Reactivity-based one-pot synthesis of the tumor-associated antigen N3 minor octasaccharide for the development of a photocleavable DIOS-MS sugar array

Article abstract of DOI:10.1002/anie.200504067

Sweet and light: Readily available thioglycosides with defined relative reactivity values were used as building blocks in a one-pot strategy to synthesize the tumor-associated carbohydrate antigen N3 minor (1). The target molecule was attached covalently to a porous silicon surface through a photocleavable linker for direct characterization in a mass spectrometer equipped with a laser source. (Chemical Equation Presented).

Full text of DOI:10.1002/anie.200504067

Angewandte
Chemie
Synthesis Design
DOI: 10.1002/anie.200504067
Reactivity-Based One-Pot Synthesis of the
Tumor-Associated Antigen N3 Minor
Octasaccharide for the Development of a
Photocleavable DIOS-MS Sugar Array**
Jinq-Chyi Lee, Chung-Yi Wu, Junefredo V. Apon,
Gary Siuzdak, and Chi-Huey Wong*
Human milk contains a variety of biologically important
oligosaccharides which reflect the lactating motherꢀs Lewis
blood group.[1] Antigens N3, known as N3 major and N3
minor, are examples of such milk-derived octasaccharides
(Scheme 1).[2] N3major, difucosyllacto- N-hexose (1), is the
a
major component of N3antigens containing Le and Lex
moieties. N3minor, difucosyllacto- N-neohexaose (2), con-
tains two Lex units and a lactose unit. The trisaccharide Lex is
one of the carbohydrate determinants of blood groups and is
known to be a tumor-related antigen.[3] The N3antigens
containing one or two Lex units were also overexpressed in
various tumors, including gastrointestinal cancer cells.
Although the synthesis of 1 and 2 has been reported,[4,5]
little is know regarding the function of 2. We describe here
the synthesis of 2 by using the one-pot strategy based on
anomeric reactivity,[6] and the use of this glycan as a model to
develop photocleavable arrays on porous silicon suitable for
direct characterization by mass spectrometry.
Carbohydrate arrays are becoming powerful tools in
glycobiology and glycomics.[7] We have recently reported the
formation of a covalent array of complex oligosaccharides on
glass slides[8] and microtiter plates for high-throughput
analysis of sugar–protein interactions.[9] The microplate
[*] Dr. J.-C. Lee, Dr. C.-Y. Wu, Prof. C.-H. Wong
Department ofChemistry and
The Skaggs Institute for Chemical Biology
The Scripps Research Institute
10550 North Torrey Pines Road, La Jolla, CA 92037 (USA)
Fax: (+1)858-784-2409
E-mail: wong@scripps.edu
Dr. C.-Y. Wu, Prof. C.-H. Wong
The Genomics Research Center
Academia Sinica
No. 128 Academia Road Section 2, Nan-Kang
Taipei, 11529 (Taiwan)
J. V. Apon, Prof. G. Siuzdak
Department ofMolecular Biology and Chemistry and
The Scripps Center for Mass Spectrometry
The Scripps Research Institute
10550 North Torrey Pines Road, La Jolla, CA 92037 (USA)
[**] J.-C.L. and C.-Y.W. contributed equally. This work was supported by
the NIH. C.-Y.W. thanks the National Science Council ofTaiwan and
the Genomics Research Center, Academia Sinica for financial
support. DIOS-MS=desorption/ionization on silicon mass spec-
trometry.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
Angew. Chem. Int. Ed. 2006, 45, 2753 –2757
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2753

Communications
Scheme 2. Retrosynthetic analysis ofthe antigen N3 minor glycoside 3.
Bn=benzyl, Bz=benzoyl, Cbz=benzyloxycarbonyl, Tol=p-methyl-
phenyl, Troc=2,2,2-trichloroethoxycarbonyl.
Scheme 1. The structures ofantigens N3 1, 2, and the amine-contain-
ing analogue 3.
methodology incorporates a cleavable disulfide linker which
allows characterization and quantitative analysis of the array.
However, chemical cleavage of the disulfide bonds is not ideal
for the glass-slide format. Furthermore, microtiter plate
arrays require larger amounts of glycans than glass-slide
arrays do, although microtiter plate arrays have advantages in
certain applications.
