Stereoselective Preparation of Deuterium-Labeled Sugars: (6R)-(6-2H1)-N-Acetylglucosamine Derivatives

Article abstract of DOI:10.1021/jo980781a

The preparation of methyl (6R)-(6-²H₁)-2-deoxy-2-N-acetamido-α-D-glucose (8α-d) and methyl (6R)-(6-²H₁)-2-deoxy-2-N-acetamido-β-D-glucose (8β-d) is described. The key step in the synthesis was the stereoselective reduction of a C6-aldehydo-GlcNAc derivative with (R)-(+)-Alpine-Borane-d. Reduction of either methyl 6-aldehydo-3,4-di-O-benzyl-α-GlcNAc (6α) or methyl 6-aldehydo-3,4-di-O-benzyl-β-GlcNAc (6β) using (R)-(+)-Alpine-Borane-d in CH₂Cl₂ was significantly more stereoselective (>15:1 stereoselectivity for both anomers) than was reduction with NaBD₄ in MeOH. The absolute stereochemistry at C6 of the deuterated GlcNAc derivatives was determined from ¹H NMR analysis of the conformationally locked sugars, methyl (6R)-(6-²H ₁)-4,6-O-benzylidene-2-deoxy-2-N-acetamido-α-D-glucose (9α-d) and methyl (6R)-(6-²H ₁)-4,6-O-benzylidene-2-deoxy-2-N-acetamido-β-D-glucose (9β-d). Comparison of ³J_H5,H6 values and ¹H-¹H NOEs for the nondeuterated and deuterated benzylidene derivatives showed that reduction with (R)-(+)-Alpine-Borane-d gave the (6R)-(G-²H₁) epimer as the major product for both the GlcNAc α and β methyl glycosides. This stereoselective reduction enabled the ¹H NMR signals for the prochiral H6 and H6′ protons in a series of GlcNAc derivatives to be assigned.

Full text of DOI:10.1021/jo980781a

J . Org. Chem. 1998, 63, 5555-5561
5555
Ster eoselective P r ep a r a tion of Deu ter iu m -La beled Su ga r s:
(6R)-(6-2H1)-N-Acetylglu cosa m in e Der iva tives
Margaret L. Falcone-Hindley and J effery T. Davis*
Department of Chemistry and Biochemistry, University of Maryland at College Park,
College Park, Maryland 20742
Received April 28, 1998
The preparation of methyl (6R)-(6-2H1)-2-deoxy-2-N-acetamido-R-D-glucose (8R-d) and methyl (6R)-
(6-2H1)-2-deoxy-2-N-acetamido-â-D-glucose (8â-d) is described. The key step in the synthesis was
the stereoselective reduction of a C6-aldehydo-GlcNAc derivative with (R)-(+)-Alpine-Borane-d.
Reduction of either methyl 6-aldehydo-3,4-di-O-benzyl-R-GlcNAc (6R) or methyl 6-aldehydo-3,4-
di-O-benzyl-â-GlcNAc (6â) using (R)-(+)-Alpine-Borane-d in CH2Cl2 was significantly more stereo-
selective (>15:1 stereoselectivity for both anomers) than was reduction with NaBD4 in MeOH. The
1
absolute stereochemistry at C6 of the deuterated GlcNAc derivatives was determined from H NMR
analysis of the conformationally locked sugars, methyl (6R)-(6-2H1)-4,6-O-benzylidene-2-deoxy-2-
N-acetamido-R-D-glucose (9R-d) and methyl (6R)-(6-2H1)-4,6-O-benzylidene-2-deoxy-2-N-acetamido-
3
â-D-glucose (9â-d). Comparison of J H5,H6 values and 1H-1H NOEs for the nondeuterated and
deuterated benzylidene derivatives showed that reduction with (R)-(+)-Alpine-Borane-d gave the
(6R)-(6-2H1) epimer as the major product for both the GlcNAc R and â methyl glycosides. This
1
stereoselective reduction enabled the H NMR signals for the prochiral H6 and H6′ protons in a
series of GlcNAc derivatives to be assigned.
In tr od u ction
(â1-N-GlcNAc)Leu-Thr-NH2 (1) had peptide-sugar NOEs
indicating intramolecular interactions between the pep-
tide and carbohydrate domains (Figure 1B).5a These
peptide-sugar interactions for 1 were sequence-specific
in that the homologous Ac-Gln(â1-N-GlcNAc)Leu-Thr-
NH2, an unnatural N-glycopeptide, did not exhibit long-
range NOEs. We concluded that the N-linked sugar in
glycopeptide 1 stabilized a side-chain-main-chain pep-
tide interaction known as the Asx-turn.8 The carbohy-
drate’s C5-C6 side chain may be important in stabilizing
structure in glycopeptide 1, particularly since an NOE
was observed from the C-terminal cis amide NH to one
of the two prochiral GlcNAc H6 protons (Figure 1B).
While our initial NMR studies addressed the peptide
conformation in glycopeptide 1, we were unable to
determine the GlcNAc C5-C6 side-chain conformation
without the stereospecific assignment of the sugar’s
hydroxymethylene protons.
Figure 2 shows Newman projections for the three
major staggered conformations about the GlcNAc C5-
C6 bond. These rotamers are designated gt, gg, and tg
where the first letter designates the relative orientation
between O6 and O5 and the second letter designates the
relative orientation between O6 and C4. Solution 1H
NMR spectroscopy has shown that the preference for the
C5-C6 conformation in most glucopyranoses is gg > gt
. tg, independent of solvent.9
Asparagine glycosylation (N-glycosylation) of the Asn-
Xaa-Ser/Thr consensus sequence is a cotranslational
process important in many biological functions,1 includ-
ing protein folding.2 NMR studies on intact glycopro-
teins,3 glycopeptide fragments,4 and smaller glycopep-
tides5 have shown that N-glycosylation can influence
peptide structure. What has been less well documented,
however, is the influence that the peptide domain might
have on carbohydrate conformation. While some NMR
studies have concluded that carbohydrate structure is not
affected by the attached peptide,6 other papers suggest
that the sugar conformation can be modulated by pep-
tide-carbohydrate interactions.3,4b,7
We have previously used 1H NMR spectroscopy to
study the conformation of N-glycopeptides in both water
and in organic solvents.4b,5a Recent ROESY experiments
in an organic solvent revealed that glycopeptide Ac-Asn-
* To whom correspondence should be addressed.
(1) Varki, A. Glycobiology 1993, 3, 97-130.
(2) O’Connor, S. E.; Imperiali, B. Chem. Biol. 1996, 3, 803-812.
(3) (a) Wyss, D. F.; Choi, J . S.; Wagner, G. Biochemistry 1995, 34,
1622-1634. (b) Wyss, D. F.; Choi, J . S.; Li, J .; Knoppers, M. H.; Willis,
K. J .; Arulanandam, A. R. N.; Smolyar, A.; Reinharz, E. L.; Wagner,
G. Science 1995, 269, 1273-1278.
(4) (a) Wormald, M. R.; Wooten, E. W.; Bazzo, R.; Edge, C.; Feinstein,
A.; Rademacher, T. W.; Dwek, R. A. Eur. J . Biochem. 1991, 198, 131-
139. (b) Davis, J . T.; Hirani, S.; Bartlett, C.; Reid, B. R. J . Biol. Chem.
