Synthesis of a galacto-configured C-ketoside-based γ-sugar-amino acid and its use in peptide coupling reactions

Article abstract of DOI:10.1016/j.carres.2006.03.006

γ-Sugar-amino acid analogues in the form of C-ketosides can be prepared in 5-6 steps starting from d-galactono-1,5-lactone. The key step in the synthesis is the trimethylsilyl trifluoromethanesulfonate (TMSOTf) promoted C-glycosylation of 2-deoxy-3-ulopyranosonates with trimethylsilyl cyanide. Hydrogenation of the resulting β-cyano esters provides C-ketoside-based γ-sugar-amino acids that serve as building blocks for the synthesis of unnatural neoglycopeptides.

Full text of DOI:10.1016/j.carres.2006.03.006

Carbohydrate Research 341 (2006) 1730–1736
Note
Synthesis of a galacto-configured C-ketoside-based
c-sugar-amino acid and its use in peptide coupling reactions
b
Frank Schweizera,b, and Ole Hindsgaul
*
aDepartment of Chemistry, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2
bDepartment of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2
Received 20 January 2006; received in revised form 28 February 2006; accepted 7 March 2006
Available online 17 April 2006
Abstract—c-Sugar-amino acid analogues in the form of C-ketosides can be prepared in 5–6 steps starting from D-galactono-1,5-
lactone. The key step in the synthesis is the trimethylsilyl trifluoromethanesulfonate (TMSOTf) promoted C-glycosylation of
2-deoxy-3-ulopyranosonates with trimethylsilyl cyanide. Hydrogenation of the resulting b-cyano esters provides C-ketoside-based
c-sugar-amino acids that serve as building blocks for the synthesis of unnatural neoglycopeptides.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Keywords: Glycoconjugates; C-Ketosides; Neoglycopeptides; Sugar-amino acids; Glycomimetics
Molecular recognition of carbohydrates by proteins
plays an important role in many intracellular and inter-
cellular processes, such as the trafficking of proteins,
bacterial and viral infection, normal cell differentiation,
tumour progression and metastasis.1 Besides these bio-
logical functions, carbohydrates have also been recog-
nized as highly functionalized synthetic scaffolds,
which if modified can demonstrate interesting proper-
ties.2 In particular, the introduction of an amino and a
carboxylic acid function into the carbohydrate platform,
leading to glycosamino acids (GAAs), sugar-amino
acids (SAAs) and sugar-amino acid hybrids (SAAHs),
has been pursued by several groups.3,4 GAAs, SAAs
and SAAHs can easily undergo oligomerization to ‘sacc-
charide peptide hybrids’, ‘glycopeptoids’ or ‘glycotides’
via well-developed peptide chemistry and represent ideal
building blocks for combinatorial synthesis.3b,5
binding proteins6,7 using the appendages AA1,. . .,AAn
and AA21,. . .,AA2n (AA = amino acid), which may be
hydrophobic, hydrophilic, neutral or charged. The
unchanged carbohydrate core encompassed by C3–C8 in
1 and 2 may allow unperturbed binding of the carbo-
hydrate moiety to the protein, which often takes place
via the non-reducing end of the sugar.7 In addition,
many sugar binding proteins (with the exception of gly-
cosidases) recognize both monosaccharidic a- and b-gly-
cosides.8 The synthesis of scaffolds 1 and 2 requires
access to unprotected C-ketoside based c-amino acid
building blocks 3 and 4. Here we describe the synthesis
of compounds 3 and 4 and their use in peptide coupling
reactions. A preliminary account without experimental
details on the synthesis of unprotected sugar-fused
GABA analogues was recently communicated.9
The synthesis of compound 3 is outlined in Scheme 2.
The commercially available lactone 5 was converted
into ketose 613 on treatment with excess tert-butylace-
tate enolate (4 equiv) at ꢀ78 ꢁC in 90% yield as previ-
ously described.14 Addition of a second enolate was
not observed. The tert-butyl group was cleaved with tri-
fluoroacetic acid in dichloromethane and the free acid
was esterified to either the methyl or benzyl ester
(Cs2CO3, MeI or BnBr) to afford the ketoses 7 and 8
As part of our ongoing studies on carbohydrate–pro-
tein interactions, we became interested in the design and
synthesis of novel galacto-configured unnatural glyco-
peptides 1 and 2 (Scheme 1). Scaffolds 1 and 2 are
designed to probe the binding-site periphery of galactose
*
Corresponding author. Fax: +1 204 474 7608; e-mail addresses:
schweize@cc.umanitoba.ca; schweize@ms.umanitoba.ca
0008-6215/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2006.03.006

F. Schweizer, O. Hindsgaul / Carbohydrate Research 341 (2006) 1730–1736
1731
OH
O
in yields averaging 75%. The cyano function was incor-
porated into the acetals 7 and 8 by trimethylsilyl triflu-
OH
O
HO
HO
HO
HO
OMe
OH
oromethanesulfonate
(TMSOTf)
promoted
C-
HO
HO
glycosylation with trimethylsilyl cyanide (TMSCN) in
acetonitrile to yield the C-ketosides 9 and 10 in 75%
and 66% yields, respectively. Analysis of the product
mixture after a reaction time of 36 h revealed in both
cases the presence of unreacted starting material
(approximately 20%). Increasing the reaction time, fur-
ther addition of reagents (promoter and nucleophile) or
changing the solvent (CH2Cl2) did not improve the
yields. The stereochemistry of the cyano compounds 9
and 10 was established by measuring the heteronuclear
3JC,H coupling constants between the cyano carbon
and H-4 as well as between C-2 and H-4 as previously
reported.9 In summary, the observed coupling constants
O
O
FmocHN
NH3
Cl
3
4
(peptide
synthesis)
(peptide
synthesis)
8
OH
OH
O
HO
HO
HO
H
7
6
HO
2
O
1
3
N
OMe
AA1
4
HO
AAn
5
HO
H
3
3JC-2,H-4 <4 Hz and JCN,H-4 >7 Hz for compounds 9
O
O
N
N
H
and 10 require the axial orientation of the cyano group.10
Subsequently, the cyano compound 10 was hydroge-
nated using Pearlman’s catalyst in acidified (HCl) meth-
anol for 48 h and the generated amino function was
protected as 9-fluorenylmethoxycarbamate by treatment
with Fmoc pentafluorophenylester (Fmoc-OPfp) in an
acetone/water mixture. Sugar-c-amino acid 3 was
isolated in 42% yield. To demonstrate the use of
O
O
AA21
AA2n
1
AA1
AAn
2
Scheme 1. General structure of C-ketoside based glycopeptides 1 and 2
from c-SAA-building blocks 3 and 4.
