Efficient synthesis of L-galactose from D-galactose

Article abstract of DOI:10.1080/07328303.2015.1115514

Racemic protein crystallography is an emerging methodology to obtain high-resolution protein structures. Preparation of both a mirror image of protein and a mirror image of glycan is essential to apply the new methodology to glycoprotein. This article describes an efficient synthesis of L-galactose that is amirror image of D-galactose. The developed method takes only 6 steps from D-galactose without any chromatographic purification.

Full text of DOI:10.1080/07328303.2015.1115514

Journal of Carbohydrate Chemistry
ISSN: 0732-8303 (Print) 1532-2327 (Online) Journal homepage: http://www.tandfonline.com/loi/lcar20
Efficient Synthesis of L-galactose from D-galactose
Ryo Orii, Masayuki Izumi, Yasuhiro Kajihara & Ryo Okamoto
To cite this article: Ryo Orii, Masayuki Izumi, Yasuhiro Kajihara & Ryo Okamoto (2015) Efficient
Synthesis of L-galactose from D-galactose, Journal of Carbohydrate Chemistry, 34:9, 560-566,
DOI: 10.1080/07328303.2015.1115514
To link to this article: http://dx.doi.org/10.1080/07328303.2015.1115514
View supplementary material
Published online: 14 Dec 2015.
Submit your article to this journal
Article views: 13
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at
http://www.tandfonline.com/action/journalInformation?journalCode=lcar20
Download by: [Library Services City University London]
Date: 17 December 2015, At: 23:53

Journal of Carbohydrate Chemistry, 34:560–566, 2015
ꢀ
C
Copyright Taylor & Francis Group, LLC
ISSN: 0732-8303 print / 1532-2327 online
DOI: 10.1080/07328303.2015.1115514
Efficient Synthesis of
L-galactose from D-galactose
Ryo Orii, Masayuki Izumi, Yasuhiro Kajihara, and Ryo Okamoto
Department of Chemistry, Graduate School of Science, Osaka University, 1-1,
Machikaneyama, Toyonaka, 560-0043 Japan
GRAPHICAL ABSTRACT
Racemic protein crystallography is an emerging methodology to obtain high-resolution
protein structures. Preparation of both a mirror image of protein and a mirror image of
glycan is essential to apply the new methodology to glycoprotein. This article describes
an efficient synthesis of L-galactose that is a mirror image of D-galactose. The developed
method takes only 6 steps from D-galactose without any chromatographic purification.
Keywords L-galactose; D-galactose; Mirror image oligosaccharide
INTRODUCTION
Recently we have demonstrated a facile crystallization using quasi-racemic
glycoprotein.[1] This glycoprotein derivative is a mixture of equimolar amounts
of a native glycoprotein and the mirror image isomer of the non-glycosylated
native protein (the D-protein) consisting of D-amino acids and achiral amino
acid glycine. The unique crystallization would be a powerful approach for the
X-ray structural analysis of glycoproteins. However, the resolution obtained
˚
was still moderate (2.1 A). The structural information with higher resolution
is essential to precisely elucidate the role of the glycosylation on the protein
function.
Received October 8, 2015; accepted October 29, 2015.
Address correspondence to Ryo Okamoto, Department of Chemistry, Graduate School
of Science, Osaka University, 1-1, Machikaneyama, Toyonaka, 560-0043 Japan. E-mail:
rokamoto@chem.sci.osaka-u.ac.jp
560