1994, 269, 3331-3338. (c) Rickert, K. W.; Imperiali, B. Chem. Biol.
1995, 2, 751-759. (d) O’Connor, S. E., Imperiali, B. J . Am. Chem. Soc.
1997, 119, 225-226.
One of our goals is to determine if the peptide chain
in N-linked glycopeptides influences the global conforma-
tion of the sugar’s C5-C6 bond by altering the relative
population of the three major rotamers. Without the
assignment of the diastereotopic hydroxymethylene pro-
(5) (a) Lee, K. C.; Falcone, M. L.; Davis, J . T. J . Org. Chem. 1996,
61, 4198-4199. (b) Live, D. H.; Kumar, R. A.; Beebe, X., Danishefsky,
S. J . Proc. Nat. Acad. Sci. U.S.A. 1996, 93, 12759-12761.
(6) (a) Brockbank, R. L.; Vogel, H. J . Biochemistry 1990, 29, 5574-
5583. (b) Lommerse, J . P. M.; Kroon-Batenburg, L. M. J .; Kroon, J .;
Kamerling, J . P.; Vliegenthart, J . F. G. J . Biomol. NMR 1995, 5, 79-
94. (c) Lu, J .; van Halbeek, H. Carbohydr. Res. 1996, 296, 1-21. (d)
Weller, C. T.; Seshradi, K.; Chadwick, C. A.; Kolthoff, C. E.; Ramnarain,
S.; Pollack, S.; Canfield, R.; Homans, S. W. Biochemistry 1996, 35,
8815-8823.
1
tons, however, H NMR spectroscopy cannot unequivo-
cally distinguish between the gt and tg rotamers.
(8) Abbadi, A.; Mcharfi, M.; Aubry, A.; Premilat, S.; Boussard, G.;
Marraud, M. J . Am. Chem. Soc. 1991, 113, 2729-2735.
(9) Nishida, Y.; Ohrui, H.; Meguro, H. Tetrahedron Lett. 1984, 25,
1575-1578.
(7) Paulsen, H. Angew. Chem., Int. Ed. Engl. 1990, 29, 823-839.
S0022-3263(98)00781-6 CCC: $15.00 © 1998 American Chemical Society
Published on Web 07/21/1998

5556 J . Org. Chem., Vol. 63, No. 16, 1998
Falcone and Davis
Resu lts a n d Discu ssion
P r elim in a r y Stu d ies. Our approach involves the
stereoselective reduction of a C6-aldehydo GlcNAc de-
rivative. After several failed approaches, we recognized
that appropriate protecting groups are important for the
smooth oxidation of the GlcNAc hydroxymethylene. Ini-
tially, we used acetates to protect O1, O3, and O4. How-
ever, the basic Swern conditions15 used to oxidize 1,3,4-
tri-O-acetyl-R-GlcNAc 2R16 led to R,â-elimination of the
C4-acetate to give the unsaturated aldehyde 3R as the
major product.17 Attempts to suppress R,â-elimination
with neutral variants of the Swern oxidation were un-
successful. Thus, treatment of 2R with the Moffat modi-
fication (DMSO, DCC, pyr-CF3COOH)18 resulted in pri-
marily 4,6-O-acyl migration and no oxidation.
F igu r e 1. (A) Sequence and B) secondary structure of
glycopeptide 1. Peptide-sugar NOEs are indicated by arrows.
The peptide’s C-terminal NHcis has an NOE to one of the
diastereotopic C6 hydroxymethylene protons (ref 5a).
Benzyl groups are not as prone as O-acetates toward
R,â-elimination. As outlined in Scheme 1, the use of the
3,4-O-benzyl protecting groups enabled straightforward
preparation of the deuterated GlcNAc derivatives. We
investigated reduction of the separate anomers, methyl
3,4-di-O-benzyl-6 aldehydo R-GlcNAc 6R and methyl 3,4-
di-O-benzyl-6-aldehydo â-GlcNAc 6â (see Scheme 2), so
as to minimize the complexity of the NMR analysis of
the products, 7R-d and 7â-d. We also wanted to deter-
mine if reduction would give similar stereoselectivities
and the same absolute configuration for the two anomeric
methyl glycosides. Indeed, reduction with (R)-(+)-Alpine-
Borane-d provides excellent yields of the (6R)-(6-2H1)-
GlcNAc derivative in both cases.
Syn t h esis of Met h yl (6-R)-(6-2H 1)-2-Deoxy-2-N-
a ceta m id o-r-glu cose. Acid-promoted cleavage of the
tritylgroup from methyl3,4-di-O-benzyl-6-O-tritylR-GlcNAc
4R19 gave 5R in 80% yield. Aldehyde 6R was prepared
from 5R in 96% yield by oxidation under the standard
Swern conditions.15 While easily hydrated,20 azeotropic
drying with toluene provided 6R in the aldehyde form,
F igu r e 2. Newman projections of the three major rotamers
about the GlcNAc C5-C6 bond.
1
with no hydrate detectable by H NMR or IR spectros-
This assignment can be achieved by stereoselective
monodeuteration of the GlcNAc hydroxymethylene. Ohrui
has made an R,â mixture of (6S)-(6-2H1)-GlcNAc10 from
a 1,6-anhydroglucose derivative.11 We sought a more
direct approach for preparation of C6-deuterated GlcNAc
from GlcNAc itself. The key step is the stereoselective
reduction of 6-aldehydo GlcNAc derivatives using the
homochiral and deuterated reagent, (R)-(+)-Alpine-Bo-
rane-d.12 Aldehyde reduction by (R)-(+)-Alpine-Borane-d
proceeds with excellent stereoselectivity to give mono-
deuterated primary alcohols with the (R-2H1) configura-
tion.13 This reagent has been recently used to prepare a
labeled sugar, (6R)-(6-2H1)-1,2:3,4-di-O-isopropylidene-
galactose in 20:1 diasteroselectivity.14 Below, we describe
the preparation and NMR analysis of various (6R)-(6-
2H1)-GlcNAc derivatives.
copy. The 1H NMR of 6R in CDCl3 showed
a
(12) Midland, M. M.; Greer, S. Synthesis 1978, 845-846.
(13) (a) Midland, M. M.; Greer, S.; Tramontano, A.; Zderic, S. A. J .
Am. Chem. Soc. 1979, 101, 2352-2355. (b) Midland, M. M.; Tramon-
tano, A.; Zderic, S. A. J . Am. Chem. Soc. 1977, 99, 5211-5213. (c)
Midland, M. M.; Kwon, Y. C. Tetrahedron Lett. 1984, 25, 5981-5984.
(d) Kobayashi, K.; J adhav, P. K.; Zydowsky, T. M.; Floss, H. G. J . Org.
Chem. 1983, 48, 3510-3512. (e) Rogic, M. M. J . Org. Chem. 1996, 61,
1341-1346. (f) For a review on Alpine-Borane and other chiral boranes,
see: Brown, H. C.; Ramachandran, P. V. Acc. Chem. Res. 1992, 25,
16-24.
(14) Midland, M. M.; Asirwatham, G.; Cheng, J . C.; Millers, J . A.;
Morell, L. A. J . Org. Chem. 1994, 59, 4438-4442.
(15) Swern, D.; Omura, K. Tetrahedron 1978, 34, 1651-1653.
