Chemoenzymatic synthesis of neoglycopeptides: Application to an α-gal-terminated neoglycopeptide

Article abstract of DOI:10.1021/jo001439c

A novel methodology for the enzymatic preparation from suitably derivatized oligosaccharides of N-linked neoglycopeptides using the microbial glutaminyl-peptide γ-glutamyl transferase, transglutaminase (TGase), is described. N-Allyl glycosides of various oligosaccharides were photochemically coupled with cysteamine to yield amino-terminated thioether spacers, which were accepted by transglutaminase to transamidate the side-chain γ-carboxamide group in the dipeptide Z-Gln-Gly.

Full text of DOI:10.1021/jo001439c

2948
J . Org. Chem. 2001, 66, 2948-2956
Ch em oen zym a tic Syn th esis of Neoglycop ep tid es: Ap p lica tion to
a n r-Ga l-Ter m in a ted Neoglycop ep tid e†
Delphine Ramos,‡ Patrick Rollin,§ and Werner Klaffke*,‡
Unilever Research Laboratory Vlaardingen, Olivier van Noortlaan 120,
NL-3313AT Vlaardingen, The Netherlands, Organisch-Chemisches Institut, Universita¨t Mu¨nster,
48149 Mu¨nster, Germany, and ICOA/ Universite´ d’Orle´ans, B.P. 6759, F-45067 Orle´ans Cedex 2, France
werner.klaffke@uni-muenster.de
Received October 5, 2000
A novel methodology for the enzymatic preparation from suitably derivatized oligosaccharides of
N-linked neoglycopeptides using the microbial glutaminyl-peptide γ-glutamyl transferase, trans-
glutaminase (TGase), is described. N-Allyl glycosides of various oligosaccharides were photochemi-
cally coupled with cysteamine to yield amino-terminated thioether spacers, which were accepted
by transglutaminase to transamidate the side-chain γ-carboxamide group in the dipeptide Z-Gln-
Gly.
In tr od u ction
This might be conceived as a limitation for the use of
transglutaminase in synthetic applications;, however, as
this study has shown, the enzyme accepts a wide range
of primary amines. This allowed us to use the enzyme
for the construction of a synthetic target, a neoglycopep-
tide, the oligosaccharide moiety of it would be furnished
at the reducing end with an amino-terminated spacer.11
To make the method applicable to a broader range of
saccharide structures, a direct and simple way to func-
tionalize the sugar backbone was sought (Scheme 1). The
hemiacetal is N-glycosylated by using allylamine and is
subsequently converted into the corresponding â-config-
ured N-acetyl allyl glycoside. This anomeric protection
allows for further manipulation of the glycoside (depicted
as pathway b) prior to the assembly of the amino-
terminated spacer.
All such synthesized compounds are substrates to
transglutaminase-catalyzed reactions and they readily
react with the model acceptor, Z-protected peptide Z-Gln-
Gly, as depicted in Scheme 2.
The here-described structures will differ fundamentally
from natural N-linked glycoproteins, in that they not only
have a heteroatom linker region but also a glutamine
attachment site. However, it is hoped that conformational
effects by the saccharide on the protein folding can be
studied and signaling functionality of the glycoprotein
can be retained.
The prominent importance of glycopeptides in intra-
cellular processes is still not sufficiently paralleled by
facile methods of preparation. In principle, there are two
main alternatives, (i) the addition of glycosylated amino
acids as building blocks in either solid- or solution-phase
peptide synthesis or (ii) the blockwise addition of a pre-
assembled saccharide moiety to a peptide. We were
seeking for a solution to the latter problem, aiming at
the chemoselective addition to the peptide of a function-
alized oligosaccharide.
Microbial glutaminyl-peptide γ-glutamyl transferase
(transglutaminase, TGase) cross-links proteins by cata-
lyzing an acyl-transfer reaction. The enzyme is specific
for the glutamine γ-carboxamide group, which acts as the
acyl donor. Although it is conceivable that certain
sequence requirements must exist for the enzyme to
accept the substrate, research has hitherto not revealed
any general rule for the peptide sequence.1-10
Nevertheless, glutamine residues in proteins which are
accepted by TGase are mostly situated in flexible and
solvent-accessible terminal polypeptide domains, or in
surface loops.
† Dedicated to our friend Professor J oachim Thiem on the occasion
of his 60th birthday.
‡ Unilever Research Laboratory Vlaardingen and Universita¨t Mu¨n-
ster.
§ ICOA/Universite´ d’Orle´ans.
(1) Ando, H.; Adachi, M.; Umeda, K.; Matsuura, A.; Nonaka, M.;
Uchio, R.; Tanaka, H.; Motroki, M. Agric. Biol. Chem. 1989, 53, 2613-
2617.
Resu lts a n d Discu ssion
(2) Folk, J . E.; Finlayson, J . S. Adv. Prot. Chem. 1977, 31, 1-133.
(3) Gorman, J . J .; Folk, J . E. J . Biol. Chem. 1981, 256(6), 2712-
2715.
(4) Gorman, J . J .; Folk, J . E. J . Biol. Chem. 1984, 259(14), 9007-
9010.
(5) Grootjans, J . J .; Groenen, P. J . T. A.; J ong, W. d. J . Biol. Chem.
1995, 270(39), 22855-58.
(6) Groenen, P. J . T. A. Eur. J . Biochem. 1992, 205, 671-674.
(7) Mahul, B. FEBS Lett. 1995, 360, 160-164.
(8) Pastor, M. T.; Diez, A.; Perez-Paya, E.; Abad, C. FEBS Lett. 1999,
451, 231-234.
In the first entry, GlcNAc, maltose, cellobiose, and
lactose (1-4) were chosen as model compounds (Scheme
3). The four hemiacetals were reacted with allylamine12
and subsequently per-O-acetylated to yield the stable
â-configured anomers 5-8, which could be easily purified
at this stage. Upon deacetylation, compounds 9-12 and
cysteamine hydrochloride13-16 were irradiated (254 nm),
(9) Coussons, P. J .; Price, N. C.; Kelly, S. M.; Smith, B.; Sawyer, L.
Biochem J . 1992, 282, 929-930.
(10) Coussons, P. J .; Kelly, S. M.; Price, N. C.; J ohnson, C. M.; Smith,
B.; Sawyers, L. Biochem. J . 1991, 273, 73-78.
(11) Ramos, D.; Rollin, P.; Klaffke, W. Angew. Chem. 2000, 112,
406-408; Angew. Chem., Int. Ed. 2000, 39(2), 396-398.
(12) Spevak, W.; Dasgupta, F.; Hobbs, C. J .; Nagy, J . O. J . Org.
Chem. 1996, 61, 3417-3422.
10.1021/jo001439c CCC: $20.00 © 2001 American Chemical Society
Published on Web 04/12/2001

Chemoenzymatic Synthesis of Neoglycopeptides
J . Org. Chem., Vol. 66, No. 9, 2001 2949
Sch em e 1. Ver sa tile Meth od for th e F u n ction a liza tion of th e Su ga r Ba ck bon ea
a
Key: (a) direct activation by photochemical coupling with cysteamine; (b) modifcation on the sugar part by conventional glycoside
synthesis prior to the photochemical coupling step.
Sch em e 2. En zym a tic Cr oss-Lin k in g of
Am in o-F u n ction a lized Su ga r s a n d
Glu ta m in e-Con ta in in g P ep tid es
feasibility of this methodology, lactoside 12 was used as
the building block for the synthesis of the R-Gal trisac-
charide derivative (17). Per-O-benzylated thioglycoside
19 was chosen as glycosyl donor. Lactoside 12 was
subjected to regioselective monoalkylation at the C-3′
position with p-methoxybenzyl chloride (PMB-Cl). Com-
pound 12 was therefore treated with dibutyltin oxide in
methanol, involving the generation of a dibutylstan-
nylene acetal as intermediate.17-18 After concentration,
the residue was suspended in dry benzene and reacted
with tetrabutylammonium iodide (TBAI) and p-meth-
oxybenzyl chloride to give 21 (30%) (Scheme 4). Acety-
lation of the remaining hydroxyl groups and oxidative
cleavage of the p-methoxy benzyl group with cerium(IV)
ammonium nitrate (CAN)19 afforded compound 20 (90%).
Coupling of thioglycoside 1920 with the lactoside 20 in
dichloromethane at room temperature, using methyl
trifluoromethanesulfonate as promotor,21 gave the R-con-
figured trisaccharide 18 in good yield (90%), with no
â-anomer present.
Sch em e 3. Gen er a l Sch em e for th e Syn th esis of
Sp a cer -Br id ged Ca r boh yd r a tes, Sta r tin g fr om
GlcNAc (1), Ma ltose (2), Cellobiose (3), a n d La ctose
(4)a
Trisaccharide 18 was deacetylated and irradiated (254
nm) with cysteamine hydrochloride in methanol to yield
the partially unprotected trisacchararide 24. Attempts
to remove the benzyl ether groups by either hydrogena-
tion in the presence of Pd/C or reduction with Raney
nickel22 were unsuccessful, probably due to the presence
of the thioether. Finally, radical reduction23,24 employing
sodium in liquid ammonia afforded the desired compound
25 in high purity. This fact was essential for the further
process, since all attempted purification procedures failed
for this compound. Thus, it was used without further
purification as a starting material for the enzymatic
coupling step.
The activity of TGase toward the artificial substrates
13-16 and 25 was measured and compared to the
hydroxylamine standard (Scheme 5). The ammonia liber-
ated during the enzymatic reaction, which is indirectly
proportional to the amount of product, was estimated
using the Sigma diagnostics method for plasma ammonia.
a
Key: (a) allylamine, rt, 72 h; (b) Ac2O/pyridine, DMAP (60%
to quantitative yields); (c) MeONa/MeOH (90% to quantitative
yields); (d) cysteamine hydrochloride (5 equiv), MeOH, hν (254 nm),
argon, 8 h (70-80%).
yielding the amino-terminated thioethers 13-16 (70-
80% yield). The basic concept of this two-step process, (i)
the synthesis of the allylamine and (ii) the final activation
toward the primary amine, was to have a stable inter-
mediate at hand which could be further functionalized
prior to the final coupling to a protein. To prove the
(17) David, S.; Hanessian, S. Tetrahedron 1985, 41(4), 643-663.