Desorption/ionization on silicon mass spectrometry
(DIOS-MS) is an ionization method that uses a porous silicon
surface to generate gas-phase ions of small molecules
(< 3000 Da) without a matrix.[10] With this technique, the
process of sample manipulation is minimized and it only
requires very small volumes for analysis. To test the feasibility
of this system for sugar arrays, we prepared the amine-
containing antigen N3minor 3 and printed it on the surface of
a modified porous silicon with a photocleavable linker, which
could be cleaved by a laser (l = 337 nm) in the mass
spectrometric analysis to detect the carbohydrate on the
porous silicon. A similar photocleavable linker on beads was
used by Gerdes and Waldmann previously.[11]
oligosaccharide 4, which could be assembled by one-pot
glycosylation with three key building blocks: fucosyl unit 5,
lactosaminyl disaccharide unit 6, and lactosyl unit 7. Since the
reactivities of these three building blocks decrease progres-
sively, they can react sequentially in a single flask. The fucosyl
building block 5 (RRV= 7.2 ” 104) and the reducing end unit 7
(RRV= 0) have the highest and the least reactivity, respec-
tively. The lactosaminyl diasccharide 6 (RRV= 41), with a
reactivity falling between 5 and 7, was prepared from
galactosyl donor 8 and acceptor 9 (Scheme 3).
We used the programmable one-pot synthesis strategy to
prepare antigen N3minor 3, since the method is rapid and has
been used in the synthesis of many complex oligosacchar-
ides.[12–16] The methodology is based on the use of designer
thioglycoside building blocks with defined relative reactivity
values (RRVs) that are collected in the Optimer database.[6]
Our retrosynthetic plan of the antigen N3minor octasac-
charide 3 is shown in Scheme 2. The target molecule 3 can be
prepared through a global deprotection of the fully protected
Scheme 3. Relative reactivity values (RRV) ofbuilding blocks 5–9.
Lev=levulinoyl.
The preparation of perbenzylated fucosyl thioglycoside 5
and galactosyl thioglycoside 8 followed literature proce-
dures.[6,17] The synthesis of lactosaminyl disaccharide building
block 6 is illustrated in Scheme 4. Thioglycoside 10,[6]
2754
www.angewandte.org
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2006, 45, 2753 –2757

Angewandte
Chemie
bromide in acetic acid and further coupled with N-(5-
hydroxypentyl)carbamate in the presence of silver carbonate
to provide an inseparable mixture of the expected b-lactoside
and its orthoester by-product. The mixture was subjected to
deacetylation to obtain the desired b-lactoside 13 in 46%
yield over three steps. Regioselective allylation of compound
13 at O3’ with dibutyltin oxide and allyl bromide afforded the
allyl ether product,[19] from which the fully protected lactoside
14 was obtained through 4,6-O-benzylidenation and perben-
zylation (54% over 4 steps). The benzylidene acetal group of
14 was removed under acidic conditions and the primary
hydroxy group of the generated diol was regioselectively
silylated in excellent yield followed by benzylation at O4’ to
yield 15. The TBDPS group was cleaved by treatment with
TBAFand the C3’ allyl ether was removed by using a two-step
sequence. Isomerization of the terminal double bond with
Scheme 4. Synthesis oflactosaminyl disaccharide building block 6.
a) 1. NaOMe, MeOH/CH2Cl2; 2. PhCH(OMe)2, cat. CSA, CH3CN;
AHCTREUNG
3. levulinic acid, DCC, DMAP, 76% over 3 steps; b) 1. TFA/CH2Cl2/
H2O=4:10:1; 2. BzCl, pyridine, CH2Cl2, 73% in 2 steps; c) 1. 8, BSP,
Tf2O, AW-300 molecular sieves, CH2Cl2, 54%. d) H2NNH2/xH2O,
pyridine, AcOH, 88%. CSA=camphorsulfonic acid, DCC=1,3-dicyclo-
hexylcarbodiimide, DMAP=N,N’-dimethylaminopyridine, BSP=ben-
Wilkinsonꢀs catalyst ([RhCl(PPh3)3]) and DBU, with concom-
A
itant hydrolysis of the resulted enol ether using a mixture of
zenesulfinyl piperidine, Tf2O=trifluoromethanesulfonic anhydride,
TFA=trifluoroacetic acid.
