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Monitoring the Progress of Solid-Phase Oligosaccharide Synthesis by High-Resolution Magic Angle Spinning NMR Observations of Enhanced Selectivity for -Glycoside Formation from -1 2-Anhydrosugar Donors in Solid-Phase Couplings.

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The development of efficient enantioselective methodologies
for the construction of tetra-o-phenylenes and less sterically
constrained biphenylene dimers is in progress in our laboratory.
Received September 4, 1996 [Z 9528IEl
German version A n g w Chrm 1997, 109, 504-507
Keywords: arenes
chirality
- double helices
*
polymers
[ l ] E. L. Eliel, S . H. Wilen, Sterrorhemisfry qf Organic Compound.s, Wiley. New
York, 1994; Chapter 14.
[2] J. D. Wallis. A. Karrer, J. D. Dunitz, Hrlv. Chim.A c f a 1986, 69. 69.
131 M. M W. Langeveld-Voss, R. A. J. Janssen, M. P. T. Christiaans, S. C. J.
Meskers, H. P. J. M. Dekkers, E. W. Meijer, J. Am. Cheni. SOC.1996,118,4908.
[4] Y. Miyamoto. S. G . Louie, M. L. Cohen, Phjs. Rev. Lett. 1996, 76, 2121.
[5] J. Gibson. M. Holohan, H. L. Riley, 1 Chem. Soc. 1946, 456; M. OKeeffe.
G. B. Adams. 0. F. Sankey. Phys. Rev. Left. 1992, 68. 2325; F. Diederich, Y
Rubin, AnyPiL. Clrem. 1992. 104. 1123; Angrw. Chrm. Int. Ed. Engl. 1992, 31.
1101.
[6] D. L. Gin. V. P. Conticello, R . H. Grubbs, J. A m . Chem. Sor. 1994,116,10934.
M A. Keegstra, S. D. Feyter, F. C. D. Schryver, K. Mhllen, Angew. Cheni.
1996, 108, 830. Angew. Cheni. Int. Ed. Engl. 1996, 35, 774.
[7] V. Enkelmanii. J Physique 1983,44, C3. G. Grem, G . Leditzky, B. Ulrich, G .
Leising. A h . Afotrr. 1992, 4, 36; M. Hamaguchi, H. Sawada, J. Kyokane, K.
Yoshino, Chrni.L e t / . 1996, 527.
[El A barrier for racemization in excess of 60 kcdl mol was reported for a tetra-ophenylene derivative: P. Rashidi-Ranjbar, Y -M. Man, J. Sandstrom, H. N. C .
Wong. J Org. Chem. 1989, 54, 4888
191 J. -M. Lehn, Anycw. Chcm. 1990, 102, 1347; Angew. Chcm. Znf. Ed. Engl. 1990,
29. 1304. J. -M Lehn, Supramolecular Chemrstrj, VCH, New York, 1995.
[lo] D. B. Amabilino. J. F. Stoddart. Chern. Rrv. 1995. 95, 2725.
[l I ] A. Rajca. A. Safronov, S. Rajca, C. R . Ross, 11, J. J. Stezowski, J. Am. Chem.
Soc 1996, 118. 1272.
[I21 G. Wittig, G Klar, Liebigs Ann. Chem. 1967, 704. 91.
[13] A small amount of biphenylene product is typically formed hut not isolated.
2.2'-Dibromo-4.4-di-terr-butylbiphenyl:
M. Tashiro, T. Yamato, J. Org. Chem.
1979. 44. 3037.
[14] For a review o n chemistry of tetra-o-phenylenes, see: T. C. W Mak, H. N. C.
Wong. Rip. ('urr. Chem. 1987, 140, 141.
1151 U . Schubert. W. Neugebauer. P. Yon R. Schleyer. J. Chenz. Soc. Chem. Comniun. 1982. 11x4: W. Neugebauer, A. J. Kos, P. von R. Schleyer, J Orgunomrt.
C h m 1982. 228. 107.
[16] A. Streitwiesei-. Act. Cheni. Re>. 1984, 17. 353.
[I71 0. Desponds. M. Schlosser, Tetrahedron 1994, 50, 5881.
1181 The largest crystals of 1 grown so far (90 x 90 x 180 mm) were too small for
analysis with a conventional X-ray diffractometer.
[I91 A. A . Bothner-By. R. W. Jeanloz. J. Lee, R. L. Stephens, C. D. Warren. J. An?.
Chm?.Sor. 1984, 106. 81 1 ; A. Bax. D. Davis, J. M a p , Reson. 63. 207, 1985.
[20] H - H distances in 1 are taken from an M N D O calculation on the parent
compound (with hydrogen atoms replacing tBu groups); AHc =
222.4 kcal mol (D,symmetry, grad norm = 2.6). M. .I.
