Reductive amination of glycosyl aldoses: Synthesis of N-glycosylated β-glycosyl amino alcohols and their enzyme inhibitory effect

Article abstract of DOI:10.1081/CAR-200046779

Reductive amination of glycosyl aldehydes (1a-c, 2) with glycosyl amino esters (3a-c, 4) in the presence of sodium borohydride gave diglycosylated amino esters (5-15) in good yield. N -Glycosyl-glycosylated amino esters were reduced to the respective digl

Full text of DOI:10.1081/CAR-200046779

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Reductive Amination of Glycosyl Aldoses:
Synthesis of N‐Glycosylated β‐Glycosyl
Amino Alcohols and their Enzyme
Inhibitory Effect
Shyam Sunder Verma a , Ram Chandra Mishra a , Akhilesh Kumar
Tamarakar b , Brajendra Kumar Tripathi b , Arvind Kumar Srivastava b
& Rama Pati Tripathi a
a Division of Medicinal & Process Chemistry , Central Drug Research
Institute , Lucknow, 226001, India
b Division of Biochemistry , Central Drug Research Institute ,
Lucknow, India
Published online: 09 Aug 2006.
To cite this article: Shyam Sunder Verma , Ram Chandra Mishra , Akhilesh Kumar Tamarakar ,
Brajendra Kumar Tripathi , Arvind Kumar Srivastava & Rama Pati Tripathi (2004) Reductive Amination
of Glycosyl Aldoses: Synthesis of N‐Glycosylated β‐Glycosyl Amino Alcohols and their Enzyme
Inhibitory Effect, Journal of Carbohydrate Chemistry, 23:8-9, 493-511, DOI: 10.1081/CAR-200046779
To link to this article: http://dx.doi.org/10.1081/CAR-200046779
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JOURNAL OF CARBOHYDRATE CHEMISTRY
Vol. 23, No. 8–9, pp. 493–511, 2004
Reductive Amination of Glycosyl Aldoses: Synthesis
of N-Glycosylated b-Glycosyl Amino Alcohols
and their Enzyme Inhibitory Effect#
Shyam Sunder Verma,1 Ram Chandra Mishra,1 Akhilesh Kumar
Tamarakar,2 Brajendra Kumar Tripathi,2 Arvind Kumar Srivastava,2
and Rama Pati Tripathi1,
*
1Division of Medicinal & Process Chemistry, Central Drug Research Institute,
Lucknow, India
2Division of Biochemistry, Central Drug Research Institute, Lucknow, India
CONTENTS
ABSTRACT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 494
I. INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 494
II. RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . 495
III. EXPERIMENTAL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 498
A. General Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 498
B. General Procedure for the Preparation of the Compounds (5–15) . . 499
C. General Procedure for the Preparation of the Compounds (16–26) . 504
ACKNOWLEDGMENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . 509
REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 509
#CDRI Communication No. 6605.
*
Correspondence: Rama Pati Tripathi, Division of Medicinal & Process Chemistry, Central Drug
Research Institute, Lucknow 226001, India; E-mail: rpt_56@yahoo.com.
493
DOI: 10.1081/CAR-200046779
0732-8303 (Print); 1532-2327 (Online)
www.dekker.com
Copyright # 2004 by Marcel Dekker, Inc.

494
Verma et al.
ABSTRACT
Reductive amination of glycosyl aldehydes (1a–c, 2) with glycosyl amino esters (3a–
c, 4) in the presence of sodium borohydride gave diglycosylated amino esters (5–15) in
good yield. N-Glycosyl-glycosylated amino esters were reduced to the respective
diglycosyl amino alcohols (16–26) with LiAlH4 in good yield. All the synthesized
compounds were studied for their inhibitory effect, if any, against hepatic glucose-6-
phosphatase, glycogen phosphorylase, and intestinal brush border membrane a-gluco-
sidase; among these compounds 7, 21, and 25 have shown marked inhibition on these
enzymes, respectively.
INTRODUCTION
Apart from classical carbohydrate chemistry of O-glycosides[1] from many years in
medicinal chemistry for the development of many therapeutics, an increase in the devel-
opment of a biological or non-natural glycoside with a C–C bond or C–N bond is gaining
momentum at the molecular level for the roles of carbohydrates in glycolipids and glyco-
proteins.[2] Identification of lead compounds from sugars for drug discovery against
various diseases are being pursued by different groups, including ours,[3] because of tre-
mendous potential of structural diversity in sugars.
Aza disaccharides are known to inhibit various glycosyl hydrolases and glycosidases and
are therefore important for drug development.[4] Polyhydroxylated pyrrolidines and piper-
idines are potent inhibitors of glycosidase, and few of them act as broad-spectrum enzyme
inhibitors. The glycosidase enzyme inhibitory activity has been exploited in the development
of many chemotherapeutics such as antiviral, anticancer, antimicrobial, antibacterial, and
antiparasitic agents for the treatment of many diseases. Many compounds from natural and
synthetic origins containing an aglycon moiety attached to a glycosyl cation mimetics act
as selective inhibitors[5] of a-glucosidases. Disaccharide analogs linked through spacers
having nitrogen atom have recently been found to bind with 16S RNA, indicating their use-
fulness as amino glycoside antibiotics. Moreover, certain disachharides with aminoalkyl
functionality were proved to be mechanism-based inactivators of bacterial aminoglycoside
30-phosphotransferases.[6] Dideoxyiminoalditols linked to other sugars by nonhydrolysable
links have much better specificity towards glycosidases than dideoxyiminoalditols them-
selves; therefore, different synthetic strategies have been developed for their synthesis.[7]
Nitrogen- and sulphur-linked pseudodisaccharides as relatively stable glycosidase inhibitors
have been reported recently.[8] In our ongoing program to develop carbohydrates as chemo-
therapeutic agents, we have recently synthesized glycosyl urea and certain C-nucleoside
analogs through amination of aldehydes with amino sugars as a-glucosidase inhibitors.[9]
a-Glycosidase, glycogen phosphorylase, and glucose-6-phosphatase are important
enzymes for the development of new drugs against many metabolic disorders, and the
most important among them is diabetes. Glycogen phosphorylase breaks down glycogen
to glucose-6-phosphate; this is the source of glucose in liver but not in muscles. The
glucose-6-phosphatase removes phosphate from glucose and releases it in the blood. Thus,
inhibitors of this enzyme may be helpful in controlling the state of hyperglycemia.
Keeping in mind the above, we were prompted to synthesize certain aza-linked
pseudo disaccharide analogs having both pyranose and furanose sugar rings and evaluate
their efficacy against the above three enzymes responsible for diabetes.

