Ritter-based glycoconjugation of amino acids and peptides-access to novel glycoconjugates displaying a β-amide linkage between amino acid and sugar moiety

Article abstract of DOI:10.1016/j.carres.2006.11.006

β-Peptidic-d-gluco-, d-galacto-, and l-fuco-configured glycosyl amino acids can be prepared from the corresponding 2-deoxy-oct-3-ulopyranosonic acids via a one-pot intramolecular Ritter reaction. Initially, a ketopyranoside-based acid condenses under Lewi

Full text of DOI:10.1016/j.carres.2006.11.006

Carbohydrate Research 342 (2007) 7–15
Ritter-based glycoconjugation of amino acids and peptides—access
to novel glycoconjugates displaying a b-amide linkage between
amino acid and sugar moiety
Marlin Penner and Frank Schweizer*
Department of Chemistry, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2
Received 18 July 2006; received in revised form 26 October 2006; accepted 7 November 2006
Available online 11 November 2006
Abstract—b-Peptidic-D-gluco-, D-galacto-, and L-fuco-configured glycosyl amino acids can be prepared from the corresponding 2-
deoxy-oct-3-ulopyranosonic acids via a one-pot intramolecular Ritter reaction. Initially, a ketopyranoside-based acid condenses
under Lewis acid promoted conditions with nitriles (PhCN, MeCN) and a partially protected diamino ester (Boc-DAB-O-t-Bu,
Boc-Orn-O-t-Bu) to form a b-peptidic glycosyl amino t-butylesters. The glycosyl amino t-butylesters can be converted into
Fmoc-protected glycosyl amino acids that are suitably protected for solid-phase glycopeptide synthesis. Furthermore, replacement
of the protected diamino ester by immobilized peptide amines permits post-synthetic N-terminal- and N(e)-glycoconjugation of
peptides on the solid phase.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Keywords: Glycoconjugates; C-Ketosides; Neoglycopeptides; Glycosyl amino acids; Glycomimetics
1. Introduction
the synthesis of unnatural glycosyl amino acids in which
the side-chain of DAB (diaminobutyric acid) or orni-
thine is conjugated via an b-amide linkage to a mono-
saccharide (glucose, galactose, fucose) using a one-pot
Ritter-based condensation (Fig. 1). The building blocks
can be converted into suitably protected glycosyl amino
acids for use in solid-phase glycopeptide synthesis. In
addition, we demonstrate that the Ritter condensation
permits (a) incorporation of carbohydrate binding
sites into the N-terminal position of resin-immobilized
Glycoconjugates involved in biological events such as
tumor metastasis, inflammation, and immune response
have a significant pharmaceutical potential.1 Of particu-
lar interest are glycoproteins and glycopeptides contain-
ing modified glycosyl amino acids, thus exhibiting new
properties. Artificial glycopeptide linkages such as C-
glycosyl amino acids,2 glycopeptoids,3 retro amides,4
and oxime-linked glycosyl amino acids5 have been devel-
oped to overcome the inherent metabolic instability of
glycoconjugates. Recently, it was reported that side-
chain modified lysine glycoconjugates incorporated into
the RGD tripeptide pharmacophore improved the
receptor-subtype selectivity and pharmacokinetic prop-
erties of RGD peptides.6 Similar approaches may also
be applicable to other peptides but require access to
unnatural glycosyl amino acids. In this paper, we report
O
O
NH
RO
O
NHBoc
n
NH
R
O
O
n = 1,2
R = C H , CH
3
6
5
*
Figure 1. Glycosyl amino acids containing a b-amide linkage between
Corresponding author. Fax: +1 204 474 7608; e-mail addresses:
carbohydrate and the side chain of diaminobutyric acid and ornithine.
schweize@cc.umanitoba.ca; schweize@ms.umanitoba.ca
0008-6215/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2006.11.006

8
M. Penner, F. Schweizer / Carbohydrate Research 342 (2007) 7–15
galacto-, and
L
-fuco-based b-peptidic glycosyl amino
OBn
O
BnO
BnO
OBn
O
acids was recently communicated.7
TMSOTf
R CN
BnO
BnO
1
O
Recently, we reported that a-D-galacto-2-deoxy-oct-3-
ulopyranosonic acid 1 can be converted into galactose-
configured-b-amides with high stereoselectivity using a
Ritter-based three-component condensation. Initially,
acid 1 was condensed with a monofunctionalized nitrile
(aromatic and aliphatic) under Lewis-acid promoted
conditions to form a cyclic imino anhydride 2 that
undergoes a second condensation with excess of mono-
functionalized amines to form galactosyl-b-amides 3
(Scheme 1).8,9 We then became interested in expanding
this three-component condensation to D-gluco-, D-man-
no, L-fuco-, and L-rhamno-based ulosonic acids, as well
as to polyfunctionalized amino acids and immobilized
peptidic amines.
OH
BnO
N
O
(-H O)
2
BnO OH
O
1
R
2
1
2
R NH
2
(excess)
OBn
O
BnO
H
N
R
BnO
2
BnO
NH
O
O
1
R
3
2. Results and discussion
Scheme 1. Ritter-based one-pot three-component condensation that
provides galactosyl-b-amides 3. 2-Deoxy-a-D-galacto-oct-3-ulopyrano-
sonic acid 1 condenses with a monofunctionalized nitrile under Lewis-
acid promoted conditions to form imino anhydride 2. Exposure of 2 to
monofunctional amines yields galactosyl-b-amides 3.