Efficient Synthesis of L-galactose
561
Figure 1: Retrosynthetic analysis of L-galactose 1.
In contrast to the quasi-racemic glycoprotein crystals, the racemic protein
˚
˚
crystals give rise to ca. 1.5 A resolution in average (0.95 A for the highest
resolution) among 11 structures reported.[2] The racemic protein is a 1:1 mix-
ture of a native protein and the D-protein. To circumvent the relatively low-
resolution structure from the quasi-racemic glycoprotein crystals, the perfect
racemic glycoprotein crystallization would be a promising approach. In this
case, the preparation of the mirror image of natural oligosaccharide was to be
essential to conduct such a new approach.
Among the diverse oligosaccharides on naturally occurring glycopro-
teins, we were interested in T-antigen consisting of D-galactose and N-
acetyl-D-galactosamine. T-Antigen is a typical O-linked oligosaccharide and is
known as a cancer antigen. We envisioned that the racemic O-glycoproteins
prepared by using the synthetic mirror image T-antigen would give us
high-resolution structures. Such unprecedented structural information con-
tributes to the designing of cancer vaccines.[3-6] To synthesize mirror im-
age O-glycoproteins in sufficient amount, stable supplying of L-galactose
and N-acetyl-L-galactosamine are essential. Of these compounds, N-acetyl-L-
galactosamine can be prepared from L-galactose through Lemieux’s protocol.[7]
Therefore, we set out to develop a practical synthetic protocol for L-galactose.
Herein we report the detail of the practical synthetic protocol for L-galactose 1
from D-galactose 2 in 6 steps without any chromatographic purification.
RESULTS AND DISCUSSION
Owing to the inherent symmetric configuration of D-galactose 2, the synthesis
of L-galactose 1 has been conventionally achieved from D-galactose 2 through
a reduction reaction of the anomeric position followed by a selective oxidation
of a hydroxy group at the 6 position (6-OH) to afford a new anomeric position
for L-galactose.[8-10] Figure 1 shows our retrosynthetic analysis of L-galactose
1 from D-galactose 2. We selected trityl group for the protection of the 6-OH
of D-galactose 2.[11,12] After the reduction reaction at the anomeric position, all
hydroxy groups were designed to be protected with acetyl group. Importantly,
we expected that the use of trityl group would confer the crystalline nature to
the intermediates, simplifying each purification step.

R. Orii et al.
562
The selective O-tritylation of D-galactose 2 was carried out using trityl
chloride in pyridine (Sch. 1). The reaction mixture was initially a suspension
but it became a clear solution with progress of the reaction. After evapora-
tion of pyridine, remaining D-galactose was removed by extraction. This sim-
ple manipulation gave 6-O-trityl-D-galactose 3 with sufficient purity that was
suitable to be used for the next reaction without further purification. The crude
3 was then treated with NaBH4 for the reduction of the anomeric position. We
found that washing of the resultant residue with acidic aqueous solution af-
ter the reaction was essential because we observed only broad spectrum in the
absence of any particular 1H NMR signals without the washing step. We specu-
lated that NaBH4 formed boronate ester with sugar hydroxy groups leading to
putative oligomeric form of the galactose derivatives. Indeed after washing of
the crude product with 50 mM HCl solution, it gave clear NMR signal of 4 with
sufficient purity. The subsequent per-acetylation of remaining hydroxy groups
of compound 4 gave the desired protected galactitol derivative 5 in a 33% yield,
corresponding to 3.85 g of 5 from 4.85 g of D-galactose 2 after crystallization.
In order to make a new anomeric aldehyde of L-galactose 1, we conducted
the selective removal of the trityl group followed by an oxidation reaction (Sch.
1). The removal of the trityl group was successfully performed using NaI and
trimethylsilyl chloride (TMSCl)[13] without considerable side reactions such as
acetyl migration. The subsequent oxidation of compound 6 with Dess-Martin
periodinane yielded aldehyde 7. These two reactions gave each desired com-
pound almost quantitatively without silica gel column chromatography but
only with extraction to remove the residual reagents. Finally, Zemple´n deacety-
lation afforded the desired L-galactose 1. The final product 1 was obtained in
excellent yield (63%, corresponding to 895 mg of 1 from 5 g of 5) over 3 steps
after crystallization.
Scheme 1: Synthesis of L-galactose 1 from D-galactose 2.

