UCHP method for oligosaccharide combinatorial library synthesis

Article abstract of DOI:10.1080/07328300903100661

A new methodology for oligosaccharide combinatorial library synthesis using a special hydroxy protecting group, the uni-chemo hydroxy protection (UCHP) group, was developed. The UCHP group was composed of oligomeric amino acid derivatives. The amino termi

Full text of DOI:10.1080/07328300903100661

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UCHP Method for Oligosaccharide
Combinatorial Library Synthesis
Shiro Komba a b & Sachiko Machida a
a Biomolecular Engineering Laboratory, National Food Research
Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki, 305-8642, Japan
b PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho,
Kawaguchi, Saitama, 332-0012, Japan
Version of record first published: 24 Jul 2009.
To cite this article: Shiro Komba & Sachiko Machida (2009): UCHP Method for Oligosaccharide
Combinatorial Library Synthesis, Journal of Carbohydrate Chemistry, 28:6, 369-393
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Journal of Carbohydrate Chemistry, 28:369–393, 2009
ꢀC
Copyright Taylor & Francis Group, LLC
ISSN: 0732-8303 print / 1532-2327 online
DOI: 10.1080/07328300903100661
UCHP Method for
Oligosaccharide
Combinatorial Library
Synthesis
Shiro Komba1 and Sachiko Machida2
1Biomolecular Engineering Laboratory, National Food Research Institute, 2-1-12 Kan-
nondai, Tsukuba, Ibaraki, 305-8642, Japan; and PRESTO, Japan Science and Technol-
ogy Agency, 4-1-8 Honcho, Kawaguchi, Saitama, 332-0012, Japan
2Biomolecular Engineering Laboratory, National Food Research Institute, 2-1-12 Kan-
nondai, Tsukuba, Ibaraki, 305-8642, Japan
A new methodology for oligosaccharide combinatorial library synthesis using a spe-
cial hydroxy protecting group, the uni-chemo hydroxy protection (UCHP) group, was
developed. The UCHP group was composed of oligomeric amino acid derivatives. The
amino terminals of UCHP groups were protected by either Boc or Fmoc groups. By
using these two types of UCHP, five kinds of trigalactoses [Galβ1-3Galβ1-3Gal, Galβ1-
3(Galβ1-4)Gal, Galβ1-4Galβ1-3Gal, Galβ1-3Galβ1-4Gal, and Galβ1-4Galβ1-4Gal] were
successfully synthesized on a solid support as a model of oligosaccharide combinatorial
library. Each step of all reactions was also successfully monitored using a combination
of two colorimetric tests, chloranil and methyl red-DIC.
Keywords UCHP; Oligosaccharide combinatorial library; Solid-phase synthesis
INTRODUCTION
Three important bio-functional chain types maintain homeostasis of living
organisms: nucleic acids (DNA and RNA), proteins (peptides), and oligosac-
charides. In the assembly of both nucleic acids (polynucleotides) and pro-
teins (polypeptides), no stereochemistry exists in the repeating bond construc-
tions (phosphate and amide, respectively), resulting in no need for selection.
In contrast, in the assembly of oligosaccharides (polysaccharides), selection
Received October 21, 2008; accepted June 8, 2009.
Address correspondence to Shiro Komba, Biomolecular Engineering Laboratory, Na-
tional Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki, 305-8642, Japan.
E-mail: skomba@affrc.go.jp
369

S. Komba & S. Machida
370
is necessary for desired bond constructions (location of glycoside bond and
anomeric configuration). Each glycosidic bond to be fashioned in a growing
oligosaccharide constitutes a new stereogenic center and a locus of possible
complexity. Because of the branching structure of oligosaccharides, the buildup
of the target structure requires complicated orthogonal protecting groups for
each hydroxyl group.[1] Recently, we applied the uni-chemo protection (UCP)
method[2] to oligosaccharide synthesis.[3,4] The UCP is composed of an amino
acid oligomer, which was originally designed to protect the amino group on a
peptide. We applied this method for hydroxy protection, naming it uni-chemo
hydroxy protection (UCHP). The individual hydroxyl groups on the carbohy-
drates were protected with a different oligomerization degree of amino acids by
ester linkage. Deprotection of this UCHP group was performed using the Ed-
man degradation reaction,[5] which removes only the N-terminal mono-amino
acid from all UCHP groups. The general Edman degradation reaction for the
UCHP method consists of four steps: the first step is deprotection of the amino
protecting group (de-Fmoc or de-Boc step); the second step is phenyl isothio-
cyanate (PITC: Edman reagent) coupling to all free amino terminals (PITC
coupling step); the third step is cleaving of the N-terminal phenyl thiocar-
bamoyl mono-amino acid derivative by using trifluoroacetic acid (TFA) to de-
crease the oligomerization degree (TFA treatment step); and the fourth step is
reprotection by the Boc group to the newly exposed N-terminal amino group
(re-Boc step). However, the first and second steps are sufficient conditions for
deprotection of monomeric-UCHP because the phenyl thiocarbamoyl mono-
amino acid derivative is removed immediately from the hydroxyl group by
the electron withdrawing effect at such basic PITC coupling conditions. De-
pending on the oligomerization degree of UCHP, each hydroxyl group was
characterized.
To demonstrate the efficiency of this UCHP method in the synthesis of
an oligosaccharide combinatorial library, five kinds of trigalactoses [Galβ1-
3Galβ1-3Gal (40), Galβ1-3(Galβ1-4)Gal (42), Galβ1-4Galβ1-3Gal (44), Galβ1-
3Galβ1-4Gal (46), and Galβ1-4Galβ1-4Gal (48)] have been synthesized on a
solid support as a model of oligosaccharide combinatorial library. The glyco-
side bonds selected were β 1-3 and β 1-4 for the second and third galactoses,
respectively, to demonstrate selective deprotection of the UCHP group even
if it protected to the adjacent hydroxyl groups. Moreover, each reaction step
was monitored by the combination of two colorimetric tests, chloranil[6] and
methyl red-DIC.[7] The chloranil test was used for the detection of the amino
group. The color of the resin changes to dark blue in the presence of an amino
group. The methyl red-DIC test is a newly developed method from our group
for the detection of the hydroxyl group. The resin reddens in the presence of a
hydroxyl group.

Oligosaccharide Combinatorial Library Synthesis
371
RESULTS AND DISCUSSION
One of the disadvantages of the UCHP method is an increase in steric hin-
drance. The oligomerization level of UCHP rises as the number of hydroxyl
groups that protect UCHP increases. As a result, the steric hindrance in-
creases. To decrease the steric hindrance, two types of UCHP were designed.
One is the Fmoc (base-removable amino protecting group)-protected UCHP;
the other is the Boc (acid-removable amino protecting group)-protected one.
Fmoc and Boc are orthogonal amino protecting groups. If the amino termi-
nal of UCHP is protected by one kind of amino protecting group, it is nec-
essary to protect two hydroxyl groups by a monomeric-UCHP and dimeric-
UCHP combination. However, if the UCHP amino terminals are protected by
both the Fmoc group and the Boc group, two hydroxyl groups can be pro-
tected by two monomeric-UCHP groups of the kind that would create the
least steric hindrance. Interestingly, the dimeric-UCHP protected with Fmoc
group was removed from the hydroxyl group at the de-Fmoc step (by using
20% piperidine/DMF for 20 min, the dimeric-UCHP removes quantitatively)
because dimeric-UCHP with a free amino group cyclized immediately by such
a basic condition and cleaved from the hydroxyl group. As a result, the Fmoc
protection was applied only for monomeric UCHP. On the other hand, the Boc
protection bears no restriction for usage (Sch. 1).
The galactose donors for trigalactose combinatorial library were des-
ignated as 12 and 13 in Scheme 2. SPh (thiophenyl) was chosen
as the anomeric leaving group for easy activation by either DMTST
[dimethyl(methylthio)sulfonium triflate][8] or NIS (N-iodosuccinimide)-TfOH
(trifluoromethanesulfonic acid).[8] 2-OH on galactose was protected by the
Scheme 1. Preparation of UCHP groups. (a) THF, rt, 2 h, 92%; (b) 1.5 equiv. Boc2O,
1,4-dioxane/saturated aq NaHCO3, rt, 2 h; (c) 10 N NaOH, MeOH/H2O, 40◦C, 4 h, 94% (two
steps); (d) 4.0 equiv. DIC, 0.4 equiv. DMAP, CH2Cl2, 6◦C, 12 h; (e) 10 N NaOH, MeOH/H2O,
40◦C, 2 h, 57% (two steps).

S. Komba & S. Machida
372
Scheme 2. Preparation of galactose donors. (a) 1.2 equiv. 10, 1.1 equiv. DIC, 0.1 equiv.
DMAP, CH2Cl2, -40◦C, 2 days, 93%; (b) 1.2 equiv. 5, 2.0 equiv. DIC, 0.1 equiv. DMAP, CH2Cl2,
0◦C, 2 h, 99%; (c) 1.2 equiv. 7, 2.0 equiv. DIC, 0.1 equiv. DMAP, CH2Cl2, 0◦C, 2 h, 95%.
benzoyl (Bz) group as a neighboring effect group for selective β-glycosidation.
3-OH on galactose was protected by monomeric-UCHP protected with an
Fmoc group. 4-OH was protected by either monomeric-UCHP protected with
a Boc group or dimeric-UCHP protected with a Boc group. To synthe-
size these two kinds of galactose donors, phenyl 2,6-di-O-benzoyl-1-thio-β-D-
galactopyranoside (9)[9] was used as the starting compound. The monomeric-
UCHP protected with an Fmoc group (10)[3,4] selectively protected 3-OH on
compound 9 by using DIC and DMAP at –40◦C. Subsequently, by using DIC
and DMAP at 0◦C, its 4-OH was protected by either monomeric-UCHP pro-
tected with a Boc group (5) or dimeric-UCHP protected with a Boc group (7).
The solid support used was HMBA-AM resin (14, 1.16 mmol/g; Novabiochem,
Merck Ltd.), a hydroxy functionalized polystyrene resin (Sch. 3). HMBA-Gly
{2-[4-(hydroxymethyl)benzamido]acetic acid} was designed as the linker,[3]
which bound to the hydroxyl group on HMBA-AM resin via ester linkage.
Scheme 3. Preparation of linker connected solid support. (a) 14 (1.0 g, 1.16 mmol/g), 3.0
equiv. 15, 3.0 equiv. DIC, 0.1 equiv. DMAP, CH2Cl2, rt, 1 day, then Ac2O, 0.1 equiv. DMAP, pyr,
CH2Cl2, rt, 1 day. The loading was determined to be 0.8447 mmol/g from UV absorbance of
the eluate of Fmoc group; (b) 20% piperidine in DMF, rt, 18 min; (c) 3.0 equiv. 18, 6.0 equiv.
NEM, 2.9 equiv. TBTU, DMF, rt, 4 h, then Ac2O, DMF, rt, 20 min; (d) 50% TFA in CH2Cl2, rt, 5 min, 6
times.