OBn
O
OBn
O
BnO
BnO
BnO
BnO
BnO
BnO
OBn
O
a)
b)
O
R
O
BnO
BnO
OBn
O
OH
O
OH
5
6
R1 = OMe
8 R1 = OBn
7
c)
OH
O
HO
HO
BnO 8
HO
HO
OBn
OH
d)
R1
e)
2
O
O
R1
1
OMe
BnO
4
HO
3
O
HO
BnO
O
O
NHFmoc
CN
H3N
Cl
R1 = OH
3
11
12
R1 = OMe
R2 = OBn
9
10
R1 = OMe
4
f)
R1 = OBn
g)
OH
HO
HO
H
N
O
O
OMe
OH
HO
HO
H
N
O
HO
h)
O
Me
O
H
N
OMe
O
HO
NHFmoc
O
Me
NHFmoc
13
14
Scheme 2. Synthesis of glycotripeptide 14 incorporating a c-SAA. Reagents and conditions: (a) LiCH2COOt-Bu (4 equiv), THF, ꢀ78 ꢁC to rt, 1 h,
85–95%; (b) 50% TFA in CH2Cl2 then Cs2CO3 (1.3 equiv), MeI or BnBr (3 equiv), DMF, rt, 1 h, 70–80%; (c) TMSOTf (3 equiv), TMSCN (5 equiv),
CH3CN, 0 ꢁC to rt, 36 h, 66–75%; (d) C/Pd(OH)2, H2, HCl (48–60 h), 80–90%; (e) then Fmoc-OPfp (4 equiv), H2O/acetone 1:2, NaHCO3 (1.2 equiv),
42%; (f) Cs2(CO3) (1.3 equiv), BnBr (3 equiv) or MeI (2 equiv), DMF, rt, 64% or 50%; (g) 3, H2N-Ala-OMe (3.5 equiv), DCC (5 equiv), HOBt
(3.5 equiv), DMF, 6 h, 72%; (h) 40% morpholine in DMF, 1 h, then Fmoc-OPfp (3 equiv), HOBt (3 equiv), DMF, 6 h, 92%.

1732
F. Schweizer, O. Hindsgaul / Carbohydrate Research 341 (2006) 1730–1736
sugar-c-amino acid building block 3 in peptide coupling
reactions we subjected it to a double peptide coupling
cycle. Initially, 3 was coupled to alanine (DCC,
ClꢀNH3þ-Ala-OMe, HOBt, DMF, 48 h) to afford gly-
codipeptide 13 in 72% yield (Scheme 2). Removal of
the Fmoc group (morpholine, DMF, 1 h) followed by
additional peptide coupling using activated phenylala-
nine (Fmoc-Phe-OPfp, HOBt, DMF, 14 h) yielded the
glycotripeptide 14 in 92% isolated yield (Scheme 2).
Similar coupling of 4 with phenylalanine (Fmoc-Phe-
OPfp) gave dipeptide 15 in 65% isolated yield along
with a small amount (ꢁ5%) of the corresponding lactam
(Scheme 3).
Encouraged by our solution-phase results, we immo-
bilized the dipeptide 15 on the Merrifield trityl-chloride
resin via the carbohydrate scaffold11 and coupled the
resin-bound dipeptide 16 to glycine (Fmoc-Gly-OPfp,
DMF). After cleavage from the resin (1% TFA,
10 min), we obtained glycotripeptide 17 as the only
product in 60% isolated yield (Scheme 3). Attempts to
use the corresponding immobilized (trityl resin) sugar-
c-amino esters 18 and 19 as building blocks for addi-
tional peptide couplings failed (Scheme 4).12 This failure
was attributed to a very efficient intramolecular cycliza-
tion because removal of the Fmoc group of resin-bound
11 and 12 (25% morpholine in DMF) and subsequent
peptide coupling provided lactam 20 in quantitative
yield after resin cleavage (Scheme 4). Similarly, peptide
coupling of 4 to Pfp-activated phenylalanine using basic
O
OH
O
O
R
HO
HO
O
R
HO
HO
n, o, p
m
O
20
HO
HO
NHFmoc
NHFmoc
11
18
19
R = OMe
R = OMe
R = OBn
12 R = OBn
Scheme 4. Attempted solid-phase glycopeptide synthesis of sugar-c-
amino esters 11 and 12. Reagents and conditions: (m) trityl-chloride
resin (1.3 equiv), DIEA (3 equiv), DMF, 2 h then quenching by the
addition of CH3OH/DIEA/CH2Cl2 1:2:17, 5 min; (n) 50% morpholine
in DMF, 30 min; (o) Fmoc-Gly-OPfp (3 equiv), HOBt, DIEA
(3 equiv); (p) TFA (3%) in CH2Cl2, 10 min.
conditions (diisopropylethylamine (DIEA)) produced
lactam 20 in quantitative yield (Scheme 3).
In summary, we have demonstrated the suitability of
sugar-c-amino acids 3 and sugar-c-amino ester 4 as
building blocks for unnatural glycopeptide synthesis.