Reductive Amination of Glycosyl Aldoses
RESULTS AND DISCUSSION
495
Starting with the reaction of glycosyl aldehyde (1a) with glycosyl amino ester (3a)[10]
(Sch. 1) resulted in the formation of an intermediate (imine), which, on subsequent in situ
reduction with sodium borohydride, gave the disaccharide analog 5 (Sch. 2). The structure
of compound 5 was determined on the basis of its spectroscopic data and analysis. IR spec-
trum of the compound showed an absorption band at 3753 cm21 corresponding to NH-
stretching, while in MS (FAB) spectrum, a peak at m/z 628 corresponded to [M þ H]þ.
1
In H NMR spectrum of the compound 5, the anomeric protons (H1 and H-1’) of the
two furanoses S1 and S2 were observed as d at d 5.92 and 5.90 with J ¼ 3.9 and 3.6 Hz,
respectively. H-2 and H-2’ in both S1 and S2 appeared as multiplet along with CH2
protons of CH2Ph group at d 4.69–4.42; H-4 appeared as m at d 4.26 in S1 and while
in S2 it appeared as m at d 4.16. A multiplet at d 3.90 accounted for H-3 and H-3’ of
the sugar rings, while methylene protons of NCH2 attached to S2 appeared as m at d
3.04. The protons for CH2 and CH3 in the carbethoxy group appeared at d 4.04 and
1.19 as a quartet and triplet, respectively, with J value of 6.9 Hz besides other usual
signals. In 13C NMR spectrum, compound 5 showed characteristic signals for NHCH2,
OCH2, and CH3 carbons at d 45.8, 60.7, and 14.5, respectively. Because, the stereo-
chemistry has already been assigned to be S in the glycosyl amino ester (3a) used in
this reaction, this is maintained in the final compounds as well, because C-5 is not involved
in the reaction.
We have extended this work using galactopyranosyl aldehydes (2) and galactopyra-
nosyl amino esters (4) to synthesize compounds having pyranose ring. Thus, the reaction
of glycosyl amino ester 3a with glycosyl aldehydes 2 gives N-galactopyranosylated
Scheme 1. Glycosyl aldehydes and amino esters.

496
Verma et al.
Scheme 2. Synthesis of N-bridged disaccharides.
glycosyl amino ester (8) in good yield. IR spectrum of compound 8 showed absorption
band at 3655 cm21 corresponding to NH-stretching. In MS (FAB) spectrum, peak at
1
m/z 608 corresponded to [M þ H]þ. In H NMR spectrum of this compound, the two
doublets for anomeric protons (H-1 and H-1’) of the furanose and pyranose sugars were
observed at d 5.96 and 5.53 with J ¼ 3.6 and 5.1 Hz, respectively. The signals for
protons of CH2 and CH3 in carbethoxy group appeared at d 3.95 and 1.24 as a quartet
(J ¼ 6.8 Hz) and triplet (J ¼ 6.8 Hz), respectively, besides other usual signals. In 13C
NMR spectrum, compound 8 showed characteristic peaks of NHCH2, OCH2, and CH3
carbons at d 47.1, 60.7 and 14.5, respectively, besides other usual signals of both the
sugars. Similarly, reaction of galactopyranosyl amino ester 4[11] with galactopyranosyl
aldehyde (2) resulted in N-galactopyranosyl galactopyranosylated amino ester (15) in
good yield. The structure of this compound was also established on the basis of its spectro-
scopic data and analysis. IR spectrum of this compound (15) showed absorption band at
3679 cm21 corresponding to NH stretching; MS (FAB) spectrum showed a peak at m/z
1
588 corresponding to [M þ H]þ. In H NMR spectrum, the anomeric protons of the two
sugar rings were observed as d at d 5.55 and 5.50 with J ¼ 5.0 Hz, besides other usual
signals. In 13C NMR spectrum, of 15 quaternary carbon of ester group appeared at d
172.7 while those of isopropylidene groups were observed at d 109.5, 109.3, 108.8, and
108.7. The anomeric carbons (C-1 and C-1’) appeared at d 97.9 and 96.7, respectively,
besides other usual signals (Table 1).
Similarly, structures of all the compounds were established on the basis of spectral
data and analysis. In all the compounds anomeric protons corresponding to furanose
and pyranose sugars appeared at around d 5.9 and 5.6 as d, with J ꢀ 3.7 and 5.0 Hz,
respectively. H-2 for the furanose sugar appeared as d at around d 4.6 in furanose
sugar, while the same in pyranose sugar appeared as m merged with H-5 or as dd with
J ꢀ 5.0 and 2.2 Hz at around d 4.3 in the disaccharide analogs. The characteristic –
NH-linkage between two sugars has been characterized both by IR (ꢀ3650, N-H
1
stretching) and H NMR (br s at around d 1.6). In 13C NMR spectra, the characteristic
signals for C-1, C-2, C-3, and C-4 in glycofuranose appeared at around d 105, 83, 82, and
80, respectively, while in the case of glycopyranose, the signals corresponding to C-1,
C-2, C-3, C-4, and C-5 appeared at around d 97, 72, 71, 70, and 62, besides other usual
signals.

