-D-glu cop yr a n -
osyl-(1f4)-r-D-glucopyranosyl-(1f4)-â-D-glucopyranosyl]-L-
a sp a r a gin e ter t-Bu t yl E st er (3). To a solution of R-D-
glucopyranosyl-(1f4)-R-D-glucopyranosyl-(1f4)-â-D-gluco-
pyranosyl azide (2, 1.4 g, 2.64 mmol) in 20 mL of dry DMF
was added 10% Pd/C (100 mg) under H2 atmosphere. The
mixture was stirred at room temperature for 2 h. TLC showed
that the azide was completely transformed to the glycosyl-
amine. The catalyst was then filtered off and the resulting
solution was directly used in the next step without other
treatment.
Similarly, N-terminal deprotection of 5 and treatment of the
deprotected product with Fmoc-Gly-OPfp (200 µmol), which
was followed by sequential N-deprotection and coupling to
Fmoc-Glu(Trt)-OPfp (200 µmol) of the resulting glycotripep-
tide, afforded the glycosyl tetrapeptide that was then treated
with 20% TFA/DCM as described to give 6 (64 mg, overall yield
82%). FABMS: calcd for C46H62N6O24 1082.4, found 1083.4 (M
In the meantime, Fmoc-Asp(OBt)-OBut was prepared by
reaction of Fmoc-Asp-OBut (1.7 g, 4.1 mmol) with HOBt (0.68
g, 5.0 mmol) and DCC (1.0 g, 5.0 mmol) in CH2Cl2 (20 mL)
and DMF (1 mL) at room temperature for 2 h. The resulting
side product (DCU) as a precipitate was filtered off and the
solution was concentrated into syrup under high vacuum. To
this syrup was added the above solution of glycosylamine, and
the solution was stirred at room temperature overnight. The
reaction mixture was concentrated to a small volume under
vacuum, and the product was purified with a silica gel column
to give pure 3 as a white solid (1.0 g, 1.12 mmol, yield: 42%).
[R]D +31.8 (c 0.6, MeOH). TLC: Rf 0.35 (eluent: MeOH, DCM,
and AcOH, 3:6:1). FABMS: calcd for C41H56N2O20 896.4, found
897.4 (M + H+), 919.3 (M + Na+). HRFABMS: calcd for
1
+ H+). H NMR (CD3OD, 600 MHz) δ 7.77 (d, J ) 7.2 Hz, 2
H), 7.66 (t, J ) 7.2 Hz, 2 H), 7.37 (t, J 7.2 ) Hz, 2 H), 7.30 (t,
J ) 7.2 Hz, 2 H), 5.14 (d, J ) 3.8 Hz, 1 H), 5.13 (d, J ) 3.8 Hz,
1 H), 4.86 (d, J ) 9.0 Hz, 1 H), 4.75 (t, J ) 6.0 Hz, 1 H), 4.40
(m, 2 H), 4.21 (t, J ) 6.6 Hz, 1 H), 4.09 (t, J ) 7.2 Hz, 1 H),
3.88 (s, 4 H), 2.80 (m, 2 H), 2.29 (t, J ) 7.8 Hz, 2 H), 2.07 (m,
1 H), 1.94 (m, 1 H).
Finally, N-terminal deprotection of 6 by the general protocol
described above afforded the free glycopeptide 1. After pre-
cipitation and washing with ether, the precipitate product was
further purified by gel filtration column chromatography on a
Bio Gel A4 column (2.5 × 35 cm) with distilled water as the
eluent and then by HPLC on a semipreparative Discovery C18
column (25 × 1 cm) with an eluent of 0.2% 2-propanol in water
at a 2.0 mL/min flow rate to produce the pure product 1 (36
C
41H56N2O20Na (M + Na+) 919.3324, found 919.3371. 1H NMR
(CD3OD, 300 MHz) δ 7.80 (d, J ) 7.7 Hz, 2 H), 7.67 (d, J )
7.7 Hz, 2 H), 7.40 (t, J ) 7.2 Hz, 2 H), 7.32 (t, J ) 7.2 Hz, 2
H), 5.18 (d, J ) 3.8 Hz, 1 H), 5.16 (d, J ) 3.8 Hz, 1 H), 4.92 (d,
J ) 9.2 Hz, 1 H), 4.48 (t, J ) 6.0 Hz, 1 H), 4.20-4.40 (m, 3 H),
2.78 (br s, 2 H), 1.46 (s, 9 H).