Amino acid 3 can be incorporated into peptides via the
usual C!N fashion without lactam formation. Amino
ester 4 can be elongated into glycopeptides with minimal
lactam formation under neutral conditions. However,
basic conditions result in intramolecular lactam forma-
tion. In addition, immobilization of the sugar-c-peptides
on Merrifield-modified trityl resin via the unprotected
carbohydrate scaffold may be an attractive strategy for
the combinatorial synthesis of neoglycopeptides libraries
OH
O
HO
HO
OH
O
OMe
O
HO
HO
i)
O
OMe
l)
HO
NH3+Cl-
O
HO
NHFmoc
N
H
4
OH
O
HO
HO
15
O
HO
j)
N
H
20
OH
O
OMe
O
HO
HO
O
k)
HO
HO
O
OMe
O
O
H
N
O
HO
N
H
NHFmoc
HO
NHFmoc
N
H
O
17
16
Scheme 3. Solid-phase glycopeptide synthesis via immobilization of the carbohydrate scaffold. Reagents and conditions: (i) Fmoc-Phe-OPfp
(3 equiv), NaHCO3 (1.3 equiv), H2O/acetone 3:1, 13 h, 65%; (j) trityl-chloride resin (1.3 equiv), DIEA (3 equiv), DMF, 2 h then quenching by the
addition of CH3OH/DIEA/CH2Cl2 1:2:17, 5 min; (k) 50% morpholine in DMF, 1 h then Fmoc-Gly-OPfp (3 equiv), DMF then TFA (3%) in CH2Cl2,
10 min, 60%; (l) Fmoc-Phe-OPfp (3 equiv), DIEA (2 equiv), DMF, quant.

F. Schweizer, O. Hindsgaul / Carbohydrate Research 341 (2006) 1730–1736
1733
and may find future use in the synthesis of conformation-
ally constrained cyclic neoglycopeptides.
1.3. Benzyl (4,5,6,8-tetra-O-benzyl-2-deoxy-a-D-galacto-
3-octulopyranosid)onate (8)
Acetal 613 (1.07 g, 1.63 mmol) was dissolved in a (1:1)
mixture containing CH2Cl2 and trifluoroacetic acid
(25 mL) and stirred for 2 h at 0 ꢁC. Toluene (30 mL)
was added and the mixture was concentrated under
reduced pressure and codistilled with toluene
(2 · 10 mL). The residue was dissolved in dry DMF
(30 mL), and caesium carbonate (743 mg, 2.28 mmol)
and benzyl bromide (542 lŁ, 4.56 mmol) were added.
The mixture was stirred for 2 h. The residue was concen-
trated under reduced pressure, and H2O (40 mL) and
CH2Cl2 (40 mL) were added. The aqueous layer was ex-
tracted with CH2Cl2 (3 · 20 mL). The combined organic
layer was dried (Na2SO4) and concentrated. Chromato-
graphic purification using 5% EtOAc in toluene as eluant
gave 8 (809 mg, 72%) as a colourless oil. 1H NMR
(CDCl3): d 7.40–7.20 (m, 25H, aromatic), 5.24 (s br,
1H, OH), 5.08 (ABq, 2H, Jgem = 12,3 Hz, OCH2Ph),
5.00 (d, 1H, Jgem = 11.4 Hz, OCH2Ph), 4.95 (d, 1H,
1. Experimental
1.1. General methods
CH2Cl2 was distilled from calcium hydride. Organic
solutions were concentrated under diminished pressure
at <40 ꢁC (bath temperature). NMR spectra were
1
recorded at 360 or 500 MHz for H and at 100 MHz
for 13C. Chemical shifts are reported relative to CHCl3
[dH 7.26, dC (centre of triplet) 77.0] or to CH3OH [dH
73.35, dC (centre of septet) 49.0] or to acetone as the
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. Trityl resin
was purchased from Novabiochem.
Jgem = 11.5 Hz, OCH2Ph), 4.77 (ABq, 2H, Jgem
=
1.2. Methyl (4,5,6,8-tetra-O-benzyl-2-deoxy-a-D-galacto-
3-octulopyranosid)onate (7)
11.7 Hz, OCH2Ph), 4.67 (d, 1H, Jgem = 11.4 Hz, OCH2Ph),
4.61 (d, 1H, Jgem = 11.5 Hz, OCH2Ph), 4.44 (ABq, 2H,
Jgem = 11,8 Hz, OCH2Ph), 4.17 (m, 1H, H-7), 4.10 (dd,
1H, J4,5 = 9.8 Hz, J5,7 = 2.7 Hz, H-5), 4.00 (dd, 1H,
J6,7 = 1.2 Hz, H-6), 3.82 (d, 1H, H-4), 3.60 (dd, 1H,
J7,8a = 6.2 Hz, J8a,8b = 9.2 Hz, H-8a). 3.43 (dd, 1H,
J7,8b = 5.5 Hz, H-8b), 2.87 (d, 1H, J2a,2b = 14.6 Hz, H-
2a), 2.43 (d, 1H, H-2b). The signal at 5.24 disappeared
by addition of D2O (15 lL); ESIMS m/z calcd for
C43H44O8Na [M+Na]+: 711.29338. Found 711.29395.
Acetal 613 (1.00 g, 1.52 mmol) was dissolved in a 1:1
mixture containing CH2Cl2 and trifluoroacetic acid
(25 mL) and stirred for 2 h at 0 ꢁC. Toluene (30 mL)
was added and the mixture was concentrated under
reduced pressure and codistilled with toluene
(2 · 10 mL). The residue was dissolved in dry DMF
(30 mL) and caesium carbonate (743 mg, 2.28 mmol)
and methyl iodide (284 lL, 4.56 mmol) were added.