Oligosaccharide Combinatorial Library Synthesis
373
Scheme 4. Condensation of first galactose. (a) 6.0 equiv. 12, 18.0 equiv. DMTST, CH2Cl2, rt, 1
day; (b) NaOMe, CH2Cl2, rt, 1 h, then added H2O and MeOH, rt, 1 h. The coupling yield was
60%. The respective retention times for 22 and 23 were 5.0 and 20.3 min.
The products of the oligosaccharide linker complex could be cleaved off from the
resin at basic conditions and could be detected by UV on HPLC. The theoretical
loading value of hydroxyl groups on linker-connected resin (20) was calculated
as 0.9126 mmol/g from Fmoc loading value of compound 16 (0.8447 mmol/g).[10]
The first galactose donor (12) was coupled with a hydroxyl group on the resin
(20) by using DMTST as a promoter in CH2Cl2 at rt (Sch. 4). After 1 day of the
coupling reaction, an aliquot of the resin was treated with base; next, the elu-
ate was analyzed by analytical normal-phase HPLC. Analytical normal-phase
HPLC separation was performed using an HPLC system (Shimadzu Corp.)
with a TSK-gel Amide-80 column (4.6 × 250 mm, 5 µm; Tosoh Co. Ltd.) at a
flow rate of 1 mL/min and detection at 254 nm. Both the unglycosylated linker
(22, retention time of 5.0 min) and glycosylated linker (23, retention time of
20.3 min) were detected, and the coupling yield was calculated as 60% (Fig. 1).
Because the Boc group was cleaved during the glycosidation reaction, the re-
sultant exposed amino group on terminal UCHP was protected by a Boc group
again (re-Boc) (Sch. 5). Re-Boc reaction was monitored by chloranil test for
amino detection. This reaction was continued until the chloranil test produced
a negative response. After the unglycosylated hydroxyl group had been pro-
tected by the benzoyl group, the monomeric-UCHP protected with Fmoc group
Figure 1. HPLC trace. After the coupling of the first galactose (12) to the resin (20). The
5.0-min peak indicated the presence of a linker (22). The 20.3-min peak indicated
compound 23. The coupling yield was estimated to be 60% based on the peak area. HPLC
condition A was used.

S. Komba & S. Machida
374
Scheme 5. Deprotection of UCHP groups by Edman degradation. (a) Boc2O, saturated aq
NaHCO3, DMF, rt, 8 h, then BzOH, DMAP, DIC, CH2Cl2, rt, 12 h; (b) 20% piperidine in DMF, rt, 18
min; (c) PITC, NMM, DMF, rt, 30 min; (d) BzOH, DMAP, DIC, CH2Cl2, rt, 12 h; (e) 20% TFA in
CH2Cl2, rt, 30 min, twice; (f) PITC, NMM, DMF, rt, 30 min.
that bound to the hydroxyl group at the 3-position was selectively removed
by one cycle of the Edman degradation reaction (de-Fmoc and PITC coupling).
Consequently, the galactose acceptor with a selectively free hydroxyl group at
the 3-position (26) was selectively obtained. The Bz capping step was moni-
tored by the methyl red-DIC test, and the de-Fmoc step of the first step in the
Edman degradation reaction was monitored by the chloranil test. The PITC
coupling step of the second step in the Edman degradation reaction was mon-
itored by both the chloranil test and the methyl red-DIC test. All steps were
successfully monitored by a combination of chloranil and methyl red-DIC tests.
For the synthesis of compounds 40, 42, and 44, the resultant galactose acceptor
with a selectively free hydroxyl group at the 3-position (26) was coupled with
a second galactose donor (13). For the synthesis of compounds 46 and 48, the
resin (26) was capped with a benzoyl group. Following that, the monomeric-
UCHP protected with a Boc group at the 4-position was selectively removed by
one cycle of the Edman degradation reaction (de-Boc and PITC coupling). As
a result, the galactose acceptor with a selectively free hydroxyl group at the
4-position (29) was selectively obtained. All steps were also successfully moni-
tored by the combination of chloranil and methyl red-DIC tests. This galactose
acceptor with a selectively free hydroxyl group at the 4-position (29) was cou-
pled with a second galactose donor (12).
For the coupling of the second galactose, DMTST was used as a coupling
promoter (Sch. 6). The second galactose donor (13) was coupled with 3-OH of
the galactose on the resin (26) by using DMTST in CH2Cl2 at rt for 1 day, iden-
tically to the previously described procedure. However, in this case, the glyco-
sylation yield was sufficiently low to warrant repeating the coupling procedure

Oligosaccharide Combinatorial Library Synthesis
375
Scheme 6. Condensation of second galactose. (a) 6.0 equiv. 13, 18.0 equiv. DMTST, CH2Cl2,
rt, 1 day, three times; (b) NaOMe, CH2Cl2, rt, 1 h, then added H2O and MeOH, rt, 1 h; (c) 6.0
equiv. 12, 18.0 equiv. NIS, 18.0 equiv. TfOH, CH2Cl2, -30◦C, 1 day; (d) NaOMe, CH2Cl2, rt, 1 h,
then added H2O and MeOH, rt, 1 h. The respective retention times for 31 and 33 were 23.7
and 23.2 min. The respective coupling yields for 31 and 33 were 50% (triple coupling) and
53% (single coupling).
three times in all. Using an analytical normal-phase HPLC at the same con-
ditions as previously described, the single, double, and triple coupling yields
were estimated as 17%, 34%, and 50%, respectively (Fig. 2i). On the other
hand, in the case of coupling the second galactose donor (12) with 4-OH of
the galactose on the resin (29) by using DMTST, as in the previously described
procedure, the product of the disaccharide was obtained in less than 10% yield.
To increase the coupling yield, the promoter was changed to NIS-TfOH from
DMTST and the reaction temperature was changed to –30◦C. As a result, the
coupling yield dramatically increased, reaching 53% with only a single cou-
pling, based on analysis of analytical normal-phase HPLC at the same condi-
tions as previously described (Fig. 2ii). The disaccharide of Galβ1-3Gal resin
(30) was subsequently treated by re-Boc, Bz capping, and one cycle of Edman
degradation reactions (de-Fmoc and PITC coupling) (Sch. 7). This selectively
Figure 2. HPLC trace. (i) After the triple coupling of the second galactose (13) to the resin
(26). The 20.3-min peak indicated un-reacted monogalactose (23). The 23.7-min peak
indicated Galβ1-3Gal (31). The coupling yield was estimated to be 50% based on the peak
area; (ii) After single coupling of the second galactose (12) to the resin (29). The 20.1-min
peak indicated un-reacted monogalactose (23). The 23.2-min peak indicated Galβ1-4Gal
(33). The coupling yield was estimated to be 53% based on the peak area. HPLC condition A
was used.

S. Komba & S. Machida
376
Scheme 7. Deprotection of UCHP groups by Edman degradation. (a) Boc2O, saturated aq
NaHCO3, DMF, rt, 8 h; (b) BzOH, DMAP, DIC, CH2Cl2, rt, 12 h; (c) 20% piperidine in DMF, rt, 18
min; (d) PITC, NMM, DMF, rt, 30 min; (e) 20% TFA in CH2Cl2, rt, 30 min, twice.
produced the digalactose acceptor with a selectively free hydroxyl group at the
3ꢁ-position (34) for the synthesis of 40. For the synthesis of both 42 and 44, the
above resin (34) was then capped by a Bz group. It was then treated by one cy-
cle of Edman degradation reaction (de-Boc, PITC coupling, TFA treating, and
re-Boc). Consequently, the digalactose acceptor with a selectively free hydroxyl
group at the 4-position (35) for the synthesis of 42 was selectively obtained.
For the synthesis of 44, this resin (35) was subsequently treated by Bz capping
and one cycle of Edman degradation reactions (de-Boc and PITC coupling). As
a product, the digalactose acceptor with a selectively free hydroxyl group at
the 4ꢁ-position (36) for the synthesis of 44 was selectively obtained. When the
disaccharide of Galβ1-4Gal resin (32) was treated with re-Boc, Bz capping, and
one cycle of Edman degradation reactions (de-Fmoc and PITC coupling), the di-
galactose acceptor with a selectively free hydroxyl group at the 3ꢁ-position (37)
for the synthesis of 46 was selectively obtained. In the next step of the syn-
thesis of 48, this resin (37) was treated by Bz capping and one cycle of Edman
degradation reactions (de-Boc and PITC coupling). As a result, the digalactose
acceptor with a selectively free hydroxyl group at the 4ꢁ-position (38) for the
synthesis of 48 was selectively obtained. All steps were also successfully mon-
itored by the combination of chloranil and methyl red-DIC tests.
The next step was the coupling of the third galactose (Sch. 8). For the
synthesis of Galβ1-3Galβ1-3Gal (40), the digalactose acceptor with a selec-
tively free hydroxyl group on 3ꢁ-postion (34) was coupled with a third galac-
tose donor (12) by using DMTST, as in the previously described procedure.
This coupling procedure was repeated two times in all. For the synthesis of
Galβ1-3(Galβ1-4)Gal (42), Galβ1-4Galβ1-3Gal (44), Galβ1-3Galβ1-4Gal (46),
and Galβ1-4Galβ1-4Gal (48), the digalactose acceptors with selectively free
hydroxyl groups at the 4-postion (35), 4ꢁ-position (36), 3ꢁ-position (37), and 4ꢁ-
position (38), respectively, were coupled with the third galactose donor (12) us-
ing NIS-TfOH at –30◦C, and one use of the coupling procedure was sufficient

Oligosaccharide Combinatorial Library Synthesis
377
Scheme 8. Condensation of third galactose and cleavage from the resin. (a) 6.0 equiv. 12,
18.0 equiv. DMTST, CH2Cl2, rt, 1 day, twice; (b) NaOMe, CH2Cl2, MeOH, rt, 1 day, then added
H2O and MeOH, rt, 1 day; (c) 6.0 equiv. 12, 18.0 equiv. NIS, 18.0 equiv. TfOH, CH2Cl2, -30◦C, 1
day. The respective retention times for 40, 42, 44, 46, and 48 were 25.4, 25.3, 25.1, 25.1, and
24.8 min. Coupling yields of the third galactose for 40, 42, 44, 46, and 48 were, respectively,
46%, 52%, 59%, 70%, and 42%. Total yields from 16 for 40, 42, 44, 46, and 48 were, respectively,
8%, 6%, 8 %, 14%, and 13%.
to reach satisfactory coupling yields. In the case of the coupling of the third
galactose with the resin (34) for the synthesis of Galβ1-3Galβ1-3Gal (40), the
NIS-TfOH procedure was also applicable, and a single coupling yield was al-
most identical to the double coupling yield of DMTST (data not shown). All
trigalactose derivatives were cleaved from the resin by basic conditions, and