aqueous HCl and acetone provided 7 in good yield.[20]
With the desired building blocks 5–7 in hand, we
proceeded to synthesize the antigen N3minor octasaccharide
3 by a reactivity-based one-pot strategy, as outlined in
Scheme 6. Coupling of fucosyl thioglycoside 5 with the less
reactive lactosaminyl disaccharide unit 6 using BSP/Tf2O as a
promoter proceeded cleanly to give the Lex trisaccharide
fragment. After completion of this reaction, without workup,
the lactosyl building block 7 was added in the same pot,
followed by addition of NIS and TfOH as activators to
introduce two trisaccharide moieties at the same time. The
generated from d-glucosamine hydrochloride in three steps,
underwent sequential deacetylation, benzylidenation, and
levulinoylation at O3to yield compound 11. Hydrolysis of 11
gave the corresponding 4,6-diol intermediate, which upon
subsequent selective O6-benzoylation with benzoyl chloride
afforded the acceptor 9. Coupling of the galactosyl thioglyco-
side 8 and 9, with activation by benzenesulfinyl piperidine/
trifluoromethanesulfonic anhydride (BSP/Tf2O) gave the b-
linked disaccharide 12 in 54% yield.[15] The levulinoate group
was finally removed with hydrazine hydrate in an acetic acid/
pyridine mixture and the disaccharide building block 6 was
isolated in 88% yield.[18]
The lactosyl building block 7 was prepared from b-lactose
octaacetate (Scheme 5), which was first transformed into the
corresponding lactosyl bromide by using 33% hydrogen
Scheme 5. Synthesis oflactosyl building block 7. a) 1. Bu2SnO, C6H6;
2. AllBr, TBAI, C6H6; 3. PhCH(OMe)2, cat. CSA, CH3CN; 4. BnBr, 60%
U
NaH, DMF, 54% over 4 steps; b) 1. TFA/CH2Cl2/H2O=4:10:1;
2. TBDPSCl, Et3N, CH2Cl2; 3. BnBr, 60% NaH, DMF, 52% over
3 steps; c) 1. TBAF,THF; 2. Ph
A
Scheme 6. One-pot synthesis ofantigen N3 minor 3. a) 1. BSP, Tf2O,
AW-300 molecular sieves, CH2Cl2, À70 to À108C; 2. NIS, TfOH, 08C
to RT; b) 1. Zn, AcOH, RT; 2. Ac2O, DMAP, pyridine, 08C to RT;
3. NaOMe, MeOH, RT; 4. Pd-black, H2 (1 atm), MeOH with 5% formic
acid, 11% from (a). NIS=N-iodosuccinimide.
H2O=8:3:1; 3. 1m HClaq/acetone=1:9, 69% over 3 steps. All=allyl,
TBDPS=tert-butyldiphenylsilyl, TBAI=tetrabutylammonium iodide,
TBAF=tetrabutylammonium fluoride, DBU=1,8-diazabicyclo-
A
Angew. Chem. Int. Ed. 2006, 45, 2753 –2757
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
2755

Communications
Scheme 7. A) Preparation ofporous silicon sugar array and the resulting DIOS-MS spectrum ofantigen N3 minor 3. a) NaBH4, MeOH, 08C to RT,
97%; b) methyl 4-bromobutyrate, TBAF, RT, 88%; c) 1. LiOH, MeOH, H2O, 08C to RT; 2. DCC, N-hydroxysuccinimide, THF, RT, 82% over 2 steps;
d) 1. Et3N, toluene, RT; 2. N,N’-disuccinimidyl carbonate, Et3N, CH3CN, RT; e) 1. antigen N3 minor 3, Et3N; 2. wash with MeOH and H2O.
B) Control experiment: N3 minor 3 was noncovalentaly deposited on the silylated amino surface 20, and showed no signal by DIOS-MS after it
had been washed.