(18) Koeman, F. A. W.; Meissner, J . W. G.; Ritter, H. R. P.v.;
Kamerling, J . P.; Vliegenhart, J . F. G. J . Carbohydr. Chem. 1994, 13(1),
1-25.
(19) J ohansson, R.; Samuelsson, B. J . Chem. Soc., Perkin Trans. 1
1984, 2371-2374.
(20) Garegg, P. J .; Hultberg, H.; Lindberg, C. Carbohydr. Res. 1980,
83, 157-162.
(13) Lee, R. T.; Lee, Y. C. Carbohydr. Res. 1974, 37, 193-201.
(14) Roy, R.; Laferriere, C. A.; Gamian, A.; J ennings, H. J . J .
Carbohydr. Chem. 1987, 6(1), 161-165.
(15) Roy, R.; Tropper, F. D. Chem. Commun. 1988, 1058-1060.
(16) Kieburg, C.; Dubber, M.; Lindhorst, T. K. Synlett 1997, 1447-
1449.
(21) Lo¨nn, H. Carbohydr. Res. 1985, 139, 105-113.
(22) Horita, K.; Yoshioka, T.; Tanaka, T.; Oikawa, Y.; Yonemitsu,
O. Tetrahedron 1986, 42(11), 3021-3028.
(23) Reist, E. J .; Bartuska, V. J .; Goodman, L. J . Org. Chem. 1964,
29, 3725-3726.
(24) Scho¨n, I. Chem. Rev. 1984, 84, 287-297.

2950 J . Org. Chem., Vol. 66, No. 9, 2001
Sch em e 4. Syn th esis of a Der iva tive of a n r-Ga la ctosyl Tr isa cch a r id ea
Ramos et al.
a
(a) (i) Bu2SnO (1.1 equiv), MeOH (anhyd), molecular sieves 4 Å, argon, reflux, 20 h; (ii) TBAI (1 equiv), PMB-Cl (1.1 equiv), benzene
(anhyd), molecular sieves 4 Å, argon, reflux, 4-5 h; (b) Ac2O/pyridine, DMAP (22% from 12); (c) CAN (3 equiv), acetonitrile/water (9/1),
O°C f rt (88%); (d) thiophenyl 2,3,4,6-tetra-O-benzyl-â-D-galactopyranoside 19 (1.6 equiv), CH2Cl2 (anhyd), pulverized molecular sieves
4 Å, methyl trifluoromethanesulfonate (5 equiv), argon, rt (90%); (e) MeONa/MeOH (94%); (f) cysteamine hydrochloride, MeOH, hν (254
nm), 8 h (75%); (g) Na/NH3, -78 °C (quantitative yields).
Sch em e 5. P r in cip le of th e Deter m in a tion of
Am m on ia in th e Assa y
No major difference was observed between the three
disaccharide derivatives (14-16), whereas the GlcNAc
derivative yielded very low values for Vmax and Km,
indicating a good binding of 13 to transglutaminase and
a slow coupling reaction. Further investigation on the
structure-function relationship is necessary to rational-
ize all these observations.
For the transacylations on a preparative scale, com-
pounds 13-16 and 25 were allowed to react with the
dipeptide benzyloxycarbonyl-glutamine-glycine (Z-Gln-
Gly) and TGase in a buffered solution (pH 6), at 50 °C
with shaking. Except for the GlcNAc derivative (13), all
products could be isolated and characterized as the result
of a covalent cross-linking between the saccharide and
the peptide. Yields of the coupling reactions varied from
one substrate to the other (50 to 70%), mainly due to the
laborious separation of the products from maltodextrins,
which are added to the enzyme preparation by the
manufacturer for stability reasons.
The enzyme (TGase, 10 g/L), peptide (Z-Gln-Gly, 15
mM) and substrate (NH2OH, 13-16 and 25, 15 mM) were
mixed in phosphate buffer (NaH2PO4/Na2HPO4, 0.2 M,
pH 6) and incubated at 50 °C for 5 min (Scheme 6). The
enzyme was thermally inactivated, and the level of
ammonia was measured for each substrate. Kinetic
studies were also conducted using different concentra-
tions of peptide and substrate at a constant ratio. The
results are depicted in Table 1. For unknown reasons,
no Km or Vmax values could be determined for trisaccha-
ride 25. Nevertheless, its relative activity was found to
be two to four times higher than for the other substrates.
Con clu sion s/Su m m a r y
The above-described method is able to transfer a
preassembled oligosaccharide block onto a peptide. Un-
like the biosynthesis of natural glycopeptides, it does not

Chemoenzymatic Synthesis of Neoglycopeptides
J . Org. Chem., Vol. 66, No. 9, 2001 2951
Sch em e 6. En zym a tic Syn th esis of Neoglycop ep tid es
Ta ble 1. Resu lts of th e Kin etic Mea su r em en ts for th e
Transglutaminase was a generous gift from Ajinomoto
Gmbh, Hamburg, Germany. The enzyme supplied was stabi-
lized in maltodextrines, the concentration of which is un-
known.
Ar tificia l Su bstr a tes (NH2OH, 13-16 a n d 25)a
sub-
Vmax
Km
Vmax/Km
activity
rel activity
(%)
strate (nmol/s) (10-3 M) (10-9
)
(µmol/s/g enz)
Meth od 1. P er -O-a cetyla ted â-NAc-a llyl Glycosid es.
The sugar was dissolved in allylamine (0.1 M solution) and
stirred at room temperature for ca. 72 h. To monitor the
reaction, an aliquot of the reaction mixture was extracted and
per-O-acetylated. Upon completion, the allylamine was evapo-
rated under vacuum and co-distilled with toluene. The crude
residue was dissolved in pyridine, a catalytic amount of DMAP
was added, and at 0 °C, acetanhydride, pyridine/Ac2O, (2:1,
v/v) was added slowly. The reaction mixture was then allowed
to reach rt and further stirred upon complete per-O-acetylation
as judged by TLC (∼24 h). The reaction mixture was poured
on ice-water and extracted with dichloromethane. The organic
layer was successively washed with hydrochloric acid (1 N)
and water. The extract was dried over MgSO4, filtered,
concentrated under vacuum, and co-distilled with toluene.
Meth od 2. De-O-a cetyla tion . The per-O-acetylated com-
pound was dissolved in methanol, and sodium methoxide (30
wt % solution in methanol) was added until pH 10 was
reached. The mixture was stirred for 1 h, and then Dowex resin
(ion exchanger H+ form, 50Wx8) was added to neutralize the
solution, and the suspension was further stirred for 1 h. The
mixture was filtered, and methanol was concentrated under
vacuum and then co-distilled with toluene.
Meth od 3. Ad d ition of Cystea m in e. The respective
â-NAc-allyl glycoside and cysteamine (5 equiv) were dissolved
in a minimum amount of methanol and transferred to a
photochemical cell. The mixture was stirred at room temper-
ature, under argon, and irradiated (254 nm) during 8 h. Upon
completion, as judged by TLC, the reaction mixture was
concentrated.
Meth od 4. P r ep a r a tive Tr a n sa cyla tion w ith Tr a n s-
glu ta m in a se. The sugar derivative (30 mM) was shaken with
Z-Gln-Gly (30 mM) and TGase (20 g/L) in a phosphate buffer
(Na2HPO4/NaH2PO4 0.2 M, pH 6) at 50 °C. The reaction was
followed by TLC (EtOAc/AcOH/H2O, 3:1:1). The enzyme was
deactivated at 100 °C (2 min), and the resulting reaction
mixture was lyophilized. The residue was first applied to SiO2
chromatography (MPLC) then to Sephadex G-10 chromatog-
raphy. Appropriate fractions were pooled and lyophilized to
yield the product.
NH2OH 1.310
6.96
3.99
14.90
10.02
13.82
188
16
13
19
16
0.885
0.050
0.109
0.127
0.112
0.222
100
6
12
14
13
25
13
14
15
16
25
0.064
0.194
0.194
0.216
a
All the assays were conducted with a concentration of enzyme
of 10 g/L. The relative activities (V0obsd/V0 OH) were estimated
NH2
against hydroxylamine (standard 100%).
require dolichyl pyrophosphate activation; however, it
differs in the chemoselectivity of the biosynthetic process,
since it attaches the glycoside to glutamine instead of
asparagine.
Research is presently under way to screen for a broader
range of acceptors.
Exp er im en ta l Section
Gen er a l P r oced u r es. Reactions were monitored by TLC
on silica plates (Merck Kieselgel 60 F254) and detected by UV
absorption and charring with naphthoresorcin (200 mg) in
sulfuric acid (2 N, 100 mL) and ethanol (100 mL) or molybdato
phosphoric acid (25 g) and Ce(IV) sulfate (10 g) in water (940
mL) containing concentrated sulfuric acid (60 mL). Free amine
was detected by spraying with a solution of ninhydrine (2%
in butanol, 95 mL, containing acetic acid 10%, 5 mL). Column
chromatography was performed on Kieselgel (Merck, 230-400
mesh, 0.063-0.200 µm). Medium-pressure liquid chromatog-
raphy (MPLC, pressure 3-5 bar) was performed on Kieselgel
(Merck, 230-400 mesh, 0.040-0.063 µm).
Anhydrous solvents (CH2Cl2, MeOH, CHCl3) were prepared
by passing solvents (analytical grade, p.a.) through molecular
sieves (4 Å) under argon.
1H and 13C NMR spectra were recorded with a Bruker WM
300 (1H, 300.1 MHz; 13C, 75.5 MHz) or with a Bruker AMX400
(1H, 400 MHz; 13C, 100.6 MHz). Two-dimensional correlation
spectra (GCOSY and GHSQC) were measured with a Varian
Unity plus 600 (1H, 599.2 MHz; 13C, 150.7 MHz). Chemical
shifts (δ) are given in ppm relative to the signal for internal
Me4Si, CDCl3 (1H, 7.24; 13C, 77.0), DMSO (d6) (1H, 2.49
(quintet); 13C, 39.7), MeOH (d4) (1H, 4.7; 13C, 49.3), and D2O
(1H, 4.78).
Indications for methylene groups in compounds 16, 17, 24,
and 27-29 is as follows: R-NAc-CH2(a)-CH2(b)-CH2(c)-S-CH2-
(d)-CH2(e)-NH2 and R-NAc-CH2(a)-CH2(b)-CH2(c)-S-CH2(d)-
CH2(e)-NH-CO-CH2(f)-CH2(g)-CH(Gln)-CO-NH-CH2(h)-CO-
OH. In all following assignments, “mc” stands for “multiplet
centered”.