Ulosonic esters 4–810 (Scheme 2) were prepared by the
reaction of t-butylacetate enolate with the correspond-
ing sugar lactone as previously described8 and isolated
in high yields averaging 90%. Initially, we were inter-
ested in studying whether polyfunctionalized amines
such as side-chain deprotected a,c-diaminobutyric
t-butylester (Boc-DAB-O-t-Bu) and ornithine (Boc-
Orn-O-t-Bu) are compatible with this Ritter-based
peptides and (b) post-synthetic internal N(e)glycoconju-
gation of lysine-containing peptides on the solid phase.
A preliminary account on the synthesis of D-gluco-, D-
OBn
R2
R4
O
OBn
R2
7
O
R1
BnO
1
R4
NH
a)
b)
O
tBu
3
5
R1
BnO
O
n
R3 NH
O
OtBu
NHBoc
O
R3 OH
O
R5
4 R1 = H, R2 = OBn, R3 = OBn, R4 = H
5 R1 = OBn, R2 = H, R3 = OBn, R4 = H
6 R1 = OBn, R2 = H, R3 = H, R4 = OBn
9
R1 = H, R2 = OBn, R3 = OBn, R4 = H, R5 = Ph; n = 1;
10 R1 = H, R2 = OBn, R3 = OBn, R4 = H, R5 = Ph; n = 2;
11 R1 = H, R2 = OBn, R3 = OBn, R4 = H, R5 = Me; n = 2;
12 R1 = OBn, R2 = H, R3 = OBn, R4 = H, R5 = Ph; n = 1;
13 R1 = OBn, R2 = H, R3 = OBn, R4 = H, R5 = Ph; n = 2;
14 R1 = OBn, R2 = H, R3 = OBn, R4 = H, R5 = Me; n = 2;
O
OH
a)
b)
O
O
OtBu
O
R1
NH
R1
R3
7
OBn
R2
OBn 1
O
NH
R2
R4
4
OBn
7 R1 = H, R2 = OBn, R3 = OBn, R4 = H
8 R1 = OBn, R2 = H, R3 = H, R4 = OBn
BnO
15 R1, R2 = H
16 R1 = COOtbutyl, R2 = NHBoc
Scheme 2. Synthesis of glycosyl amino acids 9–16. Reagents and conditions: (a) 50% TFA in CH2Cl2, 2 h; (b) sugar (4 equiv), TMSOTf (12 equiv),
nitrile (MeCN or PhCN, 16 equiv), CH2Cl2, 2 h, 0 ꢁC; quenching with DIPEA; then addition of Boc-DAB-O-t-Bu or Boc-Orn-O-t-Bu or C3H7NH2,
12 h, 18–95%.

M. Penner, F. Schweizer / Carbohydrate Research 342 (2007) 7–15
9
three-component condensation. Removal of the t-butyl
group of the galactose-based ulosonic ester 4 with triflu-
oroacetic acid generated 1 in situ. Treatment of 1 with
from ulosonic ester 5 with benzonitrile and Boc-Orn-
O-t-Bu, did not afford the corresponding mannose-con-
figured amino ester. Next we studied the use of the
L-fucose- and L-rhamno-based ulosonic esters 7 and 8
in the three-component condensation (Scheme 2). The
Ritter reaction of the L-fucose-based ulosonic acid with
PhCN and trapping of the intermediate imino anhydride
with propylamine and Boc-Dab-O-t-Bu provided the
fucose-configured amide 15 and fucosyl amino acid 16
in 40% and 18% yield, respectively. This contrasts with
the reaction of L-rhamno-based acid derived from ulo-
sonic ester 8, which did not provide the corresponding
glycosyl amino ester. We speculate that the axial substi-
tuent at C-4 in the D-manno- and L-rhamno-based ulo-
sonic acid destabilizes the cyclic form and favors the
open ketone form, thereby preventing the formation of
the corresponding b-peptidic glycosyl amino acids
(Scheme 3).
The absolute stereochemistry at C-3 of the protected
glycosyl amino esters 9–16 was assigned based on
inter-proton effects of H-7 (>0.6% NOE) measured rela-
tive to the singlet amide resonance signal. For instance,
subjection of the singlet amide signal in 9 to a one-
dimensional GOESY11 experiment showed inter-proton
effect to H-7 (0.8% NOE) measured relative to the
singlet amide signal (Fig. 2). This is only compatible
with an axial orientation of the anomeric amide group.
Similar inter-proton NOE effects between H-7 and the
singlet amide signal were observed for compounds
10–16 (Fig. 2).
trimethylsilyl-trifluoromethanesulfonate
(TMSOTf)
and excess benzonitrile (PhCN) for 2 h, followed by
quenching the reaction by the addition of excess diiso-
propylethylamine (DIPEA) and addition of Boc-DAB-
O-t-Bu or Boc-Orn-O-t-Bu, produced the protected
galactose-configured amino esters 9 and 10 as single
stereoisomers in 75% and 64% yield, respectively
(Scheme 2). Replacement of PhCN by acetonitrile
(MeCN) and exposure to Boc-Orn-O-t-Bu provided
the galactose-configured amino ester 11 in 61% yield.