S. Dewar. W Thiel. J.
An7. Chmi. Soc. 1977, Y9,489Y; J. J. P. Stewart. MOPAC93.00 Manual, Fujitsu
Limited, Tokyo, 1993.
[21] The spectral envelopes for 1 and 2 have some resemblance to those of sterically
crowded biphenyls. for example, 2,2'-dimethylbipheny1; see ref. [22].
[?2] H.-H. Perkampus, C'V-VJS.4rl~1.snjOrgunirCompounds, 2nd ed., VCH. Weinhelm. 1992, Part 2.
[23] H. Schwager. S.Spyrondis, K P. C. Vollhardt, J Organomel. Chem. 1990,382.
I91 and references therein.
[24] The control reaction of 2,7-di-tert-butylbiphenylene
under the conditions given
in ref. [23] resulted in a clean mixture of 2 and its isomer. A. Rajca, A.
Safronov. unpublished results.
~
Monitoring the Progress of Solid-Phase
Oligosaccharide Synthesis by High-Resolution
Magic Angle Spinning NMR: Observations of
Enhanced Selectivity for /?-Glycoside Formation
from a-1,2-Anhydrosugar Donors in Solid-Phase
Couplings**
Peter H. Seeberger,* Xenia Beebe,
George D. Sukenick, Susan Pochapsky, and
Samuel J. Danishefsky
The vital roles played by oligosaccharides in cell-cell signaling and adhesion have led to greatly increased interest and appreciation of this class of compounds. It is now recognized that
these complex biomolecules, in the form of glycoprotein and
glycolipid conjugates, carry detailed structural information,
which serves to mediate a variety of biological events including
inflammation,['] immunological response,[21and meta~tasis.[~]
Furthermore, cell-surface carbohydrates act as biological markers for various tumors.[41
Of the three major classes of biooligomers, polysaccharides
have proven to be the most difficult to synthesize. The synthesis
of structurally defined ~ligopeptides[~]
and oligonucleotides[61
has greatly benefited from assembly on polymer supports. In
most cases the preparation of peptides and oligonucleotides can
be carried out on automated synthesizers, which allow rapid
formation of the target compounds. To simplify the very laborintensive solution-phase synthesis of carbohydrates, considerable efforts have been directed toward developing strategies and
sequences for solid-phase synthesis.[71Recent advances have
demonstrated that methodologies useful for glycosidation in
solution can be applicable on a polymer support. Indeed, large,
biologically important oligosaccharides have been prepared by
solid-phase methods.[81
Glycals have proven to be effective building blocks for the
synthesis of increasingly complex oligosaccharides.[91The approach is applicable to the synthesis of both glycopeptides
and oligosaccharides on the solid support." O1 While significant
progress has been made, a generally applicable method for the
rapid assembly of oligosaccharides and glycopeptides by automated solid-phase synthesis strategies has not yet been developed.
A major limitation is the difficulty in characterizing the reaction products or intermediates as they evolve during the course
of the synthesis. Currently, it is necessary to cleave these products from the resin to investigate them by classical spectroscopic
methods (for example solution NMR and mass spectrometry).
This is time-consuming, expensive, and wasteful for multistep
syntheses. Full characterization of the products while they are
still hound to the resin would hold many advantages.
[*I
Dr. P. H. Seeberger, Dr. X. Beebe, Prof. S.J. Danishefsky
Laboratory for Bio-Organic Chemistry
Sloan-Kettering Institute for Cancer Research
Box 106, 1275 York Avenue, New York, NY 10021 (USA)
Fax: Int. code +(212)772-8691
e-mail: p-seeberger(a ski.mskcc.org
Further address: Department of Chemistry, COlUmbld Unicersity
Dr. G. D . Sukenick
N M R Analytical Core Facility
Sloan-Kettering Institute for Cancer Research. New York (USA)
Dr. S. Pochapsky
Bruker Instruments Inc., Billerica, MA (USA)
[**I This research was supported by the U . S. National Institutes of Health (grant
no. HL-25848). X. B. gratefully acknowledges the NIH for a postdoctoral
training grant (no. T32CA62948).
Anyew. Chem. In1 Ed. Enxl. 1997. 36. No. 5
'C)
VCH Erlagsg~.dlschafrmbH. D-69451 W>inheim, 1997
057O-(lR33,'97,'3605-04916 I 5 . 0 0 i .25 I/
491
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In recent years there have been numerous reports on the use
of NMR for characterizing compounds bound to solvent-swollen resins. Among the measurements described are
'H, I9F,
'H, and 31Pdeterminations.["' The quality of the spectra obtained greatly depends on the type of resin used. Not surprisingly, 'H NMR resonances are generally broad when the experiments are conducted in traditional high-resolution probes,
which makes characterization by NMR spectroscopy difficult.