Meth od 5. Estim a tion of th e Am m on ia a n d Deter m i-
n a tion of th e Activity. This estimation was made using the
Sigma diagnostics “Ammonia detection kit” ([No 171-A]).
Equal volumes of peptide (Z-Gln-Gly, 15 mM), substrate
(NH2OH, 13-16 and 25, 15 mM) and transglutaminase (10
g/l) in solution in buffer (NaH2PO4/Na2HPO4, 0.2 M, pH 6) were
mixed, incubated (50 °C), and shaken. Aliquots of the mixture
(200 µL) were extracted at different times, and poured into
boiling water (1 min), to denature the enzyme and stop the
reaction. The latter extracts (100 µL) were then added to the
ammonia reagent (1 mL) into the cuvette which was gently
mixed. The absorbance A1 was measured after 3 min, at 340
nm, against the blank (buffer replaces the transglutaminase).
l-GLDH (l-glutamate dehydrogenase, 10 µL) was then added,
the cuvette was gently mixed and the absorbance A2 was read
after 5 min at 340 nm, against the blank.
Optical rotations were measured at 20 °C at 589 nm (Na)
with a Perkin-Elmer 241 polarimeter using a 10 cm/1 mL cell.
Mass spectra and exact mass were measured by ESI with a
Micromass Quattro LC-Z spectrometer.

2952 J . Org. Chem., Vol. 66, No. 9, 2001
Ramos et al.
The activity, defined as the number of micromoles of NH3
liberated in 1 s per gram of enzyme, was calculated as follows:
SiO2 chromatography and eluted with a linear toluene/acetone
gradient from 5:1 to 3:1, and appropriate fractions were pooled
and concentrated, from which 7 (52.30 g, 72.8 mmol, quantita-
tive yields) was isolated as a colorless foam. [R]20 ) -13 (c
[∆A/min] ) (A2 - A1)/t (t in min)
D
1.0, CHCl3). 1H NMR (400 MHz, CDCl3): δ ) 5.89 (d, H-1),
5.73 (dddd-oct, -CHdCH2), 5.28 (dd-t, H-3), 5.19-5.11 (m,
3H, -CHdCH2 and H-3′), 5.07 (dd-t, H-4′), 4.99 (dd-t, H-2),
4.93 (dd, H-2′), 4.54 (d, H-1′), 4.53 (dd, H-6a), 4.37 (dd, H-6′a),
4.09 (dd, H-6b), 4.06 (dd, H-6′b), 3.93-3.63 (m, 5H, -CH2CHd
CH2, H-4, H-5 and H-5′), 2.12-1.97 (8*s, 24H, 8*-CH3 (Ac));
J 1,2 ) 9.5, J 2,3 ) 9, J 3,4 ) 9, J 5,6a ) 1.5, J 5,6b ) 4.6, J 6a,6b ) 12,
J 1′,2′ ) 8.5, J 2′,3′ ) 9, J 3′,4′ ) 9.5, J 5′,6′a ) 4.5, J 5′,6′b ) 2.3, J 6′a,6′b
) 12.3 Hz. 13C NMR (100.6 MHz, CDCl3): δ ) 172.09-168.73
(8*-CdO), 134.75 (-CHdCH2), 116.81 (-CHdCH2), 100.44 (C-
1′), 80.04 (C-1), 74.96 (C-4), 72.80 (C-3 and C-3′), 71.95 (C-5′
and C-5′), 71.55 (C-2′), 68.87 (C-2), 67.83 (C-4′), 61.52 (C-6 and
C-6′), 46.24 (-CH2CHdCH2), 21.96-20.33 (8*-CH3 (Ac)) ppm.
MS (ESI): m/z 718 [M + H]+, 740 [M + Na]+. Exact mass calcd
for C31H43NO18Na+: 740.2378, found 740.2388.
[NH3] produced ) ([∆A/min]/ꢀ)(1.11/0.1) mol/L/min
(ꢀ ) 6200 (1000 cm2/mol) )
molar absorptivity of NADPH at 340 nm)
nNH ) [([∆A/min] × 106 × 10-4)/ꢀ](1.11/0.1) µmol/min
3
) [([∆A/min] × 102)/(ꢀ × 60)](1.11/0.1) µmol/s
) [([∆A/min] × 102)/(6200 × 60)](1.11/0.1) µmol/s
) [[∆A/min]/(62 × 60)](1.11/0.1) µmol/s
For a concentration of enzyme of 10 g/L, the extract of 100
µL contains 1 mg of enzyme and the activity per gram of
enzyme is then expressed by the following formula:
N-Acet yl-N-a llyl-2,3,4,6-t et r a -O-a cet yl-â-D-ga la ct op y-
r an osyl-(1f4)-2,3,6-tr i-O-acetyl-â-D-glu copyr an osylam in e
(8). Lactose (5.06 g, 14.8 mmol) was subjected to the general
methods to react with allylamine (150 mL). After concentra-
tion, the residue was per-O-acetylated (TLC toluene/acetone,
7:3). The crude product was applied to a SiO2 chromatography
and eluted by toluene/acetone 4:1. The product 8 (10.57 g,
14.73 mmol, quantitative yields) was yielded as a white foam.
activity ) [([∆A/min] × 1000)/
(62 × 60)](1.11/0.1) µmol/s/g of enzyme
N-Acetyl-N-allyl-2-acetam ido-2-deoxy-3,4,6-tr i-O-acetyl-
â-D-glu cop yr a n osyla m in e (5). 2-Acetamido-2-deoxy-D-glu-
cose (5 g, 22.6 mmol) was reacted with allylamine (150 mL)
according to method 1. After concentration, the residue was
per-O-acetylated (TLC EtOAc/MeOH, 20:1). The residue was
recrystallized from ethyl acetate to yield pure compound 5
1
[R]20D: -3 (c ) 1.0, CHCl3). H NMR (400 MHz, CDCl3): δ )
5.90 (d, H-1), 5.73 (dddd-oct, -CHdCH2), 5.35 (d, H-4′), 5.30
(dd-t, H-3), 5.14 (mc, 2H, -CHdCH2), 5.11 (dd, H-2′), 4.97
(mc, 2H, H-2 and H-3′), 4.51 (2*d, 2H, H-1′ and H-6a), 4.20-
4.06 (m, 3H, H-6b, H-6′a, and H-6′b), 3.96-3.66 (m, 5H, H-5′,
H-4, H-5, and -CH2-CHdCH2), 2.24-1.96 (m, 24H, 8*-CH3
(Ac)); J 1,2 ) 9.5, J 2,3 ) 10, J 3,4 ) 8, J 5,6a ) 1.8, J 5,6b ) 6.4, J 6a,6b
) 11.2, J 1′,2′ ) 8, J 2′,3′ ) 10.5, J 3′,4′ ) 3.3, J 4′,5′ ) 0.9, J 5′,6′a ) 5,
J 5′,6′b ) 7 J 6′a,6′b ) 11.5 Hz. 13C NMR (100.6 MHz, CDCl3): δ )
170.14-169.82 (8*-CdO), 134.72 (-CHdCH2), 116.80 (-CHd
CH2), 100.73 (C-1′), 79.99 (C-1), 75.88 (C-5), 74.94 (C-4), 73.06
(C-3), 70.85 and 70.69 (C-5′ and C-3′), 69.08 and 68.96 (C-2′
and C-2), 66.59 (C-4′), 61.64 (C-6), 60.75 (C-6′), 46.23 (-CH2-
CHdCH2), 21.98-20.29 (8*-CH3 (Ac)) ppm. MS (ESI): m/z 718
[M + H]+, 740 [M + Na]+.
(5.80 g, 13.5 mmol, 60%) as a white powder. [R]20 ) -2 (c )
D
1.0, CHCl3). 1H NMR (300 MHz, CDCl3): δ ) 6.34 (d, H-1),
5.80 (d, -NH), 5.76 (dddd-oct, -CHdCH2), 5.20-5.08 (m, 3H,
-CHdCH2 and H-3 or H-4), 5.02 (dd-t, H-3 or H-4), 4.26 (ddd,
H-2), 4.15-4.10 (m, 2H, H-6), 3.98-3.77 (m, 2H, -CH2CHd
CH2), 3.74 (ddd, H-5), 2.05, 2.02, 1.99, 1.98, and 1.82 (5*s, 15H,
5*-CH3 (Ac)); J 1,2 ) 9.7, J 2,3 ) 9.7, J 2,NH ) 9.7, J 3,4 ) 9.7, J 5,6a
) 2.8, J 5,6b ) 4.3, Hz. 13C NMR (75 MHz, CDCl3): δ ) 172.70,
170.68, 170.36, 170.18, and 169.20 (5*-C)O), 134.95 (-CHd
CH2), 116.94 (-CHdCH2), 81.08 (C-1), 74.21, and 73.40 (C-4
and C-3), 68.42 (C-5), 62.04 (C-6), 51.13 (C-2), 46.57 (-CH2-
CHdCH2), 22.82-20.44 (5*-CH3(Ac)) ppm. MS (ESI): m/z 429
[M + H]+, 451 [M + Na]+.
N-Acetyl-N-a llyl-2-a ceta m id o-2-d eoxy-â-D-glu cop yr a n -
osyla m in e (9). Compound 5 (5.17 g, 12.06 mmol) was deacety-
lated according to the general method. The residue was
recrystallized from EtOAc/MeOH 3:1 to yield compound 9 (3.28
N-Acetyl-N-a llyl-2,3,4,6-tetr a -O-a cetyl-r-D-glu cop yr a n -
osyl-(1f4)-2,3,6-tr i-O-acetyl-â-D-glu copyr an osylam in e (6).
According to the general method 1, maltose (5 g, 14.6 mmol)
was reacted with allylamine (150 mL). After concentration,
the residue was per-O-acetylated (TLC: toluene/acetone, 7:3).
The crude product was applied to a SiO2-chromatography and
eluted with a linear gradient of toluene/acetone from 5:1 to
3:1. Appropriate fractions were combined, concentrated and 6
g, 10.8 mmol, 90%) as a colorless powder. [R]20 ) 14 (c ) 1.0,
D
MeOH). MS (ESI): m/z 325 [M + Na]+.