After demonstrating the compatibility of polyfunc-
tionalized amines in this three-component reaction, we
explored the one-pot Ritter-based condensation using
the glucose-based ulosonic acid derived from 5 by acidic
ester hydrolysis. TMSOTf-promoted condensation of
the acid with PhCN or MeCN and subsequent quench-
ing with two amines (Boc-Dab-O-t-Bu or Boc-Orn-O-t-
Bu) afforded the glucosyl amino esters 12–14 in 95%,
79%, and 55% yield, respectively (Scheme 2). By com-
parison, TMSOTf-promoted condensation of D-manno-
based ulosonic acid, derived by acidic ester hydrolysis
TMSOTf
CH Cl
2
2
BnO
BnO
RCN
OTMS
O
1
O
OH
BnO
OH
BnO
4
O
OH
O
To convert glycosyl amino ester 9 into a building
block suitable for solid-phase glycopeptide synthesis,
we applied a four-step deprotection/protection protocol
(Scheme 4). First, the O-benzyl protecting groups on the
galactose-moiety were removed by catalytic hydrogena-
tion followed by acetylation of the free hydroxyl groups.
Then the t-butylcarbamate and the t-butyl ester protect-
ing groups were cleaved by treatment with TFA before
selective deprotection of the amino function using
9-fluorenylmethyl pentafluorophenyl carbonate (Fmoc-
OPfp), producing the Fmoc-protected galactose-based
amino acid 19 in 76% overall yield (Scheme 4).
ketone form
cyclic ulosonic acid
X
imino
anhydride
Scheme 3. Destabilization of cyclic ulosonic acids bearing axial
substituents at C-4. The axial substituent at C-4 in D-manno- and L-
rhamno-based ulosonic acids destabilizes the cyclic form and leads to
the formation of the acyclic ketone form, thereby preventing imino
anhydride formation.
OBn
a
b
O
> 0.6%
O
1
a
NOE
O
O
NH
H
BnO
OtBu
3
R1
BnO
N
H-7
O
O
NHBoc
H-7
N
1
R2
O
NH
H
OBn
> 0.6%
4
OBn
a
BnO
NOE
a
Figure 2. Conformationally relevant NOE interactions observed for compounds 9–14 (a) and compounds 15 and 16 (b); in CDCl3.

10
M. Penner, F. Schweizer / Carbohydrate Research 342 (2007) 7–15
Compound 17 is suitably protected for solid-phase
glycopeptide synthesis.
OBn
O
BnO
BnO
O
Next we studied the use of immobilized peptidic
amines in this three-component reaction. At first we
looked at N-terminal glycoconjugation of the immobi-
lized tripeptide H-Ala-Gly-Ala-resin 18 synthesized by
standard solid-phase peptide synthesis on the Rink-resin
(Scheme 5). Exposure of tripeptide 18 to excess of galac-
tose-based cyclic imino anhydride 2 provided immobi-
lized glycopeptide 19 that was released from the resin
by treatment with trifluoroacetic acid. Glycopeptide 20
was obtained in 35% isolated yield after chromato-
graphic purification. Encouraged by these results, we
explored Ritter-based side-chain glycoconjugation of the
immobilized tripeptide Ac-Ala-Lys(H)-Ala-resin 23 syn-
thesized by solid-phase peptide synthesis using ortho-
gonally protected lysine building block 26 (Scheme 6).
Exposure of immobilized tripeptide 23 to excess imino-
anhydride 2 produced protected glycotripeptide 25 after
resin cleavage (34% yield).
NH
OtBu
BnO NH
O
O
NHBoc
9
c)
d)
e)
OAc
AcO
AcO
O
O
NH
OH
AcO NH
O
O
NHFmoc
Our modified Ritter-based method allows fast access
to glycosyl amino acids bearing a b-amide linkage
between glucose, galactose, and fucose and the side
chain of ornithine and diaminobutyric acid. The result-
ing glycosyl amino acids can be converted into Fmoc-
protected glycosyl amino acid for use in solid-phase gly-
copeptide synthesis. Furthermore, our procedure allows
17
Scheme 4. Deprotection of glycosyl amino acid 9. Reagents and
conditions: (c) Pd(OH)2, MeOH, H2, then Ac2O, C5H5N, 20 h; (d) 50%
TFA in CH2Cl2, 0 ꢁC to rt, 8 h; (e), THF–H2O (2:1), Fmoc-OPfp
(2 equiv), NaHCO3, 12 h, 76% (over three steps).
solid phase
peptide synthesis
O
O
H
H N
N
2
FmocHN
N
HN
H
O
"Rink resin"
18
f)
OBn
BnO
O
O
O
H
N
H
N
BnO
HN
N
H
BnO
O
NH
O
O
19
g)
OBn
BnO
BnO
O
O
O
H
H
N
N
N
NH
2
H
BnO
NH
O
O
O
20
Scheme 5. Ritter-based N-terminal glycoconjugation of immobilized tripeptide H-Ala-Gly-Ala-resin 18. Reagents and conditions: (f) (i) 1 (4 equiv),
50% TFA in CH2Cl2, 2 h, codistillation with toluene; (ii) TMSOTf (14 equiv), PhCN (40 equiv), CH2Cl2, 2 h, 0 ꢁC, then addition of DIPEA
(24 equiv), 2 min, then addition of 18 (1 equiv), 12 h; (g) 95% TFA in CH2Cl2, 20 min.