Efficient Synthesis of L-galactose
563
In summary, here we reported an efficient synthesis of L-galactose from D-
galactose in only 6 steps without any chromatographic purification. This prac-
tical protocol will give us opportunities to synthesize mirror image oligosaccha-
rides and consequent racemic glycoproteins for the X-ray structural analysis
of glycoproteins.
EXPERIMENTAL SECTION
General Procedures
All reactions were monitored by TLC on Silica gel 60 F254 (Merck TLC
plates) and visualizations were performed with UV light (254 nm) and/or
sulfuric acid stain (3% H2SO4 in CH3OH). All reagents were purchased from
commercial suppliers and were used without further purification. All the
reaction solvents were dried over activated molecular sieves prior to use.
◦
1
Solvents were removed under reduced pressure at <40 C. H and 13C NMR
spectra were recorded on Bruker Avance III 400 MHz spectrometer. All NMR
1
signals were assigned on the basis of H NMR, COSY, HMBC and HSQC.
HRMS were recorded on FT-ICR ESI-MS by electron spray ionization method.
Synthesis of 1,2,3,4,5-O-acetyl-6-O-trityl-D-galactitol (5)
To a suspension of D-galactose 2 (4.85 g, 26.9 mmol) in pyridine (49 mL)
was added trityl chloride (5.00 g, 17.9 mmol). The mixture was then warmed
up to 50◦C and stirred for 48 h under argon atmosphere at 50◦C. The reac-
tion mixture was then concentrated in vacuo and the residue was dissolved in
1-butanol. The solution was washed with water (ca. 100 mL × 2), brine (ca.
100 mL), dried over Na2SO4, and concentrated in vacuo to give crude 6-O-
trityl-D-galactose (3) and used for the next step without further purification
(see Fig. S1 for the 1H NMR of the crude material).
The crude compound 3 was dissolved in CH3OH (60 mL) and chilled in
ice-bath. To the solution was added NaBH4 (0.75 g, 19.8 mmol) portionwise
over 15 min under stirring in ice-bath. The reaction mixture was allowed to
warm up to room temperature and stirred for 2 h. To the solution was added
additional NaBH4 (0.68 g, 17.4 mmol) and resulted in suspension which was
stirred for additional 2 h. Then the reaction mixture was acidified by the addi-
tion of 10% acetic acid/CH3OH (v/v) to quench the NaBH4 until the suspension
turned into a clear solution. The solution was diluted with 50 mM HCl solu-
tion (ca. 80 mL) to afford the product as precipitate. The resultant precipitate
was separated by centrifuge (4000 rpm for 10 min) and washed with 50 mM
HCl solution (ca. 60 mL). This washing was repeated for 5 times, then the
resultant precipitate was washed with water (ca. 60 mL) by the same manner

R. Orii et al.
564
for three times. To the final precipitate was added water and the resultant sus-
pension was subjected to lyophilization to give crude 6-O-trityl-D-galactitol (4).
The lyophilized crude compound 4 was used for the next step without further
purification (Fig. S2 for the 1H NMR of the obtained material).
The crude compound 4 was dissolved in pyridine (39 mL) and acetic anhy-
dride (39 mL). The mixture was stirred for 50 h at room temperature under
argon. The mixture was allowed to cool to 0◦C and CH3OH was added drop-
wisely. After concentration of the solution in vacuo, the residue was dissolved
in ethyl acetate. The organic layer was washed with 1 M HCl solution, satu-
rated aqueous NaHCO3 solution, brine, dried over Na2SO4 and concentrated in
vacuo. The resultant crude material was crystallized from ethanol/petroleum
ether and gave 1,2,3,4,5-penta-O-acetyl-6-O-trityl-D-galactitol (3.85 g, 33%
over three steps). Analytical data for 5: mp. 135◦C-138◦C, [α]D = −30.0
17
(c 1.0, CHCl3); Rf=0.32 (ethyl acetate-hexane, 1:2, v/v); 1H NMR (CDCl3):
δ7.41-7.20 (m, 15H, aromatic), 5.43 (dd, 1H, J4,3 9.8 Hz, J4,5 2.0 Hz, H-4), 5.34
(ddd, 1H, J5,4 2.0 Hz, J5,6a 5.9 Hz, J5,6b 7.4 Hz , H-5), 5.31 (dd, 1H, J3,2 1.8 Hz,
J3,4 9.8 Hz, H-3), 5.18 (ddd, 1H, J2,1a 11.7 Hz, J2,1b 5.0 Hz, J2,3 1.8 Hz , H-2),
4.23 (dd, 1H, J1b,1a 11.7 Hz, J1b,2 5.0 Hz, H-1b), 3.84 (dd, 1H, J1a,1b 11.7 Hz,
J1a,2 7.5 Hz, H-1a), 3.11 (dd, 1H, J6b,6a 9.9 Hz, J6b,5 7.4 Hz, H-6b), 3.00 (dd, 1H,
J6a,6b 9.9 Hz, J6a,5 5.9 Hz, H-6a), 2.13 (s, 3H, CH3CO), 2.08 (s, 3H, CH3CO),
2.07 (s, 3H, CH3CO), 2.01 (s, 3H, CH3CO), 1.81 (s, 3H, CH3CO); 13C NMR
(CDCl3): δ 170.45 (C=O), 170.25 (2 C=O), 169.84 (C=O), 169.23 (C=O), 143.37
(Cquat), 128.64 (Ph), 127.82 (Ph), 127.14 (Ph), 87.00 (CPh3), 68.85 (C-5), 67.77
(C-2), 67.58 (C-3), 67.55 (C-4), 62.18 (C-1), 61.52 (C-6), 20.90 (CH3CO), 20.75
(CH3CO), 20.70 (CH3CO), 20.66 (CH3CO), 20.31 (CH3CO). ESI-HRMS calcd
for C35H38O11Na [M+Na]+, 657.2306; found 657.2298.
Synthesis of L-galactose (1)
Compound 5 (5 g, 7.9 mmol) was dissolved in CH3CN (158 mL) and cooled
at −20◦C. To the solution was added NaI (15.43 g, 102.4 mmol) and trimethylsi-
lyl chloride (13 mL, 102.4 mmol) and stirred for 80 min at −20◦C under ar-
gon atmosphere. Then, the reaction mixture was allowed to warm up to 0◦C
and distilled water (50 mL) was added to the mixture. The reaction mixture
was stirred for 15 min at 0◦C, then diluted with CH2Cl2. The organic layer
was washed with 5% aqueous Na2S2O3 solution, water, dried over Na2SO4,
and concentrated in vacuo to give the desired crude 1,2,3,4,5-penta-O-acetyl-
D-galactitol (6). The resultant crude compound 6 was used for the next step
without further purification (see Fig. S5 for the 1H NMR of the obtained crude
material).
To a solution of the crude compound 6 in CH2Cl2 (55 mL) was added a
suspension of Dess-Martin periodinane (5.03 g, 11.9 mmol) in CH2Cl2 (34 mL,

