Chemoselective and microwave-assisted synthesis of glycopeptoids

Article abstract of DOI:10.1021/ol9021468

The chemoselective glycosylation of N-alkylaminooxy side chains with unprotected reducing sugars has proven useful for the synthesis of glycopeptides. Herein, we extend the N-alkylaminooxy strategy to the synthesis of glycopeptoids. A N-methylaminooxy sub

Full text of DOI:10.1021/ol9021468

ORGANIC
LETTERS
2009
Vol. 11, No. 22
5210-5213
Chemoselective and Microwave-Assisted
Synthesis of Glycopeptoids
Jiwon Seo,† Nairie Michaelian,‡ Shawn C. Owens,‡ Scott T. Dashner,‡
Amanda J. Wong,‡ Annelise E. Barron,† and Michael R. Carrasco*,‡
Department of Bioengineering, Stanford UniVersity, 318 Campus DriVe,
Stanford, California 94305-5440, and Department of Chemistry and Biochemistry,
Santa Clara UniVersity, Santa Clara, California 95053-0270
mcarrasco@scu.edu
Received September 17, 2009
ABSTRACT
The chemoselective glycosylation of N-alkylaminooxy side chains with unprotected reducing sugars has proven useful for the synthesis of
glycopeptides. Herein, we extend the N-alkylaminooxy strategy to the synthesis of glycopeptoids. A N-methylaminooxy submonomer was
efficiently synthesized and incorporated into peptoids. Glycosylation of the peptoids proceeded chemoselectively and site-specifically at the
N-methylaminooxy moieties. Employing microwave irradiation significantly increased the degree of glycosylation and shortened the reaction
times.
Glycosylation, a ubiquitous post-translational modification
in proteins, plays critical roles in protein folding,1 stabiliza-
tion,2 trafficking, and recognition.3 Owing to the inherent
complexity of carbohydrates, glycosylation can produce
enormous structural diversity in proteins and induce a variety
of functional changes. In an attempt to decipher these
structure-function relationships, protein and peptide chemists
have developed various chemical and enzymatic methods for
the synthesis of homogeneous and well-defined glycopro-
teins.4
Chemoselective glycosylation methods have enabled the
convergent and site-specific modification of unprotected
peptides. Orthogonal ligation techniques employing ami-
nooxy,5 hydrazine,6 hydrazide,7 and chemoenzymatic8 strate-
gies have been utilized. Among these ligation methods, the
N-alkylaminooxy strategy first demonstrated by Peri and co-
(4) (a) Peri, F.; Nicotra, F. Chem. Commun. 2004, 623–627. (b) Pratt,
M. R.; Bertozzi, C. R. Chem. Soc. ReV. 2005, 34, 58–68. (c) Gamblin, D. P.;
Scanlan, E. M.; Davis, B. G. Chem. ReV. 2009, 109, 131–163.
(5) (a) Cervigni, S. E.; Dumy, P.; Mutter, M. Angew. Chem., Int. Ed.
Engl. 1996, 35, 1230–1232. (b) Zhao, Y.; Kent, S. B. H.; Chait, B. T. Proc.
Natl. Acad. Sci. U.S.A. 1997, 94, 1629–1633. (c) Peri, F.; Dumy, P.; Mutter,
M. Tetrahedron 1998, 54, 12269–12278. (d) Marcaurelle, L. A.; Rodriguez,
E. C.; Bertozzi, C. R. Tetrahedron Lett. 1998, 39, 8417–8420. (e) Peluso,
S.; Imperiali, B. Tetrahedron Lett. 2001, 42, 2085–2087.
(6) Grandjean, C.; Rommens, C.; Gras-Masse, H.; Melnyk, O. Angew.
Chem., Int. Ed. 2000, 39, 1068–1072.
† Stanford University.
‡ Santa Clara University.
(1) (a) Live, D. H.; Kumar, R. A.; Beebe, X.; Danishefsky, S. J. Proc.