S. Komba & S. Machida
378
Figure 3. HPLC trace. (i) After the double coupling of the third galactose (12) to the resin
(34). The 19.9-min peak indicated monogalactose (23). The 23.7-min peak indicated
Galβ1-3Gal (31). The 25.4-min peak indicated Galβ1-3Galβ1-3Gal (40); (ii) After the single
coupling of the third galactose (12) to the resin (35). The 20.2-min peak indicated
monogalactose (23). The 23.7-min peak indicated Galβ1-3Gal (31). The 25.3-min peak
indicated Galβ1-3(Galβ1-4)Gal (42). For the synthesis of the branched form, the compound
that uncoupled from the second Gal formed 3-OBz-4-Boc-mono-UCHP on mono-Gal after
the second Gal coupling and capping. The 4-OH mono-Gal was synthesized selectively
when it was treated with the next Edman degradation for the removal of the Boc protected
mono-UCHP. This was coupled with the third Gal donor to obtain Galβ1-4Gal disaccharide
(33, 23.2-min peak); (iii) After the single coupling of the third galactose (12) to the resin (36).
The 20.2-min peak indicated monogalactose (23). The 23.7-min peak indicated Galβ1-3Gal
(31). The 25.1-min peak indicated Galβ1-4Galβ1-3Gal (44); (iv) After the single coupling of
the third galactose (12) to the resin (37). The 20.3-min peak indicated monogalactose (23).
The 23.2-min peak indicated Galβ1-4Gal (33). The 25.1-min peak indicated
Galβ1-3Galβ1-4Gal (46); (v) After the single coupling of the third galactose (12) to the resin
(38). The 20.3-min peak indicated monogalactose (23). The 23.2-min peak indicated
Galβ1-4Gal (33). The 24.8-min peak indicated Galβ1-4Galβ1-4Gal (48). HPLC condition A
was used.
the eluates were purified by HPLC using the same conditions as those em-
ployed for analytical HPLC. All five kinds of trigalactoses as target compounds
were obtained as main peaks within subpeaks (Fig. 3). The final glycosidation
yields for 40, 42, 44, 46, and 48 were determined to be 46%, 52%, 59%, 70%,
and 42%, respectively. The total yields from 16 for 40, 42, 44, 46, and 48 were
determined to be 8% (11 steps), 6% (16 steps), 8% (19 steps), 14% (14 steps),
and 13% (17 steps), respectively. From the HMBC-NMR analysis for 40, 42,
44, 46, and 48, all glycoside linkages were confirmed.
In summary, by using the UCHP method, a model of an oligosaccha-
ride combinatorial library was successfully synthesized. NIS-TfOH was a
more suitable coupling promoter for solid-phase oligosaccharide synthesis than
DMTST, although it required a lower temperature. Through repetition of only
one cycle of the Edman degradation reaction and a capping reaction, the hy-
droxyl groups on carbohydrate were sequentially selectively deprotected. This
simple method for selective deprotection of each hydroxyl group facilitated

Oligosaccharide Combinatorial Library Synthesis
379
oligosaccharide library synthesis. To advance oligosaccharide combinatorial li-
brary synthesis even more, we will pursue research to increase the reaction
yield and to establish selective α-glycosidation.
EXPERIMENTAL SECTION
General
1H NMR and 13C NMR spectra were obtained using a spectrometer
(AVANCE 800, AVANCE 500, or AV 400, Bruker BioSpin) in CDCl3 or D2O.
Chemical shifts are expressed in parts per million relative to the signal of
either CHCl3 or Me4Si in CDCl3 solvent, adjusted, respectively, to 7.24 or
0.00 ppm, or MeOH in D2O solvent, adjusted to 3.30 ppm. ESI-FT-MS spec-
tra were recorded in the positive ion mode (APEX II 70e, Bruker Daltonics).
Optical rotations (P-1020-GT, Jasco Inc.) were determined in CHCl3, H2O, or
MeOH at ambient temperature. Both analytical normal-phase HPLC separa-
tion and preparative normal-phase HPLC separation were performed using
same HPLC system (Shimadzu Corp.) with a TSK-gel Amide-80 column (4.6 ×
250 mm, 5 µm; Tosoh Co. Ltd.) at a flow rate of 1 mL/min and detection
at 254 nm. For HPLC condition A, the solvent system was as follows: sol-
vent A, TFA/water (1:1000); solvent B, TFA/acetonitrile/water (1:900:100). Ini-
tially, 100% solvent B was performed isocratically for 10 min at a flow rate
of 1 mL/min. Then, a linear gradient of 0% to 67% of solvent A was applied
for 20 min at a flow rate of 1 mL/min. All reagents and solvents were reagent
grade.
Abbreviations used
Ac, acetyl; Ac2O, acetic anhydride; aq, aqueous; Boc, tert-butoxycarbonyl;
Boc2O, di-tert-butyl dicarbonate; tBu, tert-butyl; Bz, benzoyl; calcd., calcu-
lated; DIC, 1,3-diisopropylcarbodiimide; DMAP, 4-(dimethylamino)pyridine;
DMF, N, N-dimethylformamide; DMTST, dimethyl(methylthio)sulfonium
triflate; equiv., equivalent; Fmoc, 9H-fluoren-9-ylmethoxycarbonyl; Gal,
D-galactose; NEM, N-ethylmorpholine; NIS, N-iodosuccinimide; NMM, N-
methylmorpholine; PITC, phenyl isothiocyanate; pyr, pyridine; rt, room
temperature; SPh, thiophenyl; TBTU, O-(benzotriazol-1-yl)-N, N, Nꢁ, Nꢁ-
tetramethyluronium tetrafluoroborate; TFA, trifluoroacetic acid; TfOH,
trifluoromethanesulfonic acid; THF, tetrahydrofuran; TMSOTf, trimethylsilyl
trifluoromethanesulfonate; Tr, trityl.
Methyl 2-(1-Ethylpropylamino)acetate (3)
A solution of methyl bromoacetate (2, 28.53 mL, 0.301 mol) in THF
(160 mL) was added drop-wise to a cooled solution of 3-amino pentane (1,

S. Komba & S. Machida
380
77.28 mL, 0.663 mol) in THF (230 mL) over 5 min. The solution was stirred for
2 h at rt and concentrated. The product was suspended in diethyl ether. The
residue of the hydrobromide salt was removed by filtration and washed with di-
ethyl ether; the filtrate and washings were collected and concentrated in vacuo
1
to give 3 (44.22 g, 92%) as a colorless oil. [α]D+2.9 (c 0.10, MeOH); H NMR
(400 MHz, CDCl3): δ = 0.90 (t, 6H, JCH3,CH = JCH3,CHꢁ = 7.5 Hz, (CH3CH2)2CH),
ꢁ
ꢁꢁ
1.43 (m, 4H, (CH3CH2)2CH), 2.37 (quintet, 1H, JCH,CH = JCH,CH = JCH,CH
=
ꢁꢁꢁ
JCH,CH = JCH,NH = 6.0 Hz, (CH3CH2)2CH), 3.42 (s, 2H, NCH2CO), 3.73 (s,
3H, COOMe); 13C NMR (100 MHz, CDCl3): δ = 9.7 ((CH3CH2)2CH), 25.7
((CH3CH2)2CH), 48.4 (NCH2CO), 51.8 (COOMe), 59.8 ((CH3CH2)2CH); ESI-
FT-MS, calcd. for C8H18NO+2 (M + H)+ : 160.1332, found: 160.1293.
2-[(tert-Butoxycarbonyl)(1-ethylpropyl)amino]acetic Acid (5)
A solution of di-tert-butyl dicarbonate (96.22 g, 0.441 mol) in 1,4-dioxane
(350 mL) was added drop-wise to a cooled solution of 3 (46.8 g, 0.293 mol)
in saturated aq NaHCO3 (350 mL) over 5 min. After stirring for 2 h at rt,
ethyl acetate was added, and the mixture was washed with water and 2 N
HCl, dried (Na2SO4), and concentrated. The residue was dissolved in methanol
(1000 mL) and water (100 mL), and then 10 N NaOH was added until the mix-
ture reached a pH of 14. After stirring for 4 h at 40◦C, the unsolved material
was filtered through paper and washed with methanol. The filtrate and wash-
ings were collected and concentrated. The residue was purified using column
chromatography (chloroform/methanol 10:1) on silica gel to afford 5 (67.60 g,
94%, two steps). [α]D+1.6 (c 0.8, MeOH); 1H NMR (400 MHz, CDCl3): δ = 0.86,
ꢁ
0.88 (each t, 6H, JCH3,CH = JCH3,CH = 7.4, 7.5 Hz, 2CH3CH2), 1.31–1.43 (m, 4H,
2CH3CH2), 1.41, 1.43 (each s, 9H, Me3O), 3.61 (s, 2H, NCH2CO), 3.72, 3.93
(each m, 1H, (CH3CH2)2CH), 10.16 (broad s, 1H, COOH); 13C NMR (100 MHz,
CDCl3): δ = 11.0, 11.2 (CH3CH2), 25.8, 26.1 (CH3CH2), 28.3, 28.5 (Me3C), 44.3,
45.4 (NCH2CO), 58.3, 60.3 ((CH3CH2)2CH), 79.6, 80.1 (Me3CO), 156.1, 157.5
(NCOO), 175.6, 176.0 (COOH); ESI-FT-MS, calcd. for C12H23NO4Na+ (M +
Na)+ : 268.15193, found: 268.14971.
2-[{2-[(tert-Butoxycarbonyl)(1-ethylpropyl)amino]acetyl}(1-
ethylpropyl)amino]acetic Acid (7)
DIC (52.03 mL, 0.336 mol) and DMAP (4.11 g, 0.034 mol) were added
to a mixture of 5 (20.61 g, 0.084 mol) and 3 (13.38 g, 0.084 mol) in CH2Cl2
(77.4 mL) at 0◦C. The mixture was stirred for 12 h at 6◦C. The product was dis-
solved in chloroform; the solution was washed with 0.5 N HCl and saturated aq
NaHCO3, dried (Na2SO4), and concentrated. Column chromatography (ethyl
acetate/hexane 1:3) of the residue on silica gel gave roughly purified 6. The
roughly purified 6 was dissolved in methanol (508 mL) and water (50.8 mL),
following which, 10 N NaOH was added to attain a pH of 14. After stirring for