M. Penner, F. Schweizer / Carbohydrate Research 342 (2007) 7–15
11
O
O
HN
solid phase
peptide synthesis
Fmoc-strategy
O
O
H
RHN
N
FmocHN
"Rink resin"
N
HN
i)
H
O
21: R = H
22: R = COCH
h)
3
O
H N
2
OBn
O
BnO
BnO
HN
O
H
N
O
O
j)
H
BnO
NH
O
NH
AcHN
N
N
H
HN
O
O
O
24
AcHN
23
k)
O
O
O
OBn
BnO
BnO
HN
O
H
FmocHN
O
O
N
BnO
O
NH
O
NH
OH
O
25
26
AcHN
Scheme 6. Ritter-based side-chain glycoconjugation of immobilized tripeptide Ac-Ala-Lys(H)-Ala-resin 23. Reagents and conditions: (h)
CH3COOH, TBTU, DIPEA; (i) 2% N2H4 · H2O hydrazine in DMF; (j) (i) 1 (4 equiv), 50% TFA in CH2Cl2, 2 h, codistillation with toluene; (ii)
TMSOTf (14 equiv), PhCN (40 equiv), CH2Cl2, 2 h, 0 ꢁC, then addition of DIPEA (24 equiv), 2 min, then addition of 23, 12 h; (k) 95% TFA in
CH2Cl2, 20 min.
N-terminal glycoconjugation as well as internal N(e)gly-
coconjugation of lysine-containing peptides immobilized
on the Rink amide resin. The commercial availability of
large numbers of nitriles, amino acids, and immobilized
peptidic amines suggests that the methodology pre-
sented here should be attractive for the combinatorial
synthesis of unnatural glycosyl amino acids and the
preparation of neoglycopeptide libraries.12
recorded at 360 or 500 MHz for 1H and at 75 MHz
for 13C. Chemical shifts are reported relative to CHCl3
(dH 7.26, dC (center of triplet) 77.0 ppm) or to CH3OH
(dH 73.35, dC (center of septet) 49.0 ppm) or to acetone
as internal standard (D2O). TLC was performed on E.
Merck Silica Gel 60 F254 with detection by charring
with 8% H2SO4 acid. Silica gel (0.040–0.063 mm) was
used for column chromatography. Lactone 5 was pur-
chased from Toronto Research Chemicals. Rink-amide
MBHA resin was purchased from Novabiochem; com-
pound 26 is commercially available from BACHEM.
3. Experimental
3.1. General methods
3.2. General procedure for the synthesis of compounds
9–14, 15, and 16
CH2Cl2 was distilled from calcium hydride. Organic
solutions were concentrated under diminished pressure
at <40 ꢁC (bath temperature). NMR spectra were
The ketol (any of compounds 4–8, 4.3 mmol) was dis-
solved in 1:1 mixture containing trifluoroacetic acid

12
M. Penner, F. Schweizer / Carbohydrate Research 342 (2007) 7–15
and CH2Cl2 (30 mL) at 0 ꢁC for 2 h. The solvent was
removed under reduced pressure and codistilled with
toluene (3 · 10 mL) to dryness. The oily residue was
redissolved in CH2Cl2 (15 mL), PhCN or MeCN
(17.2 mmol), and TMSOTf (12.9 mmol) was added at
0 ꢁC. After 2 h, the reaction was quenched with DIPEA
(16.12 mmol) and the amino acid (Boc-DAB-O-t-
Bu · HCl or Boc-Orn-O-t-Bu · HCl; 1 mmol) dissolved
in a 1:1 mixture of CH2Cl2 and DIPEA (4 mL) was
added. Aqueous work-up after 12 h followed by chro-
matographic purification using hexane–EtOAc 1.5:1
(reaction using PhCN) or hexane–EtOAc 1:2.5 (reaction
using MeCN) afforded protected glycosylamino ester in
18–95% yield.
79.5, 80.7, 81.7, 87.0, 126.9–128.7 (aromatic carbons),
131.9, 134.7, 137.7, 137.8, 138.4, 155.4, 166.7, 168.7,
171.8. ESIMS m/z Calcd for C57H70N3O11 [M+H]+:
972.50104. Found: 972.50109.
3.5. (S)-t-Butyl 5-(2-((2S,3R,4S,5R,6R)-2-acetamido-
3,4,5-tris-(benzyloxy)-6-benzyloxymethyl-tetrahydro-
2H-pyran-2-yl)acetamido)-2-(t-butoxycarbonyl-
amino)pentanoate (11)
Compound 11 was obtained in 61% yield after chro-
matographic purification using hexane–EtOAc 1:2.5 as
1
eluent. H NMR (300 MHz, CDCl3, rt, TMS): d 1.18–
1.30 (m, 2H), 1.30–1.38 (m, 1H), 1.45 (s, 9H), 1.46 (s,
9H), 1.47–1.46 (m, 1H), 1.95 (s, 3H), 2.32–2.51 (m,
1H), 2.63 (d, 1H, J = 15.6 Hz), 3.05–3.21 (m, 1H),
3.59–3.78 (m, 4H), 3.80–3.89 (m, 1H), 3.97–4.13 (m,
3H), 4.45–5.00 (m, 8H + 1 · NH), 6.06 (s, NH), 6.81
(dd, 1H, J = 6.4, 4.9 Hz, NH), 7.20–7.45 (m, 20H); 13C
NMR (75 MHz, CDCl3, rt): d 24.6, 25.5, 28.0, 28.4,
30.2, 38.7, 43.0, 53.8, 67.6, 70.7, 72.3, 73.1, 73.7, 75.0,
76.4, 77.0, 79.5, 80.9, 81.7, 86.7, 127.5–128.5 (aromatic
carbons), 137.6, 137.7, 137.9, 138.4, 155.4, 168.7,
169.9, 171.8. ESIMS m/z Calcd for C52H68N3O11
[M+H]+: 910.48539. Found: 910.48532.