Natl. Acad. Sci. U.S.A. 1996, 93, 12759–12761. (b) Imperiali, B.; O’Connor,
S. E. Curr. Opin. Chem. Biol. 1999, 3, 643–649. (c) Helenius, A.; Aebi,
M. Science 2001, 291, 2364–2369.
(7) Rodriguez, E. C.; Marcaurelle, L. A.; Bertozzi, C. R. J. Org. Chem.
1998, 63, 7134–7135.
(2) Russell, D.; Oldham, N. J.; Davis, B. G. Carbohydr. Res. 2009, 344,
1508–1514.
(8) (a) Rodriguez, E. C.; Winans, K. A.; King, D. S.; Bertozzi, C. R.
J. Am. Chem. Soc. 1997, 119, 9905–9906. (b) Fu, X.; Albermann, C.; Jiang,
J.; Liao, J.; Zhang, C.; Thorson, J. S. Nat. Biotechnol. 2003, 21, 1467–
1469.
(3) Sears, P.; Wong, C. H. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 12086–
12093.
10.1021/ol9021468 CCC: $40.75
Published on Web 10/27/2009
 2009 American Chemical Society

workers,5c wherein peptides containing N-alkylaminooxy
groups are glycosylated in mildly acidic aqueous solutions
with native, completely unprotected reducing sugars, is
particularly attractive. The two key advantages are that
attached sugars maintain cyclic conformations and that no
synthetic sugar chemistry is required. Previously, we have
synthesized N-methylaminooxy-containing amino acids suit-
able for Boc or Fmoc solid-phase peptide synthesis.9 These
amino acids were successfully incorporated into peptides,
and their chemoselective glycosylation yielded neoglyco-
peptides. We now extend this N-alkylaminooxy strategy to
the synthesis of glycopeptoids.
Peptoids are a unique class of peptide mimics based on
oligoglycine scaffolding.10 While their sequence of backbone
atoms is identical to that of a peptide, non-natural N-
substituents render them protease-stable.11 Using submono-
mer protocols developed by Zuckermann et al.,12 diverse
oligopeptoids are readily synthesized with a variety of
different primary amines and bromoacetic acid (Figure 1);
able pharmacological properties has fueled interest in pep-
toids, and many examples of biologically active peptoids
have been reported recently.14
The advantages of peptoids can be carried to their
glycosylated counterparts, glycopeptoids, which have great
potential both as glycopeptide mimics15 and as novel
carbohydrate-presenting materials. Glycopeptoids have been
synthesized by different strategies.16 Herein, we report the
synthesis of glycopeptoids by the chemoselective ligation
of N-methylaminooxy-containing peptoid oligomers with
unprotected reducing sugars. In addition, we report that
microwave irradiation greatly enhances N-alkylaminooxy
glycosylation reactions. Thus, we present a simple and
practical strategy for the rapid generation of a wide range
of glycopeptoids.
The synthesis of N-methylaminooxy-containing peptoid
oligomers was enabled by designing and making the N-
methylaminooxy submonomer 3 (Scheme 1). Its synthesis
Scheme 1. Synthesis of N-Methylaminooxy Submonomer 3
was accomplished in two steps and 53% overall yield.
N-Boc,N-methylhydroxylamine, 1,17 was deprotonated with
sodium hydride and used to monoalkylate 3-iodo-1-bro-
mopropane to produce 2. Conversion of the chloride to an
azide followed by reduction with triphenylphosphine then
afforded the desired amine 3.
Figure 1. (A) Structures of an R-peptide and a peptoid, and (B)
peptoid submonomer synthesis protocol (DIC ) N,N′-diisopropy-
lcarbodiimide).