The mixture was stirred for 4 h. The reaction flask was
opened and left in the fumehood overnight to remove
excess of methyl iodide. The residue was concentrated
under reduced pressure, and H2O (40 mL) and CH2Cl2
(40 mL) were added. The aqueous layer was extracted
with CH2Cl2 (3 · 20 mL). The combined organic layers
were dried (Na2SO4) and concentrated. Chromato-
graphic purification using 5% EtOAc in toluene as elu-
1.4. Methyl (3,7-anhydro-4,5,6,8-tetra-O-benzyl-3-cyano-
2-deoxy-D-glycero-L-manno-oct)onate (9)
Acetal 7 (20 mg, 32.6 lmol) was dissolved under an inert
argon atmosphere in dry acetonitrile (1 mL). TMSCN
(13 lL, 97.5 lmol) and TMSOTf (19 lL, 98 lmol) were
added at 0 ꢁC. The temperature was raised to rt and the
mixture was stirred for an additional 8 h. CH2Cl2
(2 mL) and satd aq NaHCO3 (2 mL) were added. The
aqueous layer was extracted with CH2Cl2 (3 · 2 mL)
and the combined organic layer was dried (Na2SO4),
concentrated and chromatographically purified using
5% EtOAc in toluene as the eluant. Compound 9
(15 mg, 75%) was obtained as a colourless syrup. [a]D
+37 (c 1.5, CDCl3); 1H NMR (CDCl3): d 7.40–7.20
(m, 20H, aromatic), 5.05 (d, 1H, Jgem = 11.7 Hz,
OCH2Ph), 4.90 (d, 1H, Jgem = 11.3 Hz, OCH2Ph), 4.71
(ABq, 2H, Jgem = 11.4 Hz, OCH2Ph), 4.70 (d, 1H,
Jgem = 11.7 Hz, OCH2Ph), 4.55 (d, 1H, Jgem = 11.3 Hz,
OCH2Ph), 4.44 (ABq, 2H, Jgem = 11.8 Hz, OCH2Ph),
4.07–4.03 (m, 2H, H-6, H-7), 4.01 (d, 1H,
J4,5 = 9.7 Hz, H-4), 3.92 (dd, 1H, J4,5 = 2.6 Hz), 3.59
(dd, 1H, J7,8a = 8.0 Hz, J8a,8b = 9.3 Hz, H-8a), 3.57 (s,
1
ant gave 7 (766 mg, 82%) as a colourless oil. H NMR
(CDCl3): d 7.40–7.20 (m, 20H, aromatics), 5.43 (s br,
1H, OH), 4.95 (d, 1H, Jgem = 11.5 Hz, OCH2Ph), 4.91
(d, 1H, Jgem = 11.6 Hz, OCH2Ph), 4.74 (ABq, 2H,
Jgem = 11.7 Hz, OCH2Ph), 4.65 (d, 1H, Jgem = 11.5 Hz,
OCH2Ph), 4.58 (d, 1H, Jgem = 11.5 Hz, OCH2Ph), 4.41
(ABq, 2H, Jgem = 11.8 Hz, OCH2Ph), 4.15 (m, 1H, H-
7), 4.08 (dd, 1H, J4,5 = 9.8 Hz, J5,6 = 2.8 Hz, H-5),
4.00 (dd, 1H, J6,7 = 1.2 Hz, H-6), 3.75 (d, 1H, H-4),
3.60, (s, 3H, OCH3), 3.59 (dd, 1H, J7,8a = 7.7 Hz,
J8a,8b = 9.2 Hz, H-8), 3.45 (dd, 1H, J7,8b = 5.6 Hz,
H-8b), 2.75 (d, 1H, J2a,2b = 15.6 Hz, H-2a), 2.33 (d,
1H, H-2b). The signal at 5.43 disappeared by addition
of D2O; ESIMS m/z calcd for C37H41O8 [M+H]+:
613.28014. Found 613.28033.

1734
F. Schweizer, O. Hindsgaul / Carbohydrate Research 341 (2006) 1730–1736
3H, OCH3), 3.53 (dd, 1H, J7,8b = 5.5 Hz, H-8b), 2.94 (d,
1H, J2a,2b = 15.2 Hz, H-2a), 2.64 (d, 1H, H-2b); 13C
NMR (CDCl3): d 176.86 (C@O), 138.51, 137.79 · 2,
137.58, 128.60–127.66 (C–H, aromatic), 116.52 (CN),
81.94, 76.63, 76.19, 75.37, 75.18, 74.85, 74.49, 73.43,
reaction was stirred for 10 min. Then water (10 mL)
was added and EtOAc (3 · 10 mL) was used to extract
the product. Product 3 was purified with an EtOAc
equilibrated column using EtOAc/CH3OH/H2O, 12:2:1
as the eluant. The product 3 (72 mg, 42%) was obtained
as an oil. 1H NMR (1:1, CD3OD/CDCl3): d 7.78 (d, 2H,
J = 7.0 Hz, Fmoc), 7.65 (d, 2H, J = 7 Hz, Fmoc), 7.40–
7.26 (m, 4H, Fmoc), 4.36 (d, 2H, J = 6.9 Hz, OCH2,
Fmoc), 4.20 (t, 1H, J = 6.9 Hz, CHCH2, Fmoc), 4.15
(d, 1H, J4,5 = 10.0 Hz, H-4), 3.82 (d, 1H, J5,6 = 2.8 Hz,
H-6), 3.78–3.53 (m, 6H), 2.70 (d, 1H, J2a,2b = 15.1 Hz,
H-2a), 2.62 (d, 1H, H-2b); ESIMS m/z calcd for
C24H27NO9Na [M+Na]+: 496.15835. Found 496.15869.
72.80, 67.72, 52.04, 41.21; HMBC (CDCl3): JH4,CN
=
7.3 Hz, JH4,C2 = 3.6 Hz; ESIMS m/z calcd for C38H40-
NO7 [M+H]+: 622.28047. Found 622.28095. Anal.