3.3. (S)-t-Butyl 4-(2-((2S,3R,4S,5R,6R)-2-benzamido-
3,4,5-tris-(benzyloxy)-6-benzyloxymethyl-tetrahydro-
2H-pyran-2-yl)acetamido)-2-(t-butoxycarbonyl-
amino)butanoate (9)
Compound 9 was obtained in 75% yield after chromato-
graphic purification using hexane–EtOAc 1.5:1 as elu-
1
ent. H NMR (300 MHz, CDCl3, rt, TMS): d 1.44 (s,
9H), 1.46 (s, 9H), 1.51–1.65 (m, 1H), 1.70–1.85 (m,
1H), 2.74 (d, 1H, J = 15.3 Hz), 2.84–3.01 (m, 1H),
3.03–3.24 (m, 1H), 3.64–3.81 (m, 4H), 3.86–3.94 (m,
1H), 3.96–4.07 (m, 1H), 4.11 (dd, J = 2.0, <1 Hz, 1H),
4.21 (d, 1H, J = 9.6 Hz), 4.41–5.09 (m, 8H + NH),
6.86 (s, 1H, NH), 7.01 (t, 1H, J = 5.3 Hz, NH), 7.22–
7.66 (m, 25H); 13C NMR (75 MHz, CDCl3, rt): d 28.0,
28.4, 31.2, 36.1, 43.0, 52.3, 67.8, 71.0, 72.1, 72.8, 73.6,
74.7, 76.1, 77.0, 79.6, 80.7, 81.8, 87.0, 126.9–128.7,
131.9, 134.6, 137.8 (·2), 138.3, 155.5, 166.7, 169.0,
171.5. ESIMS m/z Calcd for C56H68N3O11 [M+H]+:
958.48539. Found: 958.48542. Anal. Calcd C, 70.20;
H, 7.05; N, 4.39. Found: C, 70.44; H, 7.08; N, 4.35.
3.6. (S)-t-Butyl 4-(2-((2S,3R,4S,5S,6R)-2-benzamido-
3,4,5-tris-(benzyloxy)-6-benzyloxymethyl-tetrahydro-
2H-pyran-2-yl)acetamido)-2-(t-butoxycarbonyl-
amino)butanoate (12)
Compound 12 was obtained in 95% yield after chro-
matographic purification using hexane–EtOAc 1.5:1 as
1
eluent. H NMR (300 MHz, CDCl3, rt, TMS): d 1.45
(s, 9H), 1.46 (s, 9H), 1.58–1.76 (m, 1H), 1.88–2.06 (m,
1H), 2.75 (d, 1H, J = 14.5 Hz), 3.05–3.34 (m, 2H),
3.64–3.96 (m, 7H), 4.06–4.20 (m, 1H), 4.44–4.68 (m,
3H), 4.76–5.07 (m, 5H), 5.11 (d, 1H, J = 8.0 Hz, NH),
6.87 (s, 1H, NH), 6.97–7.73 (m, 25H + 1 · NH); 13C
NMR (75 MHz, CDCl3, rt): d 28.0, 28.3, 32.6, 36.3,
42.8, 52.3, 68.0, 72.4, 73.4, 75.1, 75.7, 75.9, 77.4, 79.7,
80.4, 82.0, 83.4, 86.6, 126.9–128.7 (aromatic carbons),
132.0, 134.3, 137.7, 137.9, 138.2, 155.6, 166.7, 169.1,
171.6. ESIMS m/z Calcd for C56H68N3O11 [M+H]+:
958.48539. Found: 958.48545. Anal. Calcd C, 70.20;
H, 7.05; N, 4.39. Found: C, 70.38; H, 7.11; N, 4.44.