Oligosaccharide Combinatorial Library Synthesis
381
2 h at 40◦C, the undissolved material was filtered through paper and washed
with methanol. The filtrate and washings were collected and concentrated.
The residue was purified using column chromatography (chloroform/methanol
10:1) on silica gel to afford 7 (17.85 g, 57%, two steps). [α]D+1.0 (c 0.7, MeOH);
1H NMR (400 MHz, CDCl3): δ = 0.82–0.97 (m, 12H, 2(CH3CH2)2CH), 1.29–
1.62 (m, 8H, 2(CH3CH2)2CH), 1.41, 1.47 (each s, 9H, Me3C), 3.43, 3.56, 3.71,
3.98 (4m, 2H, 2(CH3CH2)2CH), 3.86, 3.88, 3.91, 3.94 (4s, 4H, 2NCH2CO); 13C
NMR (100 MHz, CDCl3) δ = 11.1, 11.1, 11.4 (2(CH3CH2)2CH), 25.7, 26.1, 26.2,
26.3 (2(CH3CH2)2CH), 28.2, 28.5 (Me3C), 43.7, 43.9, 44.0, 44.3 (2NCH2CO),
58.3, 60.4, 60.7, 61.1 (2(CH3CH2)2CH), 79.8, 80.2 (Me3C), 156.0, 156.6 (2NCO),
171.8, 172.1 (COOH); ESI-FT-MS, calcd. for C19H36N2O5Na+ (M + Na)+
:
395.2516, found: 395.2518.
Phenyl 2,6-Di-O-benzoyl-3-O-{2-[(1-ethylpropyl)(9H-fluoren-9-
ylmethoxycarbonyl)amino]acetyl}-1-thio-β-D-
galactopyranoside (11)
DIC (4.3 mL, 27.5 mmol) and DMAP (305 mg, 2.5 mmol) were added to
a solution of phenyl 2,6-di-O-benzoyl-1-thio-β-D-galactopyranoside (9, 12.0 g,
25.0 mmol) in CH2Cl2 (240 mL) and cooled to –40◦C. A solution of 2-[(1-
ethylpropyl)(9H-fluoren-9-ylmethoxycarbonyl)amino]acetic acid[3,4] (10, 11.0 g,
29.9 mmol) in CH2Cl2 (100 mL) was added to the mixture at –40◦C, and the
mixture was stirred for 2 days at –40◦C. After methanol (100 mL) was added
to stop the reaction, the solution was concentrated. Column chromatography
(ethyl acetate/toluene 1:10) of the residue on silica gel gave 11 (19.28 g, 93%).
1
[α]D+20.2 (c 1.3, CHCl3); H NMR (400 MHz, CDCl3): δ = 0.56–0.66 (m, 6H,
2CH3CH2), 0.89–1.29 (m, 4H, 2CH3CH2), 3.48–3.56 (m, 1H, (CH3CH2)2CH),
ꢁ
3.64 (d, 2H, Jgem = 2.2 Hz, NCH2CO), 4.02 (t, 1H, J5,6 = J5,6 = 6.2 Hz, H-
ꢁ
5), 4.22 (t, 1H, J9,CH = J9,CH = 6.0 Hz, H-9 Fmoc), 4.31, 4.35, 4.47, 4.53 (4dd,
2H, J9,CH2 = 5.9, 6.7, 6.1, 5.8 Hz, Jgem = 10.9, 11.1, 10.6, 10.6 Hz, CH2 Fmoc),
4.40 (m, 1H, H-4), 4.66 (d, 2H, J5,6 = 6.1 Hz, H-6), 4.84, 4.86 (2d, 1H, J1,2
=
10.1, 10.1 Hz, H-1), 5.12, 5.21 (2dd, 1H, J2,3 = 9.8, 9.7 Hz, J3,4 = 3.2, 3.2
Hz, H-3), 5.51, 5.67 (2t, 1H, J1,2 = J2,3 = 9.9 Hz, H-2), 7.04–8.06 (m, 23H,
Ar Fmoc, 2PhCO, and PhS); 13C NMR (100 MHz, CDCl3): δ = 10.7, 10.7
(2CH3CH2), 25.6, 25.9 (2CH3CH2), 44.4 (NCH2CO), 47.2, 47.4 (C-9 Fmoc),
59.3, 59.9 ((CH3CH2)2CH), 64.0 (C-6), 66.3 (C-4), 67.6, 67.7 (CH2 Fmoc), 67.9,
68.2 (C-2), 76.0 (C-3), 76.2 (C-5), 86.7, 87.2 (C-1), 119.9, 119.9, 124.8, 125.0,
125.3, 126.9, 127.1, 127.2, 127.6, 127.7, 128.1, 128.2, 128.4, 128.5, 128.8, 128.9,
129.0, 129.6, 129.8, 129.9, 131.8, 132.5, 133.2, 133.3, 133.4, 133.7 (C1-8-H Fmoc,
2PhCOO, and PhS), 141.4, 141.5 (C4a and C4b Fmoc), 143.7, 143.9 (C8a and C9a
Fmoc), 157.8 (NCOO), 165.1 (PhCOO at C-2), 166.3 (PhCOO at C-6), 169.0
(NCH2COO); ESI-FT-MS, calcd. for C48H47NO10SNa+ (M + Na)+ : 852.2813,
found: 852.2815.

S. Komba & S. Machida
382
Phenyl 2,6-Di-O-benzoyl-4-O-{2-[(tert-butoxycarbonyl)(1-
ethylpropyl)amino]acetyl}-3-O-{2-[(1-ethylpropyl)(9H-
fluoren-9-ylmethoxycarbonyl)amino]acetyl}-1-thio-β-D-
galactopyranoside (12)
DIC (3.77 mL, 24.1 mmol) and DMAP (147 mg, 1.2 mmol) were added to a
mixture of 11 (10 g, 12.0 mmol) and 5 (3.5 g, 14.3 mmol) in CH2Cl2 (50 mL) at
0◦C, and the mixture was stirred for 2 h at 0◦C. After methanol (50 mL) was
added to stop the reaction, the solution was concentrated. Column chromatog-
raphy (ethyl acetate/hexane 1:4) of the residue on silica gel gave 12 (12.60
1
g, 99%). [α]D+23.1 (c 1.6, CHCl3); H NMR (400 MHz, CDCl3): δ = 0.50–0.96
(m, 12H, 4CH3CH2), 1.09–1.55 (m, 8H, 4CH3CH2), 1.40, 1.42, 1.43, 1.45, (4s,
9H, Boc), 3.50–4.03 (m, 2H, 2(CH3CH2)2CH), 3.52–4.02 (m, 4H, 2NCH2COO),
3.87, 4.12 (each m, 1H, H-9 Fmoc), 4.04, 4.25 (each m, 2H, CH2 Fmoc), 4.30–
4.60 (m, 2H, H-6), 4.90 (m, 1H, H-1), 5.34, 5.44 (m, 1H, H-3), 5.51–5.60 (m,
1H, H-2), 5.62, 5.68 (each d, 1H, J3,4 3.1, 3.2 Hz, H-4), 7.04–8.04 (m, 23H, Ar
Fmoc, 2PhCO, and PhS); 13C NMR (100 MHz, CDCl3): δ = 11.1, 11.1, 11.2, 11.2,
11.3, 11.3 (4CH3CH2), 25.4, 25.5, 25.7, 25.8, 25.9, 26.0, 26.0, 26.3, 26.3, 26.4,
26.6 (4CH3CH2), 28.3, 28.3, 28.3, 28.4, (Me3CO), 43.1, 43.2, 43.5 (2NCH2COO),
46.9, 47.2 (C-9 Fmoc), 58.3, 58.5, 59.5, 59.6, 59.8, 59.9, 60.0 (2(CH3CH2)2CH),
62.1, 62.2, 62.3 (C-6), 67.1, 67.2, 68.4 (CH2 Fmoc), 67.7, 67.8, 67.9, 68.0 (C-
2 and C-4), 72.3, 72.5, 72.8 (C-3), 74.7, 74.7, 74.8 (C-5), 80.0, 80.0, 80.2, 80.2
(Me3CO), 87.3, 87.3 (C-1), 119.7, 119.9, 119.9, 124.9, 124.9, 125.1, 125.2, 126.9,
126.9, 127.0, 127.4, 127.5, 127.6, 127.6, 127.9, 128.0, 128.1, 128.2, 128.2, 128.3,
128.4, 128.5, 128.7, 128.8, 128.9, 129.1, 129.3, 129.3, 129.4, 129.5, 129.5, 129.6,
129.8, 129.8, 129.9, 130.0, 132.2, 132.3, 132.5, 132.6, 132.7, 133.0, 133.2, 133.2,
133.2, 133.4, 133.5, 141.1, 141.2, 141.3, 141.4, 143.9, 144.0, 144.1, 144.1, 144.2,
144.3 (Ar Fmoc, 2PhCOO, and PhS), 155.7, 156.2, 156.2, 156.6, 156.7 (2NCOO),
165.0, 165.4 (PhCOO at C-2), 165.9, 166.0 (PhCOO at C-6), 169.2, 169.4, 169.5,
169.6, 169.6, 170.0 (2NCH2COO); ESI-FT-MS, calcd. for C60H68N2O13SNa+ (M
+ Na)+ : 1079.4334, found: 1079.4331.
Phenyl 2,6-Di-O-benzoyl-4-O-{2-[{2-[(tert-butoxycarbonyl)(1-
ethylpropyl)amino]acetyl}(1-ethylpropyl)amino]acetyl}-3-O-
{2-[(1-ethylpropyl)(9H-fluoren-9-
ylmethoxycarbonyl)amino]acetyl}-1-thio-β-D-
galactopyranoside (13)
DIC (1.89 mL, 12.1 mmol) and DMAP (74 mg, 0.6 mmol) were added to a
mixture of 11 (5 g, 6.0 mmol) and 7 (2.7 g, 7.2 mmol) in CH2Cl2 (25 mL) at
0◦C. The mixture was stirred for 2 h at 0◦C. After methanol (25 mL) was added
to stop the reaction, the solution was concentrated. Column chromatography