Recourse to magic angle spinning (MAS) combined with a
high-resolution probe has, in some instances, resulted in highquality 'H and I3C NMR spectra of small resin-bound compounds.["] We report here the use of high-resolution MAS
NMR (HR-MAS) for characterizing a trisaccharide glycal
bound to a Merrifield resin. This compound, which is the
product of a multistep synthesis, is one of the largest moieties
analyzed by HR-MAS to date.
The trisaccharide 2-0-acetyl-3,4-carbonyl-~-galactopyranose( 1+6)-~-~-2-0-acetyl-3,4-carbonyl-galactopyranose-(l
+6)/?-~-3,4-dibenzyl-glucalwas assembled by the glycal solid-support
synthesis strategy previously reported." O1 Polystyrene polymer
crosslinked with 1 % divinylbenzene was used as the polymeric
support due to its high loading capacity and stability towards a
broad range of conditions. This type of resin gives significantly
broader lines in the 'H and I3C NMR spectra than resins with
longer tethers, for example, the increasingly popular, but more
expensive, TentaGel resins, which have a lower loading capacity.
Polymeric support-bound trisaccharide 3 was assembled in five
steps (Scheme 1) and examined by 'H, I3C, and 'H- I3C HMQC
NMR. Spin-echo techniques['31were used to reduce the signal of
the polystyrene backbone observed in the 'H NMR spectrum
(Figure la). Although the lines in the obtained spectrum are
170 160 150 140 I30120 110 100 90 80 70 60 50 40 30 20 10
broadened (relative to those for the resin-cleaved sample), several
A
S
indications of a successful coupling reaction can be readily identiFigure
1
a)
'H
N
M
R
spectrum
of
3
usmg
spm-echo
techniquer to reduce the supfied. The resonance at 6 % 6.4 is characteristic of the glucal C1
port signals, b)
NMR spectrum of 3
proton. This signal, in conjunction with four doublets at 6 = 4.83,
4.69,4.64, and 4.55 for two methylene units of the benzyl protecting groups, this signal strongly suggests that glucal had indeed
been successfully incorporated. Acetylation of the C2 hydroxyl
silyl linker, through which the first galactose is connected to the
group of the coupled product is indicated by a methyl signal
polystyrene support. The broad signals in the region of S = 1.3
at 6 % 2.10. The resonances in the range of 6 = 0.8-1.2 are
to 1.9 originate from the polymer and may also contain impuriassignable to the protons of the isopropyl group attached to the
ties. A two-hour I3C experiment (Figure lb) confirmed the presence of two acetyl-type carbonyl groups (6 =
169.3 and 169.1) as well as two carbonate carbonyls (6 = 154.3 and 153.6). Furthermore, a two1. DMDO
hour 'H- I3C HMQC experiment provided confirmation of the proposed structure (Figure 2 ) . All
signals are clearly distinguishable, and several diZnCI,
agnostic signals could be assigned based on their
chemical shifts. The two anomeric carbon atoms
and the C2/H2 of the glycal can be identified in the
1
range of 6 = 90-100. Although not all of the ring
protons and carbons could be rigorously assigned,
the total number of signals confirms the presence
of a trisaccharide. Correct assignment of every sig1. DMDO
nal, which is not required for the general utility of
HR-MAS monitoring of synthetic progress, would
&:ni ' 0 n
be possible by performing TOCSY and COSY ex+
periments on the support-bound material. More
ZnClp
important is that detailed information about the
3.AQO, DMAP,
success of the chemical transformations on the
collidine
solid support can be obtained from a few relatively
short NMR experiments. To collect data of comparable quality, it would have been necessary to
Scheme 1. Solid-phase synthesis of 3 by the glycal assembly method [lo]. @ = 1 % divinylbenzene
cleave the reaction product from the support and
cross-linked polystyrene polymer. DMDO = dimethyldioxirane, DMAP = 4-(dimethylamino)then characterize it.
pyridine.