3.4. (S)-t-Butyl 5-(2-((2S,3R,4S,5R,6R)-2-benzamido-
3,4,5-tris-(benzyloxy)-6-benzyloxymethyl-tetrahydro-
2H-pyran-2-yl)acetamido)-2-(t-butoxycarbonyl-
amino)pentanoate (10)
Compound 10 was obtained in 64% yield after chro-
matographic purification using hexane–EtOAc 1.5:1 as
1
eluent. H NMR (300 MHz, CDCl3, rt, TMS): d 1.24–
1.32 (m, 2H), 1.32–1.42 (m, 1H), 1.46 (s, 9H), 1.47 (s,
9H), 1.48–1.61 (m, 1H), 2.38–2.55 (m, 1H), 2.71 (d,
1H, J = 15.6 Hz), 3.09–3.23 (m, 1H), 3.65–3.85 (m,
4H), 3.88–3.96 (m, 1H), 3.99–4.10 (m, 1H), 4.13 (dd,
1H, J = 2.2, <1 Hz), 4.19 (d, 1H, J = 9.6 Hz), 4.49 (d,
1H, J = 11.6 Hz), 4.55 (d, 1H, J = 10.6 Hz), 4.57
(d, 1H, J = 11.6 Hz), 4.68 (d, 1H, J = 11.7 Hz), 4.80
(d, 1H, J = 11.7 Hz), 4.85–4.97 (m, 3H + NH), 6.78–
6.92 (m, 2H, 2 · NH), 7.23–7.66 (m, 25H); 13C NMR
(75 MHz, CDCl3, rt): d 25.6, 28.0, 28.4, 30.2, 38.8,
43.1, 53.8, 67.7, 70.9, 72.1, 73.1, 73.6, 75.0, 76.2, 77.1,
3.7. (S)-t-Butyl 5-(2-((2S,3R,4S,5S,6R)-2-benzamido-
3,4,5-tris-(benzyloxy)-6-benzyloxymethyl-tetrahydro-
2H-pyran-2-yl)acetamido)-2-(t-butoxycarbonyl-
amino)pentanoate (13)
Compound 13 was obtained in 79% yield after chro-
matographic purification using hexane–EtOAc 1.5:1 as
1
eluent. H NMR (300 MHz, CDCl3, rt, TMS): d 1.37–
1.50 (m, 3H), 1.42 (s, 9H), 1.46 (s, 9H), 1.55–1.73 (m,

M. Penner, F. Schweizer / Carbohydrate Research 342 (2007) 7–15
13
1
1H), 2.73 (d, 1H, J = 14.5 Hz), 2.81–2.98 (m, 1H), 3.07–
3.26 (m, 1H), 3.65–3.90 (m, 7H), 4.02–4.18 (m, 1H),
4.43–4.66 (m, 3H), 4.81–5.13 (m, 5H + 1 · NH), 6.83
(s, 1H, NH), 7.06 (t, 1H, J = 5.8 Hz, NH), 7.11–7.71
(m, 25H); 13C NMR (75 MHz, CDCl3, rt): d 25.8,
28.0, 28.4, 30.3, 39.1, 42.9, 53.9, 68.1, 72.2, 73.5, 75.2,
75.8, 76.0, 77.3, 79.5, 80.5, 81.7, 83.3, 86.6, 126.9–
128.7 (aromatic carbons), 132.0, 134.4, 137.8, 137.9,
138.1, 155.5, 166.6, 168.9, 171.8. ESIMS m/z Calcd for
C57H70N3O11 [M+H]+: 972.50104. Found: 972.50111.
eluent. H NMR (300 MHz, CDCl3, rt, TMS): d 1.20–
1.50 (m, 5H), 1.45–1.55 (m, 18H), 2.70–2.80 (m, 2H),
3.25–3.35 (m, 1H), 3.68–3.90 (m, 4H), 4.0–4.1 (m, 1H),
4.18 (d, 1H, J = 9.3 Hz), 4.65 (d, 1H, J = 10.9 Hz),
4.70 (d, 1H, J = 11.0 Hz), 4.80 (d, 1H, J = 11.0 Hz),
4.88–5.02 (m, 3H), 5.15 (d, 1H, J = 7.5 Hz), 6.81 (s,
1H, NH), 7.20 (t, 1H, NH), 7.23–7.65 (m, 20H). ESIMS
m/z Calcd for C49H62N3O10 [M+H]+: 852.44352.
Found: 852.44344.
3.11. (S)-4-(2-((2S,3R,4S,5R,6R)-2-Benzamido-3,4,5-
tris-(acetoxy)-6-acetoxymethyl-tetrahydro-2H-pyran-2-
yl)acetamido)-2-(9-fluorenylmethoxycarbonylamino)buta-
noic acid (17)
3.8. (S)-t-Butyl 5-(2-((2S,3R,4S,5S,6R)-2-acetamido-
3,4,5-tris-(benzyloxy)-6-benzyloxymethyl-tetrahydro-
2H-pyran-2-yl)acetamido)-2-(t-butoxycarbonyl-
amino)pentanoate (14)
Compound 9 (120 mg, 0.13 mmol) and Pearlman’s cata-
lyst (30 mg) were hydrogenated in MeOH (10 mL) at
atmospheric pressure for 8 h. The solution was filtered
and evaporated to dryness. The crude mixture was dis-
solved in a 1:1 mixture containing pyridine and acetic
anhydride and left for 12 h, evaporated to dryness,
and codistilled with toluene (3 · 10 mL) and purified
by chromatography using EtOAc–hexane 3:1 as eluent
to yield 70 mg of purified ester. The purified ester was
dissolved in CH2Cl2 (2 mL) and trifluoroacetic acid
(2 mL) and left for 90 min. The crude material was
evaporated to dryness and codistilled with toluene