Oligosaccharide Combinatorial Library Synthesis
383
(ethyl acetate/toluene 1:10) of the residue on silica gel gave 13 (6.79 g, 95%).
1
[α]D+25.2 (c 1.2, CHCl3); H NMR (400 MHz, CDCl3): δ = 0.65–1.07 (m, 18H,
6CH3CH2), 1.10–1.62 (m, 12H, 6CH3CH2), 1.39, 1.40, 1.43 (each s, 9H, Boc),
3.43–3.97 (m, 3H, 3(CH3CH2)2CH), 3.56–4.00 (m, 6H, 3NCH2CO), 3.89–4.12
(each m, 1H, H-9 Fmoc), 4.03–4.31 (m, 2H, CH2 Fmoc), 4.15 (m, 1H, H-5),
4.46–4.66 (m, 2H, H-6), 4.84–4.94 (m, 1H, H-1), 5.31–5.37 (m, 1H, H-3), 5.44,
5.63 (each d, 1H, J3,4 = 3.0, 3.2 Hz, H-4), 5.48–5.61 (m, 1H, H-2), 6.98–8.06 (m,
23H, Ar Fmoc, 2PhCO, and PhS); 13C NMR (100 MHz, CDCl3): δ = 10.8, 10.9,
11.2, 11.2, 11.4, 11.5 (6CH3CH2), 25.5, 25.6, 25.7, 25.8, 26.2, 26.5, 26.6, 26.7
(6CH3CH2), 28.3, 28.5, (Me3CO), 42.5, 42.8, 43.0, 43.3, 43.5, 43.7 (3NCH2CO),
47.0, 47.2 (C-9 Fmoc), 58.3, 59.6, 59.9, 60.0, 60.1, 60.3, 60.7 (3(CH3CH2)2CH),
62.6, 62.8 (C-6), 67.1, 67.8 (CH2 Fmoc), 68.0, 68.1, 68.3 (C-2 and C-4), 72.2, 72.3
(C-3), 75.1, 75.4 (C-5), 79.5, 79.6 (Me3CO), 87.1, 87.3 (C-1), 119.9, 124.9, 125.1,
125.3, 126.9, 127.0, 127.6, 127.8, 127.9, 128.2, 128.3, 128.4, 128.7, 128.8, 128.9,
129.0, 129.4, 129.6, 129.7, 129.8, 129.9, 130.0, 132.2, 132.2, 132.3, 133.2, 141.3,
144.0, 144.1, 144.1, 144.3 (Ar Fmoc, 2PhCOO, and PhS), 156.1, 156.2, 156.3,
156.5, 156.6, 156.6, 156.8 (2NCOO), 165.1 (PhCOO at C-2), 166.1 (PhCOO at
C-6), 168.6, 168.9, 168.9, 169.0, 169.1, 169.3 (2NCH2COO); ESI-FT-MS, calcd.
for C67H81N3O14SNa+ (M + Na)+ : 1206.5332, found: 1206.5334.
[4-(Carboxymethylcarbamoyl)phenyl]methyl O-
(β-D-Galactopyranosyl)-(1→3)-O-(β-D-galactopyranosyl)-
(1→3)-β-D-galactopyranoside (40)
The HMBA-AM resin (14, 1.0 g, 1.16 mmol/g; Novabiochem, Merck Ltd.)
was packed into a 20-mL disposable chromatography column (Econo-Pac, Bio-
Rad Laboratories Inc., Hercules, CA). The column was connected to a suction
flask using a Teflon tube with a manual two-way Teflon valve; excess reagents,
DMF, etc., were removed by applying a vacuum. The resin was derivatized with
the HMBA-Gly linker via the following procedure. First, the resin was swollen
with dichloromethane (12 mL). N-α-Fmoc-glycine (15, 1.03 g, 3.46 mmol),
DMAP (14 mg, 0.115 mmol), and DIC (545 µL, 3.48 mmol) were then added
and the mixture was agitated. After 1 day, the solution was removed, and the
resin was washed thoroughly with MeOH (5 × 10 mL) and dichloromethane
(5 × 10 mL). Subsequently, dichloromethane (8 mL), acetic anhydride (4 mL),
pyridine (2 mL), and DMAP (14 mg, 0.115 mmol) were added to the resin and
the mixture was again agitated. After 1 day, the solution was removed; the
resin was then washed thoroughly with MeOH (5 × 10 mL), DMF (5 × 10 mL),
dichloromethane (5 × 10 mL), and diethyl ether (3 × 10 mL), and dried in
vacuo. The loading was determined as 0.8447 mmol/g from UV absorbance of
the eluate of the Fmoc group.[10] The Fmoc group was cleaved by 20% piperi-
dine in DMF (12 mL) for 18 min. Then 4-(trityloxymethyl)benzoic acid[3] (18,
Tr-HMBA, 1.4 g, 3.549 mmol), NEM (886 µL, 6.962 mmol), and TBTU (1.08 g,

S. Komba & S. Machida
384
3.364 mmol) were dissolved in DMF. After 5 min, the mixture was added to the
resin and agitated. After 4 h, the solution was removed. The resin was washed
with DMF (6 × 10 mL) and a solution of acetic anhydride/DMF (2 mL/14 mL)
was added and the mixture was agitated. After 20 min, the resin was washed
thoroughly with DMF (5 × 10 mL) and dichloromethane (5 × 10 mL). Depro-
tection of the trityl group from the resin was performed using dichloromethane
(6 mL) and TFA (6 mL) at rt. After agitation for 5 min, the resin was washed
thoroughly with dichloromethane (1 × 10 mL). This trityl deprotection pro-
cedure was repeated six times in all. The resin was then washed thoroughly
with DMF (5 × 10 mL), dichloromethane (5 × 10 mL), and diethyl ether (3 ×
10 mL) and dried in vacuo. Consequently, hydroxy functionalized HMBA-Gly-
HMBA-AM resin (20) was obtained (theoretical hydroxyl group loading was
0.9126 mmol/g).
The hydroxy functionalized HMBA-Gly-HMBA-AM resin (20, 35 mg,
0.032 mmol, 0.9126 mmol/g) was packed into a disposable chromatography
column (mini column, SARSTEDT AG & Co.) with a rubber septum for inert
atmosphere. This hydroxy functionalized resin was glycosylated with a galac-
tose donor (12, 200 mg, 0.189 mmol) using DMTST (147 mg, 0.569 mmol)
in dichloromethane (4 mL) at rt. After agitation for 1 day at rt, the solu-
tion was removed. The resin was washed thoroughly with DMF (5 × 10 mL)
and dichloromethane (5 × 10 mL). To determine the coupling yield, aliquots
of the resin were taken and treated with a solution of dichloromethane/25
wt% NaOMe in MeOH (200 µL/30 µL) for 1 h. Subsequently, with added wa-
ter/MeOH (30 µL/200 µL), the reaction mixture was agitated for an additional
1 h. The resin was then filtered and neutralized with acetic acid, and the elu-
ate was analyzed using normal-phase HPLC condition A. The peak of mono-
glycosylated compound 23 was detected at 20.3 min, and the glycosylation yield
was determined as 60%, as calculated from the area under the peaks. Unfortu-
nately, the Boc group was removed during the coupling condition. The exposed
amino group was protected by the Boc group again (re-Boc) using DMF (2 mL),
Boc2O (100 mg, 0.458 mmol), and saturated aq NaHCO3 (100 µL). After agita-
tion for 8 h, the solution was removed; the resin was washed thoroughly with
aq 50% DMF (5 × 2 mL), DMF (5 × 2 mL), dichloromethane (5 × 2 mL), and
diethyl ether (3 × 2 mL). Then it was dried in vacuo. The chloranil colorimet-
ric test to detect secondary amines was used to detect the reaction completion.
To protect the unreacted hydroxyl group, benzoylation was performed (Bz cap-
ping) with benzoic acid (40 mg, 0.328 mmol), DIC (100 µL, 0.646 mmol), and
DMAP (10 mg, 0.082 mmol) in CH2Cl2 (2.4 mL). After agitation for 12 h, the
solution was removed. The resin was washed thoroughly using aq 50% DMF
(5 × 2 mL), DMF (5 × 2 mL), dichloromethane (5 × 2 mL), and diethyl ether
(3 × 2 mL). Then it was dried in vacuo. The newly developed methyl red-DIC
colorimetric test for detecting hydroxyl groups was used to detect the reac-
tion completion. It is very important to protect the unreacted hydroxyl group

Oligosaccharide Combinatorial Library Synthesis
385
completely; otherwise, the synthesized oligosaccharide will be obtained as a
complicated mixture. The next step was Edman degradation to selectively de-
protect the hydroxyl group at the 3-position of Gal. Selective deprotection of the
monomeric-UCHP protected with Fmoc group by one cycle of Edman degrada-
tion reaction (de-Fmoc and PITC coupling) was performed: (1) De-Fmoc step:
Deprotection of the Fmoc group was performed using 20% piperidine/DMF
(4 mL) at rt. After agitation for 18 min, the solution was removed, and the resin
was washed thoroughly with DMF (6 × 10 mL). The chloranil test changed to
positive because of exposure of the amino group. (2) PITC coupling step: PITC
coupling reaction was performed using PITC (1 mL) and NMM (240 µL) in
DMF (2 mL). After agitation for 30 min, the solution was removed; the resin
was washed thoroughly with DMF (5 × 10 mL), dichloromethane (5 × 2 mL),
and diethyl ether (3 × 2 mL). Then it was dried in vacuo. At this time, the
results of the chloranil test changed to negative because of PITC coupling to
the amino group. Furthermore, the methyl red-DIC test gave a positive result
due to the existence of a free hydroxyl group. As a result of this reaction, a
Gal acceptor with a selectively free hydroxyl group at the 3-position (26) was
synthesized selectively.
The next step was coupling with a second galactose. The previous resul-
tant 3-OH resin (26) was glycosylated with a galactose donor (13, 224 mg,
0.1891 mmol) using DMTST (110 mg, 0.425 mmol) in dichloromethane (3 mL)
at rt. After agitation for 1 day at rt, the solution was removed. The resin was
washed thoroughly with DMF (5 × 10 mL) and dichloromethane (5 × 10 mL).
Galβ1-3Gal (31) was detected at 23.7 min, and the glycosylation yield was de-
termined to be 17% by analytical HPLC as described previously. To increase
the coupling yield, the same coupling reaction was repeated three times in all:
the double coupling yield was estimated as 34%; the triple coupling yield was
estimated as 50%. Next, the exposed amino group on digalactose (30, di-Gal)
was protected by the Boc group (re-Boc) and the unreacted hydroxyl group
on monogalactose (26, mono-Gal) was capped by benzoyl group (Bz capping).
Again, to deprotect monomeric-UCHP protected with Fmoc group, one cycle
of Edman degradation (de-Fmoc and PITC coupling) was performed to selec-
tively synthesize the di-Gal acceptor with a selectively free hydroxyl group at
the 3ꢁ-position (34).
The last step was coupling with a third galactose. The previously resul-
tant 3ꢁ-OH resin (34) was glycosylated with a galactose donor (12, 200 mg,
0.189 mmol) using DMTST (147 mg, 0.569 mmol) in dichloromethane (4 mL)
at rt. After agitation for 1 day at rt, the solution was removed; the resin was
washed thoroughly with DMF (5 × 10 mL), dichloromethane (5 × 10 mL), and
diethyl ether (3 × 2 mL). Then it was dried in vacuo. The coupling reaction was
repeated once to increase the coupling yield. The resin was treated with a solu-
tion of dichloromethane/MeOH/25 wt% NaOMe in MeOH (1 mL/100 µL/50 µL)
for 1 day to cleave off the target compound from the resin. Water/MeOH