mg, 91%). HPLC: RT ) 6.20 min. FABMS: calcd for
1
31H52N6O22 860.3, found 861.4 (M + H+). H NMR (D2O, 600
A Typ ica l P r oced u r e for th e N-Ter m in a l Dep r otection
(of F m oc). A glycosyl Fmoc-amino acid or N-terminal pro-
tected glycopeptide (56 µmol) was dissolved in a 2-mL mixture
C
MHz) δ 5.42 (d, J ) 3.8 Hz, 1 H), 5.41 (d, J ) 3.8 Hz, 1 H),
4.98 (d, J ) 9.0 Hz, 1 H), 4.75 (t, J ) 6.0 Hz, 1 H), 4.57 (dd, J
) 7.2, 4.2 Hz, 1 H), 4.03 (s, 2 H), 3.99 (s, 2 H), 2.87 (dd, J )
15.6, 4.8 Hz, 1 H), 2.73 (dd, J ) 15.6, 8.4 Hz, 1 H), 2.37 (t, J
) 7.2 Hz, 2 H), 1.98 (m, 1 H), 1.93 (m, 1 H).
(30) Blanzat, M.; Turrin, C.-O.; Perez, E.; Rico-Lattes, I.; Caminade,
A. M.; Majoral, J .-P. Chem. Commun. 2002, 1864-1865.
2718 J . Org. Chem., Vol. 68, No. 7, 2003

Efficient Synthesis of Complex Glycopeptides
L-Glu t a m in yl-glycyl-glycyl-{N-[r-D-glu cop yr a n osyl-
(1f4)-r-D-glu cop yr a n osyl-(1f4)-â-D-glu cop yr a n osyl]}-L-
a sp a r a gin yl-L-tyr osyl-glycyl-glycin e Meth yl Ester (11).
After the mixture of 6 (5 mg, 4.6 µmol), HOBt (1.9 mg, 13.8
µmol), and DCC (2.8 mg, 13.8 µmol) in 0.5 mL of NMP was
stirred at room temperature for 2 h, 12 (7.1 mg, 23 µmol) was
added to the solution. The stirring was continued for another
day, and then diethyl ether was added to precipitate the
product. The precipitate was collected following centrifugation,
washed with ether, and dried under vacuum to afford the crude
product 11 (4.7 mg, 89%). The product was studied with HPLC
(on a Discovery C18 25 × 1 cm column with an eluent of 8%
2-propanol in water at a 2.0 mL/min flow rate), which did not
show any impurity peak. HPLC: RT ) 5.18 min. FABMS:
calcd for C45H69N9O26 1151.4, found 1152.4 (M + H+), 1174.3
2.86 (dd, J ) 15.0, 4.2 Hz, 1 H), 2.82 (m, 1 H), 2.80 (dd, J )
15.0, 7.2 Hz, 1 H), 2.41 (t, J ) 7.2 Hz, 2 H), 2.17 (m, 1 H), 2.05
(m, 1 H).
Glycyl-glycyl-L-glu t a m in yl-{N-[r-D-glu cop yr a n osyl-
(1f4)-r-D-glu cop yr a n osyl-(1f4)-â-D-glu cop yr a n osyl]}-L-
a sp a r a gin yl-{N-[r-D-glu cop yr a n osyl-(1f4)-r-D-glu cop y-
r a n osyl-(1f4)-â-D-glu cop yr a n osyl]}-L-a sp a r a gin yl-{N-[r-
D-glu cop yr a n osyl-(1f4)-r-D-glu cop yr a n osyl-(1f4)-â-D-
glu cop yr a n osyl]}-L-a sp a r a gin yl-{N-[r-D-glu cop yr a n osyl-
(1f4)-r-D-glu cop yr a n osyl-(1f4)-â-D-glu cop yr a n osyl]}-L-
a sp a r a gin e (17). The tert-butyl group in 20 (20 mg, 13.4 µmol)
was deprotected by treatment with 25% TFA/DCM (3 mL) at
room temperature for 2 h. The reaction mixture was condensed
and the residue was washed with ether to produce 21 that
was directly used in the coupling reaction. At the same time,
20 mg of 20 was treated with 20% piperidine/NMP (2 mL) at
room temperature for 2 h to give the N-terminal free product
22 that was isolated by the precipitation method. After 21 was
stirred with DCC (8.3 mg, 40 µmol) and HOBt (5.4 mg, 40
µmol) in NMP (1 mL) for 2 h, 22 was added to the mixture.