(3 · 20 mL). The solid material was dissolved in tetra-
hydrofuran (2 mL), and 9-fluorenylmethyl pentafluoro-
phenyl carbonate (74 mg, 0.26 mmol), and an aqueous
solution containing sodium bicarbonate (46 mg,
0.55 mmol) were added. After 12 h, formic acid
(0.2 mL) was added. The residue was concentrated
under reduced pressure and water (10 mL) and CH2Cl2
(10 mL) were added. The aqueous layer was extracted
with CH2Cl2 (3 · 10 mL). The combined organic layers
were dried (Na2SO4) and concentrated. Chromato-
graphic purification using 15% MeOH in EtOAc affor-
Compound 14 was obtained in 55% yield after chro-
matographic purification using hexane–EtOAc 1:2.5 as
1
eluent. H NMR (300 MHz, CDCl3, rt, TMS): d 1.34–
1.50 (br m, 3H), 1.41 (s, 9H), 1.45 (s, 9H), 1.57–1.67
(m, 1H), 1.96 (s, 3H), 2.64 (d, 1H, J = 14.7 Hz), 2.77–
2.94 (m, 1H), 3.05–3.21 (m, 1H), 3.58–3.87 (m, 7H),
4.01–4.16 (m, 1H), 4.43–4.96 (m, 8H), 5.03 (d, 1H,
J = 8.0 Hz, NH), 6.02 (s, 1H, NH), 6.96 (t, 1H,
J = 5.8 Hz, NH), 7.13–7.39 (m, 20H); 13C NMR
(75 MHz, CDCl3, rt): d 24.6, 25.8, 28.0, 28.3, 30.2,
39.1, 42.9, 53.9, 68.0, 72.0, 73.5, 75.2, 75.8, 76.2, 77.3,
79.5, 80.4, 81.7, 83.3, 86.2, 127.5–129.2 (aromatic car-
bons), 137.6, 137.9 (·2), 138.2, 155.5, 168.9, 169.8,
171.8. ESIMS m/z Calcd for C52H68N3O11 [M+H]+:
910.48539. Found: 910.48532.
3.9. ((2R,3S,4R,5S,6S)-2-Benzamido-3,4,5-tris-(benzyl-
oxy)-6-benzyloxymethyl-tetrahydro-2H-pyran-2-yl)acet-
amido)propane (15)
Compound 15 was obtained in 40% yield after chro-
matographic purification using hexane–EtOAc 1.5:1 as
1
eluent. H NMR (300 MHz, CDCl3, rt, TMS): d 0.78
1
ded compound 17 (76 mg, 84%). H NMR (DMSO-d6,
(t, 3H), 1.20–1.37 (m, 5H), 2.75 (d, 1H, J = 15.1 Hz),
2.76–2.85 (m, 1H), 3.03–3.18 (m, 1H), 3.75–3.85 (m,
3H), 3.85–3.90 (m, 1H), 4.20 (d, 1H, J = 9.2 Hz), 4.65
(d, 1H, J = 10.9 Hz), 4.70 (d, 1H, J = 11.0 Hz), 4.80
(d, 1H, J = 11.0 Hz), 4.90–5.05 (m, 3H), 6.85 (s, 1H,
NH), 7.05 (t, 1H, J = 7 Hz, NH), 7.30–7.70 (m, 20H).
ESIMS m/z Calcd for C39H45N2O6 [M+H]+:
637.32776. Found: 637.32771. Anal. Calcd C, 73.56;
H, 6.96; N, 4.40. Found: C, 73.84; H, 7.05; N, 4.48.
300 MHz, rt): d 1.58–1.87 (m, 2H), 1.89 (s, 3H), 1.96
(s, 3H), 1.99 (s, 3H), 2.12 (s, 3H), 2.81 (d, 1H,
J = 14.6 Hz), 2.87–3.20 (m, 3H), 3.59–3.72 (m, 2H),
3.97–4.10 (m, 4H), 4.14–4.29 (m, 1H), 4.34 (t, 1H,
J = 6.6 Hz), 5.31 (d, 1H, J = 10.3 Hz), 5.34 (d, 1H,
J = 3.8 Hz), 5.83 (dd, 1H, J = 10.3, 3.7 Hz), 6.58–6.77
(m, 1H, NH), 7.26–7.92 (m, 13 H), 7.92–8.06 (m, 1H,
NH), 8.60 (s, 1H). ESIMS m/z Calcd for C42H46N3O15
[M+H]+: 832.29290. Found: 832.29285.
3.10. (S)-t-Butyl 4-(2-((2R,3S,4R,5S,6S)-2-benzamido-
3,4,5-tris-(benzyloxy)-6-benzyloxymethyl-tetrahydro-
2H-pyran-2-yl)acetamido)-2-(t-butoxycarbonyl-
amino)butanoate (16)
3.12. General procedures for glycopeptide synthesis
The solid-phase synthesis of 18 and 22 was carried out
manually using Fmoc chemistry. Building block 26
was used for the synthesis of tripeptide 23. Starting with
unprotected Rink-amide resin (loading level 0.22 mmol/g),
Compound 16 was obtained in 18% yield after chro-
matographic purification using hexane–EtOAc 1.5:1 as

14
M. Penner, F. Schweizer / Carbohydrate Research 342 (2007) 7–15
the next amino acid (4 equiv), activated with TBTU
(4 equiv), and DIPEA (8 equiv) in DMF, was added.