Calcd for C38H39NO7: C, 73.41; H, 6.32; N, 2.25.
Found: C, 73.19; H, 6.20; N, 2.30.
1.5. Benzyl (3,7-anhydro-4,5,6,8-tetra-O-benzyl-3-cyano-
2-deoxy-D-glycero-L-manno-oct)onate (10)
Acetal 7 (99.7 mg, 144.8 lmol) was dissolved under an
argon atmosphere in dry acetonitrile (1 mL). TMSCN
(58 lL, 435 lmol) and TMSOTf (19 lL, 98 lmol) were
added at 0 ꢁC. The temperature was raised to rt and
the mixture was stirred for an additional 6 h. CH2Cl2
(6 mL) and satd aq NaHCO3 (4 mL) were added. The
aqueous layer was extracted with CH2Cl2 (3 · 5 mL)
and the combined organic layer were dried (Na2SO4),
concentrated and chromatographically purified using
3% EtOAc in toluene as the eluant. Compound 10
(67 mg, 66%) was obtained as a white powder [a]D
+33 (c 1.1, CDCl3); 1H NMR (CDCl3): d 7.40–7.20
(m, 25H, aromatic), 5.06 (ABq, 2H, Jgem = 12.4 Hz,
OCH2Ph), 5.0 (d, 1H, Jgem = 12.6 Hz, OCH2Ph), 4.90
(d, 1H, Jgem = 11.3 Hz, OCH2Ph), 4.71 (ABq, 2H,
Jgem = 11.5 Hz, OCH2Ph), 4.70 (d, 1H, Jgem = 11.6 Hz,
OCH2Ph), 4.55 (d, 1H, Jgem = 11.6 Hz, OCH2Ph), 4.43
(ABq, 2H, Jgem = 11.8 Hz, OCH2Ph), 4.07–4.02 (m,
2H, H-6, H-7), 4.03 (d, 1H, J4,5 = 9.9 Hz, H-4), 3.92
1.7. Methyl [3,7-anhydro-2-deoxy-3-C-(aminomethyl-
(N-9-fluorenylmethoxycarbonyl))-D-glycero-L-manno-
oct]onate (11)
Compound
3 (55 mg, 182.3 lmol), CH3I (23 lL,
364 lmol) and sodium bicarbonate (30.6 mg,
364.6 lmol) were stirred in DMF for 4 h. The solvent
was removed under reduced pressure redissolved in
CH3OH (10 mL) and filtered over Celite. The residue
was purified with a gradient silica chromatography
using 9–12.5% CH3OH in ethyl acetate as eluant. Prod-
uct 11 (57 mg, 64%) was obtained as a white powder. 1H
NMR (CD3OD): d 7.78 (d, 2H, J = 7.0 Hz, Fmoc), 7.65
(d, 2H, J = 7.0 Hz, Fmoc), 7.40–7.26 (m, 4H, Fmoc),
4.36 (d, 2H, J = 6.9 Hz, OCH2, Fmoc), 4.20 (t, 1H,
J = 6.9 Hz, CHCH2, Fmoc), 4.15 (d, 1H, J4,5
10.0 Hz, H-4), 3.82 (d, 1H, J5,6 = 2.8 Hz, H-6), 3.78–
3.53 (m, 9H including s at 3.64), 2.70 (d, 1H, J2a,2b
=
=
15.1 Hz, H-2a), 2.62 (d, 1H, H-2b); ESIMS m/z calcd
(dd, 1H, J5,6 = 2.7 Hz, H-5), 3.56 (dd, 1H, J7,8b
7.9 Hz, J8a,8b = 9.2 Hz, H-8a), 3.45 (dd, 1H, J7,8b
=
=
for C25H30NO9 [M+H]+: 488.19205. Found 488.19187.
5.2 Hz, H-8b), 3.00 (d, 1H, J2a,2b = 15.1 Hz, H-2a),
2.70 (d, 1H, H-2b); 13C NMR (CDCl3): d 116.3 (CN);
HMBC (CDCl3); JH4,CN = 8.5 Hz, JH4,C2 = 3.8 Hz;
ESIMS m/z calcd for C44H44NO7 [M+H]+: 698.31177.
Found 698.31213. Anal. Calcd for C44H44NO7: C,
75.73; H, 6.21; N, 2.01. Found: C, 75.52; H, 6.09; N,
2.03.
1.8. Benzyl [3,7-anhydro-2-deoxy-3-C-(aminomethyl(N-9-
fluorenylmethoxycarbonyl))-D-glycero-L-manno-oct]onate
(12)
Compound 3 (8 mg, 17 lmol), benzyl bromide (50 lL,
170 lmol) and sodium bicarbonate (16.8 mg,
0.20 mmol) were dissolved in DMF (1 mL) and stirred
for 4 h. Filtration (Celite) and chromatographic purifi-
cation with a gradient silica gel chromatography using
9–12.5% CH3OH in EtOAc as eluant gave product 12
1.6. 3,7-Anhydro-2-deoxy-3-C-(aminomethyl(N-9-fluoren-
ylmethoxycarbonyl))-D-glycero-L-gluco-octonic acid (3)
1
Compound 10 (100 mg, 143.0 lmol), hydrochloric acid
(200 lmol) and Pearlman’s catalyst (Pd(OH)2/C,
85 mg) were added to dry CH3OH (15 mL) and hydro-
genated (1 atm) for 48 h. The residue was filtered
through a Millipore filter and concentrated under
reduced pressure. The residue was dissolved in a mixture
of water (5 mL) and acetone (10 mL), sodium bicarbon-
ate powder (61 mg) and Fmoc-pentafluorophenylester
(Fmoc-OPfp, 450 mg) were added. After stirring the
reaction for 12 h, formic acid (0.5 mL) was added. The
(5.2 mg, 50%) as a white powder. H NMR (CD3OD):
d 7.78 (d, 2H, J = 7.0 Hz, Fmoc), 7.65 (d, 2H,
J = 7.0 Hz, Fmoc), 7.40–7.24 (m, 9H, Fmoc, Ph), 5.10
(ABq, 2H, Jgem = 12.0 Hz, OCH2Ph), 4.35 (d, 2H,
J = 6.9 Hz, OCH2, Fmoc), 4.19 (t, 1H, J = 6.9 Hz,
CHCH2, Fmoc), 4.18 (d, 1H, J4,5 = 9.0 Hz, H-4), 3.83
(d, 1H, J5,6 = 2.7 Hz, H-6), 3.78–3.53 (m, 6H), 2.74
(d, 1H, J2a,2b = 14.6 Hz, H-2a), 2.66 (d, 1H, H-2b);
ESIMS m/z calcd for C31H33NO9Na [M+Na]+:
586.20530. Found 586.20579.

F. Schweizer, O. Hindsgaul / Carbohydrate Research 341 (2006) 1730–1736
1735
1.9. 3,7-Anhydro-2-deoxy-3-C-(aminomethyl(N-9-fluo-
renylmethoxycarbonyl))-D-glycero-L-manno-octon-
(L-Ala-OCH3)amide (13)
130.39–120.87 (C–H aromatics), 79.56, 74.51, 72.45,
71.93, 71.07, 68.13, 63.29, 58.17, 52.77, 49.65, 48.33,
42.98, 38.81, 38.64, 17.26; ESIMS m/z calcd for
C37H44N3O11 [M+H]+: 706.29758. Found 706.29773.
Acid 3 (6 mg, 12.7 lmol), N,N0-1,3 dicyclohexylcarbo-
diimide (16 mg, 78 lmol), alanine methyl ester hydro-
chloride (12 mg, 85 lmol) and hydroxybenzotriazole
(3 mg, 22 lmol) were dissolved in DMF (1.5 mL) and
stirred for 48 h. The urea precipitate was filtered (Celite)
and the residue was concentrated under reduced pres-
sure. The residue was chromatographed twice using
10% CH3OH in EtOAc as the eluant. Product 13
(5 mg, 72%) was obtained as a white powder. 1H
NMR (7:1, CDCl3/CD3OD): d 7.81–7.75 (d br, 1H,
J = 7 Hz, NH(Ala)), 7.70 (d, 2H, J = 7.6 Hz, Fmoc),
7.53 (d, 2H, J = 7.5 Hz, Fmoc), 7.33 (t, 2H,
J = 7.6 Hz, Fmoc), 7.23 (t, 2H, J = 7.6 Hz, Fmoc),
6.10–6.00 (t br, 1H, J ꢁ 6 Hz, NH(Fmoc)), 4.15 (t, 1H,
J = 6.7 Hz, Fmoc), 3.86 (d, 1H, J5,6 = 3 Hz, H-6), 3.84
(d, 1H, J4,5 = 9.9 Hz, H-4), 3.66 (s, 3H, OCH3), 3.52
(dd, 1H, Jgem = 14.8 Hz, Jvic = 6.0 Hz, CH2NH), 3.38
(dd, 1H, CH2NH), 2.44 (d, 1H, J2a,2b = 15.1 Hz, H-
2a), 2.47 (d, 1H, H-2b), H-2b; 1.33 (d, 3H, CH3
(Ala)); The signal at 7.81 and 6.10 disappeared under
this conditions over a period of 2 days; MALDI-MS
m/z calcd for C28H34N2O10Na [M+Na]+: 581.2. Found
581.3.
1.11. Methyl [3,7-anhydro-2-deoxy-3-C-(aminomethyl-
(N-L-Phe-Na(Fmoc)amide))-D-glycero-L-manno-oct]onate
(15)
Cyano compound 9 (50 mg, 80 lmol), hydrochloric acid
(110 lmol) and Pd(OH)2/C (Pearlman’s catalyst) were
added to CH3OH (8 mL) and hydrogenated for 60 h.
The residue was filtered through a Millipore filter and
concentrated under reduced pressure to yield 4 (23 mg,
90%), which was directly used for peptide coupling reac-
tions. Compound 4 (23 mg, 76 lmol) sodium bicarbon-
ate (13.5 mg, 160 lmol) and Fmoc-Phe-OPfp (126 mg,
227 lmol) were dissolved in a mixture of 20% H2O in
acetone (10 mL) and stirred for 13 h. The solvent was
removed under reduced pressure and the residue was
purified by chromatography using 7% CH3OH in
EtOAc as the eluant on a EtOAc equilibrated silica col-
umn. Product 15 (31.2 mg, 65%) was obtained as a white
1
powder. H NMR (CDCl3): d 7.70 (d, 2H, J = 7.5 Hz,
Fmoc), 7.53 (d, 2H, J = 7.4 Hz, Fmoc), 7.33 (t, 2H,
Fmoc), 7.23 (t, 2H, J = 7.4 Hz, Fmoc), 7.30–7.15 (m,
5H, Ph), 7.00 (t br, 1H, J = 6.5 Hz, NH, Fmoc), 6.00
(d br, 1H, NH, Phe), 4.37 (t, 2H, J = 6.7 Hz, CHCH2,
Fmoc), 4.10 (t, 1H, J = 6.7 Hz, CHCH2, Fmoc), 4.12–
4.08 (m, 1H, Ha(Phe)), 3.86 (d, 1H, J4,5 = 9.6 Hz,
H-4), 3.78 (d, 1H, J5,6 = 3 Hz, H-6), 3.65–3.32 (m,
9H), 3.05–2.87 (m, 2H, CH2Ph), 2.80–2.60 (s br, 4H,
4 · OH), 2.50 (d, 1H, J2a,2b = 15.4 Hz, H-2a), 2.37 (d,
1H, H-2b). The signals at 7.05, 6.10 and 2.80–2.60 disap-
peared by addition of D2O; MALDI-MS m/z calcd for
C38H41N2O10Na [M+Na]+: 657.2. Found 657.6.