S. Komba & S. Machida
386
(300 µL/1 mL) were then added, and the reaction mixture was agitated for an
additional 1 day. Subsequently, the mixture was neutralized with acetic acid
and the resin filtered, which was washed with MeOH and water. The filtrate
and washings were concentrated, and the residue was analyzed and purified
using normal-phase HPLC condition A. The target trigalactose Galβ1-3Galβ1-
3Gal (40, 2.03 mg, retention time of 25.4 min) was synthesized and purified.
The glycosylation yield was determined to be 46%, which was calculated from
the area under the peaks. The total yield was 8% (11 steps from 16). At the
same time, the monogalactose (23, 2.9 mg, retention time of 19.9 min) and
digalactose Galβ1-3Gal (31, 1.99 mg, retention time of 23.7 min) were also pu-
rified.
Compound 40 (Galβ1-3Galβ1-3Galβ1-linker); [α]D+1.5 (c 0.07, MeOH); 1H
NMR (500 MHz, D2O): δ = 3.50 (dd, 1H, J1,2 = 7.6 Hz, J2,3 = 9.9 Hz, H-2c), 3.56
(dd, 1H, J2,3 = 10.1 Hz, J3,4 = 3.3 Hz, H-3c), 3.55–3.62 (m, 3H, H-5a, H-5b, and
H-5c), 3.55–3.70 (m, 6H, H-6a, H-6b, and H-6c), 3.64 (m, 1H, H-2a), 3.66 (m,
1H, H-2b), 3.67–3.73 (m, 2H, H-3a and H-3b), 3.81 (d, 1H, J3,4 = 3.2 Hz, H-4c),
4.07 (s, 2H, NCH2CO), 4.09 (d, 2H, J3,4 = 3.1 Hz, H-4a and H-4b), 4.43 (d, 1H,
J1,2 = 7.9 Hz H-1a), 4.51 (d, 1H, J1,2 = 7.6 Hz, H-1c), 4.56 (d, 1H, J1,2 = 7.7
Hz, H-1b), 4.74, 4.92 (each d, 2H, Jgem = 12.3 Hz, C6H4-CH2), 7.49, 7.74 (each
d, 4H, Ar); 13C NMR (125 MHz, D2O): δ = 41.8 (NCH2CO), 60.8, 60.9 (C-6a,
C-6b, and C-6c), 68.3, 68.4, 68.5 (C-4a, C-4b, and C-4c), 69.9 (C-2a), 70.2 (C-
2b), 70.5 (C6H4-CH2), 71.0 (C-2c), 72.5 (C-3c), 74.6, 74.8, 75.0 (C-5a, C-5b, and
C-5c), 82.0 (C-3b), 82.2 (C-3a), 101.5 (C-1a), 104.0 (C-1b), 104.3 (C-1c), 127.5,
128.6 (CAr-H), 132.6, 141.1 (CAr), 170.8 (CONH), 173.9 (COOH); HMBC NMR
(500 MHz, D2O): H-1a → C6H4-CH2, H-1b → C-3a, H-1c → C-3b; ESI-FT-MS,
calcd. for C28H40NO−19 (M - H)− : 694.2200, found: 694.2203.
Compound 31 (Galβ1-3Galβ1-linker); [α]D –0.7 (c 0.1, MeOH); 1H NMR
(500 MHz, D2O): δ = 3.50 (dd, 1H, J1,2 = 7.6 Hz, J2,3 = 9.9 Hz, H-2b), 3.55
(dd, 1H, J2,3 = 10.2 Hz, J3,4 = 3.3 Hz, H-3b), 3.55–3.62 (m, 2H, H-5a and H-
5b), 3.59–3.65 (m, 1H, H-2a), 3.60–3.71 (m, 4H, H-6a and H-6b), 3.69 (m, 1H,
H-3a), 3.81 (d, 1H, J3,4 = 3.3 Hz, H-4b), 4.06 (s, 2H, NCH2CO), 4.09 (d, 1H,
J3,4 = 3.2 Hz, H-4a), 4.43 (d, 1H, J1,2 = 7.7 Hz, H-1a), 4.49 (d, 1H, J1,2 = 7.6
Hz, H-1b), 4.74, 4.92 (each d, 2H, Jgem = 12.3 Hz, C6H4-CH2), 7.49, 7.74 (each
d, 4H, Ar); 13C NMR (125 MHz, D2O): δ = 41.8 (NCH2CO), 60.8, 60.9 (C-6a
and C-6b), 68.4 (C-4a), 68.5 (C-4b), 69.9 (C-2a), 70.5 (C6H4-CH2), 71.0 (C-2b),
72.4 (C-3b), 74.8, 75.0 (C-5a and C-5b), 82.3 (C-3a), 101.5 (C-1a), 104.3 (C-1b),
127.5, 128.6 (CAr-H), 132.6, 141.1 (CAr), 170.8 (CONH), 173.9 (COOH); HMBC
NMR (500 MHz, D2O): H-1a → C6H4-CH2, H-1b → C-3a; ESI-FT-MS, calcd.
for C22H30NO−14 (M - H)− : 532.1672, found: 532.1668.
Compound 23 (Galβ1-linker); [α]D –6.5 (c 0.2, MeOH); 1H NMR (400 MHz,
D2O): δ = 3.50 (dd, 1H, J1,2 = 7.5 Hz, J2,3 = 9.9 Hz, H-2), 3.56 (dd, 1H, J2,3
=
ꢁ
9.9 Hz, J3,4 = 3.3 Hz, H-3), 3.61 (dddd, 1H, J4,5 = 0.9 Hz, J5,6 = 4.4 Hz, J5,6
=
7.8 Hz, H-5), 3.68 (dd, 1H, J5,6 = 4.4 Hz, Jgem = 11.7 Hz, H-6), 3.74 (dd, 1H,

Oligosaccharide Combinatorial Library Synthesis
387
J5,6 = 7.8 Hz, Jgem = 11.7 Hz, H-6ꢁ), 3.86 (dd, 1H, J3,4 = 3.3 Hz, J4,5 = 0.8 Hz,
H-4), 4.00 (s, 2H, NCH2CO), 4.41 (d, 1H, J1,2 = 7.5 Hz, H-1), 4.77, 4.94 (each d,
2H, Jgem = 12.3 Hz, C6H4-CH2), 7.52, 7.77 (each d, 4H, Jo,m = 8.3 Hz, Ar); 13C
NMR (200 MHz, D2O): δ = 42.7 (NCH2CO), 60.9 (C-6), 68.6 (C-4), 70.6 (C6H4-
CH2), 70.7 (C-2), 72.7 (C-3), 75.2 (C-5), 101.9 (C-1), 127.4, 128.5 (CAr-H), 132.9,
141.0 (CAr), 170.5 (NCO), 175.3 (COOH); HMBC NMR (800 MHz, D2O): H-1a
→ C6H4-CH2; ESI-FT-MS, calcd. for C16H21NO9Na+ (M + Na)+ : 394.1109,
found: 394.1108.
ꢁ
[4-(Carboxymethylcarbamoyl)phenyl]methyl O-
(β-D-Galactopyranosyl)-(1→3)-O-[(β-D-galactopyranosyl)-
(1→3)]-β-D-galactopyranoside (42)
To synthesize compound 42, similar to the synthesis of compound 40, the
hydroxy functionalized HMBA-Gly-HMBA-AM resin (20, 35 mg, 0.032 mmol,
0.9126 mmol/g) was used. The reaction conditions were identical up to the syn-
thesis of the di-Gal acceptor with a selectively free hydroxyl group at the 3ꢁ-
position (34). To cap the hydroxyl group at the 3ꢁ-position, benzoylation was
performed (Bz capping) with benzoic acid (40 mg, 0.328 mmol), DIC (100 µL,
0.646 mmol), and DMAP (10 mg, 0.082 mmol) in CH2Cl2 (2.4 mL). After ag-
itation for 12 h, the solution was removed, and the resin was washed thor-
oughly with aq 50% DMF (5 × 2 mL), DMF (5 × 2 mL), dichloromethane
(5 × 2 mL), and diethyl ether (3 × 2 mL). Then it was dried in vacuo. The
methyl red-DIC test was used to detect the reaction completion. The next step
was Edman degradation for selective deprotection of the hydroxyl group at
the 4-position of di-Gal. To selectively deprotect the monomeric-UCHP pro-
tected with Boc group and to simultaneously decrease the polymerization de-
gree of the dimeric-UCHP protected with Boc group to the monomeric-UCHP
protected with Boc group, one cycle of Edman degradation was performed: (1)
De-Boc: Deprotection of the Boc group was performed using 20% TFA/CH2Cl2
(3 mL) at rt. After agitation for 30 min, the solution was removed; the reaction
was then repeated once more. Next, the resin was washed thoroughly with
dichloromethane (5 × 2 mL) and DMF (5 × 10 mL). The chloranil test changed
to positive because of the exposed amino group. (2) PITC coupling step: The
PITC coupling reaction was performed similarly to the method previously de-
scribed, using PITC (1 mL) and NMM (240 µL) in DMF (2 mL). After agitation
for 30 min, the solution was removed, and the resin was washed thoroughly
with DMF (5 × 10 mL), dichloromethane (5 × 2 mL), and diethyl ether (3 ×
2 mL). Then it was dried in vacuo. At this time, the chloranil test changed
to negative because of the coupling with PITC to the amino group. As a re-
sult of this reaction, the 4-position was selectively deprotected. (3) TFA treat-
ment step: To decrease the polymerization degree of dimeric-UCHP at the 4ꢁ-
position to monomeric-UCHP, N-terminal phenyl thiocarbamoyl mono-amino