N-Acetyl-N-allyl-r-D-glu copyr an osyl-(1f4)-â-D-glu copy-
r a n osyla m in e (10). Compound 6 (5 g, 6.9 mmol) was deacety-
lated according to the general method. The residue was
purified by MPLC and eluted with EtOAc/MeOH/AcOH, 60:
15:5 (TLC EtOAc/i-PrOH/H2O, 9:4:2) yielding compound 10
(2.9 g, 6.8 mmol, quantitative yields) as a white foamy
(9.85 g, 13.7 mmol, 94%) was isolated as a colorless foam. [R]20
D
1
) 71 (c ) 1.0, CHCl3). H NMR (300 MHz, CDCl3): δ ) 5.86
compound. [R]20 ) 77 (c ) 1.0, CH3OH). MS (ESI): m/z: 446
D
(d, H-1), 5.74 (dddd-oct., -CHdCH2), 5.36 (d, H-1′), 5.34 (dd-
t, H-3), 5.31 (dd, H-3′), 5.10 (d, 2H, -CHdCH2), 5.01 (dd-t,
H-4′), 4.87 (dd-t, H-2), 4.82 (dd, H-2′), 4.40 (dd, H-6′a), 4.20
(dd, H-6′b), 4.15 (dd, H-6a), 4.03 (dd, H-6b), 4.00-3.85 (m, 2H,
H-4 and H-5′), 3.83-3.68 (m, 3H, H-5 and -CH2CHdCH2),
2.09-1.90 (8*s, 24H, 8*-CH3 (Ac)); J 1,2 ) 9.5, J 2,3 ) 9, J 3,4 ) 9,
[M + Na]+, 284 [(M - Glc) + Na]+ (daughter ion of 446). Exact
mass calcd for C17H29NO11Na+ 446.1638, found 446.1634.
N-Acetyl-N-a llylâ-D-glu cop yr a n osyl-(1f4)-â-D-glu cop y-
r a n osyla m in e (11). Compound 7 (10 g, 13.93 mmol) was
deacetylated according to the general method. The residue was
purified by MPLC and eluted with EtOAc/MeOH/AcOH, 60:
15:5 (TLC EtOAc/iPrOH/H2O, 9:4:2) and recrystallized from
EtOAc/MeOH, yielding compound 11 (5.85 g, 13.8 mmol,
J 5,6a ) 4, J 5,6b ) 2.5, J 6a,6b ) 12.4, J 1′,2′ ) 4, J 2′,3′ ) 10.4, J 3′,4′
)
9.6, J 4′,5′ ) 10, J 5′,6′a ) 2.5, J 5′,6′b ) 4, J 6′a,6′b ) 12.3 Hz. 13C NMR
(75.5 MHz, CDCl3): δ ) 171.00-170.00 (8*-C)O), 135.17
(-CHdCH2), 117.43 (-CHdCH2), 96.00 (C-1′), 80.34 (C-1),
76.27 (C-3), 74.98 (C-5), 73.04 (C-4), 71.32 (C-2), 70.49 (C-2′),
69.80 (C-3′), 69.03 (C-4′), 68.55 (C-5′), 62.99 (C-6), 61.95 (C-
6′), 46.62 (-CH2CHdCH2), 22.00-20.92 (8*-CH3 (Ac)) ppm. MS
quantitative yields) as a white foamy compound. [R]20 ) 9 (c
D
1
1.0, CH3OH). H NMR (400 MHz, D2O): δ ) 5.89 (dddd-oct,
-CHdCH2), 5.51 (d, H-1), 5.39-5.00 (m, 2H, -CHdCH2), 4.52
(d, H-1′), 4.06 (mc, 2H, -CH2CHdCH2), 3.92 (mc, 2H, H-6′),
3.84 (mc, 2H, H-6), 3.71 (mc, 4H, H-2, H-3, H-4, and H-5), 3.56-
3.30 (m, 4H, H-3′, H-5′, H-4′, and H-2′), 2.26 and 2.20 (2*s,
3H, -CH3 (Ac)); J 1,2 ) 8.4, J 6a,6b ) 11.9, J 1′,2′ ) 7.9, J 2′,3′ ) 9.5,
J 3′,4′ ) 9.3, J 6′a,6′b ) 12.7 Hz. 13C NMR (100.6 MHz, CDCl3): δ
) 177.08 and 175.61 (-CdO), 134.54 (-CHdCH2), 117.16
(-CHdCH2), 103.01 (C-1′), 82.95 (C-1), 78.52, 77.27, 76.56,
76.07, and 75.62 (C-2, C-3, C-3′, C-5, and C-5′), 73.71 (C-2′),
70.53 and 70.04 (C-4 and C-4′), 61.19 (C-6′), 60.55 (C-6), 44.32
(ESI) m/z: 740 [M + Na]+. Exact mass calcd for C31H43NO18
-
Na+: 740.2378, found 740.2406.
N-Acetyl-N-a llyl-2,3,4,6-tetr a -O-a cetyl-â-D-glu cop yr a n -
osyl-(1f4)-2,3,6-tr i-O-acetyl-â-D-glu copyr an osylam in e (7).
Cellobiose (25 g, 73 mmol) was reacted with allylamine (450
mL). After concentration, the residue was per-O-acetylated
(TLC toluene/acetone, 7:3). The crude product was applied to

Chemoenzymatic Synthesis of Neoglycopeptides
J . Org. Chem., Vol. 66, No. 9, 2001 2953
(-CH2CHdCH2), 22.26 and 21.78 (-CH3 (Ac)) ppm. MS
(ESI): m/z 424 [M + H]+, 446 [M + Na]+. Exact mass calcd
for C17H29NO11Na+ 446.1638, found 446.1661. Anal. Calcd for
of acetonitrile and water (9:1, 100 mL) and cooled to 0 °C. At
this temperature, CAN (13.71 g, 25 mmol, 3 equiv) was added
in four aliquots within 2 h. The reaction mixture was then
allowed to reach rt and was further stirred for 0.5 h (TLC,
toluene/acetone, 1:1). The reaction mixture was diluted with
CH2Cl2 (300 mL) and washed with a saturated NH4HCO3
solution. The aqueous layer was extracted once with CH2Cl2,
and the combined organic layers were then washed with water,
dried over MgSO4, filtered, and concentrated to yield a
yellowish foam, which was subjected to SiO2 chromatography
(toluene/acetone, 7:2 to 2:1), to yield pure 20 as a white fluffy
C
17H29NO11 + CH3OH (1 mol): C, 47.47; H, 7.30; N, 3.08.
Found: C, 47.33; H, 7.42; N, 3.08.
N-Acet yl-N-a llyl-â-D-ga la ct op yr a n osyl-(1f4)-â-D-glu -
cop yr a n osyla m in e (12). Compound 8 (3.8 g, 5.3 mmol) was
deacetylated according to the general method (TLC EtOAc/
iPrOH/H2O, 9:4:2). The residue was recrystallized from EtOAc/
MeOH, 3:1 and washed with ethyl acetate and diethyl ether
yielding compound 12 (2.15 g, 5.08 mmol, 96%) as a white
powder. [R]20 ) -6 (c 1.0, CH3OH). 1H NMR (400 MHz,
compound (4.95 g, 7.32 mmol, 88%). [R]20 ) -2 (c ) 1.0,
D
D
CHCl3). 1H NMR (400 MHz, CDCl3): δ ) 5.90 (d, H-1), 5.73
(dddd-oct, -CHdCH2), 5.31 (d, H-4′), 5.28 (mc, H-3), 5.11 (mc,
2H, -CHdCH2), 4.98 (dd, H-2), 4.87 (dd-t, H-2′), 4.52 (d,
H-6a), 4.44 (d, H-1′), 4.16 (d, H-6b), 4.09 (mc, 2H, H-6′a and
H-6′b), 3.94-3.66 (m, 6H, H-3′, H-5′, H-5, H-4, and -CH2CHd
CH2), 2.22-1.96 (7*s, 21H, 7*-CH3 (Ac)); J 1,2 ) 9.1, J 2,3 ) 9.1,
J 6a,6b ) 11.7, J 1′,2′ ) 8.4, J 2′,3′ ) 10, J 3′,4′ ) 3.5 Hz. 13C NMR
(100.6 MHz, CDCl3): δ ) 170.48-169.36 (7*-C)O), 134.70
(-CHdCH2), 116.79 (-CHdCH2), 100.45 (C-1′), 80.03 (C-1),
75.83 and 75.01 (C-4 and C-5), 72.83 (C-2′), 71.22 and 70.97
(C-3′ and C-5′), 69.20 and 68.92 (C-3, C-4′ and C-2), 61.81 (C-
6), 61.40 (C-6′), 46.27 (-CH2CHdCH2), 21.93-20.37 (7*-CH3
(Ac)) ppm. MS (ESI): m/z 676 [M + H]+, 698 [M + Na]+. Exact
mass calcd for C29H41NO17Na+ 698.2272, found 698.2264.
D2O): δ ) 6.05 (dddd-oct, -CHdCH2), 5.67 (d, H-1), 5.53-
5.29 (m, 2H, -CHdCH2), 5.23 (d, H-4′), 4.62 (d, H-1′), 4.32-
4.15 (m, 2H, -CH2CHdCH2), 4.14-4.04 (m, 2H, H-6a and
H-3), 4.03-3.78 (m, 8H, H-6b, H-6′a, H-6′b, H-4, H-3′, H-2,
H-5, H-5′), 3.72 (dd-t, H-2′), 2.41 and 2.36 (2*s, 3H, -CH3);
J 1,2 ) 8.3, J 2,3 ) 9, J 1′,2′ ) 8, J 2′,3′ ) 10.5, J 3′,4′ ) 8 Hz. 13C NMR
(100.6 MHz, D2O): δ ) 177.12 and 175.66 (-CdO), 134.59
(-CHdCH2), 117.93 (-CHdCH2), 103.43 (C-1′), 87.47 (C-4′),
83.00 (C-1), 78.28, 77.45, 75.49, and 73.11 (C-2, C-3, C-5, and
C-5′), 71.52 (C-2′), 70.47 (C-3′), 69.15 (C-4), 61.62 (C-6), 60.59
(C-6′), 44.33 (-CH2CHdCH2), 22.27-21.79 (-CH3(Ac)) ppm.