The protected N-methylaminooxy submonomer 3 was
readily incorporated into oligopeptoids using standard solid-
phase peptoid synthesis procedures, and no distinct side
products were observed. In this study we synthesized four
different model peptoids (4 - 7, Figure 2; see Supporting
Information for synthetic details).
therefore, rapid and convenient access to a wide array of
peptoid sequences is possible.13 The combination of ease of
synthesis, ability to display diverse functionality, and favor-
(9) (a) Carrasco, M. R.; Nguyen, M. J.; Burnell, D. R.; MacLaren, M. D.;
Hengel, S. M. Tetrahedron Lett. 2002, 43, 5727–5729. (b) Carrasco, M. R.;
Brown, R. T.; Serafimona, I. M.; Silva, O. J. Org. Chem. 2003, 68, 195–
197. (c) Carrasco, M. R.; Brown, R. T. J. Org. Chem. 2003, 68, 8853–
8858. (d) Carrasco, M. R.; Brown, R. T.; Doan, V. H.; Kandel, S. M.; Lee,
F. C. Biopolymers 2006, 84, 414–420.
(14) (a) Chongsiriwatana, N. P.; Patch, J. A.; Czyzewski, A. M.; Dohm,
M. T.; Ivankin, A.; Gidalevitz, D.; Zuckermann, R. N.; Barron, A. E. Proc.
Natl. Acad. Sci. U.S.A. 2008, 105, 2794–2799. (b) Brown, N. J.; Wu, C. W.;
Seurynck-Servoss, S. L.; Barron, A. E. Biochemistry 2008, 47, 1808–1818.
(c) Murphy, J. E.; Uno, T.; Hamer, J. D.; Cohen, F. E.; Dwarki, V.;
Zuckermann, R. N. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 2794–2799.
(15) Marcaurelle, L. A.; Bertozzi, C. R. Chem.sEur. J. 1999, 5, 1384–
1390.
(10) Simon, R. J.; Kania, R. S.; Zuckermann, R. N.; Huebner, V. D.;
Jewell, D. A.; Banville, S.; Ng, S.; Wang, L.; Rosenberg, S.; Marlowe,
C. K.; Spellmeyer, D. C.; Tan, R.; Frankel, A. D.; Santi, D. V.; Cohen,
F. E.; Bartlett, P. A. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 9367–9371.
(11) Miller, S. M.; Simon, R. J.; Ng, S.; Zuckermann, R. N.; Kerr, J. M.;
Moos, W. H. Drug DeV. Res. 1995, 35, 20–32.
(16) (a) Saha, U. K.; Roy, R. Tetrahedron Lett. 1995, 36, 3635–3638.
(b) Yuasa, H.; Kamata, Y.; Kurono, S.; Hashimoto, H. Bioorg. Med. Chem.
Lett. 1998, 8, 2139–2144. (c) Dechantsreiter, M. A.; Burkhart, F.; Kessler,
H. Tetrahedron Lett. 1998, 39, 253–254. (d) Burger, K.; Boettcher, C.;
Radics, G.; Hennig, L. Tetrahedron Lett. 2001, 42, 3061–3063. (e) Norgren,
A. S.; Budke, C.; Majer, Z.; Heggemann, C.; Koop, T.; Sewald, N. Synthesis
2009, 3, 488–494. (f) Comegna, D.; Riccardis, F. D. Org. Lett. 2009, 11,
3898–3901.
(12) Zuckermann, R. N.; Kerr, J. M.; Kent, S. B. H.; Moos, W. H. J. Am.
Chem. Soc. 1992, 114, 10646–10647.
(13) (a) Zuckermann, R. N.; Martin, E. J.; Spellmeyer, D. C.; Stauber,
G. B.; Shoemaker, K. R.; Kerr, J. M.; Figliozzi, G. M.; Goff, D. A.; Siani,
M. A.; Simon, R. J.; Banville, S. C.; Brown, E. G.; Wang, L.; Richter,
L. S.; Moos, W. H. J. Med. Chem. 1994, 37, 2678–2685. (b) Nguyen, J. T.;
Turck, C. W.; Cohen, F. E.; Zuckermann, R. N.; Lim, W. A. Science 1998,
282, 2088–2092.