Reductive Amination of Glycosyl Aldoses
497
Table 1. Compounds synthesized.
R1
Entry
Compound
R2
R3
1
2
5
6
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH3
CH2Ph
CH3
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
3
7
CH2CH ¼ CH2
4
8
–
5
9
–
CH2Ph
6
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
CH2CH ¼ CH2
7
CH2CH ¼ CH2
CH3
CH2Ph
8
–
9
–
CH3
CH2CH ¼ CH2
10
11
12
13
14
15
16
17
18
19
20
21
22
–
–
CH2Ph
–
CH2Ph
CH2Ph
CH2Ph
CH3
CH2CH ¼ CH2
CH2Ph
CH3
–
–
CH2Ph
CH2CH ¼ CH2
CH2CH ¼ CH2
CH3
CH2Ph
–
–
–
–
CH3
CH2CH ¼ CH2
–
Lithium aluminiumhydride (LiAlH4) reduction of glycosyl amino esters 5–15 was
carried out at 08C to ambient temperature, resulting in the formation of respective N-gly-
cosyl glycosylated amino alcohols 16–26 in good yield (Fig. 1). The formation of alcohol
from the respective amino esters was evidenced by their spectroscopic data and analysis.
FAB MS of all of the above amino alcohols showed peaks corresponding to [M þ H]þ, and
in IR spectrum, appearance of a broad signal around 3400 cm21 and disappearance of
signal at around 1725 cm21 indicated the reduction of ester functionality to the alcohol.
1
In H NMR spectrum of the diglycosyl amino alcohols, disappearance of the q and t at
around d 4.0 and 1.25 corresponds to OCH2and OCH2CH3, respectively; and appearance
of an m at d 3.7 for the CH2OH confirmed the reduction of ester to the respective alcohol.
Further in the 13C NMR spectrum, appearance of a peak at around d 62.0 corresponding to
CH2OH carbon and disappearance of the signals for methylene and methyl carbons in
OCH2CH3 at around d 60.7, 14.5, and carbonyl carbon at around d 172 clearly confirmed
the formation of alcohol.
As evident from Table 2, out of all the compounds screened against glucose-6-phos-
phatase, glycogen phosphorylase, and a-glucosidase, only compounds 6, 10, 13, 18, 19,
21, and 25 exhibited activity against all or two of the enzymes, while other compounds
did not show any significant activity. Compound 7 inhibited only a-glucosidase to the
extent of 82%, while compounds 21 and 25 possessing allyl group as substituent in
either of the sugar rings were found to be a good inhibitor of glycogen phosphorylase.
In the present study, there were five compounds having more than 50% inhibition on

498
Verma et al.
Figure 1. N-Glycosyl-glycosylamino ester 5–15 and alcohol 16–26 synthesized.
glucose-6-phosphatase. These molecules have the potential to be developed as antidiabetic
agents, because these compounds will possibly reduce the hepatic glucose production.
These agents may suppress glucose production in liver as evidenced by reduction in the
activities of glucose-6-phosphatase.
EXPERIMENTAL
General Methods
Thin-layer chromatography was carried out on silica gel (Kiesel 60-F254, Merck),
and spots were developed in iodine vapors and by spraying with 5% sulfuric acid in
alcohol followed by heating at 1008C. Column chromatography was carried out on flash
silica gel (230–400 mesh, Merck) using the indicated eluent. IR spectra were recorded
as thin films on KBr plates with a Perkin Elmer 881 spectrophotometer. NMR spectra
were recorded on Bruker spectrometers 200 and 300 MHz and reference used was CDCl3.
Chemical shifts were given as d ppm values, and J values were given in Hertz (Hz).

Reductive Amination of Glycosyl Aldoses
499
Table 2. Biological activity of disaccharide analogs against different enzymes.
% Inhibition
Glucose-6-
phosphatase
Glycogen
phosphorylase
S. No.
Compound
a-Glucosidase
1
2
5
6
nd
22.5
31.6
nd
nd
45.9
NIL
nd
þ1.98
10.1
82.3
nd
3
7
4
8
5
9
nd
nd
nd
6
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
33.8
nd
29.7
nd
29.8
nd
7
8
nd
nd
2.83
16.0
3.65
3.68
þ3.68
nd
9
30.9
30.2
nd
16.2
nd
10
11
12
13
14
15
16
17
18
19
20
21
22
nd
nd
nd
nd
nd
30.9
35.2
nd
40.5
18.9
nd
þ26.4
56.2
nd
25.3
39.4
nd
72.9
5.40
nd
þ7.58
þ1.96
þ1.98
nd
nd
nd
27.4
nd
94.6
nd
nd
6.51
All the compounds were tested at the concentration of 100 mg/mL; nd ¼ not done.
Elemental analyses were performed on a Perkin-Elmer 2400 II elemental analyzer. The
optical rotations were measured in a 1.0 dm tube with Jasco dip-140 polarimeter in chloro-
form. The excess of the reagents or solvents were evaporated under reduced pressure at a
bath temperature between the ranges 55–608C.
General Procedure for the Preparation of the Compounds (5–15)
Ethyl 5-(50-amino-30-O-benzyl-50-deoxy-10,20-O-isopropylidene-a-D-xylofuranos-
50-yl)-3-O-benzyl-5,6-dideoxy-1,2-O-isopropylidene-b-L-ido-heptofuranuronate
˚
(5). To a magnetically stirred slurry of 4 A molecular sieve (6.0 g) in dry chloro-
form (6.0 mL), 3-O-benzyl-1,2-O-isopropylidene-a-D-xylofuranos-5-ulose, 1a (1.0 g,
3.59 mmol) in dry chloroform (5.0 mL), and ethyl 5-amino-3-O-benzyl-5,6-dideoxy-1,2-
O-isopropylidene-b-L-ido-heptofuranuronate, 2a (1.31, 3.59 mmol) in dry chloroform
(5.0 mL) was added sequentially at 08C, and stirring was continued for 30 min. Reaction
mixture was further stirred for 6 hr at ambient temperature, until the disappearance of the
aldehyde (TLC). Reaction mixture was filtered and concentrated under reduced pressure