MS (ESI): m/z 424 [M + H]+, 446 [M + Na]+. Anal. Calcd for
C
17H29NO11: C, 48.22; H, 6.90; N, 3.31. Found: C, 48.14; H,
6.86; N, 3.29.
N-Acet yl-N-a llyl-(3-O-(4-m et h oxyb en zyl)-â-D-ga la ct o-
p yr a n osyl)-(1f4)-â-D-glu cop yr a n osyla m in e (21). A sus-
pension of NAc-allyl lactoside 12 (3 g, 7.09 mmol) and
dibutyltin oxide (1.94 g, 7.8 mmol, 1.1 equiv) in anhyd
methanol (100 mL) was refluxed in a Soxhlet apparatus
containing molecular sieves (4 Å) under exclusion of moisture.
After 20 h, the reaction mixture was concentrated, and the
residue was codistilled three times with toluene. The residue
was dried in a vacuum, suspended in dry benzene containing
molecular sieves (4 Å) and tetrabutylammonium iodide (2.62
g, 7.09 mmol, 1 equiv). p-Methoxybenzyl chloride (1.06 mL,
7.8 mmol, 1.1 equiv) was added at once under argon atmo-
sphere, and the reaction mixture was refluxed in a Soxhlet
apparatus filled with molecular sieves (4 Å). Upon completion
as judged by TLC (EtOAc/i-PrOH/H2O, 9:4:2, few hours), the
mixture was cooled to room temperature, filtered through
Celite, and concentrated. The residue was applied to a silica
gel chromatography (CH2Cl2/MeOH, 20:1-10:1) to yield 21 as
a colorless powder, which was immediately reacted further.
Th iop h en yl 2,3,4,6-Tetr a -O-ben zyl-â-D-ga la ctop yr a n o-
sid e (19). 2,3,4,6-Tetra-O-acetyl-R-D-galactosyl bromide (4.75
g, 11.55 mmol, purchased from Fluka) was reacted as previ-
ously reported20 to yield pure compound 19 (4.4 g, 6.96 mmol,
1
60%, white crystals). H NMR (400 MHz, CDCl3): δ ) 7.40-
7.12 (H-arom), 4.96 and 4.59 (each d, each 1H, -CH2-Ph (on
C-4)), 4.79 and 4.73 (each d, each 1H, -CH2-Ph (on C-2)), 4.78
and 4.70 (each d, each 1H, -CH2Ph (on C-3)), 4.64 (d, H-1),
4.47 and 4.41 (each d, each 1H, -CH2Ph (on C-6)), 3.97 (dd-
d, H-4), 3.93 (dd∼t, H-2), 3.65 (mc, 2H, H-6a and H-6b), 3.60
(mc, H-5), 3.59 (dd, H-3); J 1,2 ) 9.7, J 2,3 ) 9.4, J 3,4 ) 2.6, J CH2-Ph
) 11.4, 10.0, 11.8, and 11.7 Hz.13C NMR (100.6 MHz, CDCl3):
δ ) 132.21-127.68 (C arom), 88.46 (C-1), 84.95 (C-3), 78.08,
and 78.06 (C-5 and C-2), 76.28, 75.17, 74.27 and 73.46 (4*-
CH2Ph), 74.46 (C-4), 69.53 (C-6) ppm. MS (ESI): m/z 655 [M
+ Na]+, 671 [M + K]+.
N-Acetyl-N-a llyl-2,3,4,6-tetr a -O-ben zyl-r-D-ga la ctop y-
r a n osyl-(1f3)-2,4,6-t r i-O-a cet yl-â-D-ga la ct op yr a n osyl-
(1f4)-2,3,6-tr i-O-acetyl-â-D-glu copyr an osylam in e (18). Ac-
ceptor 20 (3 g, 4.4 mmol) and donor 19 (4.42 g, 7 mmol, 1.6
equiv) were dissolved in anhyd CH2Cl2 (90 mL), pulverized
molecular sieves (4 Å, 15 g) were added, and the suspension
was stirred at room temperature under argon. After 5 min,
methyl trifluoromethanesulfonate (2.4 mL, 22 mmol, 5 equiv)
was added, and stirring was continued overnight (TLC,
toluene/acetone, 2:1). Triethylamine (3 mL, 22 mmol, 5 equiv)
was added, and stirring was continued for a further 30 min.
The suspension was filtered through a Celite pad and concen-
trated. The resulting yellow syrup was applied to MPLC
(toluene/acetone, linear gradient 10:1 to 8:1) to yield the
[R]20 ) 17 (c ) 1.0, MeOH). MS (ESI): m/z 544 [M + H]+,
D
566 [M + Na]+. Exact mass calcd for C25H37NO12Na+: 566.2214,
found 566.2239.
N-Acetyl-N-a llyl-2,4,6-tr i-O-a cetyl-3-O-(4-m eth oxyben -
zyl)-â-D-ga la ctopyr a n osyl-(1f4)-2,3,6-tr i-O-a cetyl-â-D-glu -
cop yr a n osyla m in e (22). Compound 21 was acetylated ac-
cording to the general method. The residue was applied to
silica gel chromatography (toluene/acetone, 5:1) to give 22 as
a colorless powder (1.22 g, 1.53 mmol, 22% from the unpro-
tected 13). [R]20 ) 36 (c ) 1.0, CHCl3). 1H NMR (600 MHz,
D
CDCl3): δ ) 7.12 and 6.83 (2d, 4 H-arom), 5.85 (d, H-1), 5.67
(dddd-oct, -CHdCH2), 5.42 (d, H-4′), 5.25 (dd, H-3), 5.11 (mc,
2H, -CHdCH2), 4.97 (dd, H-2′), 4.93 (dd-t, H-2), 4.56 and
4.26 (each d, each 1H, -CH2Ph), 4.42 (d, H-6a), 4.34 (d, H-1′),
4.10-4.03 (2*dd-d, 2H, H-6′a and H-6′b), 4.05 (d, H-6b), 3.73
(mc, 4H, H-5, H-5′ and -CH2CHdCH2), 3.68 (dd-d, H-4), 3.44
(dd, H-3′), 2.32 (s, 3H, Ph-OCH3), 2.16-1.92 (m, 21H, 7*-CH3
(Ac)); J 1,2 ) 9.5, J 2,3 ) 9.5, J 3,4 ) 5.7, J 6a,6b ) 12.1, J 1′,2′ ) 8,
J 2′,3′ ) 10, J 3′,4′ ) 3.2, J CH2-Ph ) 11.8 Hz. 13C NMR (150.7 MHz,
CDCl3): δ ) 172.23-168.89 (7*-CdO), 159.33, 129.34, 128.99,
and 128.17 (6*C-arom), 134.75 (-CHdCH2), 116.93 (-CHd
CH2), 100.90 (C-1′), 79.95 (C-1), 76.30 (C-3′), 75.90 (C-4), 74.98
(C-5), 73.09 (C-3), 70.90 (C-5′), 70.58 (C-2′), 68.99 (C-2), 65.38
(C-4′), 61.89 (C-6), 61.46 (C-6′), 46.31 (-CH2CHdCH2), 22.14,
21.40, 20.76, 20.74, 20.72, 20.69, and 20.57 (7*-CH3 (Ac)) ppm.
MS (ESI): m/z 818 [M + Na]+, 834 [M + K]+. Exact mass calcd
for C37H49NO18Na+: 818.2847, found 818.2847.
trisaccharide 18 as a white foam (4.77 g, 3.99 mmol, 90%).
1
[R]20 ) 43 (c ) 1.0, CHCl3). H NMR (600 MHz, CDCl3): δ )
D
7.38-7.13 (H-arom), 5.89 (d, H-1), 5.71 (dddd-oct, -CHdCH2),
5.43 (d, H-4′), 5.28 (dd-t, H-3), 5.14 (mc, 2H, -CHdCH2), 5.10
(dd, H-2′′), 5.04 (d, H-1′′), 4.96 (dd∼t, H-2), 4.91 and 4.49 (each
d, each 1H, -CH2Ph), 4.81 and 4.63 (each d, each 1H, -CH2-
Ph), 4.71 and 4.69 (each d, each 1H, -CH2Ph), 4.48 (d, H-6a),
4.45 and 4.39 (each d, each 1H, -CH2Ph), 4.34 (d, H-1′), 4.10-
3.99 (m, 3H, H-6b, H-6′a, and H-6′b), 3.98 (dd, H-2′′), 3.90-
3.75 (m, 7H, -CH2CHdCH2, H-5′, H-5′′, H-4′′, H-3′ and H-3′′),
3.71 (mc, H-4), 3.66 (mc, H-5), 3.50 (dd-d, 2H, H-6′′a, and
H-6′′b), 2.35 (s, 3H, -CH3 (Ac)), 2.10-1.80 (m, 18H, 6*-CH3
(Ac)); J 1,2 ) 9.1, J 2,3 ) 9.1, J 3,4 ) 9.1, J 6a,6b ) 12, J 1′,2′ ) 8.1,
J 2′,3′ ) 10, J 3′,4′ ) 3.2, J 1′′,2′′ ) 3.1, J 2′′,3′′ ) 10, J 6′′a,6′′b ) 6.2,
J CH ) 11.8 Hz. 13C NMR (150.7 MHz, CDCl3): δ ) 172.26-
Ph
2
168.66 (7*-CdO), 134.78 (-CHdCH2), 129.00-127.38 (C-
arom), 116.96 (-CHdCH2), 100.84 (C-1′), 95.08 (C-1′′), 80.01
(C-1), 78.38 (C-3′′), 75.68 (C-2′′), 75.55 (C-5′′), 75.05 (C-4),
N-Acetyl-N-a llyl 2,4,6-tr i-O-a cetyl-â-D-ga la ctop yr a n o-
syl-(1f4)-2,3,6-tr i-O-acetyl-â-D-glu copyr an osylam in e (20).
Compound 22 (6.63 g, 8.34 mmol) was dissolved in a mixture

2954 J . Org. Chem., Vol. 66, No. 9, 2001
Ramos et al.