(16) Chaudhary, S. K.; Hernandez, O. Tetrahedron Lett. 1979, 95-
98.
(17) For other examples of R,â-elimination during oxidation of sugar
hydroxymethylene groups, see; (a) Cree, G. M.; Mackie, D. W.; Perlin,
A. S. Can. J . Chem. 1969, 47, 511-512. (b) Singh, S.; Nambiar, S.;
Porter, R. A.; Sander, T. L.; Taylor, K. G. J . Org. Chem. 1989, 54,
2300-2307. (c) Sauerbrei, B.; Niggemann, J .; Groger, S.; Lee, S.; Floss,
H. Carbohydr. Res 1996, 280, 223-235.
(10) Nishida, Y.; Hori, H.; Ohrui, H.; Meguro, H. Carbohydr. Res.
1987, 170, 106-111.
(11) Ohrui, H.; Nishida, Y.; Meguro, H. Agric. Biol. Chem. 1983,
47, 1101-1106.
(18) Horton, D.; Nakadate, M.; Tronchet, J . M. J . Carbohydr. Res
1968, 7, 56-65.

Preparation of Deuterium-Labeled Sugars
J . Org. Chem., Vol. 63, No. 16, 1998 5557
Sch em e 1a
Ta ble 1. Ster eoselectivity of Red u ction of Ald eh yd e 6r
%
%
reductant
solventa
CH2Cl2
(6R)-7R-d
(6S)-7R-d
6R:6Sb
(R)-(+)-Alpine-
Borane-d
94
6
16:1
NaBD4
MeOH
37
63
1:1.6
a
b
Both reactions were carried out at room temperature. Ratios
were determined from 2H NMR analysis in 1:1 CDCl3/CD3CN at
60 °C.
1
2
into the C6 position of 7R-d was confirmed by H, H,
and 13C (J CD ) 21.8 Hz) NMR spectroscopy and by FAB
mass spectrometry. Although clearly excellent, the ste-
reoselectivity of deuterium incorporation was difficult to
1
determine using H NMR since the residual H6R reso-
nance for the minor product (δ 3.72 ppm) overlapped with
the H3 and H4 resonances. The stereoselectivity was
2
better determined by integration of the H NMR spec-
trum of 7R. Separate 2H signals for the two epimers,
(6R)-(6-2H1)-7R-d (δ 3.72 ppm) and (6S)-(6-2H1)-7R-d (δ
3.80 ppm), were resolved in CDCl3/CD3CN (1:1) at 60 °C
(see Figure 2 in the Supporting Information). Integration
a
Key: (i) HBr/HOAc, 80%; (ii) DMSO, (CO2)Cl2, 96%; (iii) (R)-
Alpine-Borane-d, CH2Cl2, 90%; (iv) H2, Pd(OH)2/C, EtOH 85%; (v)
PhCH(OMe)2, p-TsOH, DMF.
2
of the H NMR showed a 16:1 ratio of (6R)-(6-2H1)-7R-d
and (6S)-(6-2H1)-7R-d after reduction of 6R with (R)-(+)-
Alpine-Borane-d (Table 1). Alpine-Borane-d was superior
to NaBD4 with regard to stereoselectivity. Thus, 2H NMR
analysis indicated that NaBD4 reduction of 6R gave a
1:1.6 ratio of (6R)-(6-2H1)-7R-d and (6S)-(6-2H1)-7R-d. This
low stereoselectivity is similar to that observed for the
monodeuteration of other glucosyl 6-aldehydes with
NaBD4.17c,22
Sch em e 2a
The O3 and O4 benzyl ethers were removed from 7R-d
by hydrogenation over Pearlman’s catalyst (Pd(OH)2/C)23
to give (6R)-(62H1)-methyl R-GlcNAc 8R-d in 85% yield.
The nondeuterated GlcNAc R-methyl glycoside 8R has
been previously converted into GlcNAc peracetate 10R
in high yield via acetolysis.24 Peracetate 10R is a key
intermediate in the synthesis of N-linked glycopeptides.25
Thus, access to the labeled sugar, (6R)-(62H1)-methyl
R-GlcNAc 8R-d, should enable the preparation of deu-
terated N-linked glycopeptides.
a
Key: (i) HBr/HOAc, 100%; (ii) DMSO, (CO2)Cl2, 96%; (iii) (R)-
Alpine-Borane-d, CH2Cl2, 74%; (iv) H2, Pd(OH)2/C, EtOH, 83%;
(v) PhCHO, ZnBr2.
signal at δ 9.64 ppm for the aldehyde proton, while the
13C NMR spectrum in CD2Cl2 had a carbonyl resonance
at 197.7 ppm.21 Aldehyde 6R was typically reduced
immediately after its isolation and azeotropic drying.
Reduction of GlcNAc aldehyde 6R in CH2Cl2 at room
temperature with (R)-(+)-Alpine-Borane-d, prepared from
(+)-pinene (97% ee),12 gave 7R-d in 90% yield and high
stereoselectivity. As described below, the absolute C6
configuration was determined by transformation of 7R-d
into the conformationally locked 4,6-O-benzylidene de-
rivatives 9R-d. The incorporation of a single deuterium
(19) (a) Shashkov, A. S.; Evstigneev, A. Y.; Dereviskaya, V. A. Sov.
J . Bioorg. Chem. 1979, 4, 1076-1085. (b) Roy, A.; Ray, A. K.;
Mukherjee, A.; Roy, N. Ind. J . Chem. 1987, 26B, 1165-1167. (c)
J eanloz, R. W. J . Am. Chem. Soc. 1954, 76, 558-560.
(20) Hydrate formation from related sugar aldehydes is known;
see: ref 17c and: J enkins, D. J .; Riley, A. M.; Potter, B. V. L. J . Org.
Chem. 1996, 61, 7719-7726.
(22) Gagnaire, D.; Horton, D.; Taravel, F. R. Carbohydr. Res. 1973,
27, 363-372.
(23) Hiskey, R. G.; Lorthrup, R. C. J . Am. Chem. Soc. 1961, 83,
(21) Aldehyde 6R was unstable in CDCl3, presumably due to the
presence of acid. It was, however, stable in CD2Cl2, and the 13C NMR
spectra were taken in this solvent.
4798-4800.
(24) Wang, L.-X.; Lee, Y. C. J . Chem. Soc., Perkin Trans. 1 1996,
581-591.

5558 J . Org. Chem., Vol. 63, No. 16, 1998
Falcone and Davis
upfield-shifted H6′ resonance at δ 3.77 ppm, indicating
that this was the axial H6proS resonance (Figure 3A).
As expected, there were no NOE enhancements to the
downfield-shifted H6 resonance at δ 4.27 ppm. These
NOE experiments indicated that the H6proR resonance
(δ 4.27 ppm) is downfield of the H6proS signal (δ 3.77
ppm) in the unlabeled benzylidene 9R.
Analysis of deuterated benzylidene, 9R-d, prepared
from 7R-d, showed that deuterium was incorporated at
the 6R position since the 1H NMR spectrum clearly
lacked the one-proton signal at δ 4.27 ppm (see Figure 3
in the Supporting Information). This stereochemical
assignment for 9R-d is consistent with Midland’s transi-
tion-state model, which predicts that (R)-(+)-Alpine-
Borane-d reduces aldehydes to give alcohols of R
configuration.13a On the basis of this NMR analysis, we
can assign the H6proR and H6proS signals for the
GlcNAc derivative 5R and the unlabeled methyl glycoside
8R.