500
Verma et al.
and the residue thus obtained was dissolved in methanol (15.0 mL) and stirred magneti-
cally at 08C. Sodium borohydride (0.136 g, 3.70 mmol) was added to the stirring reaction
mixture and reaction continued for 3 hr at ambient temperature. Saturated ammonium
chloride solution was added to the reaction mixture and it was filtered. The solid cake
was washed with methanol and the combined filtrate was concentrated and extracted
with ethyl acetate (2 ꢁ 50 mL) and washed with water (2 ꢁ 12.5 mL). The organic
layer was dried (Na2SO4) and concentrated under reduced pressure to give a crude
mass, which was chromatographed over SiO2 column using hexane/ethyl acetate (4 : 1)
as eluent to give compound 5 (2.18 g, 97.4%) as colorless oil; Rf 0.50 (hexane/ethyl
acetate, 13 : 7), [a]2D0 252.58 (c 0.80, chloroform); MS (FAB) ¼ m/z 628 (M þ H)þ; IR
1
(Neat) nmax cm21: 3753, 1730; H NMR (200 MHz, CDCl3): d 7.31 (m, 10H, Ar-H);
5.92 (d, J ¼ 3.9 Hz, 1H, H-1), 5.90 (d, J ¼ 3.6 Hz, 1H, H-10), 4.69–4.42 (m, 6H,
2 ꢁ CH2Ph, H-2, H-20), 4.26 (m, 1H, H-4), 4.16 (m, 1H, H-40), 4.04 (q, J ¼ 6.9 Hz, 2H,
OCH2CH3), 3.90 (m, 2H, H-3, H-30), 3.50 (m, 1H, H-5), 3.04 (m, 2H, CH2NH), 2.36
(m, 2H, H-6), 1.59 (br s, exchangeable 1H, NH), 1.47, 1.25 [s, 12H, 2 ꢁ (CH3)2C],
1.19 (t, J ¼ 6.9 Hz, 3H, OCH2CH3); 13C NMR (50 MHz, CDCl3): d 172.0 (C55O),
138.8, 137.5, 128.4, 128.2, 128.1, 128.0 (Ar-C), 111.9, 111.8 [2 ꢁ (CH3)2C], 105.3,
105.2 (C-1, C-10), 82.7, 82.6 (C-2, C-20), 82.3, 82.2 (C-4, C-40), 80.6, 80.5 (C-3, C-30),
72.2 (-OCH2Ph), 60.7 (OCH2CH3), 54.8 (C-5), 45.8 (CH2NH), 36.7 (C-6), 27.1, 26.7
[C(CH3)2], 14.5 (CH3).
Anal. Calcd. for C34H45O10N; C, 65.07, H 7.17, N, 2.20; Found: C, 65.10, H, 7.10,
N 2.17.
Ethyl 5-(50-amino-50-deoxy-10,20-O-isopropylidene-30-O-methyl-a-D-xylofuranos-
50-yl)-3-O-benzyl-5,6-dideoxy-1,2-O-isopropylidene-b-L-ido-heptofuranuronate
(6). Reaction of the amino ester 3a (1.39 g, 4.95 mmol) with the glycosyl aldehyde 1b
(1.0 g, 4.95 mmol) in presence of NaBH4 (0.139 g, 3.6 mmol) as described above, followed
by column chromatography of the crude product using ethyl acetate : hexane (1 : 4) as
eluent gave compound 6 (2.45 g, 90.0%), as colorless oil; Rf 0.50 (hexane : ethyl
acetate, 3 : 2) [a]2D0 287.08 (c 0.10, chloroform); MS (FAB) ¼ m/z 552 (M þ H)þ; IR
1
(Neat) nmax cm21: 3345, 1731; H NMR (200 MHz, CDCl3): d 7.32–7.26 (m, 5H, Ar-
H), 5.93 (d, J ¼ 3.6 Hz, 1H, H-10), 5.86 (d, J ¼ 3.8 Hz, 1H, H-1), 4.66–4.48
(d, J ¼ 11.8 Hz, 1H, CHAPh), 4.62 (d, J ¼ 3.6 Hz, 1H, H-20), 4.53 (d, J ¼ 3.8 Hz,1H,
H-2), 4.43 (d, J ¼ 11.8 Hz, 1H, CHBPh), 4.42–4.10 (m, 2H, H-4, H-40), 4.08
(q, J ¼ 7.2 Hz, 2H, OCH2CH3), 3.91 (d, J ¼ 3.2 Hz, 1H, H-3), 3.67 (d, J ¼ 3.2 Hz, 1H,
H-30), 3.40 (m, 1H, H-5), 3.36 (s, 3H, OCH3), 2.36 (m, 2H, H-6), 2.95 (d, J ¼ 6.4 Hz,
2H, CH2NH), 2.36 (m, 2H, H-6), 1.62 (br s, exchangeable 1H, -NH), 1.48, 1.30 [s, 6H,
(CH3)2C], 1.25–1.19 [m, 9H, (CH3)2C and OCH2CH3]; 13C NMR (50 MHz, CDCl3): d
172.0 (C55O), 137.4, 128.9, 128.4, 128.2 (Ar-C), 111.9, 111.7 [2 ꢁ (CH3)2C], 105.1,
105.2 (C-1, C-10), 84.4, 84.3 (C-2, C-20), 82.5, 82.3 (C-4, C-40), 80.4, 80.3 (C-3, C-30),
71.8 (OCH2Ph), 60.7 (OCH2CH3), 58.0 (–OCH3), 54.4 (C-5), 45.4 (CH2 NH), 36.5 (C-
6), 27.1, 26.6 [2 ꢁ C(CH3)2], 14.5 (CH3).
Anal. Calcd. for C28H41O10N: C, 60.90, H, 7.44, N, 2.50; Found: C, 60.92, H, 7.40,
N, 2.54.
Ethyl 5-(30-O-allyl-50-amino-50-deoxy-10,20-O-isopropylidene-a-D-xylofuranos-50-
yl)-3-O-benzyl–5,6-dideoxy-1,2-O-isopropylidene-b-L-ido-heptofuranuronate
(7). Reaction of the aldehyde 1c (1.0 g, 4.38 mmol) with amino ester, 3b (1.60 g,
4.38 mmol) in presence of NaBH4 (0.168 g, 4.38 mmol) as described above, followed by