(17) (a) House, H. O.; Richey, F. A. J. Org. Chem. 1969, 34, 1430–
1439. (b) Freeman, J. P.; Lillwitz, L. D. J. Org. Chem. 1970, 35, 3107–
3110.
Org. Lett., Vol. 11, No. 22, 2009
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Table 1. Glycosylation of 4 at 40 °C with Microwave
Irradiationa
peptoid concn sugar excess reaction convc
entry
sugar
(mM)
(molar)
time
(%)
1b
2b
3b
4
5
6
7
8
9
D-glucose
D-glucose
D-glucose
D-glucose
D-glucose
D-glucose
D-maltose
D-melibiose
maltotriose
3.8
7.6
6.3
6.3
6.3
6.3
6.3
6.3
6.3
8.0
6.3
50
50
12 h
12 h
68
78
35
83
88
94
87
81
85
89
87
100
100
100
200
100
100
100
100
100
10 min
10 min
30 min
10 min
10 min
10 min
10 min
5 h
10 D-lactose
11 GlcNAcd
5 h
a Reaction corresponds to Figure 3; 0.1 M sodium acetate buffer/
methanol ) 6:1, pH ) 4.0. b Reaction performed at 40 °C without
microwave irradiation. c Conversion based on HPLC integrations at 220
nm. d N-Acetyl-D-glucosamine.
Encouraged by these results, we next sought additional
carbohydrates that could be efficiently incorporated. D-Maltose
(D-Glc-R(1f4)-D-Glc), D-melibiose (D-Gal-ꢀ(1f4)-D-Glc), and
maltotriose (D-Glc-R(1f4)-D-Glc-R(1f4)-D-Glc), where the
hydroxyl groups of the first sugar are all equatorial, ligated
smoothly to the peptoid (Table 1, entries 7-9). Reaction
proceeded much more slowly with D-lactose (D-Gal-R(1f6)-
D-Glc) and N-acetyl-D-glucosamine (Table 1, entries 10 and 11).
D-Galactose and D-mannose, which contain axial hydroxyl
groups at their C4 and C2 positions, respectively, yielded
mixtures of glycosylation products that we attribute to the
presence of R and ꢀ stereoisomers or pyranose and furanose
sugar forms as reported for related reactions with these
sugars.5c,18 N-Acetyl-D-galactosamine behaved similarly to
D-galactose. Fructose, a monosaccharide with a ketone rather
than an aldehyde functional group, was also tried, and
virtually no reaction occurred.
Figure 2. Peptoids synthesized in this study.
Oligopeptoid 4 was used to optimize the glycosylation
conditions and to investigate the range of sugars that could
be incorporated (Figure 3). We first used our previously
described conditions (0.1 M NaOAc, pH ) 4.0, 40 °C).
Overall, the reactivity differences seen between various
sugars mirrored those reported for other N-alkylaminooxy
glycosylations, but the advantage provided by microwave
irradiation proved dramatic. As an example, Peri et al. found
the reaction of an N-alkylaminooxy-containing peptide to be
much slower with lactose than with glucose, and heating the
lactose reaction for 144 h at 60 °C was required to achieve
a 57% yield.5c Reaction with lactose was also slower in our
case, but only 5 h at 40 °C were required to achieve an 89%
conversion when microwave irradiation was employed (Table
1, entry 10).
Divalent glycosylation was attempted next with model
peptoid 5. Using the microwave-assisted reaction conditions,
5 was diglucosylated nearly completely (Figure 4).
Lastly, the chemoselectivity of the glycosylation was
investigated with peptoid 6, which contains hydroxyl, amino,
Figure 3. Glycosylation of 4. The range of sugars and conditions
investigated is detailed in Table 1.