F igu r e 3. (A) Benzylidene 9R with key NOEs to H6proS from
H4 and acetal proton indicated with arrows; (B) Newman
projection of benzylidene 9R.
Th e â-GlcNAc Ser ies. Syn th esis a n d Ster eoch em -
ica l An a lysis of Meth yl (6R)-(6-2H1)-2-Deoxy-2-N-
a ceta m id o-â-glu cose. The same approach that was
used to prepare methyl (6R)-(6-2H1)-2-deoxy-2-N-aceta-
mido-R-glucose 8R-d was also used on the â-GlcNAc series
(Scheme 2). As described below, Alpine-Borane-d reduc-
tion of aldehyde 6â gave 7â-d in excellent stereoselec-
tivity, indicating that the methyl glycoside’s anomeric
configuration does not greatly influence the stereochem-
ical course of C6 reductions with Alpine-Borane-d.
Detritylation of methyl 3,4-di-O-benzyl-6-O-trityl
â-GlcNAc 4â19 with HBr gave 5â in quantitative yield.
In contrast to 5R, both H6proR and H6proS for 5â are
resolved from other resonances. Thus, the stereoselec-
tivity of deuterium incorporation was directly measured
Deter m in a tion of Ster eoch em istr y a n d Assign -
m en t of P r och ir al H6 NMR Reson an ces. The H6proS
proton normally resonates downfield of H6proR in glu-
copyranose derivatives.17c This “rule” is often used to
assign chemical shifts for the prochiral hydroxymethyl-
ene protons in glucopyranoses. Ohrui has shown, how-
ever, that all glucopyranoses do not conform to this rule.26
In the present work, we firmly established the assign-
ment of the prochiral hydroxymethylene protons by
converting the major reduction product, (6R)-(6-2H1)-7R-
d, into the conformationally locked derivative (6R)-(6-
2H1)-9R-d. Thus, the nondeuterated and deuterated 4,6-
O-benzylidene GlcNAc derivatives, 9R27 and 9R-d, were
prepared from the corresponding methyl-R-GlcNAc, ei-
ther 8R or 8R-d, and benzaldehyde dimethyl acetal. As
described below, both 3J coupling constants and NOEs
were used to assign the H6, H6′ NMR resonances in 9R
and 9R-d. This NMR analysis confirmed that reduction
of aldehyde 6R by (R)-(+)-Alpine-Borane-d gave (6R)-[6-
2H1]-7R-d as the predominant product.
1
2
from the H NMR spectra of 7â; the use of H NMR to
determine stereoselectivity was not necessary. Swern
oxidation of 5â gave aldehyde 6â in 96% yield. Aldehyde
6â, without purification but after azeotropic drying with
toluene, was reduced with (R)-(+)-Alpine-Borane-d in
CH2Cl2 at room temperature to give 7â-d in 74% yield
after purification. The stereoselectivity of the reduction,
As shown in Figure 3, 4,6-O-benzylidene sugars, such
as 9R, adopt a trans-decalin C1 (D) conformation, with
H6proS in the axial position and H6proR in the equato-
1
as determined by H NMR, was excellent; (6R)-(6-2H1)-
7â-d was formed in a greater than 20:1 ratio (Figure 4).
As for the R-series, NaBD4 reduction of aldehyde 6â gave
only a modest stereoselectivity, with the (6S)-7â-d epimer
being the major product in a 1.9:1 ratio (Figure 4).
Again, the configuration at C6 was determined by
transformation of (6R)-(6-2H1)-7â-d into the conforma-
tionally rigid benzylidene (6R)-(6-2H1)-9â-d. The 1H NMR
spectrum of unlabeled 9â in DMSO-d6 was assigned from
a 2D 1H-1H COSY experiment, and the H6proR and
H6proS resonances in 9â were well-separated and re-
solved. The H6proR proton of benzylidene 9â has a
3
rial position. In this fixed conformation, J 5,6R does not
3
equal J 5,6S, and the diastereotopic protons can be as-
1
signed on the basis of 3J 5,6 values and intramolecular H-
1
1H NOEs. The H NMR resonances of 9R in CDCl3 were
assigned from a 2D 1H-1H COSY experiment (see spectra
in the Supporting Information). The H6 and H6′ protons
of benzylidene 9R have chemical shifts of δ 4.27 ppm (3J 5,6
3
) 2.4 Hz) and δ 3.77 ppm. The smaller J coupling for
the downfield-shifted resonance suggested that this
signal corresponds to H6proR, being located gauche to
3
3
chemical shift of δ 4.20 ppm (dd, J 5,6 ) 4.8 Hz, J 6,6′
)
3
H5 (Figure 3B). The J 5,6 value for the upfield-shifted
10.0 Hz), while H6proS has a signal at δ 3.72 ppm (t,
H6′ resonance could not be determined from the 1D NMR
3J 5,6 ) 10.0 Hz, J 6,6′ ) 10.0 Hz). NMR analysis of the
3
spectrum because of chemical shift overlap with the H5
deuterated benzylidene, (6R)-(6-2H1)-9â-d, indicated that
deuteration resulted in the loss of H6proR, the equatorial
1
signal. The 1D H-1H NOE experiments (see the Sup-
porting Information) confirmed the assignments made on
the basis of the 3J 5,6R measurement. Irradiation of either
the benzylidene acetal proton (δ 5.54 ppm) or GlcNAc H4
(δ 3.57 ppm) showed a positive NOE of 5-6% to the
3
proton with the smaller J 5,6 coupling at δ 4.20 ppm (see
Figure 4 in the Supporting Information). As expected,
(6R)-(6-2H1)-9â-d had a doublet for the 6S proton at δ
3.70 (3J 5,6 ) 10.0 Hz).28 These results confirmed that (R)-
(+)-Alpine-Borane-d reduced the â-linked aldehyde 6â
(25) Garg, G.; J eanloz, R. W. Adv. Carbohydr. Chem. Biochem. 1985,
43, 135-201.
(26) Nishida, Y.; Ohrui, H.; Meguro, H. Tetrahedron Lett. 1984, 25
1575-1578.
(27) (a) Galemno, R. N.; Horton, D. Carbohydr. Res. 1983, 119, 231-
240. (b) Kovac, P.; Edgar, K. J . J . Org. Chem. 1992, 57, 2455-2467.

Preparation of Deuterium-Labeled Sugars
J . Org. Chem., Vol. 63, No. 16, 1998 5559
pulse scheme using 1 W of Rf power. IR spectra were recorded
using either NaCl or KBr plates. Fast atom bombardment
mass spectra (FAB) used glycerol as the matrix. Melting
points are reported uncorrected. Elemental analyses were
performed by Schwartzkopf Analytical Labs, Brooklyn, NY.