Reductive Amination of Glycosyl Aldoses
501
column chromatography gave 7 (1.9 g, 75%) as colorless oil; Rf 0.52 (hexane : ethyl
acetate, 3 : 2) [a]2D0 252.08 (0.10, chloroform); MS (FAB) ¼ m/z 578 (M þ H)þ;
1
IR(Neat) nmax cm21: 3655, 1732; H NMR (200 MHz, CDCl3): d 7.32–7.27 (m, 5H,
Ar-H), 5.90–5.87 (m, 3H, H-1, H-10, CH2CH55CH2); 5.20 (m, 2H, OCH2CH55CH2),
4.71–4.41 (m, 4H, OCH2Ph, H-2, H-20), 4.20–4.06 (m, 6H, H-4, H-40, OCH2CH55CH2,
OCH2CH3), 3.90 (d, J ¼ 3.2 Hz, 1H, H-30), 3.83 (d, J ¼ 3.2, 1H, H-3), 3.30 (m, 1H, H-5),
2.90 (m, 2H, CH2NH), 1.75 (br s, exchangeable H, NH), 1.47–1.19 [m, 15H,
2 ꢁ (CH3)2C, OCH2CH3], 13C NMR (50 MHz, CDCl3): d 172.1 (C55O), 138.0, 128.8
(Ar-C), 133.95 (OCH2CH55CH2), 118.4 (OCH2CH55CH2), 111.9 [(CH3)2C], 105.2,
105.1 (C-1, C-10), 83.2, 83.1, 82.7, 82.6, 82.2 (C-2, C-20, C-4, C-40), 80.6, 80.5 (C-3,
C-30), 72.7 (OCH2Ph), 71.3 (OCH2CH55CH2), 60.8 (OCH2CH3), 54.7 (C-5), 45.9
(CH2NH), 36.7 (C-6), 27.1, 26.7 [C(CH3)2], 14.5 (CH3).
Anal. Calcd. for C30H43O10N; C, 62.40, H, 7.40, N, 2.40; Found: C, 62.45, H, 7.42,
N, 2.38.
Ethyl 5-(60-amino-60-deoxy-10,20:30,40-di-O-isopropylidene-a-D-galactoctapyra-
nos-60-yl)-3-O-benzyl-5,6-dideoxy-1,2-O-isopropylidene-b-L-ido-heptofuranuronate
(8). Reaction of amino ester 3a (2.80 g, 7.75 mmol) with the aldehyde 2 (2.09 g,
7.75 mmol) in presence of NaBH4 (0.25 g, 6.61 mmol) as described above gave compound
8 (3.6 g, 85%) as colorless oil; Rf 0.50 (hexane : ethyl acetate, 3 : 2), [a]2D0 264.08 (c 0.10,
1
chloroform); MS (FAB) ¼ m/z 608 (M þ H)þ; IR (Neat) nmax cm21: 3655, 1733; H
NMR (200 MHz, CDCl3): d 7.34–7.28 (m, 5H, Ar-H), 5.96 (d, J ¼ 3.6 Hz, 1H, H-1),
5.53 (d, J ¼ 5.1 Hz, 1H, H-10), 4.71–4.56 (m, 3H, OCHAPh, H-2, H-30), 4.49
(d, J ¼ 11.7 Hz, 1H, CHBPh), 4.30–4.20 (m, 3H, H-20, H-4 and H-40), 3.95
(q, J ¼ 6.8 Hz, 2H, OCH2), 3.80–3.70 (m, 2H, H-3, H-50), 3.50 (m, 1H, H-5), 2.90
(m, 2H, H-60), 2.30 (m, 2H, H-6), 1.69 (br s, exchangeable H, NH), 1.50, 1.44 [s, 12H,
2 ꢁ (CH3)2C], 1.32 [s, 6H, (CH3)2C], 1.24 (t, J ¼ 6.8 Hz, 3H, –OCH2CH3); 13C NMR
(50 MHz, CDCl3): d 172.2 (C55O), 137.6, 128.8, 128.3, 128.1 (Ar-C), 111.9, 109.4,
108.8 [3 ꢁ (CH3)2C], 105.3 (C-1), 96.7 (C-10), 82.4, 82.2, 82.1 (C-4, C-2, and C-3),
72.0, 71.9, 71.2, 70.0 (C-20, C-40, C-30, OCH2Ph), 67.4 (C-50), 60.7 (OCH2CH3), 54.0
(C-5), 47.1 (C-60), 36.5 (C-6), 27.1, 26.4, 24.8 [3 ꢁ C(CH3)2], 14.5 (CH3).
Anal. Calcd. for C31H45O11N; C, 61.28, H, 7.41, N, 2.31; Found: C, 61.20, H, 7.46,
N, 2.26.
Ethyl 5-(60-amino-60-deoxy-10,20:30,40-di-O-isopropylidene-a-D-galactoctapyra-
nos-60-yl)-5,6-dideoxy-1,2-O-isopropylidene-3-O-methyl-b-L-ido-heptofuranuronate
(9). Reaction of aldehyde, 2 (1.09 g, 3.87 mmol) with amino ester 3b (1.13 g, 3.87 mmol)
in presence of NaBH4 (0.15 g, 3.96 mmol) as described above gave compound 9 (1.54 g,
75.4%) as colorless oil; Rf 0.51 (hexane : ethyl acetate, 3 : 2), [a]2D0 281.38 (c 0.15, chloro-
1
form); MS (FAB) ¼ m/z 532 (M þ H)þ; IR(Neat) nmax cm21: 3757, 3346; H NMR
(200 MHz, CDCl3): d 5.90 (d, J ¼ 4.0 Hz, 1H, H-1); 5.53 (d, J ¼ 5.0 Hz, 1H, H-10),
4.60–4.56 (m, 2H, H-30, H-2), 4.31 (d, J ¼ 5.0 Hz, 1H, H-20), 4.21 (m, 3H, H-40,
OCH2CH3), 3.90 (m, 1H, H-4), 3.86–3.60 (m, 1H, H-50), 3.60 (d, J ¼ 2.0 Hz, 1H, H-3),
3.41 (s, 3H, OCH3), 3.10 (m, 1H, H-5), 2.98 (m, 2H, H-60), 1.90 (m, 2H, H-6), 1.70 (br
s, exchangeable H, –NH), 1.53, 1.48, 1.43 [s, 6H, 2 ꢁ (CH)3C], 1.23 [s, 6H,
2 ꢁ (CH3)2C], 1.26 (t, J ¼ 7.2 Hz, 3H, OCH2CH3); 13C NMR (50 MHz, CDCl3):
d 111.8, 109.6, 108.9 [3 ꢁ (CH3)2C], 104.8 (C-1), 96.7 (C-10), 84.3 (C-2), 82.2, 81.5
(C-4, C-3), 72.2 (C-30), 71.2, 70.9 (C-20, C-50), 67.9 (C-40), 62.6 (OCH2CH3), 57.8
(–OCH3), 47.8 (CH2NH), 30.2 (C-6), 27.1, 26.4, 24.8 [C(CH3)2], 14.5 (OCH2CH3).