Glycosylation proceeded well with D-glucose (Glc), and
68-78% conversion was observed after 12 h (Table 1, entries
1 and 2). We were pleased to find that the reactivity was
greatly enhanced with microwave irradiation: 83% conver-
sion was observed in 10 min with 300 W microwave
irradiation at 40 °C in a CEM MARS multimodal microwave
reactor (Table 1, entry 4; see Supporting Information for
detailed microwave conditions). The same conditions without
microwave irradiation only resulted in 35% conversion
(Table 1, entry 3). The best conversion that we observed
under microwave conditions was 94% in 10 min (Table 1,
entry 6); only approximately 5% of the starting peptoid 4
remained unreacted. In general, conversion yields were
greater when longer microwave irradiation and higher reagent
concentrations were used.
(18) (a) Peri, F.; Deutman, A.; La Ferla, B.; Nicotra, F. Chem. Commun.
2002, 1504–1505. (b) Peri, F.; Jime´nez-Barbero, J.; Garc´ıa-Aparicio, V.;
Tvarosˇka, I.; Nicotra, F. Chem.sEur. J. 2004, 10, 1433–1444. (c)
Langenhan, J. M.; Peters, N. R.; Guzei, I. A.; Hoffmann, F. M.; Thorson,
J. S. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 12305–12310
.
5212
Org. Lett., Vol. 11, No. 22, 2009

As seen with other N-alkylaminooxy glycosylation
products,9a,18c the glycopeptoids were pH responsive. They
remained stable in solutions at pH > 6; however, at pH ) 2
they rapidly returned to the parent peptoid and sugar. We
are investigating how we might take advantage of the pH-
induced reversability of the peptoid-sugar linkage for bio-
material applications.
Although our strategy is not particularly amenable to the
inclusion of different sugars within a single peptoid molecule,
it is ideal for providing differently glycosylated versions of
a single peptoid. One may synthesize a single N-alkylami-
nooxy-containing peptoid oligomer and subsequently gly-
cosylate it with a variety of sugars to provide a panel of
glycopeptoids. The glycosylation reactions are simple, fast,
and efficient, and most importantly they do not require sugar
synthesis or any expensive commercial sugar derivatives.
Furthermore, the strategy requires the synthesis of only a
single submonomer rather than a different submonomer for
each desired sugar.
Thus we have developed an efficient, new strategy to
access glycopeptoids by the use of a N-alkylaminooxy-
containing submonomer. Microwave irradiation enabled rapid
reactions with high conversion as well as divalent glycosy-
lation reactions with maintainence of chemoselectivity. We
note that the advantage provided by microwave irradiation
will also benefit those using similar strategies for the
glycosylation of peptides, proteins, and small molecule drugs.
We envision the use of this method to generate an extensive
range of biologically functional glycopeptoids. Our current
focus is on the synthesis of multivalent glycopeptoids to
investigate the influence of dense glycosylation on peptoid
secondary structure.
Figure 4. (A) Synthesis of divalent glycopeptoid 8, and (B) HPLC
analysis of the synthesis (UV detection at 220 nm). See Supporting
Information for detailed HPLC conditions.
sulfhydryl, and carboxamido functionalities in addition to
the N-methylaminooxy group. After 10 min of microwave
reaction with a large excess of D-glucose, the HPLC
chromatogram and ESI-MS analysis indicated an 88%
conversion of 6 to a monoglucosylated peptoid. No additional
glucosylation was observed. To confirm that glucosylation
occurred solely at the N-alkylaminooxy site, we synthesized
the control peptoid 7 where the N-methylaminooxy group
of 6 was replaced by methoxy. Treatment of 7 under the
same glycosylation conditions yielded only unreacted starting
material.
Acknowledgment. We are grateful to the National Insti-
tutes of Health (R01 AI072666 to A.E.B.; R15 GM081851-
01 to M.R.C.) and the Camille and Henry Dreyfus Founda-
tion for their support of this work.
Supporting Information Available: Detailed procedures
for the synthesis, characterization, and purification of all new
compounds and oligopeptoids. This material is available free
of charge via the Internet at http://pubs.acs.org.
OL9021468
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