Met h yl 3,4-Di-O-b en zyl-2-d eoxy-2-N-a cet a m id o-r-D-
glu cose (5r). To a solution of 4R19 (0.90 g, 1.37 mmol) in
HOAc/CH2Cl2 (50 mL, 1:1) at 0 °C was added a 1.38 M solution
of HBr in HOAc (1.00 mL, 1.38 mmol). The reaction mixture
was stirred at 0 °C for 5 min. Trityl chloride was removed by
filtration of the reaction mixture into ice-water. The aqueous
layer was extracted with CH2Cl2. The combined organic layers
were washed (saturated NaHCO3 and brine), dried (Na2SO4),
and filtered, and the solvent was removed in vacuo. Flash
chromatography (20% hexane/80% CHCl3) gave 5R19 as a white
solid (0.45 g, 80%). An analytical sample was recrystallized
from CHCl3/petroleum ether: mp ) 186-188 °C; [R]27
)
D
1
+97.7 (c ) 0.6, CHCl3); H NMR (400 MHz, CDCl3) δ 7.35-
3
2
7.27 (m, 10H, Ar), 5.24 (d, 1H, J ) 9.2, NH), 4.86 (d, 1H, J
2
2
) 10.9), 4.84 (d, 1H, J ) 11.7), 4.66 (d, 1H, J ) 10.9), 4.63
(d, 1H, 3J ) 3.6, H1), 4.62 (d, 1H, 2J ) 11.7), 4.17 (ddd, 1H, 3J
) 3.6, 9.6, 9.6, H2), 3.79 (m, 1H, H6proS), 3.73 (m, 1H,
H6proR), 3.69-3.62 (m, 3H, H3, H4, H5), 3.29 (s, 3H, OMe),
1.82 (s, 3H, Ac), 1.77 (dd, 3J ) 5.6, 7.6, OH); 13C NMR (100
MHz, CDCl3) δ 169.8, 138.3, 137.9, 128.5, 128.2, 128.1, 128.0,
127.8, 98.6, 79.9, 78.2, 75.1, 74.8, 71.2, 61.8, 55.0, 52.6, 23.4;
FAB MS m/z 416.3 (M+ + 1, 2.1); HRMS (M+ + 1) m/z calcd
for C23H30NO6 416.2073, found 416.2067. Anal. Calcd for
F igu r e 4. Selective Deuterium Incorporation into 7â-d. A
region of the 400 MHz 1H NMR spectra for alcohols 5â and
7â-d in CDCl3 is shown. Selected resonances are labeled. The
bottom spectrum is that for 5â. The middle spectrum is that
for (6R)-(6-2H1)-7â-d, formed by reduction of aldehyde 6â with
(R)-(+)-Alpine-Borane-d. The top spectrum shows a mixture
of (6R)-(6-2H1)-7â-d and (6S)-(6-2H1)-7â-d formed from NaBD4
reduction of 6â.
C
23H29NO6; C, 66.49; H, 7.04; N, 3.37. Found: C, 66.23; H,
7.10; N, 3.33.
Meth yl 6-Ald eh yd o-3,4-d i-O-ben zyl-2-d eoxy-2-N-a ceta -
m id o-r-D-glu cose (6r). To a solution of oxalyl chloride (0.25
mL, 2.88 mmol) in CH2Cl2 (20 mL) at -78 °C was added DMSO
(0.38 mL, 5.77 mmol). After 5 min, a solution of 5R (0.60 g,
1.44 mmol) in CH2Cl2 (30 mL) was added, and the reaction
mixture was stirred at -78 °C for 15 min. After this time,
NEt3 (1.20 mL, 8.65 mmol) was added, and the reaction
mixture was stirred for an additional 15 min. The reaction
mixture was washed (0.05% HCl, saturated NaHCO3, and
brine), dried (Na2SO4), filtered, concentrated in vacuo, and
repeatedly azeotroped from toluene to give aldehyde 6R (0.57
with the same absolute stereochemistry as it did the
R-linked aldehyde 6R.
Con clu sion
Reduction of both 6-aldehydo-R-GlcNAc and 6-alde-
hydo-â-GlcNAc methyl glycosides (6R and 6â) with (R)-
(+)-Alpine-Borane-d proceeded with excellent stereose-
lectivity to give (6R)-(6-2H1)-GlcNAc derivatives. NMR
spectroscopy was used to determine the stereoselectivity
of reduction and the absolute configuration at the deu-
terated C6 position. This methodology, in turn, allowed
us to unequivocally assign 1H NMR resonances for the
prochiral C6 hydroxymethylene protons in a series of
GlcNAc derivatives. We anticipate that these (6R)-(6-
2H1)-GlcNAc derivatives will be useful for the synthesis
and conformational analysis of specifically labeled N-
linked glycopeptides.
g, 96%) as a pale yellow solid: mp ) 176-178 °C; [R]25
)
D
1
+86.0 (c ) 0.8, CHCl3); H NMR (400 MHz, CDCl3) δ 9.64 (s,
1H, CHO), 7.32-7.28 (m, 10H), 5.31 (d, 1H, J ) 9.1, NH), 4.82
(d, 1H, 2J ) 11.7), 4.78 (d, 1H, 2J ) 10.7), 4.72 (d, 1H, 3J )
2
2
3.3, H1), 4.64 (d, 1H, J ) 10.7), 4.63 (d, 1H, J ) 11.7), 4.22
(ddd, 1H, J ) 3.3, 9.2, 9.2, H2), 4.11 (d, 1H, J ) 9.0, H5), 3.77-
3.72 (m, 2H, H3, H4), 3.35 (s, 3H, OMe), 1.81 (s, 3H, Ac); 13C
NMR (100 MHz, CD2Cl2) δ 197.7, 169.8, 128.8, 128.5, 128.4,
128.3, 117.2, 99.2, 80.1, 78.3, 75.5, 75.3, 75.1, 55.9, 52.1, 23.5;
IR (CD2Cl2): 3687, 3443, 3037, 1737, 1675, 1600 cm-1; FAB-
MS m/z 414.3 (M+ + 1, 3.6); HRMS (M+ + 1) m/z calcd for
C
23H28NO6 414.1916, found 414.1924. Anal. Calcd for C23H27-
NO6 + H2O; C, 64.02; H, 6.77; N, 3.25. Found: C, 63.40; H,
6.89; N, 2.97.
Meth yl (6R)-(6-2H1)-3,4-Di-O-ben zyl-2-d eoxy-2-N-a ceta -
m id o-r-D-glu cose ((6R)-(6-2H1)-7r-d ). Meth od A. To a
solution of aldehyde 6R (3.75 g, 9.06 mmol) in CH2Cl2 (300
mL) at room temperature was added a 1.0 M solution of (R)-
(+)-Alpine-Borane-d9a in 1:1 THF/hexane (27.2 mL, 27.2
mmol). The reaction mixture was stirred at room temperature
for 16 h, after which time acetaldehyde (15.2 mL, 27.2 mmol)
was added to quench the excess Alpine-Borane-d. After the
solution was stirred for 1 h, the CH2Cl2 was removed in vacuo
and replaced with THF. To the solution were added 3 M
NaOH (75 mL) and 30% H2O2 (75 mL). The solution was
stirred for 60 min, and the THF was removed in vacuo. The
aqueous layer was extracted with CH2Cl2, and the organic
layer was washed (H2O, 0.05% HCl, and brine), dried
(Na2SO4), and concentrated in vacuo to give an oily solid.
Excess pinene was removed by trituration with hexane. To
remove the last traces of pinene, the product was dissolved in
70 mL of CHCl3 and precipitated with petroleum ether. The
solid was filtered and dried to give 7R-d (3.41 g, 90%).