74.78, 73.27 and 73.22 (4*-CH2-Ph), 73.57 (C-3′), 73.06 (C-3),
73.02 (C-4′′), 71.05 (C-5), 70.49 (C-2′), 69.84 (C-5′), 69.07 (C-
2), 68.48 (C-6′′), 64.81 (C-4′), 61.87 (C-6), 61.37 (C-6′), 46.37
(-CH2CHdCH2), 22.18, 21.44, 20.75, 20.71, 20.66, 20.61 and
20.40 (7*-CH3 (Ac)) ppm. MS (ESI): m/z 1221 [M + Na]+. Exact
mass calcd for C63H75NO22Na+: 1220.4679, found 1220.4659.
N-Acetyl-N-a llyl-2,3,4,6-tetr a -O-ben zyl-r-D-ga la ctop y-
r a n osyl-(1f3)-â-D-ga la ct op yr a n osyl-(1f4)-â-D-glu cop y-
r a n osyla m in e (23). Compound 18 (3.67 g, 3.06 mmol) was
deacetylated according to the general method. The residue was
applied to SiO2 chromatography (EtOAc/MeOH, 20:1 then 10:
1) to yield trisaccharide 23 as a colorless foam (2.74 g, 2.89
mmol, 94%). [R]20D ) 63 (c ) 1.0, CH3OH). 1H NMR (400 MHz,
CDCl3): δ ) 7.38-7.20 (H-arom), 5.84 (dddd-oct, -CHdCH2),
5.54 (d, H-1), 5.12-4.95 (m, 2H, -CHdCH2), 2.16 and 2.02
(2*s, 3H, -CH3 (Ac)); J 1,2 ) 9.1 Hz. 13C NMR (100.6 MHz,
CDCl3): δ ) 175.52 (CdO), 128.47-127.30 (C-arom), 134.16
(-CHdCH2), 116.69 (-CHdCH2), 103.93 (C-1′), 95.80 (C-1′′),
87.37 (C-1), 70.7-70.2 (4*-CH2Ph), 22.26 (-CH3 (Ac)) ppm. MS
(ESI): m/z 946 [M + H]+, 968 [M + Na]+, 984 [M + K]+. Exact
mass calcd for C51H63NO16Na+: 968.4045, found 968.4036.
N-Acetyl-N-(3-th io-(2-am in oeth yl))pr opyl-2-acetam ido-
2-d eoxy-â-D-glu cop yr a n osyla m in e (13). Compound 9 (2 g,
6.6 mmol) and cysteamine (2.54 g, 33 mmol, 5 equiv) were
dissolved in methanol and reacted according to method 3 (TLC
EtOAc/AcOH/H2O, 3:3:1). The residue was applied to a MPLC
and eluted with EtOAc/AcOH/H2O, 3:1:1 then 3:3:1 to yield
pound 12 (1.55 g, 3.67 mmol) and cysteamine (1.41 g, 18.34
mmol, 5 equiv) were dissolved in methanol and processed
according to method 3 (TLC EtOAc/AcOH/H2O, 3:3:1). The
residue was applied to MPLC and eluted with EtOAc/AcOH/
H2O, 3:1:1 then 3:3:1 to yield 16 (1.52 g, 3 mmol, 82%). [R]20
D
) 21 (c 1.0, H2O). 1H NMR (400 MHz, D2O): δ ) 5.50 (d,
-NH2), 5.11 (d, H-1), 4.56 (d, H-1′), 4.04 (mc, H-3), 4.02 (d,
H-4′), 3.95-3.72 (m, 9H, H-4, H-5, H-5′, H-6a, H-6b, H-6′a,
H-6′b, H-2, H-3′), 3.65 (dd, H-2′), 3.50-3.38 (m, 2H, -CH2-
(a)), 3.35-3.29 (m, 2H, -CH2(e)), 3.00-2.93 (m, 2H, -CH2-
(d)), 2.77-2.67 (m, 2H, -CH2(c)), 2.34 and 2.32 (2*s, 3H, -CH3
(Ac)), 2.18-1.86 (m, -CH2(b)); J 1,2 ) 8.7, J 1′,2′ ) 8.3, J 2′,3′
)
10, J 3′,4′ ) 3.9 Hz. 13C NMR (100.6 MHz, D2O): δ ) 176.03
(-CdO), 103.50 (C-1′), 83.61 (C-1), 78.63, 77.47, 76.00, 75.80,
73.19, 71.60, and 70.52 (C-2, C-3, C-4, C-5, C-2′, C-3′, and C-5′),
69.21 (C-4′), 61.68 (C-6), 60.81 (C-6′), 39.08 (-CH2(e)), 28.94,
28.78, 28.73, and 28.53 (-CH2(c), -CH2(d), -CH2(a), and
-CH2(b)), 22.23 and 21.99 (-CH3 (Ac)) ppm. MS (ESI): m/z:
501 [M + H]+, 523 [M + Na]+. Exact mass calcd for C19H36N2O11-
SH+: 501.2118, found 501.2135.
N-Acetyl-N-(3-th io(2-a m in oeth yl))p r op yl(2,3,4,6-tetr a -
O-ben zyl-r-D-ga la ctop yr a n osyl)-(1f3)-(â-D-ga la ctop yr a n -
osyl)-(1f4)-â-D-glu cop yr a n osyla m in e (24). Compound 23
(2 g, 2.1 mmol) and cysteamine (1.2 g, 10.6 mmol, 5 equiv)
were dissolved in methanol and processed according to method
3 (TLC EtOAc/MeOH/AcOH, 3:3:1). After concentration, the
residue was dissolved in CH2Cl2 and successively washed with
brine and water. The organic layer was dried over MgSO4,
filtered, and concentrated. The residue was applied to MPLC
and eluted with EtOAc/MeOH 10:1 (to elute the starting
material first) and then a linear gradient from 1:1 to 1:5 was
applied to elute compound 24 (1.61 g, 1.57 mmol, 75%), which
compound 13 (2 g, 5.3 mmol, 80%). [R]20 ) 14 (c ) 1.0, H2O).
D
1H NMR (300 MHz, D2O): δ ) 5.80 (d, -NH2), 5.24 (d, H-1),
4.14 (dd-t, 1H, H-3 or H-4), 4.04 (dd, H-6a), 3.88 (dd, H-6b),
3.84-3.46 (m, 5H, H-2, H-3 or H-4, H-5, -CH2), 3.41-3.33 (m,
2H, -CH2), 3.06-2.97 (m, 2H, -CH2), 2.82-2.69 (m, 2H,
-CH2), 2.35 and 2.30 (2*s, 3H, -CH3 (Ac)), 2.12 and 2.10 (2*s,
3H, -CH3 (Ac)), 2.09 and 1.88 (m, 2H, -CH2); J 1,2 ) 9.3, J 5,6a
) 3.2, J 5,6b ) 5.7, J 6a,6b ) 12.5 Hz. 13C NMR (75 MHz, D2O):
δ ) 177.76-174.68 (2*-C)O), 86.29 (C-1), 78.83 and 75.09 (C-4
and C-3), 70.22 (C-5), 61.51 (C-6), 53.48 (C-2), 41.50, 38.94,
38.39, 33.98, and 29.80 (5*-CH2), 22.54, 21.86 and 21.43 (2*-
CH3 (Ac)) ppm. MS (ESI): m/z 380 [M + H]+. Exact mass calcd
for C15H29N3O6SH+: 380.1855, found 380.1835.
after concentration was yielded as a colorless foam. [R]20
)
D
63 (c ) 1.0, CH3OH). 1H NMR (300 MHz, MeOH (d4)): δ )
7.35 (mc, H-arom), 5.55 (mc, NH2), 4.56 (dd-d, H-4′), 3.40 (mc,
-CH2), 2.95, 2.75 and 2.63 (3*t, 6H, 3*-CH2), 2.27 (s, 3H, -CH3
(Ac)), 2.10-1.90 (m, 2H, -CH2); J 3′-4′ ) 3.8 Hz. 13C NMR (75.5
MHz, MeOH (d4)): δ ) 174.65 (-CdO), 140.00-128.56 (C-
arom), 105.02 (C-1′), 96.46 (C-1′′), 88.73 (C-1), 80.85, 80.49,
80.07, 78.79, 77.55, 77.38, 76.58 and 76.42 (8*-CH), 75.98,
75.03, 74.12, and 73.58 (4*-CH2-Ph), 71.67, 70.91, and 70.65
(3*-CH), 69.87 (C-6′′), 66.78 (C-4′), 62.49 (C-6), 62.14 (C-6′),
41.66, 41.04, 34.05, 30.09, and 29.74 (5*-CH2 (a-e)), 22.12
(-CH3 (Ac)) ppm. MS (ESI): m/z 1023 [M + H]+. Exact mass
calcd for C53H70N2O16SH+: 1023.4525, found 1023.4523.
N-Acetyl-N-(3-th io(2-a m in oeth yl))p r op yl(r-D-ga la cto-
p yr a n osyl)-(1f3)-(â-D-ga la ct op yr a n osyl)-(1f4)-â-D-glu -
cop yr a n osyla m in e (25). Ammonia (∼50 mL) was condensed
by passing through a dry ice condenser (-78 °C) to which 24
(749 mg, 0.73 mmol) was added. To this suspension, stirred
at -78 °C under argon, was added sodium in small aliquots,
until the blue color of the solution persisted for at least 10
min. The completion of the reaction was checked by TLC (i-
PrOH/CH3COONH4 (1 M), 2:1), after which ammonium chlo-
ride was added carefully until the blue color disappeared. The
reaction mixture was concentrated to dryness by a stream of
argon, yielding a slightly yellow residue. This was applied to
Sephadex column chromatography (d ) 25 mm, G-10) and
eluted with water. After pooling the product fractions and
lyophilization, 25 was obtained as a yellowish powder (480 mg,
0.72 mmol, quantitative yields) and could not be further
purified; it was used as such in the next step. MS (ESI): m/z
663 [M + H]+, 685 [M + Na]+; daughters of 663: 501 [(M -
Gal) + H]+, 339 [(M - 2*Gal) + H]+, 177 [(M - 2*Gal-Glu) +
H]+; daughters of 685: 523 [(M - Gal) + Na]+, 361 [(M -
2*Gal) + Na]+, 199 [(M - 2*Gal-Glu) + Na]+. Exact mass calcd
for C25H46N2O16SH+: 663.2646, found 663.2603.