The Fmoc group was then removed using 20% piperi-
dine in DMF. The coupling/deprotection cycles were re-
peated using the appropriate amino acids until the entire
peptide sequence had been assembled. The side-chain
protection of lysine in tripeptide 22 was removed by
exposure to 2% hydrazine hydrate in DMF to produce
unprotected tripeptide 23. A Quest 210 (Agilent) manual
synthesizer with 5 mL reaction vessels was used.
and washed with DMF (3 · 10 mL), MeOH (3 ·
10 mL), and CH2Cl2 (3 · 10 mL) and dried. The resin
was subjected to cleavage conditions (95% TFA, 2.5%
thioanisole and 2.5% H2O) for 20 min and the released
glycopeptide was purified using 4% MeOH in EtOAc
to afford 25 (75 mg, 34%). 1H NMR (300 MHz,
DMSO-d6, rt): d 0.94–1.34 (m, 10H), 1.35–1.64 (m,
2H, Hb Lys), 1.83 (s, 3H), 2.62–2.75 (m, 1H, He Lys),
2.79 (d, 1H, J = 14.1 Hz, H-2b), 2.85–3.00 (m, 1H, He
Lys), 3.28 (d, 1H, J = 14.1 Hz, H-2a), 3.46–3.70 (m,
2H, H-8a, H-8b), 3.89–3.99 (m, 1H, H-7), 4.02–4.33
(m, 6H, H(a)Ala, H(a)Ala, H(a)Lys, H-4, H-5, H-6),
4.99–4.39 (m, 8H, benzylic), 7.03 (s, 1H, NH), 7.22–
7.41 (m, 24 H, Ar, NH2), 7.64–7.45 (m, 3H, NH, Ar),
7.77–7.70 (m, 2H, NH, 1 · Ar), 7.92 (d, 1H,
J = 7.8 Hz, NH), 8.08 (d, 1H, J = 7.0 Hz, NH); 13C
NMR (75 MHz, DMSO-d6, rt): d 17.96, 18.33, 22.49,
22.77, 28.54, 31.26, 31.28, 42.57, 47.92, 48.44, 52.69,
68.07, 70.46, 71.10, 72.27, 73.84, 74.06, 74.44, 77.43,
79.94, 86.72, 127.12–128.26 (aromatic), 131.49, 135.37,
138.17, 138.64, 138.85, 165.25, 166.66, 167.82, 169.32,
170.97, 172.61. ESIMS m/z Calcd for C57H68N6NaO11:
1035.48438 [M+Na]+. Found: 1035.48447.
3.13. N-(2-((2S,3R,4S,5R,6R)-2-Benzamido-3,4,5-tris-
(benzyloxy)-6-benzyloxymethyl-tetrahydro-2H-pyran-2-
yl)acetyl)-Ala-Gly-Ala-NH2 (20)
Acid 1 (0.89 mmol, 0.58 g) was dissolved in CH2Cl2
(10 mL), and PhCN (8.88 mmol, 0.90 mL), and
TMSOTf (3.11 mmol, 0.56 mL) were added at 0 ꢁC.
After 2 h, the reaction was quenched with DIPEA
(16.12 mmol, 0.93 mL) and the immobilized peptide 18
(0.22 mmol) was added. After 12 h, the resin was filtered
and washed with DMF (3 · 10 mL), MeOH
(3 · 10 mL), and CH2Cl2 (3 · 10 mL) and dried. The
resin was subjected to cleavage conditions (95% TFA,
2.5% thioanisole and 2.5% H2O) for 20 min and the
released glycopeptide was purified using 4% MeOH in
Acknowledgments
1
EtOAc to afford 20 (69 mg, 35%). H NMR (300 MHz,
Financial support of this project was provided by the
Natural Sciences and Engineering Research Council of
Canada (NSERC).
DMSO-d6, rt): d 1.11 (d, 3H, J = 7.0 Hz, Me(Ala)),
1.19 (d, 3H, J = 7.2 Hz, Me(Ala)), 2.80 (d, 1H,
J = 14.4 Hz, H-2b), 3.48–3.71 (m, 5H, CH2(Gly), H-
8a, H-8b, H-2a), 3.90–3.98 (m, 1H, H-7), 4.11 (d, 1H,
J = 2.4 Hz, H-6), 4.12–4.22 (m, 2H), 4.29 (dd, 1H,
J = 2.4, 10.0 Hz, H-5), 4.38 (d, 1H, J = 10.0 Hz, H-4),
4.40 (d, 1H, J = 11.8 Hz, benzylic), 4.49–4.95 (m, 7H,
benzylic), 7.02 (s, 1H, NH), 7.22–7.40 (m, 22H), 7.43–
7.60 (m, 3H), 7.60–7.76 (m, 2H), 7.81 (d, 1H, J = 7.4,
NH), 7.92 (t, 1H, J = 5.8 Hz, NH), 8.02 (d, 1H,
J = 6.6 Hz, NH); 13C NMR (75 MHz, CD3OD, rt): d
18.12, 18.15, 42.10, 48.01, 48.70, 54.91, 68.23, 70.80,
71.24, 72.26, 73.63, 73.80, 74.77, 77.43, 79.82, 86.92,
127.22–128.45 (aromatic), 131.54, 135.23, 138.15,
138.64, 138.76, 166.70, 168.26, 168.36, 172.31, 174.18.
ESIMS m/z Calcd for C51H57N5NaO10 [M+Na]+:
922.40031. Found: 922.40029.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.carres.
2006.11.006.
References
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