Recrystallization from CHCl3/petroleum ether gave analyti-
Exp er im en ta l Section
Except where noted, all reactions were carried out under
dry N2. All solvents were distilled from drying agents.
Reagents were purchased from Aldrich, Sigma, or Acros
Organics. 1H NMR data, recorded at either 400 or 500 MHz,
are reported as follows: spin multiplicity, number of protons,
coupling constant (Hertz), peak assignment. Chemical shifts
are reported in parts per million relative to the residual
1
protonated solvent. The H NMR chemical shift assignments
were made using 2D COSY experiments. 2H NMR was
performed on a 500 MHz NMR instrument equipped with a 5
mm broad-band probe operating at 76.7 MHz. Proton broad-
band decoupling was achieved through the WALTZ composite
(28) The difference in the 1H chemical shift for the 6S proton in the
unlabeled benzylidene 9â (δ 3.72 ppm) and in the deuterated ben-
zylidene (6R)-(6-2H1)-9â-d (δ 3.70 ppm) is due to a deuterium isotope
effect. See also: Ohrui, H.; Nishida, Y.; Higuchi, H.; Hori, H.; Marguro,
H. Can. J . Chem. 1987, 65, 1145-53.

5560 J . Org. Chem., Vol. 63, No. 16, 1998
Falcone and Davis
cally pure material: mp ) 176-178 °C; [R] ) +97.1 (c ) 0.6,
CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.35-7.27 (m, 10H, Ar),
5.24 (d, 1H, 3J ) 9.2, NH), 4.86 (d, 1H, 2J ) 10.9), 4.84 (d, 1H,
CH2Cl2, washed (brine), dried (Na2SO4), filtered, and concen-
trated in vacuo. The crude product was purified by flash
chromatography (2% MeOH in CHCl3) to yield 9R-d (17.7 mg,
21.5%) as a white solid: mp ) 250-252 °C; 1H NMR (400 MHz,
CDCl3) δ 7.49-7.46 (m, 2H, Ar), 7.38-7.36 (m, 3H, Ar), 5.85
2
3
2J ) 11.7), 4.66 (d, 1H, J ) 10.9), 4.63 (d, 1H, J ) 3.6, H1),
2
3
4.62 (d, 1H, J ) 11.7), 4.17 (ddd, 1H, J ) 3.6, 9.6, 9.6), 3.78
3
3
3
(d, 1H, J ) 2.7, H6S), 3.68-3.62 (m, 3H, H3, H4, H5), 3.29
(d, 1H, J ) 8.2, NH), 5.54 (s, 1H, H7), 4.70 (d, 1H, J ) 3.7,
3 3
(s, 3H, OMe), 1.82 (s, 3H, Ac), 1.77 (s, 1H, OH); 2H NMR (76.7
MHz, CD3CN/CDCl3) δ 3.72; 13C NMR (100 MHz, CDCl3) δ
169.8, 138.3, 137.9, 128.5, 128.2, 128.1, 127.9, 127.8, 98.6, 79.9,
78.2, 75.0, 74.8, 71.2, 61.4 (t, J CD ) 21.8), 55.0, 52.6, 23.4; FAB
MS m/z 417.2 (6.6); HRMS (M+ + 1) m/z calcd for C23H29DNO6
417.2136, found 417.2132. Anal. Calcd for C23H28DNO6‚
H2O: C, 63.58; H, 6.96; N, 3.22. Found: C, 64.02; H, 6.81; N,
3.18.
H1), 4.21 (ddd, 1H, J ) 3.7, 9.0, 9.2, H2), 3.89 (ddd, 1H, J )
3
1.5, 9.0, 9.0, H3), 3.77 (m, 2H, H5, H6R), 3.57 (dd, 1H, J )
9.0, 9.2, H4), 3.39 (s, 3H, OMe), 3.06 (d, 1H, 3J ) 1.5, OH),
2.05 (s, 3H, Ac); 13C NMR (100 MHz, CDCl3) δ 171.5, 137.1,
129.2, 128.3, 126.3, 101.9, 98.8, 82.1, 70.8, 68.5 (t, J CD ) 23.6),
62.2, 55.3, 54.1, 23.3; FAB MS m/z 325.3 (M+ + 1, 100.0);
HRMS (M+ + 1) m/z calcd for C16H21DNO6 325.1510, found
325.1514.
Mixtu r e of (6R)-(6-2H1)-7r-d a n d (6S)-(6-2H1)-7r-d . To
a solution of aldehyde 6R (0.050 g, 0.120 mmol) in MeOH (10
mL) was added NaBD4 (0.007 g, 0.180 mmol). After the
solution was stirred at room temperature for 10 min, water
(10 mL) was added. The reaction mixture was extracted with
CH2Cl2. The combined organic layers were washed (0.1% HCl,
saturated NaHCO3, and brine), dried (Na2SO4), filtered, and
concentrated in vacuo to give a white solid. The crude product
was dissolved in CHCl3 and triturated with petroleum ether
to yield a mixture of (6R)-(6-2H1)-7R-d and (6S)-(6-2H1)-7R-d
(47.7 mg, 95%). 1H NMR was consistent with the structure
â-GlcNa c Ser ies. Meth yl 3,4-Di-O-ben zyl-2-d eoxy-2-N-
a ceta m id o-â-D-glu cose (5â). To a solution of 4â19 (1.53 g,
2.32 mmol) in 1:1 HOAc/CH2Cl2 (40 mL) was added a 1.38 M
solution of HBr in HOAc (1.10 mL, 4.56 mmol). The reaction
mixture was stirred for 3 min at 5 °C, followed by the addition
of water and CH2Cl2. The organic layer was separated, washed
(saturated NaHCO3, brine), dried (Na2SO4), filtered, concen-
trated in vacuo, and triturated with hexane to yield a white
solid (0.96 g, 100%). The solid was crystallized from CHCl3/
petroleum ether: mp ) 202-205 °C; [R]27 ) +16.4 (c ) 0.6,
D
CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.34-7.24 (m, 10H, Ar),
5.39 (d, 1H, 3J ) 7.8, NH), 4.84 (d, 1H, 2J ) 11.5), 4.83 (d, 1H,
2
of 7R-d, and H NMR indicated that the material was a 37:63
mixture of (6R)-(6-2H1)-7R-d (2H δ 3.72) and (6S)-(6-2H1)-7R-d
(2H δ 3.80).
2J ) 11.0), 4.73 (d, 1H, J ) 8.1, H1), 4.65 (d, 1H, J ) 11.0),
4.65 (d, 1H, 2J ) 11.5), 4.07 (t, 1H, 3J ) 9.0, H3), 3.88 (m, 1H,
H6proS), 3.73 (m, 1H, H6proR), 3.59 (t, 1H, 3J ) 9.0, H4), 3.46
(s, 3H), 3.43 (m, 1H, H5), 3.33 (m, 1H, H2), 1.89 (m, 1H, OH),
1.85 (s, 3H, Ac); 13C NMR (100 MHz, CDCl3) δ 170.4, 146.9,
138.3, 137.9, 128.5, 128.0, 127.9, 127.3, 101.0, 80.3, 78.5, 75.1,
74.8, 61.9, 57.2, 56.9, 23.6; FAB-MS m/z 438.5 (M+ + Na, 10.2),
416.5 (M+ + 1, 4.6); HRMS (M+ + 1) m/z calcd for C23H30NO6
416.2073, found 416.2073.