N-Acetyl-N-(3-th io(2-a m in oeth yl))p r op yl-(r-D-glu cop y-
r a n osyl)-(1f4)-â-D-glu cop yr a n osyla m in e (14). Compound
10 (2 g, 4,7 mmol) and cysteamine (1.82 g, 23.6 mmol, 5 equiv)
were dissolved in methanol and processed according to method
3 (TLC EtOAc/AcOH/H2O, 3:3:1). The residue was subjected
to MPLC and eluted with EtOAc/AcOH/H2O, 3:1:1 then 3:3:1
to yield compound 14 (1.88 g, 3.75 mmol, 80%). [R]20 ) 77 (c
D
) 1.0, H2O). MS (ESI): m/z: 501 [M + H]+, 523 [M + Na]+.
Exact mass calcd for
501.2120.
C
19H36N2O11SH+: 501.2118, found
N-Acetyl-N-(3-th io(2-a m in oeth yl))p r op yl(â-D-glu cop y-
r a n osyl)-(1f4)-â-D-glu cop yr a n osyla m in e (15). Compound
11 (1.0 g, 2.4 mmol) and cysteamine (910 mg, 11.8 mmol, 5
equiv) were processed according to method 3 (TLC EtOAc/
AcOH/H2O, 3:3:1). The residue was subjected to MPLC and
eluted with EtOAc/AcOH/H2O, 3:1:1 then 3:3:1. Recrystalli-
zation from EtOAc/MeOH yielded pure compound 15 (845 mg,
1.7 mmol, 71%). [R]20 ) 4 (c 1.0, H2O). 1H NMR (400 MHz,
D
D2O): δ ) 5.58 (d, -NH2), 5.20 (d, H-1), 4.69 (d, H-1′), 4.14
(dd-d, H-6a), 4.10 (dd-d, H-6′a), 4.00 (dd, H-6b), 3.96-3.80
(m, 5H, H-6′b, H-5, H-2, H-3, and H-4), 3.74 (mc, 1H, -CH2),
3.67 (mc, 2H, H-3′ and H-5′), 3.62-3.56 (m, H-4′), 3.54-3.47
(m, 2H, H-2′ and -CH2), 3.43-3.37 (m, 2H, -CH2), 3.08-3.01
(m, 2H, -CH2), 2.85-2.75 (m, 2H, -CH2), 2.41 and 2.39 (2*s,
3H, -CH3 (Ac)), 2.24-1.96 (m, 2H, -CH2); J 1,2 ) 8, J 2,3 ) 9,
J 6a,6b ) 12, J 1′,2′ ) 7.2, J 2′,3′ ) 9.5, J 6′a,6′b ) 12.5 Hz. 13C NMR
(100.6 MHz, CDCl3): δ ) 176.84 and 176.00 (-C)O), 103.12
(C-1′), 87.50 (C-1), 78.89 and 77.64 (C-2 and C-3), 76.66 and
76.18 (C-3′ and C-5′), 75.74 (C-5), 73.82 (C-2′), 70.58 (C-4),
70.16 (C-4′), 61.32 (C-6′), 60.80 (C-6), 39.05 (-CH2), 29.75
(-CH2), 28.95, 28.78, and 28.51 (3*-CH2), 22.17 and 22.01 (-CH3
(Ac)) ppm. MS (ESI): m/z: 501 [M + H]+, 523 [M + Na]+. Exact
mass calcd for C19H36N2O11SH+: 501.2118, found 501.2120.
N-Acetyl-N-(3-th io(2-a m in oeth yl))p r op yl (â-D-ga la cto-
p yr a n osyl)-(1f4)-â-D-glu cop yr a n osyla m in e (16). Com-
N-Acetyl-N-(3-th io-(2-a ceta m id oeth yl))p r op yl(2,3,4,6-
t et r a -O-a cet yl-r-D-ga la ct op yr a n osyl)-(1f3)-(3,4,6-t r i-O-
a cetyl-â-D-ga la ctop yr a n osyl)-(1f4)-2,3,6-tr i-O-a cetyl-â-D-
glu cop yr a n osyla m in e (25-Ac). Compound 25 (130 mg, 0.19
mmol) was acetylated according to the general method (TLC:
toluene/acetone, 1:1). The residue was applied to silica gel
chromatography (toluene/acetone, 2:1 to 1:2) to give 25-Ac as
a colorless powder (180 mg, 0.16 mmol, 83%). 1H NMR (600

Chemoenzymatic Synthesis of Neoglycopeptides
J . Org. Chem., Vol. 66, No. 9, 2001 2955
MHz, CDCl3): δ ) 5.43 (dd, H-4′′), 5.32 (dd∼m, H-4′), 5.23
(mc, 3H, H-2, H-3, H-1′′), 5.13 (dd-t, H-2′), 5.07 (mc, 2H, H-2′′
and H-3′′), 4.97 (d, H-1), 4.45 (dd-d, H-6a), 4.43 (d, H-1′),
4.21-4.00 (m, 6H, H-5′′, H-6b, H-6′a, H-6′b, H-6′′a and H-6′′b),
3.85-3.64 (m, 4H, H-3′, H-4, H-5, and H-5′), 3.58 (mc, 1H,
-CH2), 3.39 (mc, 2H, -CH2), 3.26 (mc, 1H, -CH2), 2.63 (mc,
2H, -CH2), 2.47 (mc, 2H, -CH2), 2.16-1.90 (m, 36 H, 12*-
(d, H-1′), 4.33-4.23 (m, 1H, -CH (Gln)), 4.11 (dd-d, H-6a),
4.07 (dd-d, H-6′a), 3.96 (dd, H-6b), 3.93-3.77 (m, 7H, H-6′b,
H-4, H-5, -CH2, H-2 and H-3), 3.70-3.45 (m, 8H, H-3′, H-5′,
H-4′, 2*-CH2 and H-2′), 2.85-2.78 (m, 2H, -CH2), 2.75-2.60
(m, 2H, -CH2), 2.54-2.46 (m, 2H, -CH2), 2.37 and 2.36 (2*s,
3H, -CH3 (Ac)), 2.30 (mc, 1H, -CH2), 2.16-2.00 (m, 3H, -CH2
+ -CH2 (1H)); J 1,2 ) 7.8, J 1′,2′ ) 8.4 J 6a,6b ) 12.0, J CH2Ph
)
12.0 Hz. 13C NMR (100.6 MHz, D2O): δ ) 176.73-174.09 (5*-
CdO), 136.24 (C arom), 129.43, 129.09 and 128.45 (5*-CH
arom), 103.15 (C-1′), 87.54 (C-1), 78.98 and 77.51 (C-2 and C-3),
77.66 and 76.25 (C-5′ and C-3′), 75.75 (C-5), 73.82 (C-2′), 70.59
(C-4), 70,14 (C-4′), 67.84 (-CH2-Ph), 61.25 (C-6), 60.88 (C-6′),
55.26 (-CH (Gln)), 43.98 (-CH2(g)) 39.21 (-CH2(e)), 32.55
(-CH2(c)), 29.13 (-CH2(d)), 28.71 (-CH2(a)), 27.90 (-CH2(b)),
22.18 and 21.97 (-CH3 (Ac)) ppm. MS (ESI): m/z: 843 [M +
CH3 (Ac)), 1.74 (mc, 2H, -CH2); J 1,2 ) 8.0, J 6a,6b ) 11.8, J 1′,2′
)
8.5, J 2′,3′ ) 9.4, J 2′′,3′′ ) 9.7, J 3′′,4′′ ) 1.5 Hz. 13C NMR (150.7
MHz, CDCl3): δ ) 170.67-168.55 (12*-C)O), 101.15 (C-1′),
93.40 (C-1′′), 85.49 (C-1), 75.61 and 75.36 (C-5 and C-5′), 72.95
(C-3), 72.73 (C-3′), 70.73 (C-4), 69.71 (C-2′), 68.88 and 67.11
(C-2′′ and C-3′′), 67.60 (C-4′′), 66.79 (C-5′′), 66.42 (C-2), 64.52
(C-4′), 61.66 (C-6), 61.20 and 60.83 (C-6′ and C-6′′), 43.09
(-CH2), 38.43 (-CH2), 30.23 (-CH2), 28.89 (-CH2), 28.37
(-CH2), 23.19-20.45 (12*-CH3 (Ac)) ppm. MS (ESI): m/z 1148
[M + Na]+.
Na]+, 865 [M + 2Na - H]+. Exact mass calcd for C34H52N4O17
-
SNa+ 843.2496, found 843.2922.
[N1-(Ben zyloxyca r bon yl)-N4-[N-a cetyl-N-(3-th io(2-a cet-
a m id oet h yl))p r op yl(2-a cet a m id o-2-d eoxy-â-D-glu cop y-
r a n osyla m in e)]-L-glu ta m yl]-L-glycin e (26). Compound 13
(300 mg, 0.79 mmol), Z-Gln-Gly (266 mg, 0.79 mmol), and
TGase (526 mg) were dissolved in buffer (26 mL) and processed
according to method 4. The coupling could only be detected by
mass spectrometry analysis on the crude mixture, the product
could not be isolated. MS (ESI): m/z 698 [M - H]-.
[N1-(Ben zyloxycar bon yl)-N4-[N-acetyl-N-(3-th io-(2-acet-
a m id oe t h yl))p r op yl((â-D -ga la ct op yr a n osyl)-(1f4)-â-
D-glu copyr an osylam in e)]-L-glu tam yl]-L-glycin e (29). Com-
pound 16 (300 mg, 0.6 mmol), Z-Gln-Gly (200 mg, 0.6 mmol),
and TGase (400 mg) were reacted according to method 4. The
buffered solution (20 mL) was shaken for few hours until no
further cross-linking was detected by TLC (EtOAc/AcOH/H2O,
3:1:1). After heat inactivation of the enzyme and lyophilization,
the residue was applied to MPLC and eluted with EtOAc/
AcOH/H2O, 6:2:1. The product-containing fraction was further
purified on Sephadex G-10 gel chromatography (eluted with
water). Appropriate fractions were lyophilized to yield white
[N 1-(Be n zyloxyca r b on yl)-N 4-[N -a ce t yl-N -(3-t h io-(2-
a ce t a m id oe t h yl))p r op yl((r-D-glu cop yr a n osyl)-(1f4)-
â-D-glu cop yr a n osyla m in e)]-L-glu t a m yl]-L-glycin e (27).