1.10. 3,7-Anhydro-2-deoxy-3-C-(aminomethyl(N-L-Phe-
Na(Fmoc)amide))-D-glycero-L-manno-octon-(L-Ala-
OMe)amide (14)
The glycodipeptide 13 (5.2 mg, 9.1 lmol) was dissolved
in DMF (1.5 mL). Morpholine (1 mL) was added and
the reaction was stirred for 1 h at rt. The solvent was
removed under reduced pressure and codistilled with
toluene (2 · 3 mL). The residue was dissolved in dry
DMF (1.5 mL), Fmoc-Phe-OPfp (14.9 mg, 27 lmol)
and 1-hydroxybenzotriazole (3 mg, 22 lmol) were
added. The reaction was stirred for 14 h. The solvent
was removed under reduced pressure and the product
14 was purified by column chromatography using 9%
CH3OH in EtOAc. The product 14 (5.8 mg, 92%) was
obtained as a white powder. 1H NMR (CD3OD): d
7.75 (d, 2H, J = 7.5 Hz, Fmoc), 7.58 (d, 2H, J =
7.5 Hz, Fmoc), 7.37 (t, 2H, J = 7.5 Hz, Fmoc), 7.28 (t,
2H, J = 7.5 Hz, Fmoc), 7.25–7.14 (m, 5H, Phe), 4.37
(dd, 1H, J = 6.0 Hz, 9.0 Hz), 4.33–4.20 (m, 3H,
H(a,Ala), OCH2 (Fmoc)), 4.14 (t, 1H, CHCH2 (Fmoc)),
1.12. Methyl [3,7-anhydro-2-deoxy-3-C-(aminomethyl-
(N-L-Phe-Gly-Na(Fmoc)amide))-D-glycero-L-manno-
oct]onate (17)
Compound 15 (46 mg, 76 lmol), tritylchloride resin
(160 mg, capacity 950 lmol/g) were added to CH2Cl2
solution (2 mL) containing diisopropylethylamine
(66 lL, 380 lmol). The mixture was stirred for 2 h while
the thin layer chromatogram showed the complete dis-
appearance of the starting material. The solvent was
removed by filtration and the resin was washed with
diisopropylethylamine/CH3OH/CH2Cl2 (2:1:17, 3 ·
10 mL), DMF (2 · 5 mL) and CH2Cl2 (2 · 10 mL)
before it was dried under vacuum overnight. A portion
of this resin 16 (35 mg) was taken and suspended in a
mixture of CH2Cl2/morpholine (1:1, 2 mL) for 2 h.
The solvent was removed by filtration and the resin
was washed with DMF (2 · 5 mL), CH2Cl2 (2 · 5 mL),
CH3OH (2 · 5 mL) and CH2Cl2 (2 · 5 mL) before it
4.00 (d, 1H, J4,5 = 9.9 Hz, H-4), 3.84 (d, 1H, J5,6
=
3.1 Hz, H-5), 3.75–3.68 (m, 2H), 3.66 (s, 3H, OCH3),
3.65–3.42 (m, 4H), 3.12 (dd, 1H, Jgem = 13.7 Hz,
Jvic = 5.6 Hz, CH2(Phe)), 2.90 (dd, 1H, Jvic = 10.2 Hz,
CH2(Phe)), 2.50 (s, 2H, H-2a, H-2b), 1.28 (d, 3H,
CH3(Ala)); 13C NMR (CD3OD): d 174.92, 174.47,
172.20 (·2), 145.26, 145.10, 142.54 (·2), 138.60,

1736
F. Schweizer, O. Hindsgaul / Carbohydrate Research 341 (2006) 1730–1736
Barbosa, J.; Liu, J., et al. J. Med. Chem. 1998, 41, 1382–
1391; (c) Nicolaou, K. C.; Trujillo, J. L.; Chibale, K.
Tetrahedron 1997, 53, 8751–8778; (d) Smith, A. B., III;
Sasho, S.; Barwis, B. A.; Sprengeler, P.; Barbosa, J.;
Hirschmann, R.; Cooperman, B. S. Bioorg. Med. Chem.
Lett. 1998, 8, 3133–3141; (e) Dinh, T. Q.; Smith, C. D.;
Du, X.; Armstrong, R. W. J. Med. Chem. 1998, 41, 981–
985; (f) Wunberg, T.; Kallus, C.; Opatz, T.; Henke, S.;
Schmidt, W.; Kunz, H. Angew. Chem., Int. Ed. 1998, 37,
2503–2505; (g) Opatz, T.; Kallus, C.; Wunberg, T.;
Schmidt, W.; Henke, S.; Kunz, H. Eur. J. Org. Chem.
was dried under vacuum overnight. The resin (30 mg),
Fmoc-Gly-OPfp (23 mg, 49.6 lmol) and 1-hydroxy-
benzotriazole (3 mg, 22 lmol) were added to a solution
of DMF (1.5 mL) and stirred for 7 h. The resin was
washed with diisopropylethylamine/CH3OH/CH2Cl2
(2:1:17, 3 · 5 mL), DMF (2 · 5 mL) and CH2Cl2 (3 ·
5 mL) before it was dried under vacuum overnight.