3
2
Met h yl (6R)-(6-2H 1)-2-Deoxy-2-N-a cet a m id o-r-D-glu -
cose (8r-d ). To a solution of (6R)-(6-2H1)-7R-d (0.83 g, 1.99
mmol) in HOAc/EtOH (15 mL/85 mL) was added Pearlman’s
catalyst (0.20 g, 20% Pd(OH)2/C). The reaction mixture was
shaken under a H2 atmosphere (50 psi) on a Parr hydrogenator
for 24 h. The catalyst was removed by filtration through
Celite, and the mixture was concentrated in vacuo. The crude
product was dissolved in EtOH and precipitated with Et2O to
yield 8R-d (0.47 g, 85%) as a colorless solid. Recrystallization
Meth yl 6-Ald eh yd o-3,4-d i-O-ben zyl-2-d eoxy-2-N-a ceta -
m id o-â-D-glu cose(6â). To a solution of oxalyl chloride (0.77
mL, 8.84 mmol) in CH2Cl2 (50 mL) was added DMSO (1.16
mL, 17.7 mmol) at -78 °C. After the solution was stirred at
-78 °C for 5 min, a solution of 5â (1.84 g, 4.42 mmol) in
CH2Cl2 (100 mL) was added. The reaction mixture was stirred
at -78 °C for an additional 15 min before NEt3 (3.68 mL, 26.5
mmol) was added. The reaction mixture was allowed to warm
to room temperature over 30 min. The reaction mixture was
washed (0.05% HCl, saturated NaHCO3, brine), dried (Na2-
SO4), filtered, concentrated in vacuo, and azeotroped from
toluene to give aldehyde 6â (1.74 g, 96%) as a pale yellow solid.
This material was used directly in the next reaction without
further purification: 1H NMR (400 MHz, CD2Cl2) δ 9.76 (s,
from EtOH afforded fine needles: mp ) 159-161 °C; [R]20
)
D
-77.1 (c ) 0.45, H2O); 1H NMR (400 MHz, CD3OD) δ 4.65 (d,
1H, 3J ) 3.4, H1), 3.80 (dd, 1H, 3J ) 3.4, 10.3, H2), 3.74 (s,
1H, H6S), 3.62-3.53 (m, 2H, H4, H5), 3.35 (t, 1H, 3J ) 9.4,
H3), 3.27 (s, 3H, OMe), 1.92 (s, 3H, Ac); 13C NMR (100 MHz,
D2O) δ 174.4, 98.1, 71.6, 71.1, 69.9, 60.2 (t, J CD ) 21.4), 55.1,
53.6, 21.8; FAB-MS m/z 237.1 (M+ + 1, 100.0); HRMS (M+
1) m/z calcd for C9H17DNO6 237.1197, found 237.1201.
+
Meth yl 4,6-O-Ben zylid en e-2-d eoxy-2-N-a ceta m id o-r-D-
glu cose (9r). To a solution of methyl-R,â-GlcNAc 8R (1.00 g,
4.25 mmol) in DMF (15 mL) was added R,R-benzaldehyde
dimethyl acetal (0.65 g, 4.25 mmol) and p-TsOH hydrate (2.5
mg, 0.013 mmol). The reaction mixture was heated at reflux
for 2 h and cooled to room temperature, and saturated
NaHCO3 was added. The product was extracted with CH2-
Cl2, washed (brine), dried (Na2SO4), filtered, and concentrated
in vacuo to give the crude product (0.452 g, 33%) as a yellow
solid. The R and â anomers were separated by flash chroma-
tography (2% MeOH in CHCl3). A pure fraction of 9R (80 mg)
3
1H), 7.37-7.25 (m, 10H), 6.32 (d, 1H, J ) 8.0), 4.69 (d, 1H,
2
2J ) 11.1), 4.63 (m, 3H), 4.59 (d, 1H, J ) 12.7), 4.22 (d, 1H,
3
3J ) 4.0), 4.11 (m, 1H), 3.96 (t, 1H, J ) 9.0), 3.75 (t, 1H, 3J )
9.0), 3.51 (s, 3H), 1.81 (s, 3H, Ac);13C NMR (100 MHz,
CD2Cl2): δ 200.5, 169.6, 138.2, 137.7, 129.0, 128.7, 128.6, 128.3,
128.1, 101.9, 79.2, 75.8, 75.1, 73.3, 72.5, 57.1, 50.1, 23.5; IR
(CD2Cl2) 3693, 3056, 2981, 2831, 2687, 1725, 1681; FAB-MS
was obtained as a white solid and recrystallized from MeOH:
m/z 446.2 (M+ + Na, 3.4), 414.2 (M+ + 1, 5.3); HRMS (M+
1) m/z calcd for C23H28NO6 414.1917, found 414.1913.
+
1
mp ) 250-252 °C; [R]25 ) +29.2 (c ) 0.5, MeOH); H NMR
D
(400 MHz, CDCl3) δ 7.49-7.47 (m, 2H, Ar), 7.38-7.36 (m, 3H,
Ar), 5.85 (d, 1H, J ) 8.2, NH), 5.54 (s, 1H), 4.70 (d, 1H, J )
3.7, H1), 4.27 (dd, 1H, J ) 3.2, 8.2, H6proR), 4.21 (ddd, 1H, J
) 3.7, 9.0, 9.2, H2), 3.89 (ddd, 1H, J ) 3.0, 9.0, 9.2, H3), 3.77
(m, 2H, H5, H6proS), 3.57 (dd, 1H, J ) 9.0, 9.2, H4), 3.39 (s,
3H, OMe), 3.01 (d, 1H, J ) 3.0, OH), 2.05 (s, 3H, Ac); 13C NMR
(100 MHz, CDCl3) δ 171.4, 137.1, 129.2, 128.3, 126.3, 102.0,
98.8, 82.1, 70.8, 68.8, 62.3, 55.3, 54.1, 23.3; FAB MS m/z 324.2
(M+ + 1, 44.8); HRMS (M+ + 1) m/z calcd C16H22NO6 324.1447,
found 324.1460.