2756
www.angewandte.org
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2006, 45, 2753 –2757

Angewandte
Chemie
reaction was stirred at room temperature to obtain the
octasaccharide 4, which has the skeleton of N3minor. The
mixture of the desired octasaccharide and the inseparable
Keywords: carbohydrates · glycan array · glycosylation ·
mass spectrometry · synthesis design
.
syl-
unknown by-product was isolated after the one-pot glyco
A
[1] A. Kobata in The Glycoconjugates, Vol. 1 (Eds.: M. I. Horowitz,
W. Pigman), Academic Press, New York, 1977, pp. 423– 440.
[2] a) A. Kobata, Methods Enzymol. 1972, 28, 262 – 271; b) A.
Kobata, V. Ginsburg, Arch. Biochem. Biophys. 1972, 150, 273–
281.
[3] S. Hakomori, Annu. Rev. Immunol. 1984, 2, 103– 126.
[4] S. J. Danishefsky, P. P. Deshpande, I. J. Kim, P. Livingston, J. K.
Hyun, G. Ragupathi, T. K. Park, Pat. WO9830190, 1998.
[5] H. M. Kim, I. J. Kim, S. J. Danishefsky, J. Am. Chem. Soc. 2001,
123, 35 – 48.
ACHTREUNG
tion and chromatography to give pure N3minor antigen (see
the Supporting Information). The two trichloroethyl carba-
mate (Troc) functional groups were removed with activated
Zn nanoparticles in acetic acid, followed by acetylation of the
free amino groups with acetic anhydride and pyridine in the
presence of catalytic amounts of DMAP to afford the
corresponding NHAc derivative. Cleavage of the O-acyl
groups under ZemplØn conditions and deprotection of the
benzyl ether and benzyl carbamate groups were accomplished
by hydrogenolysis with palladium black in 5% formic acid/
methanol solution. The target antigen N3minor 3 was finally
obtained in 11% yield based on the one-pot glycosylation of
building blocks 5–7. The characterization and configuration of
3 were confirmed by NMR spectroscopic and HRMS analysis.
The preparation of the photocleavable sugar array on
porous silicon is shown in Scheme 7. 5-Hydroxyl-2-nitro-
benzaldehyde (16) was reduced with sodium borohydride
(NaBH4) to give compound 17 (97%), which was subse-
quently subjected to regioselective alkylation with methyl 4-
bromobutyrate and TBAF to provide 18 in 88% yield.[21]
Hydrolysis of the methyl ester 18 using LiOH, followed by
reaction with N-hydroxysuccinimide gave the succinic ester
19 (82% over 2 steps). Coupling of 19 with the silylated amino
surface 20[10] furnished the photocleavable linker with a free
hydroxy group, which was transformed into the carbamate
ester 21 in the presence of N,N’-disuccinimidyl carbonate and
triethylamine. Finally, the N3minor 3 was printed directly on
the plate of 21. Extensive washing of the plate with MeOH
and H2O resulted in the formation of the photocleavable
sugar array on the porous silicon (for detailed experimental
procedures, see the Supporting Information). DIOS-MS
analysis showed a strong signal at m/z 1473([ M + Na]+) and
a signal at m/z 1327 (corresponding to the defucosylated
molecule). In the control experiment, we printed the N3
minor 3 on the silylated amino surface 20, which showed no
signal after it had been washed. A mannose array was also
prepared for the binding study with ConA, and the result
showed that nonspecific lectin-surface interactions are insig-
nificant.
[6] Z. Zhang, I. R. Ollmann, X.-S. Ye, R. Wischnat, T. Baasov, C.-H.
Wong, J. Am. Chem. Soc. 1999, 121, 734 – 753.
[7] For recent reviews: a) M. Mrksich, Chem. Biol. 2004, 11, 73 9 –
140; b) T. Feizi, F. Fazio, C. Wengang, C.-H. Wong, Curr. Opin.
Struct. Biol. 2003, 13, 637 – 645; c) K. Drickamer, M. E. Taylor,
Genomebiology 2002, 3, 1034.1 – 1034.4; d) K. R. Love, P. H.
Seeberger, Angew. Chem. 2002, 114, 3733 – 3736; Angew. Chem.
Int. Ed. 2002, 41, 3583 – 3586.