502
Verma et al.
Anal. Calcd. for C25H41O11N; C,56.49, H, 7.72, N, 2.63; Found: C, 56.40, H, 7.70,
N, 2.60.
Ethyl 5-(50-amino-30-O-benzyl-50-deoxy-10,20-O-isopropylidene-a-D-xylofuranos-
50-yl)-3-O-allyl-5,6-dideoxy-1,2-O-isopropylidene-b-L-ido-heptofuranuronate
(10). Reaction of aldehyde, 1a (1.0 g, 3.59 mmol) with amino ester 3c (1.13 g, 3.59 mmol)
in presence of NaBH4 (0.137 g, 3.70 mmol) as described above gave compound 10 (1.92 g,
93.8%) as colorless oil; Rf 0.50 (hexane : ethyl acetate, 3 : 2), [a]2D0 258.08 (c 0.10, chloro-
1
form); MS (FAB) ¼ m/z 578 (M þ H)þ; IR(Neat) nmax cm21: 1730, 3630; H NMR
(200 MHz, CDCl3): d 7.33–7.26 (m, 5H, Ar-H), 5.92–5.81 (m, 3H, H-1, H-10,
CH255CHCH2), 5.23 (dd, J ¼ 19.0 and 1.4 Hz, 2H, CH2CH55CH2), 4.75–4.52 (m, 4H,
CHAPh, CHBPh, H-2, H-20), 4.21–3.89 (m, 4H, OCH2CH3, H-4, H-40), 3.90
(d, J ¼ 2.8 Hz, 1H, H-3), 3.83–3.76 (m, 3H, H-30, OCH2CH55CH2), 3.20 (m, 1H, H-5),
3.06 (m, 2H, CH2NH), 1.70 (br s, exchangeable H, –NH), 1.57, 1.31 [s, 6H,
2 ꢁ (CH)2C], 1.22 (t, J ¼ 6.8 Hz, 3H, CH3); 13C NMR (50 MHz, CDCl3): d 137.9
(CH2CH55CH2), 133.9, 128.8, 127.9 (Ar-C), 118.5 (CH2CH ¼ CH2), 111.9
[2 ꢁ (CH3)2C], 105.2, 104.9 (C-1, C-10), 82.7, 82.5, 82.3, 81.9, 80.6 (C-2, C-4, C-40,
C-20, C-3), 80.3 (C-30), 72.0 (OCH2Ph), 62.3 (OCH2CH55CH2), 60.8 (OCH2CH3),
57.4(C-5), 44.4 (CH2NH), 30.1 (C-6), 27.1, 26.6, 24.9 [2 ꢁ C(CH3)2], 14.5 (CH3).
Anal. Calcd. for C30H43O10N C, 62.39; H, 7.45; N, 2.23; Found: C,62.30; H, 7.40;
N, 2.20.
Ethyl 5-(50-amino-50-deoxy-10,20-O-isopropylidene-30-O-methyl-a-D-xylofuranos-
50-yl)-3-O-allyl-5,6-dideoxy-1,2-O-isopropylidene-b-L-ido-heptofuranuronate
(11). Reaction of aldehyde 1b (1.0 g, 4.95 mmol) with amino ester 3c (1.56 g, 4.95 mmol)
in presence of NaBH4 (0.37 g, 5.01 mmol) as described above gave compound 11 (2.0 g,
82.2%) as colorless oil; Rf 0.48 (hexane : ethyl acetate, 3 : 2), [a]2D0 252.08 (c 0.10, chloro-
1
form); MS (FAB) ¼ m/z 502 (M þ H)þ; IR (Neat) nmax cm21: 3788, 1780; H NMR
(200 MHz, CDCl3): d 5.92 (two d, J ¼ 3.8 Hz, 2H, H-1, H-10), 5.87 (m, 1H, OCH2-
CH55CH2), 5.23 (m, 2H, OCH2CH55CH2), 4.54 (two d, 1H, J ¼ 3.8 Hz, H-2, H-20),
4.16 (m, 2H, C-4, C-40), 4.14 (q, J ¼ 6.2 Hz, 2H, OCH2CH3), 3.78 (d, J ¼ 3.2 Hz, 1H,
H-3), 3.68 (d, J ¼ 3.2 Hz, 1H, H-30), 3.39 (s, 3H, –OCH3), 3.20 (m, 1H, H-50), 3.04
(d, 1H, J ¼ 6.4 Hz, 2H, H-50), 1.70 (br s, exchangeable 1H, NH), 1.65 (m, 2H, H-6),
1.49, 1.30 [s, 12H, 2 ꢁ (CH3)2C], 1.12 (OCH2CH3); 13C NMR (50 MHz, CDCl3):
d 133.95 (CH255CHCH2), 118.4 (CH255CHCH2), 111.9, 111.8 [2 ꢁ (CH3)2C], 105.1,
104.9 (C-1, C-10), 84.5 (C-2); 82.4, 82.3, 82.2, 81.7 (C-20, C-4, C-40 C-30), 80.1 (C-3),
71.1 (OCH2CH55CH2), 60.8 (-OCH2CH3) 57.9 (OCH3), 57.0 (C-5), 44.1 (C-50), 36.5
(C-6), 27.1, 26.3 [C(CH3)2], 14.5 (OCH2CH3).
Anal. Calcd. for C23H37NO10: C, 56.66; H, 7.65; N, 2.87; Found: C, 56.32; H, 7.82;
N, 2.77.
Ethyl 6-(50-amino-30-O-benzyl-50-deoxy-10,20-O-isopropylidene-a-D-xylofuranos-
50-yl)-6,7-dideoxy-1,2 : 3,4-di-O-isopropylidene-b-L-glycero-a-D-galactoctapyranuro-
nate (12). Reaction of aldehyde 1a (2.0 g, 7.19 mmol) with amino ester 4 (2.50 g,
7.19 mmol) in presence of NaBH4 (0.270 g, 7.10 mmol) as described above gave the com-
pound 12 (3.60 g, 80%) as colorless oil; Rf 0.52 (hexane : ethyl acetate, 3 : 2), [a]D –40.08
(c 0.10, chloroform); MS (FAB) ¼ m/z 608 (M þ H)þ; IR(Neat)nmax cm21: 3370, 1725;
1H NMR (200 MHz, CDCl3): d 7.35–7.27 (m, 5H, Ar-H), 5.93 (d, J ¼ 3.9 Hz, 1H, H-10),
5.52 (d, J ¼ 5.1 Hz, 1H, H-1), 4.63–4.54 (m, 4H, CHAPh, CHBPh, H-3,H-20), 4.37–4.30
(m, 3H, H-2, H-4, H-40), 4.08 (q, J ¼ 7.2 Hz, 2H, OCH2CH3), 3.96 (d, J ¼ 3.0 Hz, 1H,