Cleavage of 16 from the resin was achieved using 1%
TFA in CH2Cl2 for 5 min. After filtration and chro-
matographic purification using 9% CH3OH in EtOAc,
peptide 17 (6.8 mg, 60%) was obtained as a white pow-
der. 1H NMR (CD3OD): d 7.80 (d, 2H, J = 7.5 Hz,
Fmoc), 7.66 (d, 2H, J = 7.4 Hz, Fmoc), 7.38 (t, 2H,
J = 7.5 Hz, Fmoc), 7.31 (t, 2H, J = 7.5 Hz, Fmoc),
7.27–7.10 (m, 5H, Ph), 4.65 (dd, 1H, J = 5.5 Hz,
J = 9.1 Hz, Ha(Phe)), 4.35 (d, 2H, J = 7.0 Hz, CH2
(Fmoc)), 4.21 (t, 1H, J = 7.0 Hz, Fmoc), 4.07 (d, 1H,
J4,5 = 9.9 Hz, H-4), 3.78 (d, 1H, J5,6 = 3.1 Hz, H-6),
3.77–3.48 (m, 11H, including s at 3.6 (3H, OCH3)),
3.17 (d, 1H, Jgem = 13.7 Hz, CH2Ph), 2.93 (dd, 1H,
CH2Ph), 2.64 (d, 1H, J2a,2b = 14.8 Hz, H-2a), 2.52 (d,
1H, H-2b); ESIMS m/z calcd for C36H42N3O11
[M+H]+: 692.28499. Found 692.28478.
2003, 1527–1536; (h) Streicher, B.; Wunsch, B. Eur. J. Org.
¨
Chem. 2001, 115–120; (i) Hirschmann, R.; Ducry, L.;
Smith, A. B., III. J. Org. Chem. 2000, 65, 8307–8316.
3. For a list of selected references: (a) Heyns, K. H.; Paulsen,
H. Chem. Ber. 1955, 88, 188–195; (b) McDevitt, J. P.;
Lansbury, P. T. J. Am. Chem. Soc. 1996, 118, 3818–3828;
(c) Graf von Roedern, E.; Lohof, E.; Hessler, G.;
Hoffmann, M.; Kessler, H. J. Am. Chem. Soc. 1996, 118,
10156–10167; (d) Graf von Roedern, E.; Kessler, H.
Angew. Chem., Int. Ed. Engl. 1994, 33, 687–689; (e)
Chakraborty, T. K.; Jayaprakash, S.; Diwan, P. V.;
Nagaraj, R.; Jampani, S. R. B.; Kunwar, A. C. J. Am.
Chem. Soc. 1998, 120, 12962–12966; (f) Lohof, E.;
Planker, E.; Mang, C.; Burkhart, F.; Dechantsreiter, M.
A.; Haubner, R.; Wester, H.-J.; Schwaiger, M.; Ho¨lze-
mann, G.; Goodman, S. L.; Kessler, H. Angew. Chem.,
Int. Ed. 2000, 39, 2761–2764; (g) Sofia, M. J.; Hunter, R.;
Chan, T. Y.; Vaughan, A.; Dulina, R.; Wang, H.; Gange,
D. J. Org. Chem. 1998, 63, 2802–2803; (h) Ghosh, M.;
Dulina, R. R.; Kakarla, R.; Sofia, M. J. J. Org. Chem.
2000, 65, 8387–8390; (i) Fuchs, T.; Schmidt, R. R.
Synthesis 1998, 753–756.
1.13. 3,7-Anhydro-2-deoxy-3-C-(aminomethyl)-D-
glycero-L-manno-octonic acid lactam (20)
1H NMR (CD3OD): d 3.85 (d, 1H, J5,6 = 3.0 Hz, H-6),
3.75 (d, 1H, J4,5 = 9.8 Hz, H-4), 3.68–3.65 (m, 4H, H-
7, H-8a, H-8b, CH2N), 3.46 (d, 1H, CH2N), 3.42 (dd,
1H, H-5), 2.78 (d, 1H, J2a,2b = 17.5 Hz, H-2a), 2.37 (d,
1H, H-2b). MALDI-MS m/z calcd for C9H16NO6
[M+H]+: 234.1. Found 234.5.
4. For recent reviews on sugar-amino acids see: (a) Dondoni,
A.; Marra, A. Chem. Rev. 2000, 100, 4395–4421; (b)
Schweizer, F. Angew. Chem., Int. Ed. 2002, 41, 231–253;
(c) Gruner, S. A. W.; Locardi, E.; Lohof, E.; Kessler, H.
Chem. Rev. 2002, 102, 491–514; (d) Chakraborty, T. K.;
Ghosh, S.; Jayaprakash, S. Curr. Med. Chem. 2002, 9,
421–435.
5. (a) Wessel, H. P.; Mitchell, C. M.; Lobato, C. M.; Schmid,
G. Angew. Chem., Int. Ed. Engl. 1995, 34, 2712–2713; (b)
Nicolaou, K. C.; Floerke, H.; Egan, M. G.; Barth, T.;
Estevez, V. A. Tetrahedron Lett. 1995, 36, 1775–1778;
(c) Kim, J. M.; Roy, R. Carbohydr. Lett. 1996, 1, 465–
468.
6. (a) Lemieux, R. U.; Du, M.-H.; Spohr, U. J. Am. Chem.
Soc. 1994, 116, 9803–9804; (b) Dietrich, H.; Schmidt, R.
R. Carbohydr. Res. 1993, 250, 161–164; (c) Toogood, P.
L.; Galliker, P. K.; Glick, G. D.; Knowles, J. R. J. Med.
Chem. 1991, 34, 3140–3143.
7. Quiocho, F. A. Annu. Rev. Biochem. 1986, 55, 287–315.
8. Goldstein, I. J.; Poretz, R. D. In The Lectins; Liener, I. E.,
Sharon, N., Goldstein, I. J., Eds.; Academic Press:
London, 1986; p 35.
9. Schweizer, F.; Otter, A.; Hindsgaul, O. Synlett 2001,
1743–1746.
Acknowledgements
Financial support of this project was provided by the
Natural Sciences and Engineering Research Council of
Canada (NSERC).
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