The reaction mixture was stirred at room temperature over-
night, and then ether (10 mL) was added. The resulting
precipitate was collected and washed by ether. Subsequent
treatment of this precipitate with 20% piperidine/NMP (2 mL)
and precipitation and washing of the product with ether (20
mL) afforded the crude 23 (33.0 mg, quantitative). A part of
this product (4.0 mg) was purified by gel filtration chroma-
tography and RP HPLC to produce pure 23 (3.0 mg). HPLC:
RT ) 5.58 min (on a Discovery 25 × 1 cm C18 column with an
eleuent of 6% 2-propanol in water at a 2.0 mL/min flow rate).
ESMS (negative mode): calcd for C92H154N8O69 2476, found
1
(M + Na+), 1190.4 (M + K+). H NMR (D2O, 600 MHz) δ 7.11
(d, J ) 8.4 Hz, 2 H), 6.81 (d, J ) 8.4 Hz, 2 H), 5.37 (d, J ) 3.6
Hz, 1 H), 5.36 (d, J ) 3.6 Hz, 1 H), 4.91 (d, J ) 9.0 Hz, 1 H),
4.70 (d, J ) 6.6 Hz, 1 H), 4.53 (t, J ) 7.8 Hz, 1 H), 4.00 (s, 2
H), 3.97 (s, 2 H), 3.88 (s, 4 H), 3.72 (s, 3 H), 3.06 (dd, J ) 13.8,
7.2 Hz, 1 H), 2.94 (dd, J ) 13.8, 8.4 Hz, 1 H), 2.79 (dd, J )
15.2, 6.0 Hz, 1 H), 2.70 (dd, J ) 15.2, 7.2 Hz, 1 H), 2.37 (t, J
) 7.8 Hz, 2 H), 2.11 (m, 1 H), 1.99 (m, 1 H).
Glycyl-L-glu t a m in yl-glycyl-{N-[r-D-glu cop yr a n osyl-
(1f4)-r-D-glu cop yr a n osyl-(1f4)-â-D-glu cop yr a n osyl]}-L-
a sp a r a gin yl-{N-[r-D-glu cop yr a n osyl-(1f4)-r-D-glu cop y-
r a n osyl-(1f4)-â-D-glu cop yr a n osyl]}-L-a sp a r a gin e (16).
After a solution of 3 (30 mg, 33 µmol) in 2 mL of 25% TFA/
DCM was stirred at room temperature for 2 h, the solvent was
evaporated under reduced pressure. The residue was washed
with ether 3 times to give the expected product 18 (25 mg,
90%). Its 1H NMR spectrum indicated the complete removal
of the tert-butyl group. This crude product was stirred with
DCC (18.3 mg, 89 µmol) and HOBt (12.0 mg, 89 µmol) in NMP
(2 mL) at room temperature for 2 h. To this reaction mixture
was then added 4 (22 mg, 33 µmol), and the solution was
stirred at room temperature overnight. The product was
precipitated by addition of ether (20 mL) and the precipitate
was washed with ether (3 × 20 mL) to give the crude 20 (49
mg, quantitative). The product was analyzed by HPLC and
characterized by NMR and MS. HPLC: RT ) 7.42 min (on a
Waters Spherisorb 5 µm NH2 250 × 4.6 mm analytic column
with an eluent of 75% acetonitrile in water at a 2.0 mL/min
flow rate). FABMS: calcd for C63H92N4O37 1497.5, found 1498.4
(M + H+), 1520.4 (M + Na+). 1H NMR (D2O, 600 MHz) δ 7.40-
7.95 (m, 8 H), 5.41 (m, 4 H), 4.98 (m, 2 H), 4.65 (m, 2 H), 4.56
(m, 1 H), 4.40 (m, 2 H), 4.00 (m, 4 H), 2.80 (m, 4 H), 1.43 (s, 9
H).