Reductive Amination of Glycosyl Aldoses
503
H-30), 3.90 (d, J ¼ 7.2 Hz, 1H, H-5), 3.40 (m, 1H, H-6), 3.0 (m, 2H, H-50), 2.60–2.40
(m, 2H, H-7), 1.63 (br s, 1H, NH) 1.50–1.22 [m, 21H, 2 ꢁ (CH)3C, CH3]; 13C NMR
(50 MHz, CDCl3): d 172.5 (C55O), 138.1, 128.9, 129.0, 127.9 (Ar-C), 111.9, 109.6,
108.6 [3 ꢁ (CH3)2C], 105.3 (C-1), 96.9 (C-10), 82.7, 82.5 (C-20,C-40), 80.4 (C-30), 72.3
(OCH2Ph), 71.7, 71.4, 71.0 (C-2, C-4, C-3), 68.8 (C-5), 60.7 (OCH2CH3), 56.1 (C-6),
45.5 (CH2NH), 35.9 (C-7), 27.1, 24.7 [C(CH3)2].
Anal. Calcd. for C31H45NO11: C, 61.27; H, 7.46; N, 2.30; Found: C, 61.22; H, 7.40;
N, 2.38.
Ethyl 6-(50-amino-50-deoxy-10,20-O-isopropylidene-30-O-methyl-a-D-xylofuranos-
50-yl)-6,7-dideoxy-1,2 : 3,4-di-O-isopropylidene-b-L-glycero-a-D-galactoctapyranuro-
nate (13). Reaction of aldehyde, 1b (1.50 g, 7.42 mmol) with amino ester 4 (2.70 g,
7.42 mmol) in presence of NaBH4 (0.250 g, 6.61 mmol) as described above gave the
above compound 13 (2.32 g, 59.2%) as colorless oil; Rf 0.54 (hexane : ethyl acetate,
3 : 2), [a]2D0 223.78 (c 0.12, chloroform); MS (FAB) ¼ m/z 532 (M þ H)þ; IR (Neat)
1
n
max cm21: 3361,1726; H NMR (200 MHz, CDCl3): d 5.87 (d, J ¼ 3.8, 1H, H-10), 5.50
(d, J ¼ 5.0, 1H, H-1), 4.61–4.49 (m, 3H, H-2, H-20, H-3), 4.28–4.25 (m, 2H, H-4,
H-40), 4.13 (q, J ¼ 6.2 Hz, 2H, OCH2CH3), 3.74 (m, 2H, H-30, H-5), 3.40 (s, 3H,
OCH3), 3.60 (m, 1H, H-6), 2.94 (m, 2H, H-50), 2.40–2.20 (m, 2H, H-7), 1.63 (br s,
exchangeable1H, -NH), 1.48–1.21 [m, 21H, (2 ꢁ CH)3C, CH3]; 13C NMR (50 MHz,
CDCl3): d 173.0 (C55O), 111.9, 109.3, 108.8 [(CH3)2C], 105.2 (C-10), 96.9 (C-1), 84.2
(C-20), 82.0 (C-40), 80.0, 79.9 (C-3, C-30), 71.2, 70.8 (C-2, C-4), 68.7 (C-5), 60.4
(OCH2), 58.1 (-OCH3), 54.4 (C-6), 44.1 (CH2NH), 34.7 (C-7), 27.1, 26.4, 24.9
[3 ꢁ C(CH3)2], 14.6 (CH3).
Anal. Calcd. for C25H41NO11:C, 56.49; H, 7.72; N, 2.63;. Found: C, 56.43; H, 7.70;
N, 2.68.
Ethyl 6-(30-O-allyl-50-amino-50-deoxy-10,20-O-isopropylidene-a-D-xylofuranos-50-
yl)-6,7-dideoxy-1,2 : 3,4-di-O-isopropylidene-b-L-glycero-a-D-galactoctapyranuro-
nate (14). Reaction of aldehyde, 1c (0.73 g, 3.30 mmol) with amino ester 4 (1.1 g,
3.30 mmol) in presence of NaBH4 (0.140 g, 5.2 mmol) as described above gave the com-
pound 14 (1.6 g, 88%) as colorless oil; Rf 0.48 (hexane : ethyl acetate, 3 : 2), [a]2D0 282.08
(c 0.10, chloroform); MS (FAB) ¼ m/z 558 (M þ H)þ; IR (Neat) nmax cm21: 3679, 1731;
1H NMR (200 MHz, CDCl3): d 5.91 (d, J ¼ 3.6 Hz, 1H, H-10), 5.55 (d, J ¼ 5.2 Hz, 1H,
H-1), 5.25 (dd, J ¼ 19.0 and 1.4 Hz, 2H, OCH2CH55CH2), 4.59–4.53 (m, 2H, H-3,
H-20), 4.33 (dd, J ¼ 5.2 and 2.0 Hz, 1H, H-2), 4.12 (q, J ¼ 6.8 Hz, 2H, OCH2), 4.00
(m, 2H, H-4, H-40), 3.90 (m, 1H, H-30), 3.11–3.05 (m, 3H, CH2NH, H-5), 2.91 (m, 1H,
H-6), 1.90–1.60 (m, 2H, H-7), 1.70 (br s, exchangeable H, -NH), 1.49, 1.44 [s, 12H,
2 ꢁ (CH)3C], 1.31 [s, 6H, (CH3)2C], 1.25 (t, J ¼ 6.8 Hz, 3H, OCH2CH3); 13C NMR
(50 MHz, CDCl3): d 172.2 (C ¼ O), 134.4 (CH255CHCH2O), 118.2 (OCH2CH55CH2),
111.8, 109.6, 108.8 [3 ꢁ (CH3)2C], 105.2 (C-10), 96.9 (C-1), 82.7, 82.1, 79.9 (C-20,C-
40,C-30), 71.4, 71.1, 70.9 (C-2, C-3, C-4), 68.8 (C-5), 60.7 (OCH2CH3), 57.7 (C-6), 43.6
(CH2NH), 28.4 (C-7), 27.1, 26.7, 24.9 [3 ꢁ C(CH3)2], 14.5 (CH3).
Anal. Calcd. for C27H43NO11: C, 58.15; H, 7.77; N, 2.51; Found: C, 58.20; H, 7.82;
N, 2.46.
Ethyl
6-(60-amino-60-deoxy-10,20:30,40-di-O-isopropylidene--a-D-galactoctapyra-
nos-60-yl)-6,7-dideoxy-1,2:3,4-di-O-isopropylidene-b-L-glycero-a-D-galactoctapyran
uronate (15). Reaction of aldehyde 2 (2.0g, 7.75mmol) with amino ester 4 (2.70 g,
7.75mmol)inpresenceof NaBH4 (0.29 g, 7.86mmol) asdescribed above gave the compound