Meth yl (6R)-(6-2H1)-3,4-Di-O-ben zyl-2-d eoxy-2-N-a ceta -
m ido-â-D-glu cose ((6R)-(6-2H1)-7â-d). Reduction with Alpine-
Borane-d was followed as described for the synthesis of (6R)-
(6-2H1)-7R-d. Reduction of aldehyde 6â (1.63 g, 3.94 mmol)
with (R)-(+)-Alpine-Borane-d provided (6R)-(6-2H1)-7R-d (0.64
g, 74%) as a white solid after trituration from hexane: mp )
218-220 °C; [R]25 ) +14.3 (c ) 0.6, CHCl3); 1H NMR (400
D
3
MHz, CDCl3) δ 7.34-7.24 (m, 10H, Ar), 5.39 (d, 1H, J ) 7.8,
2
2
NH), 4.84 (d, 1H, J ) 11.5), 4.83 (d, 1H, J ) 11.0), 4.73 (d,
1H, 3J ) 8.1, H1), 4.65 (d, 1H, 2J ) 11.0), 4.65 (d, 1H, 2J )
11.5), 4.07 (t, 1H, 3J ) 9.0, H3), 3.86 (m, 1H, H6S), 3.59 (t,
Met h yl (6R)-(6-2H 1)-4,6-O-Ben zylid en e-2-d eoxy-2-N-
a ceta m id o-r-D-glu cose (9r-d ). To a solution of 8R-d (0.060
g, 0.25 mmol) in DMF (6 mL) were added R,R-benzaldehyde
dimethyl acetal (0.038 g, 0.25 mmol) and p-TsOH (1.5 mg,
0.030 mmol). The reaction mixture was heated at reflux for
18 h and cooled to room temperature, and saturated NaHCO3
was added. The resulting white precipitate was extracted with
3
1H, J ) 9.0, H4), 3.46 (s, 3H), 3.43 (m, 1H, H5), 3.33 (m, 1H,
H2), 1.89 (m, 1H, OH), 1.85 (s, 3H, Ac); 13C NMR (100 MHz,
CDCl3) δ 170.4, 137.9, 128.5, 128.0, 127.9, 127.8, 101.0, 80.3,
78.5, 75.0, 74.8, 61.6 (t, J CD ) 21.0), 57.2, 56.9, 23.6; FAB-MS
m/z 439.3 (M+ + Na, 4.4), 417.3 (M+ + 1, 3.8); HRMS (M+
+
1) calcd for C23H29DNO6 417.2136, found 417.2126. Anal.

Preparation of Deuterium-Labeled Sugars
J . Org. Chem., Vol. 63, No. 16, 1998 5561
Calcd for C23H28DNO6 + H2O: C, 60.38; H, 6.61; N, 3.06.
Found: C, 60.05; H, 6.66; N, 3.14.
mmol) in benzaldehyde (0.15 mL) was added dry ZnCl2 (0.020
g). The resulting suspension was stirred at room temperature
for 18 h, after which time distilled water and CH2Cl2 were
added. The CH2Cl2 layer was separated and evaporated to
give a gum, which was triturated with Et2O to give a white
solid (20 mg). This solid was recrystallized from MeOH to give
9â-d (12.7 mg, 30%) as colorless needles: mp ) 278-280 °C;
1H NMR (400 MHz, DMSO-d6) δ 7.81 (d, 1H, J ) 8.5, NH),
7.44-7.35 (m, 5H, Ar), 5.59 (s,1H, CHPh), 5.27 (d, 1H, J )
5.0, OH), 4.37 (d, 1H, J ) 8.0, H1), 3.70 (d, 1H, J ) 10.0, H6S),
Meth yl (6R)-(6-2H1)-2-d eoxy-2-N-a ceta m id o-â-D-glu cose
(8â-d ). To a solution of 7â-d (1.16 g, 2.78 mmol) in HOAc/
EtOAc/EtOH (20/10/85 mL) was added Pearlman’s catalyst
(0.40 g, 20% Pd(OH)2/C). The reaction mixture was charged
and shaken in a H2 atmosphere (50 psi) on a Parr hydrogenator
for 24 h. The catalyst was removed by filtration through
Celite, and the solvent was concentrated in vacuo to give a
white solid. This material was dissolved in EtOH and
precipitated by addition of Et2O to give 8â-d (0.55 g, 83%) as
3
3.58-3.51 (m, 2H, H2, H3), 3.39 (t, 1H, J ) 10.0, H4), 3.33
a colorless solid: mp ) 187-190 °C; [R]25 ) +21.3 (c ) 0.47,
(m, 1H, H5), 3.32 (s, 3H, OMe), 1.81 (s, 3H, Ac); 13C NMR (100
MHz, DMSO-d6) δ 169.1, 137.7, 128.8, 128.0, 126.3, 102.4,
100.6, 81.2, 70.5, 67.5 (t, J CD ) 22.5), 65.9, 56.0, 55.9, 23.1;
FAB MS m/z 325.1 (M+ + 1, 100.0); HRMS (M+ + 1) m/z calcd
for C16H21DNO6 325.1510, found 325.1504.
D
1
3
H2O); H NMR (400 MHz, D2O) δ 4.30 (d, 1H, J ) 8.5, H1),
3
3.77 (s, 1H, H6S), 3.54 (t, 1H, J ) 9.0), 3.38 (m, 1H), 3.36 (s,
3H), 3.30 (m, 2H), 1.89 (s, 3H); 13C NMR (100 MHz, D2O) δ
174.7, 101.9, 75.8, 73.9, 69.9, 60.4 (t, J CD ) 21.4), 57.0, 55.4,
22.1; FAB-MS m/z (M+ + 1) 237.0 (100.0); HRMS (M+ + 1)
m/z calcd for C9H17DNO6 237.1197, found 237.1201.
Ack n ow led gm en t. We thank the Mizutani Founda-
tion for Glycoscience and the National Institutes of
Health (R29 CA66144) for financial support and Phil
DeShong, Simon Hinkley, and Marshall Werner for
helpful discussions. J .T.D. is a Camille-Dreyfus Teacher-
Scholar (1998-2003).
Meth yl-4,6-O-Ben zylid en e-2-d eoxy-2-N-a ceta m id o-â-D-
glu cose (9â). This compound was synthesized as described
for 9R, and a fraction was isolated after flash chromatography
to give a pure sample of 9â (94 mg, 7.0%). Colorless needles
were obtained by recrystallization from CHCl3: mp ) 279-
281 °C; [R]25 ) -59.3 (c ) 0.56, MeOH); 1H NMR (400 MHz,
D
DMSO-d6) δ 7.81 (d, 1H, J ) 8.5, NH), 7.44-7.35 (m, 5H, Ar),
5.59 (s, 1H, CHPh), 5.27 (d, 1H, J ) 5.0, OH), 4.37 (d, 1H, J
) 8.0, H1), 4.20 (dd, 1H, J ) 4.8, 10.0, H6proR), 3.72 (t, 1H,
Su p p or tin g In for m a tion Ava ila ble: 2D 1H-1H DQF-
3
1
J ) 10.0, H6proS), 3.58-3.51 (m, 2H, H2, H3), 3.39 (t, 1H, J
COSY spectra for 9R and 9â, 1D NOE H difference spectrum
2
for 9R, H NMR spectrum for 7R-d, and 1H NMR spectra for
) 10.0, H4), 3.33 (m, 1H, H5), 3.32 (s, 3H, OMe), 1.81 (s, 3H,
Ac); 13C NMR (100 MHz, DMSO-d6) δ 169.1, 137.7, 128.8,
128.0, 126.3, 102.4, 100.6, 81.2, 70.5, 67.5, 65.9, 56.0, 55.9, 23.1;
FAB-MS m/z 324.2 (M++ 1, 100.0); HRMS (M+ + H) m/z calcd
for C16H22NO6 324.1447, found 324.1432.
5R, 5â, 7R-d, 7â-d, 8R-d, 8â-d, 9R, 9R-d, 9â, and 9â-d (15
pages). This material is contained in libraries on microfiche,
immediately follows this article in the microfilm version of the
journal, and can be ordered from the ACS; see any current
masthead page for ordering information.
Met h yl (6R)-(6-2H 1)-4,6-O-Ben zylid en e-2-d eoxy-2-N-
a ceta m id o-â-D-glu cose (9â-d ). To a solution of methyl (6R)-
(6-2H1)-2-deoxy-2-N-acetamido-â-D-glucose 8â-d (0.020 g, 0.085
J O980781A