Y
Y
-,\
492
m
c VCH Verlagsgesellschaft mbH, 0-69451 Winheim, 1997
057U-U833/97/36UJ-U492 $lS.OU+ ,2510
AngeK'. Chem. Int. Ed. Engl. 1997, 36, No. 5
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Keywords: glycosylations . N M R spectroscopy * oligosaccharides . solid-phase synthesis . synthetic methods
I
95
~
2)
1001
C'3 x i 3
5 1 50 49 48 47 4 6 4 5 4 4 4 3 42 41 4 0 3 9 38 37
/I
-
-6
Figure 2. I h g n o s t i c region of the 'H-'.'C
H M Q C N M R spectrum o f 3
Thc spectra show that HR-MAS can bc used for monitoring
the solid-phase synthesis of oligosaccharidcs in a very sensitive
fashion. Parenthctically, the measurements tend to confirm our
earlier claim that solid-support coupling rcactions -using properly protected 1,2-anhydro sugars (at least in this series)- are
highly stereoselective. even morc so than the solution-based
couplings." 'I The original claim was based on thc finding that
no a-linkcd products could be found upon cleavage from the
polymer and subsequent purification. Of course, arguments of
this sort arc subject to uncertainties as to whethcr other stereoisomers might have been inadvertently overlooked during the
removal and purification sequcnce. The argument becomes
more persuasive as one cxamines thc "crude reaction mixture"
bound to the solid phasc. While the data we obtaincd cannot
exclude adventitious overlapping of signals from small amounts
of isomcric %-products, it certainly indicates a high degree of
control of each coupling event conducted on the solid phase.
We attribute this exccllent B-stereoselectivity in this and related
solid-state experiments19, l o ] to the relative diminution of cffective solvation upon complexation of thc oxirane by the zinc
chloridc promoter. Solvation forces can lend the anomeric
oxiranyl donor oxonium-like qualities. A donor spccies, which
is far advanced in the direction of a free oxonium ion, is likely
to be responsible for the formation of small but significant
amounts of a-glycosides from the solution-based a-epoxide
donor.
In summary, the development of novel methodologies for thc
assembly of oligosaccharides on the solid support will undoubtedly benefit from this disccrning "on-resin" analytical method.
Chemists will be able to determine whether coupling has occurred and quickly estimate the specificity of the coupling step.
Complete assignments can be madc aftcr the product is cleaved
from the solid support and deprotected.
E,qwitnenfal Section
All specti-a were obtained on a Bruker DKX500 spectrometer. operating a t
500.13 M H r ( ' H ) and 125.76 MHz ("C). equipped with a 4 m m Bruker CCA
HR-MAS probc. Trisaccharide 3 (20 mg at 0.54 nimo1g-l loading, 10.8 pmol) was
loaded into a ceramic rotor. suspended in 30 p L CDCI,, and spun at the magic angle
at 3.5 K H r ' H N M R spectra were obtained with a Carr-Purcell~Meihoom-(iill
pulse sequence [13]. 128 transients (1.64 s acquisition time. 0.5 s relaxation delay)
were accumulated. The 13C['H: spectrum was obtained in 2 h 10 i n i n
(3000 transients. 0 6 s acquisition time. 2 s relaxation delay). The phase-sensitive
(TPPI) ' H - "C H M Q C spectrum was obtained i n 2 h (16 scans per 256 increments.
0.17 s acquisition time, 1.3 s relaxation delay) with a BIRD sequence to minimi7e
resin sigiials "41. Total time for ' H , "C. and H M Q C N M R experiments was 4 h
15 inin.
Received. July 3, 1996
Revised vcrsion' October 7. 1996 [Z9291 IE]
German version: A n p i i ' . C k m . 1997. I O Y , 507 510
A. Giannic. Afigeii. U i e i i 7 1994. 106. 188: Aii,cyir. Ciiriii. / f i r Ed. h g l . 1994.
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Hakamori. J. C. Paulson, S<I(WP1990. 250. 1130. L.M Stoolman. Ceii 1989,
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Hodge. D C. Sherrington). Wiley. Chichester, 1980. p. 1.
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S. J Danishefsky. K. t McCIure, J. T. Randolph, R. B. Ruggei-i. Sci~.,mw1993.
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I I 1989. 111. 6661
Novel Carbocyclic Ring Closure of
Hex-5-enop yr anosides
Sanjoy Kumar D a s , Jean-Maurice Mallet, and
Pierre S h y *
Carbohydrates have been used as starting inaterials for thc
synthesis of an extcnsive rangc of cnantiomerically pure noncarbohydrate natural products and relatcd substances.[]' The intramolecular ring closurc of carbohydrates to form carbocyclic
compounds is an attractive transformation. which offers direct
access to highly runctionalized cyclohexane derivatives. In this
[*] Prof P Sinay. Dr. S K. Das. Dr L M . Mallet
Departement de Chirnie. URA 1686
Ecole Norrnale Suptrieure
24 rue Lhomond
F-75231 Paris Cedex 05 (France)
Fax: Int. codc +(1) 44-32-3397
e-mail . sinay:u chimenc.ens.fr
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progress, resolution, solis, selectivity, formation, couplings, high, oligosaccharides, phase, spinning, monitoring, synthesis, nmr, observations, glycosides, donor, anhydrosugar, enhance, angl, magii
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