1
2476 (M-). ESMS (positive mode): 2499 (M + Na+). H NMR
(D2O, 600 MHz) δ 5.37 (m, 8 H), 4.94 (m, 4 H), 4.75-4.73 (m,
3 H), 4.70 (t, J ) 6.0 Hz, 1 H), 3.98 (t, J ) 9.6 Hz, 4 H), 2.60-
2.90 (m, 8 H), 1.49 (s, 9 H).
Sequential elongation of the peptide chain of 23 with Fmoc-
Glu(Trt)-OPfp and Fmoc-Gly-OPfp and final deprotection of
the resulting glycopeptide following the standard procedures
gave the crude product 17 in quantitative yield. Thorough
purification of this product by gel filtration chromatography
and then RP HPLC afforded the pure 17 (19.5 mg, 68% isolated
yield from 20). [R]D +7.2 (c 0.1, H2O). HPLC: RT ) 11.9 min
(on a Discovery 25 × 1 cm C18 column with an eluent of 0.4%
2-propanol in water at a 2.0 mL/min flow rate). MALDIMS:
calcd for C97H160N12O73 2662, found 2663 (M + H+), 2685 (M
+ Na+), 2771 (M + K+). 1H NMR (D2O, 600 MHz) δ 5.41-5.43
(m, 8 H), 5.00 (m, J ) 9.0 Hz, 4 H), 4.79 (t, J ) 7.1 Hz, 1 H),
4.78 (t, J ) 7.0 Hz, 1 H), 4.76 (t, J ) 7.2 Hz, 1H), 4.51 (t, J )
6.0 Hz, 1 H), 4.38 (dd, J ) 7.8, 6.0 Hz, 1 H), 4.05 (s, 2 H), 3.98
(t, J ) 9.6 Hz, 4 H), 3.91 (s, 2 H), 2.94-2.99 (m, 3 H), 2.82-
2.87 (m, 4 H), 2.79 (dd, J ) 15.6, 7.8 Hz, 1 H), 2.39 (t, J ) 7.2
Hz, 2 H), 2.14 (m, 1 H), 2.02 (m, 1 H).
Further elongation of the peptide chain of 20 with Fmoc-
Glu(Trt)-OPfp and Fmoc-Gly-OPfp in sequence, as well as final
deprotection of the resulting glycopeptide, followed the stan-
dard procedures described above. The crude product was
eventually obtained in quantitative yield. Its purity was
determined by HPLC and NMR. Thorough purification of this
product by gel filtration chromatography and then RP HPLC
afforded the pure 16 in an isolated yield of 93%. HPLC: RT )
6.15 min (on a Discovery 25 × 1 cm C18 column with an eluent
Ack n ow led gm en t is made to the Research Corpora-
tion (Research Innovation Award RI 0663) and to the
donors of the Petroleum Research Funds administered
by the American Chemical Society (ACS PRF grant
37743-G) for their support of this research. The authors
are grateful to Dr. S. Chen and Mr. H. Faulk for the
MS measurements.
of 4% 2-propanol in water at
a 2.0 mL/min flow rate).
FABMS: calcd for C53H88N8O39 1460, found 1461 (M + H+).
1H NMR (D2O, 600 MHz) δ 5.42 (m, 4 H), 4.99 (d, J ) 9.0 Hz,
1 H), 4.98 (d, J ) 9.0 Hz, 1 H), 4.85 (dd, J ) 7.2, 6.0 Hz, 1 H),
4.52 (dd, J ) 6.0, 6.2 Hz, 1 H), 4.42 (dd, J ) 8.4, 7.2 Hz, 1 H),
3.99 (s, 2 H), 3.98 (s, 2 H), 2.93 (dd, J ) 15.6, 4.2 Hz, 1 H),
J O020570C
J . Org. Chem, Vol. 68, No. 7, 2003 2719