Efficient Synthesis of L-galactose
565
0.35 mol/L for Dess-Martin periodinane). After being stirred for 110 min at
room temperature, saturated aqueous NaHCO3 solution (70 mL) and 5% aque-
ous Na2S2O3 (120 mL) solution were added to the reaction mixture and the re-
sulting suspension was stirred for additional 20 min. The suspension was then
diluted with CH2Cl2 and washed with water, brine, dried over Na2SO4, and
concentrated in vacuo to give the desired 2,3,4,5,6-penta-O-acetyl-aldehydo-L-
galactose (7). The resultant crude 7 was used for the next step without further
purification (see Fig. S6 for the 1H NMR of the obtained crude material).
To the crude compound 7 in CH3OH (80 mL) was allowed to cool to 0◦C
under argon atmosphere. To the suspension was added NaOCH3 (213 mg,
3.94 mmol) and stirred for 5.5 h at 0◦C. The suspension was then neutral-
ized with cation exchanging resin (Amberlite IR120 [H+ form]). The resin was
removed by filtration and washed with CH3OH and water. All filtrate were
combined and concentrated in vacuo. To the residue was added H2O to afford
a precipitate of the residual compound from the trityl group deprotected at
the previous step. The precipitate was filtered off with celite-chacoal bed and
the filtrate was concentrated in vacuo. Crystallized of the residue from H2O/
CH3OH gave L-galactose (1) (895 mg, 63% over three steps). L-galactose 1:
Analytical data for 1: mp. 159◦C-161◦C, Rf=0.17 (ethyl acetate-CH3OH-H2O,
17
1
10:5:1, v/v); [α]D =−77.4 (c 1.0, H2O); H NMR (D2O): δ5.26 (d, 0.3H, J1,2
3.6 Hz, H-1α), 4.58 (d, 0.7H, J1,2 8.0 Hz, H-1β), 4.09 (t, 0.3H, H-5α), 3.99 (d,
0.7H, J4,3 3.0 Hz, H-4α), 3.93 (d, 0.7H, J4,3 3.5 Hz, H-4β), 3.86 (dd, 0.3H, J3,2
3.2 Hz, J3,4 3.0 Hz, H-3α), 3.80 (dd, 0.3H, J2,1 3.6 Hz, J2,3 3.2 Hz, H-2α), 3.78-
3.67 (m, 2.7H, H-5β, H-6aα, H-6bα, H-6aβ, H-6bβ), 3.65 (dd, 0.7H, J3,2 9.9, J3,4
3.5, H-3β), 3.49 (dd, 0.7H, J2,1 8.0, J2,3 9.9 H-2β); 13C NMR (D2O): δ 96.41 (C-
1β), 92.24 (C-1α), 75.11 (C-5β), 72.76 (C-3β), 71.83 (C-2β), 70.44 (C-5α), 69.20
(C-4α), 69.12 (C-3α), 68.70 (C-4β), 68.30 (C-2α), 61.14 (C-6α), 60.94 (C-6β),; ESI-
HRMS Cald for C6H12O6 [M+Na]+ 203.0526, found 203.0528.
FUNDING
This work was supported by JSPS KAKENHI (24750161): Grant-in-Aid-for
Young Scientists (B).
SUPPLEMENTAL MATERIAL
Supplementary data (1H NMR spectra of compounds 1, 3-7 and 13C NMR
spectra of compounds 1,4,5) associated with this article can be found on the
publisher’s website.