[8] a) M. C. Bryan, F. Fazio, H.-K. Lee, C.-Y. Huang, A. Y. Chang,
M. D. Best, D. A. Calarese, O. Blixt, J. C. Paulson, D. Burton,
I. A. Wilson, C.-H. Wong, J. Am. Chem. Soc. 2004, 126, 8640 –
8641; b) O. Blixt, S. Head, et al., Proc. Natl. Acad. Sci. USA
2004, 109, 17033 – 17038; c) C.-Y. Huang, D. A. Tharey, A. Y.
Chang, M. D. Best, J. Hoffman, S. Head, C.-H. Wong, Proc. Natl.
Acad. Sci. USA 2006, 103, 15 – 20.
[9] F. Fazio, M. C. Bryan, O. Blixt, J. C. Paulson, C.-H. Wong, J. Am.
Chem. Soc. 2002, 124, 14397 – 14402.
[10] a) J. Wei, J. M. Buriak, G. Siuzdak, Nature 1999, 399, 243– 246;
b) Z. Shen, J. J. Thomas, C. Averbuj, K. M. Broo, M. Engelhard,
J. E. Crowell, M. G. Finn, G. Siuzdak, Anal. Chem. 2001, 73,
4932 – 4937; c) S. A. Trauger, E. P. Go, Z. Shen, J. V. Apon, B. J.
Compton, E. S. P. Bouvier, M. G. Finn, G. Siuzdak, Anal. Chem.
2004, 76, 4484 – 4489.
[11] J. M. Gerdes, H. Waldmann, J. Comb. Chem. 2003, 5, 814 – 820.
[12] F. Burkhart, Z. Zhang, S. Wacowich-Sgarbi, C.-H. Wong, Angew.
Chem. 2001, 113, 1314; Angew. Chem. Int. Ed. 2001, 40, 1274 –
1277.
[13] T. K.-K. Mong, C.-H. Wong, Angew. Chem. 2002, 114, 4261;
Angew. Chem. Int. Ed. 2002, 41, 4087 – 4090.
[14] T. K.-K. Mong, C.-Y. Huang, C.-H. Wong, J. Org. Chem. 2003,
68, 2135 – 2142.
[15] T. K.-K. Mong, H.-K. Lee, S. G. Durón, C.-H. Wong, Proc. Natl.
Acad. Sci. USA 2003, 100, 797 – 802.
[16] H.-K. Lee, C. N. Scanlan, C.-Y. Huang, A. Y. Chang, D. A.
Calarese, R. A. Dwek, P. M. Rudd, D. R. Burton, I. A. Wilson,
C.-H. Wong, Angew. Chem. 2004, 116, 1018; Angew. Chem. Int.
Ed. 2004, 43, 1000 – 1003.
In summary, we have successfully synthesized the tumor-
associated antigen N3minor octasaccharide by using the
reactivity-based one-pot strategy. The strategy proved that
complex molecules can be rapidly assembled using readily
available thioglycosides with defined relative reactivity values
as building blocks. In addition, we have developed a novel
strategy for arraying synthetic carbohydrates on porous
silicon surface containing a photocleavable linker for direct
characterization by mass spectrometry. This strategy should
be useful for preparation of a large glycan array for high-
throughput analysis.
[17] K. Chayajarus, D. J. Chambers, M. J. Chughtai, A. J. Fairbanks,
Org. Lett. 2004, 6, 3797 – 3800.
[18] J. H. van Boom, P. M. J. Burgers, Tetrahedron Lett. 1976, 17,
4875 – 4878.
[19] X. Qian, K. Sujino, A. Otter, M. M. Palcic, O. Hindsgaul, J. Am.
Chem. Soc. 1999, 121, 12063– 12072.
[20] F. Roussel, M. Takhi, R. R. Schmidt, J. Org. Chem. 2001, 66,
8540 – 8548.
[21] C.-Y. Wu, A. Brik, S.-K. Wang, Y.-H. Chen, C.-H. Wong,
ChemBioChem 2005, 6, 2176 – 2180.
Received: November 15, 2005
Revised: January 7, 2006
Published online: March 20, 2006
Angew. Chem. Int. Ed. 2006, 45, 2753 –2757
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
2757