S. Komba & S. Machida
388
acid derivative was selectively cleaved using 20% TFA/CH2Cl2 (3 mL) at rt,
with identical reaction conditions to those of “de-Boc.” After agitation for 30
min, the solution was removed; then the reaction was repeated once more.
Next, the resin was washed thoroughly using dichloromethane (5 × 2 mL) and
DMF (5 × 10 mL). The chloranil test changed to positive because of the ex-
posure of the amino group on monomeric-UCHP at the 4ꢁ-position. (4) Re-Boc
step: To protect the newly exposed amino group by the Boc group, the resin
was treated with DMF (2 mL), Boc2O (100 mg, 0.458 mmol), and saturated aq
NaHCO3 (100 µL), similarly to the method described previously. After agita-
tion for 8 h, the solution was removed. The resin was washed thoroughly with
aq 50% DMF (5 × 2 mL), DMF (5 × 2 mL), dichloromethane (5 × 2 mL), and
diethyl ether (3 × 2 mL). It was then dried in vacuo. At this time, the chloranil
test changed to negative because the exposed amino group was protected by
the Boc group. Furthermore, the methyl red-DIC test gave a positive result
due to the free hydroxyl group at the 4-position. As a result of this reaction,
a di-Gal acceptor with a selectively free hydroxyl group at the 4-position (35)
was synthesized selectively.
The next step was coupling with a third galactose. To the above product
(35), a galactose donor (12, 200 mg, 0.189 mmol), NIS (128 mg, 0.569 mmol),
and dichloromethane (500 µL) were added. The mixture was then cooled at
–30◦C and TfOH (50 µL, 0.565 mmol) was added. After agitation for 1 day
at –30◦C, the solution was removed, and the resin was washed thoroughly
with DMF (5 × 10 mL) and dichloromethane (5 × 10 mL). To cleave off the
target compound from the resin, the resin was treated with a solution of
dichloromethane/MeOH/25 wt% NaOMe in MeOH (1 mL/100 µL/50 µL) for 1
day. Subsequently, with added water/MeOH (300 µL/1 mL), the reaction mix-
ture was agitated for another 1 day. Next, it was neutralized with acetic acid
and the reaction mixture was filtered. The resin was washed with MeOH and
water; the combined filtrate and washings were concentrated. The residue was
analyzed and purified using normal-phase HPLC condition A. The target tri-
galactose Galβ1-3(Galβ1-4)Galβ1-linker (42, 1.41 mg, retention time of 25.3
min) was synthesized and purified. The glycosylation yield was determined to
be 52%, which was calculated from the area under the peaks. The total yield
was 6% (16 steps from 16). At the same time, the monogalactose Galβ1-linker
(23, 1.2 mg, retention time of 20.2 min) and digalactose Galβ1-3Galβ1-linker
(31, 0.84 mg, retention time of 23.7 min) were also purified.
Compound 42 {Galβ1-3(Galβ1-4)Galβ1-linker}; [α]D+0.5 (c 0.2, MeOH); 1H
NMR (800 MHz, D2O): δ = 3.43 (dd, 1H, J1,2 = 7.9 Hz, J2,3 = 9.9 Hz, H-2c), 3.49
(dd, 1H, J1,2 = 7.8 Hz, J2,3 = 9.9 Hz, H-2b), 3.54–3.58 (m, 2H, H-3b and H-3c),
3.53–3.62 (m, 3H, H-5a, H-5b, and H-5c), 3.62–3.74 (m, 6H, H-6a, H-6b, and
H-6c), 3.75 (m, 2H, H-2a and H-3a), 3.78, 3.80 (each d, 2H, J3,4 = 3.4, 3.4 Hz,
H-4b and H-4c), 4.07 (s, 2H, NCH2CO), 4.29 (d, 1H, J3,4 = 1.8 Hz, H-4a), 4.44
(d, 1H, J1,2, = 7.7 Hz, H-1a), 4.48 (d, 1H, J1,2 = 7.7 Hz, H-1b), 4.71 (H-1c), 4.72,

Oligosaccharide Combinatorial Library Synthesis
389
4.89 (each d, 2H, Jgem = 12.3 Hz, C6H4-CH2), 7.47, 7.73 (each d, 4H, Ar); 13C
NMR (200 MHz, D2O): δ = 41.6 (NCH2CO), 60.2, 61.0, 61.1 (C-6a, C-6b, and C-
6c), 68.6, 68.6 (C-4b and C-4c), 70.2 (C-2a), 70.5 (C6H4-CH2), 71.0 (C-2b), 71.2
(C-2c), 72.4, 72.6 (C-3b and C-3c), 74.1, 74.7, 75.0 (C-5a, C-5b, and C-5c), 75.3
(C-4a), 81.8 (C-3a), 101.5 (C-1a), 102.9 (C-1c), 104.6 (C-1b), 127.5, 128.6 (CAr
-
H), 132.6, 141.1 (CAr), 170.9 (CONH), 173.7 (COOH); HMBC NMR (800 MHz,
D2O): H-1a → C6H4-CH2, H-1b → C-3a, H-1c → C-4a; ESI-FT-MS, calcd. for
C28H40NO−19 (M - H)− : 694.2200, found: 694.2197.
[4-(Carboxymethylcarbamoyl)phenyl]methyl O-
(β-D-Galactopyranosyl)-(1→4)-O-(β-D-galactopyranosyl)-
(1→3)-β-D-galactopyranoside (44)
To synthesize compound 44, similarly to the syntheses of compounds 40
and 42, the hydroxy functionalized HMBA-Gly-HMBA-AM resin (20, 35 mg,
0.032 mmol, 0.9126 mmol/g) was used in the same reaction conditions, up to
the synthesis of the di-Gal acceptor with a selectively free hydroxyl group at
the 4-position (35). To cap the hydroxyl group at the 4-position, benzoylation
was performed (Bz capping) similarly to the method described previously. The
next step was Edman degradation for selective deprotection of the hydroxyl
group at the 4ꢁ-position of di-Gal. For selective deprotection of the monomeric-
UCHP protected with Boc group, one cycle of the Edman degradation reaction
(de-Boc and PITC coupling) was performed to selectively synthesize the di-Gal
acceptor with a selectively free hydroxyl group at the 4ꢁ-position (36), similarly
to the method described previously.
The last step was coupling with a third galactose. After adding a galac-
tose donor (12, 200 mg, 0.189 mmol), NIS (128 mg, 0.569 mmol), and
dichloromethane (500 µL) to the 4ꢁ-OH resin (36), the mixture was cooled at
–30◦C, and TfOH (50 µL, 0.565 mmol) was added. Using the same reaction
conditions, workup, and purification procedures for the synthesis of compound
42, the target trigalactose Galβ1-4Galβ1-3Galβ1-linker (44, 1.74 mg, retention
time of 25.1 min) was synthesized and purified. The glycosylation yield was de-
termined to be 59%, which was calculated from the area under the peaks. The
total yield was 8% (19 steps from 16). At the same time, the monogalactose
Galβ1-linker (23, 1.2 mg, retention time of 20.2 min) and digalactose Galβ1-
3Galβ1-linker (31, 0.86 mg, retention time of 23.7 min) were also purified.
1
Compound 44 (Galβ1-4Galβ1-3Galβ1-linker); [α]D+1.9 (c 0.2, MeOH); H
NMR (800 MHz, D2O): δ = 3.46 (dd, 1H, J1,2 = 7.9 Hz, J2,3 = 9.9 Hz, H-2c),
3.55 (dd, 1H, J2,3 = 9.9 Hz, J3,4 = 3.5 Hz, H-3c), 3.55–3.62 (m, 3H, C-5a, C-5b
and C-5c), 3.57 (m, 1H, H-2b), 3.62 (m, 1H, H-2a), 3.62–3.73 (m, 6H, H-6a, H-
6b, and H-6c), 3.65 (m, 1H, H-3b), 3.68 (m, 1H, H-3a), 3.79 (d, 1H, J3,4 = 3.3
Hz, H-4c), 4.05 (d, 1H, J3,4 = 3.2 Hz, H-4a), 4.06 (d, 1H, J3,4 = 3.1 Hz H-4b),

S. Komba & S. Machida
390
4.08 (s, 2H, NCH2CO), 4.41 (d, 1H, J1,2 = 7.9 Hz, H-1a), 4.48 (d, 1H, J1,2
7.9 Hz, H-1c), 4.53 (d, 1H, J1,2 = 7.8 Hz, H-1b), 4.73, 4.90 (each d, 2H, Jgem
=
=
12.3 Hz, C6H4-CH2), 7.48, 7.73 (each d, 4H, Ar); 13C NMR (200 MHz, D2O):
δ = 41.6 (NCH2CO), 60.4, 60.8, 60.9 (C-6a, C-6b, and C-6c), 68.4 (C-4a), 68.5
(C-4c), 69.9 (C-2a), 70.5 (C6H4-CH2), 71.4, 71.4 (C-2b and C-2c), 72.7 (C-3c),
72.8 (C-3b), 74.1, 74.8, 75.1 (C-5a, C-5b, and C-5c), 77.1 (C-4b), 82.2 (C-3a),
101.5 (C-1a), 104.2 (C-1c), 104.3 (C-1b), 127.5, 128.6 (CAr-H), 132.5, 141.1 (CAr),
170.9 (CONH), 173.6 (COOH); HMBC-NMR (800 MHz, D2O): H-1a → C6H4-
CH2, H-1b → C-3a, H-1c → C-4b; ESI-FT-MS, calcd. for C28H40NO−19 (M - H)− :
694.2200, found: 694.2197.
[4-(Carboxymethylcarbamoyl)phenyl]methyl O-
(β-D-Galactopyranosyl)-(1→3)-O-(β-D-galactopyranosyl)-
(1→4)-β-D-galactopyranoside (46)
To synthesize compounds 46 and 48, the reaction was initially performed
in a single reaction vessel. At the point of diverging reaction conditions (after
synthesis of compound 37), the resin was divided. The hydroxy functionalized
HMBA-Gly-HMBA-AM resin (20, 173 mg, 0.158 mmol, 0.9126 mmol/g) was
packed into a 20-mL disposable chromatography column (Econo-Pac; Bio-Rad
Laboratories Inc., Hercules, CA). This hydroxy functionalized resin was glyco-
sylated with a galactose donor (12, 1 g, 0.946 mmol) using DMTST (734 mg,
2.84 mmol) in dichloromethane (15 mL) at rt for 1 day of agitation. Using the
same reaction conditions, workup, and HPLC analysis procedure as previously
described, the mono-glycosylated compound 23 was detected at 20.3 min, and
the glycosylation yield was determined as 59% from HPLC analysis. Using
the same reaction conditions as that previously described, the steps of (1) re-
Boc, (2) Bz capping, and (3) one cycle of Edman degradation reaction (de-Fmoc
and PITC coupling) were performed, and a Gal acceptor with a selectively free
hydroxyl group at the 3-position (26) was selectively synthesized. Aliquots of
the resin (26, 70.30 mg) were taken and used in the next reaction. Benzoyla-
tion was performed (Bz capping) similarly to the method described previously
to cap the hydroxyl group at the 3-position. The reaction was continued until
a negative result in the methyl red-DIC test. The next step was the Edman
degradation step for selective deprotection of the 4-position of hydroxyl group.
For selective deprotection of the monomeric-UCHP protected with Boc group,
one cycle of Edman degradation reaction (de-Boc and PITC coupling) was per-
formed to selectively synthesize the mono-Gal acceptor with a selectively free
hydroxyl group at the 4-position (29), similar to the method described previ-
ously. All reactions were also monitored by the combination of the chloranil
and methyl red-DIC tests.
The next step was coupling with a second galactose. A galactose donor
(12, 360 mg, 0.341 mmol), NIS (230 mg, 1.02 mmol), and dichloromethane