R. Orii et al.
566
REFERENCES
1. Okamoto, R.; Mandal, K.; Sawaya, M. R.; Kajihara, Y.; Yeates, T. O.; Kent, S. B.
H. (Quasi-)Racemic x-ray structures of glycosylated and non-glycosylated forms of the
chemokine ser-CCL1 prepared by total chemical synthesis. Angew. Chem. Int. Ed. 2014,
53, 5194–5198.
2. Yeates, T. O.; Kent, S. B. H. Racemic protein crystallography. Annu. Rev. Biophys.
2012, 41, 41–61.
3. Barchi, J. J. Mucin-type glycopeptide structure in solution: Past, present, and fu-
ture. Biopolymers 2013, 99, 713–723.
4. Gaidzik, N.; Westerlind, U.; Kunz, H. The development of synthetic antitumour
vaccines from mucin glycopeptide antigens. Chem. Soc. Rev. 2013, 42, 4421–4442.
5. Danishefsky, S. J.; Allen, J. R. From the laboratory to the clinic: A retrospective
on fully synthetic carbohydrate-based anticancer vaccines. Angew. Chem. Int. Ed. 2000,
39, 836–863.
6. Springer, G. T and Tn, general carcinoma autoantigens. Science 1984, 224,
1198–1206.
7. Lemieux, R. U.; Ratcliffe, R. M. The azidonitration of tri-O-acetyl-D-galactal. Can.
J. Chem. 1979, 57, 1244–1251.
8. Glatthaar, C.; Reichstein, T. l-Tagatose. Helv. Chim. Acta 1937, 20, 1537–1541.
9. Pizzarello, R. A.; Freudenberg, W. A new synthesis of l-galactose. The structure of
α- and β-diacetone dulcitol. J. Am. Chem. Soc. 1939, 61, 611–613.
10. Doboszewski, B.; Herdewijn, P. 1,2;3,4-Di-O-isopropylidene-l-galactose synthesis
from its d-enantiomer. Tetrahedron Lett. 2012, 53, 2253–2256.
11. Bhattacharjee, A. K.; Zissis, E.; Glaudemans, C. P. J. Synthesis of 6-O-(6-O-β-d-
galactopyranosyl-β-d-galactopyranosyl)-d-galactopyranose by use of 2,3,4-tri-O-acetyl-
6-O-(chloroacetyl)-α-d-galactopyranosyl bromide, a key intermediate for the solid-phase
synthesis of β-(1→6)-linked d-galactopyranans. Carbohydr. Res. 1981, 89, 249–254.
12. Cheng, J. M. H.; Chee, S. H.; Knight, D. A.; Acha-Orbea, H.; Hermans, I. F.; Tim-
mer, M. S. M.; Stocker, B. L. An improved synthesis of dansylated α-galactosylceramide
and its use as a fluorescent probe for the monitoring of glycolipid uptake by cells. Car-
bohydr. Res. 2011, 346, 914–926.
13. Brask, J.; Jensen, K. J. Carboproteins: A 4-α-helix bundle protein model assembled
on a d-galactopyranoside template. Bioorg. Med. Chem. Lett. 2001, 11, 697–700.