Compound 14 (300 mg, 0.6 mmol), Z-Gln-Gly (200 mg, 0.6
mmol), and TGase (400 mg) were reacted according to method
4. The buffered solution (20 mL) was shaken for few hours
until no further cross-linking was detected on TLC (EtOAc/
AcOH/H2O, 3:1:1). After inactivation of the enzyme and
lyophilization, the residue was applied to a MPLC and eluted
with EtOAc/AcOH/H2O, 6:2:1. The appropriate fractions were
pooled and concentrated, and the resulting residue was further
purified by Sephadex gel chromatography (G-10, eluted with
water). Product-containing fractions were lyophilized to yield
fluffy 29 (438 mg, 0.53 mmol, 89%). [R]20 ) 3 (c 1.0, H2O). 1H
D
NMR (400 MHz, D2O): δ ) 7.50 (mc, 5H arom), 5.50 (d, -NH),
5.27 and 5.19 (each d, each 1H, -CH2Ph), 5.11 (d, H-1), 4.55
(d, H-1′), 4.30-4.20 (m, -CH (Gln)), 4.09-3.99 (m, 2H, H-6),
3.98-3.55 (m, 15 H, H-6′, H-f, H-g, H-h, H-2, H-2′, H-3, H-3′,
H-4, H-4′, H-5, and H-5′), 3.49-3.39 (m, 2H, -CH2(e)), 2.80-
2.71 (m, 2H, -CH2(d)), 2.70-2.58 (m, 2H, -CH2(a)), 2.50-2.42
(m, 2H, -CH2(c)), 2.32 and 2.31 (2*s, 3H, -CH3(Ac)), 2.23 and
27 (336 mg, 0.4 mmol, 68%). [R]20 ) 83 (c ) 1.0, H2O). 1H
2.00 (2*mc, 2*1H, -CH2(b)); J 1,2 ) 8.3, J 6a,6b ) 12.0, J 1′,2′
)
D
7.7, J CH Ph ) 12.4 Hz. 13C NMR (150.7 MHz, D2O): δ ) 178.45-
NMR (600 MHz, D2O): δ ) 7.45-7.37 (m, 5 H-arom), 5.41-
5.36 (m, H-1′), 5.15 and 5.07 (each d, each 1 H, -CH2Ph), 4.98
(d, H-1), 4.15-4.10 (m, -CH (Gln)), 3.90-3.45 (m, 5* -CH2:
H-6, H-6′, H-f, H-g, H-h; and 7*-CH: H-2, H-2′, H-3, H-3′,
H-4′, H-5, H-5′), 3.40 (dd-t, H-4), 3.35-3.25 (m, 2H,-CH2(e)),
2.67-2.60 (m, 2H, -CH2(d)), 2.57-2.43 (m, 2H, -CH2(a)),
2.39-2.30 (m, 2H, -CH2(c)), 2.19 (2*s-s, 3H, -CH3 (Ac)), 2.13
and 1.87 (2*mc, 2*1H, -CH2(b)); J 1,2 ) 8.5, J 3,4 or J 3′,4′ ) 9.5,
J 4,5) 9.5, J 6a,6b or J 6′a,6′b ) 12.0 Hz. 13C NMR (150.7 MHz,
D2O): δ ) 175.16-175.53 (5*CdO), 136.12 (C-arom), 128.70-
127.69 (5*-CH arom), 99.57 (C-1′), 86.73 (C-1), 77.19-69.20
(8*CH: 2, 2′, 3, 3′, 4, 4′, 5, 5′; 2*-CH2), 67.17 (-CH2-Ph), 60.73
and 60.36 (C-6 and C-6′), 54.46 (-CH (Gln)), 43.33 (-CH2(g))
38.42 (-CH2(e)), 31.87 (-CH2(c)), 29.10 (-CH2(d)), 27.85
(-CH2(a)), 27.12 (-CH2(b)), 21.41 and 21.25 (-CH3 (Ac)) ppm.
MS (ESI): m/z 865 [M/Na + Na]+ (daughter 757 [(M/Na -
OCH2Ph) + Na]+), 859 [M + K]+ (daughter 751 [(M - OCH2-
Ph) + K]+, 843 [M + Na]+ (daughter 735 [(M - OCH2Ph) +
Na]+, 819 [M - H]- (daughter 711 [(M - OCH2Ph) - H]-.
2
176.12 (5*CdO), 137.27 (C arom), 131.09, 130.79, and 130.10
(5*-CH arom), 105.19 (C-1′), 89.16 (C-1), 80.45-70.88 (8*-
CH: 2, 2′, 3, 3′, 4, 4′, 5, 5′; 2*-CH2), 69.56 (-CH2Ph), 63.31
and 62.62 (C-6 and C-6′), 56.34 (-CH (Gln)), 44.79 (-CH2(g))
40.84 (-CH2(e)), 34.24 (-CH2(c)), 32.77 (-CH2(d)), 30.21 (CH2-
(a)), 29.53 (CH2(b)), 23.82 and 23.69 (-CH3(Ac)) ppm. MS
(ESI): m/z 865 [M + 2Na - H]+, 843 [M + Na]+, 821 [M +
H]+, 819 [M - H]-, 711 [M - H - OBn]- (daughter ion of 819).
Exact mass calcd for
843.2922.
C
34H52N4O17SNa+: 843.2946, found
[N 1-(Be n zyloxyca r b on yl)-N 4-[N -a ce t yl-N -(3-t h io(2-
a cet a m id oet h yl))p r op yl((r-D-ga la ct op yr a n osyl)-(1f3)-
(â-D-ga la ctop yr a n osyla m in e)-(1f4)-â-D-glu cop yr a n osyl]-
L-glu ta m yl]-L-glycin e (17). Trisaccharide 25 (100 mg, 0.15
mmol), Z-Gln-Gly (51 mg, 0.15 mmol), and TGase (100 mg)
were reacted according to method 4. The buffered solution (5
mL) was shaken for few hours until no further cross-linking
was detected by TLC (EtOAc/AcOH/H2O, 3:3:1). Upon heat
inactivation of the enzyme and concentration, the residue was
applied to silica gel chromatography (MPLC, EtOAc/AcOH/
H2O, 3:1:1). Product-containing fractions were pooled, concen-
trated and purified further by sephadex G-10 gel chromatog-
raphy (eluted with water). The fractions containing the desired
compound were lyophilized to yield 17 (93 mg, 0.095 mmol,
Exact mass calcd for
843.2984.
C
34H52N4O17SNa+ 843.2946, found
[N 1-(Be n zyloxyca r b on yl)-N 4-[N -a ce t yl-N -(3-t h io-(2-
a cet a m id oet h yl))p r op yl((â-D-glu cop yr a n osyl)-(1f4)-â-
D-glu copyr an osylam in e)]-L-glu tam yl]-L-glycin e (28). Com-
pound 15 (300 mg, 0.6 mmol), Z-Gln-Gly (200 mg, 0.6 mmol),
and TGase (400 mg) were reacted according to the general
method. The buffered solution (20 mL) was shaken for few
hours until no further cross-linking was detected on TLC
(EtOAc/AcOH/H2O, 3:1:1). After inactivation of the enzyme and
lyophilization, the residue was applied to MPLC and eluted
with EtOAc/AcOH/H2O, 6:2:1. The residue was further purified
by Sephadex G-10 gel chromatography (eluted with water).
1
63%). H NMR (600 MHz, D2O): δ ) 7.28 (mc, 5H, H-arom),
5.01 and 4.94 (each d, each 1H, -CH2Ph), 4.98 (d, H-1′′), 4.86
(d, H-1), 4.35 (d, H-1′), 4.03 (dd-t, 1H), 4.01 (dd-d, H-4′), 3.97
(mc, -CH (Gln)), 3.86 (d, 1H), 3.79 (dd, H-6a), 3.76 (mc, 1H),
3.71 (dd, H-6b), 3.68-3.25 (m, 20H, 14*-CH and 3*-CH2)), 3.17
(mc, 2H, -CH2(e)), 2.49 (mc, 2H, -CH2(d)), 2.37 (mc, 2H, -CH2-
(a)), 2.20 (mc, 2H, -CH2(c)), 2.06 and 2.05 (2*s, 3H, -CH3 (Ac)),
2.00 and 1.80 (2*mc, 2*1H, -CH2(b)); J 1,2 ) 8.6, J 5,6a ) 3.0,
Appropriate fractions were lyophilized to yield colorless fluffy
1
28 (236 mg, 0.29 mmol, 48%). [R]20 ) -8 (c ) 1.0, H2O). H
D
J 5,6b ) 4.0, J 6a,6b ) 10.3, J 1′,2′ ) 7.9, J 3′,4′ ) 2.3, J CH
) 12.4
NMR (400 MHz, D2O): δ ) 7.59 (mc, 5H arom), 5.55 (d, -NH),
5.33 and 5.25 (each d, each 1H, -CH2Ph), 5.16 (d, H-1), 4.66
Ph
2
Hz. 13C NMR (150.7 MHz, CDCl3): δ ) 178.78-173.52 (5*-Cd

2956 J . Org. Chem., Vol. 66, No. 9, 2001
Ramos et al.
O), 136.17 (C arom), 128.74, 128.40, and 127.75 (5*CH arom),
102.76 (C-1′), 95.36 (C-1′′), 86.76 (C-1), 78.07, 77.12, 76.99,
76.80, 75.39, 75.11, 74.99, 70.74, 69.75, 69.47, 69.17, 69.06,
and 68.08 (11*-CH and 2*-CH2), 67.18 (-CH2Ph), 64.74 (C-
4′), 60.93, 60.84, and 60.16 (C-6, C-6′, and C-6′′), 54.49 (-CH
(Gln)), 43.23 (-CH2(g)), 38.41 (-CH2(e)), 30.17 (-CH2(c)), 27.88
(-CH2(a)), 27.16 (-CH2(b)), 21.47 and 21.30 (-CH3 (Ac)) ppm.
MS (ESI): m/z: 981 [M - H]-, 873 [M - (BnO)]- (daughter
ion). Exact mass calcd for [C40H62N4O22S - H]- 981.3498, found
981.3531.
Ack n ow led gm en t. This work has enjoyed financial
support by Unilever Research, Vlaardingen, The Neth-
erlands.
Su p p or tin g In for m a tion Ava ila ble: Copies of 1H and 13C
NMR spectra of compounds 7, 8, 14-18, 20, 22, 24, 25-Ac,
and 27-29. This material is available free of charge via the
Internet at http://pubs.acs.org.
J O001439C