Oligosaccharide Combinatorial Library Synthesis
391
(1 mL) were then added to the 4-OH resin (29, 89.16 mg), the mixture was
cooled at –30◦C, and TfOH (90 µL, 1.02 mmol) was added. After agitation for
1 day at –30◦C, the solution was removed. The resin was washed thoroughly
with DMF (5 × 4 mL) and dichloromethane (5 × 4 mL). The coupling yield
was determined using analytical HPLC as in the previously described proce-
dure; Galβ1-4Galβ1-linker (33) was detected at 23.2 min, and the glycosyla-
tion yield was determined as 53%. Subsequently, the protected exposed amino
group on di-Gal by Boc group (re-Boc) and the unreacted hydroxyl group on
mono-Gal were capped with benzoyl groups (Bz capping). Again, to deprotect
Fmoc-protected monomeric-UCHP, one cycle of Edman degradation reaction
(de-Fmoc and PITC coupling) was performed to synthesize a di-Gal acceptor
with a selectively free hydroxyl group at the 3ꢁ-position (37, 117.96 mg). This
resin was divided into fractions of 50 mg and 63 mg for the synthesis of com-
pounds 46 and 48, respectively.
The last step was coupling with a third galactose. A galactose donor
(12, 200 mg, 0.189 mmol), NIS (128 mg, 0.569 mmol), and dichloromethane
(500 µL) were added to the 3ꢁ-OH resin (37, 50 mg), the mixture was cooled at
–30◦C, and TfOH (50 µL, 0.565 mmol) was added. Using the same reaction con-
ditions, workup, and purification procedure as for the synthesis of compound
42, the target trigalactose Galβ1-3Galβ1-4Galβ1-linker (46, 3.03 mg, retention
time of 25.1 min) was synthesized and purified. The glycosylation yield was
determined to be 70%, as calculated from the area under the peaks. The to-
tal yield was 14% (14 steps from 16). At the same time, the monogalactose
Galβ1-linker (23, 1.2 mg, retention time of 20.3 min) and digalactose Galβ1-
4Galβ1-linker (33, 0.49 mg, retention time of 23.2 min) were also purified.
1
Compound 46 (Galβ1-3Galβ1-4Galβ1-linker); [α]D+0.7 (c 0.3, MeOH); H
NMR (500 MHz, D2O): δ = 3.50 (dd, 1H, J1,2 = 7.6 Hz, J2,3 = 10.0 Hz, H-2c),
3.55 (dd, 1H, J1,2 = 7.1 Hz, J2,3 = 10.3 Hz, H-2a), 3.56 (dd, 1H, J2,3 = 10.6
Hz, J3,4 = 3.3 Hz, H-3c), 3.82 (dd, 1H, J3,4 = 3.3 Hz, J4,5 = 0.7 Hz, H-4c), 4.07
(broad d, 2H, H-4a, and H-4b), 4.09 (s, 2H, NCH2CO), 4.40 (d, 1H, J1,2 = 7.8
Hz, H-1a), 4.51 (d, 1H, J1,2 = 7.6 Hz, H-1c), 4.55 (d, 1H, J1,2 = 7.8 Hz, H-1b),
4.72, 4.90 (each d, 2H, Jgem = 12.3 Hz, C6H4-CH2), 7.48, 7.74 (each d, 4H, Ar);
13C NMR (125 MHz, D2O): δ = 41.6 (NCH2CO), 60.4, 60.9 (C-6a, C-6b, and
C-6c), 68.4 (C-4b), 68.5 (C-4c), 70.5 (C6H4-CH2), 70.6 (C-2b), 71.0 (C-2c), 71.2
(C-2a), 72.5 (C-3c), 73.2 (C-3a), 74.3, 74.7, 75.0 (C-5a, C-5b, and C-5c), 77.3 (C-
4a), 82.1 (C-3b), 101.7 (C-1a), 104.0 (C-1b), 104.3 (C-1c), 127.5, 128.5 (CAr-H),
132.5, 141.2 (CAr), 170.9, 173.6 (2C=O); HMBC NMR (500 MHz, D2O): H-1a
→ C6H4-CH2, H-1b → C-4a, H-1c → C-3b; ESI-FT-MS, calcd. for C28H40NO1−9
(M - H)− : 694.2200, found: 694.2200.
Compound 33 (Galβ1-4Galβ1-linker); [α]D –1.5 (c 0.3, MeOH); 1H NMR
(400 MHz, D2O): δ = 3.51 (dd, 1H, J1,2 = 7.7 Hz, J2,3 = 9.9 Hz, H-2b), 3.57
(dd, 1H, J1,2 = 8.0 Hz, J2,3 = 9.7 Hz, H-2a), 3.58 (m, 1H, H-3b), 3.59–3.67 (m,
2H, H-5a and H-5b), 3.64 (m, 1H, H-3a), 3.64–3.80 (m, 4H, H-6a and H-6b),

S. Komba & S. Machida
392
3.82 (d, 1H, J3,4 = 3.1 Hz, H-4b), 3.91 (s, 2H, NCH2CO), 4.09 (d, 1H, J3,4
=
2.9 Hz, H-4a), 4.42 (d, 1H, J1,2 = 7.7 Hz, H-1a), 4.51 (d, 1H, J1,2 = 7.7 Hz, H-
1b), 4.74, 4.92 (each d, 2H, Jgem = 12.3 Hz, C6H4-CH2), 7.49, 7.76 (each d, 4H,
Jo,m = 8.2 Hz, Ar); 13C NMR (200 MHz, D2O): δ = 43.4 (NCH2CO), 60.4 (C-6a),
60.9 (C-6b), 68.5 (C-4b), 70.5 (C6H4-CH2), 71.2 (C-2a), 71.3 (C-2b), 72.7 (C-3b),
73.1 (C-3a), 74.2 (C-5a), 75.0 (C-5b), 77.0 (C-4a), 101.7 (C-1a), 104.2 (C-1b),
127.4, 128.5 (CAr-H), 133.1, 140.8 (CAr), 170.3 (NCO), 176.4 (COOH); HMBC
NMR (800 MHz, D2O): H-1a → C6H4-CH2, H-1b → C-4a; ESI-FT-MS, calcd.
for C22H31NO14Na+ (M + Na)+ : 556.1637, found: 556.1640.
[4-(Carboxymethylcarbamoyl)phenyl]methyl O-
(β-D-Galactopyranosyl)-(1→4)-O-(β-D-galactopyranosyl)-
(1→4)-β-D-galactopyranoside (48)
To synthesize compound 48, the di-Gal acceptor with a selectively free
hydroxyl group at the 3ꢁ-position (37, 63.48 mg) that was synthesized for
compound 46 was used. To protect the hydroxyl group at the 3ꢁ-position, ben-
zoylation was performed (Bz capping) similarly to the method described previ-
ously. The next step was Edman degradation for selective deprotection of the
hydroxyl group at the 4ꢁ-position of di-Gal. For selective deprotection of the
monomeric-UCHP protected with Boc group, one cycle of the Edman degrada-
tion reaction (de-Boc and PITC coupling) was performed to selectively synthe-
size the di-Gal acceptor with a selectively free hydroxyl group at the 4ꢁ-position
(38), similar to the method described previously.
The last step was coupling with a third galactose. A galactose donor
(12, 200 mg, 0.189 mmol), NIS (128 mg, 0.569 mmol), and dichloromethane
(500 µL) were next added to the 4ꢁ-OH resin (38), the mixture was cooled at
–30◦C, and TfOH (50 µL, 0.565 mmol) was added. Using the same reaction
condition, workup, and purification procedure as for the synthesis of compound
42, the target trigalactose Galβ1-4Galβ1-4Galβ1-linker (48, 2.90 mg, retention
time of 24.8 min) was synthesized and purified. The glycosylation yield was de-
termined as 42%, as calculated from the area under the peaks. The total yield
was 13% (17 steps from 16). At the same time, the monogalactose Galβ1-linker
(23, 1.0 mg, retention time of 20.3 min) and digalactose Galβ1-4Galβ1-linker
(33, 1.56 mg, retention time of 23.2 min) were also purified.
Compound 48 (Galβ1-4Galβ1-4Galβ1-linker); [α]D+3.9 (c 0.3, H2O); 1H
NMR (500 MHz, D2O): δ = 3.49 (dd, 1H, J1,2 = 7.8 Hz, J2,3 = 9.9 Hz, H-2c),
3.80 (d, 1H, J3,4 = 3.4 Hz, H-4c), 4.07 (broad s, 2H, H-4a and H-4b), 4.09 (s, 2H,
NCH2CO), 4.40 (d, 1H, J1,2 = 7.8 Hz, H-1a), 4.49 (d, 1H, J1,2 = 7.8 Hz, H-1c),
4.54 (d, 1H, J1,2 = 7.9 Hz, H-1b), 4.72, 4.90 (each d, 2H, Jgem = 12.3 Hz, C6H4-
CH2), 7.48, 7.74 (each d, 4H, Jo,m = 8.3 Hz, Ar); 13C NMR (125 MHz, D2O):
δ = 41.6 (NCH2CO), 60.5, 60.6, 60.9 (C-6a, C-6b, and C-6c), 68.6 (C-4c), 70.6
(C6H4-CH2), 71.2 (C-2a), 71.3 (C-2c), 71.8 (C-2b), 72.7 (C-3c), 73.2, 73.2 (C-3a

Oligosaccharide Combinatorial Library Synthesis
393
and C-3b), 74.4, 74.4, 75.1 (C-5a, C-5b, and C-5c), 77.1 (C-4b), 77.6 (C-4a),
101.8 (C-1a), 104.3 (C-1c), 104.3 (C-1b), 127.5, 128.6 (CAr-H), 132.5, 141.2 (CAr),
170.9, 173.5 (2C=O); HMBC NMR (500 MHz, D2O): H-1a → C6H4-CH2, H-1b
→ C-4a, H-1c → C-4b; ESI-FT-MS, calcd. for C28H40NO−19 (M - H)− : 694.2200,
found: 694.2198.
ACKNOWLEDGMENTS
This work was supported by the Program for Promotion of Basic Research Ac-
tivities for Innovative Bioscience (PROBRAIN) and Precursory Research for
Embryonic Science and Technology (PRESTO). We thank Dr. T. Terauchi, Ms.
S. Sasaki, Ms. J. Maru, Ms. E. Moriwaki, Ms. H. Kunifuda, Ms. A. Utsugi, and
Ms. T. Watanabe for technical assistance.
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