504
Verma et al.
15 (4.10 g, 90%) as colorless oil; Rf 0.50 (hexane : ethyl acetate, 3 : 2), [a]2D0 254.08 (c 0.10,
chloroform); MS (FAB) ¼ m/z 588 (M þ H)þ; IR (Neat) nmax cm21: 3679, 1726; 1H NMR
(200MHz, CDCl3): d 5.55, 5.50 (two d, J ¼ 5.0 Hz, 2H, H-1, H-10), 4.58 (m, 2H, H-3,
H-30), 4.37–4.26 (m, 4H, H-2, H-20, H-4, H-40), 4.13 (q, J ¼ 6.8 Hz, 2H, OCH2), 3.80
(d, J ¼ 6.6 Hz, 2H, H-5, H-50), 3.32 (m, 1H, H-6), 2.86–2.33 (m, 4H, H-60, H-7), 1.85 (br
s, exchangeable 1H, NH), 1.53–1.21 [m, 27H, 4 ꢁ (CH3)2C, CH3]; 13C NMR (50 MHz,
CDCl3): 172.7 (C55O), 109.5, 109.3, 108.8, 108.7 [(CH3)2C], 97.9, 96.7 (C-1, C-10), 72.0,
71.8, 71.2, 71.0, 70.9, 68.6, 62.6 (C-2, C-20 C-4, C-3, C-30, C-50, C-5), 60.0 (OCH2), 55.4
(C-6), 46.5 (CH2NH), 36.0 (C-7), 26.4, 25.3, 24.8, 24.7 [(CH3)2C]; 14.5 (CH3).
Anal. Calcd. for C28H45NO12 C, 57.23; H, 7.72; N, 2.38;. Found: C, 57.20; H, 7.76;
N, 2.40.
General Procedure for the Preparation of the Compounds (16–26)
5-(50-Amino-30-O-benzyl-50-deoxy-10,20-O-isopropylidene-a-D-xylofuranos-50-yl)-
3-O-benzyl-5,6-dideoxy-1,2-O-isopropylidene-b-L-ido-heptanol (16). To a magnetically
stirred slurry of LiAlH4 in anhydrous THF (2 mL), a solution of ethyl 5-(50-amino-30-
O-benzyl-50-deoxy-10,20-O-isopropylidene-a-D-xylofuranos-50-yl)-3-O-benzyl-5,6-dideoxy-
1,2-O-sopropylidene-b-L-ido-heptofuranuronate 5 (1.5 g, 2.39 mmol) in anhydrous THF
(5.0 mL) was added drop-wise at 08C, and stirring continued for 30 min at 08C. The reac-
tion mixture was further stirred magnetically for 5 hr at ambient temperature. Excess
LiAlH4 was quenched by adding saturated aqueous sodium sulphate solution, and the reac-
tion mixture was filtered. The solid cake was washed with THF and the filtrate concen-
trated under reduced pressure. The later was extracted with chloroform (2 ꢁ 25 mL)
and water (12.5 mL) and dried (Na2SO4). Organic layer was concentrated under
reduced pressure to give a crude mass, which was chromatographed over SiO2 column
using chloroform : methanol (98 : 2) as eluent to give 16 (1.12 g, 80%) as colorless oil;
Rf 0.5 (chloroform : methanol, 24 : 1); [a]2D0 261.88 (c 0.22, chloroform); MS
1
(FAB) ¼ m/z 586 (M þ H)þ; IR (Neat): nmax cm21 3332, 3754; H NMR (200 MHz,
CDCl3): d 7.31 (m, 10H, Ar-H); 5.90 (two d, J ¼ 3.9 and 3.6 Hz, 2H, H-1, H-10), 4.71–
4.51 (m, 6H, 2 ꢁ CH2Ph, H-2, H-20), 4.17 (m, 2H, H-4, H-40), 3.90 (d, J ¼ 3.2 Hz, 1H,
H-3), 3.82 (d, J ¼ 3.2 Hz, 1H, H-30), 3.72 (m, 2H, H-7), 3.30 (m, 1H, H-5), 3.0 (m, 2H,
H-50), 1.91 (br s, exchangeable 1H, NH), 1.47 [s, 6H, (CH3)2C], 1.32–1.25 [m, 8H,
(CH3)2C, H-6]; 13C NMR (50 MHz, CDCl3): d 137.9, 128.9, 128.4, 128.1 (Ar-C),
111.9, 111.8 [2 ꢁ (CH3)2C], 105.2, 105.1 (C-1, C-10), 82.9, 82.6, 82.3, 81.9, 81.7, 82.2
(C-2, C-20 C-4, C-40 C-3, C-30), 72.3 (CH2Ph), 62.5 (C-7), 57.2 (C-5), 44.2 (CH2NH),
30.0 (C-6), 27.1, 26.7 [C(CH3)2].
Anal. Calcd. for C32H43NO9: C, 65.64; H, 7.35; N, 2.39; Found: C, 65.10; H, 7.25;
N, 2.38.
5-(50-Amino-50-deoxy-10,20-O-isopropylidene-30-O-methyl-a-D-xylofuranos-50-yl)-
3-O-benzyl-5,6-dideoxy-1,2-O-isopropylidene-b-L-ido-heptanol (17). Reduction of
amino ester 6 (4.0 g, 7.85 mmol) with LiAlH4, (0.59 g, 15.7 mmol) and work up as described
above afforded glycosyl amino alcohol 17 (1.56 g, 77.5%) as colorless oil;. Rf, 0.5 (chloro-
form/methanol, 24 : 1); [a]2D0 243.08 (c 0.10, chloroform); MS (FAB) ¼ m/z 510
1
(M þ H)þ; IR (Neat) nmax cm21 3339, 3754; H NMR (200 MHz, CDCl3): d 7.33–7.26
(m, 5H, Ar-H), 5.93 and 5.87 (two d, 1H, J ¼ 3.7 Hz, 2H, H-1, and H-10), 4.66–4.63

Reductive Amination of Glycosyl Aldoses
505
(m, 2H, CHAPh, and H-20), 4.55 (d, J ¼ 3.7 Hz, 1H, H-2), 4.42 (d, J ¼ 11.6 Hz, 1H,
OCHBPh), 4.22–4.10 (m, 2H, H-4 and H-40), 3.81 (d, J ¼ 3.2 Hz, 1H, H-3), 3.70 (m, 2H,
H-7), 3.67 (d, J ¼ 3.2 Hz, 1H, H-30), 3.37 (s, 3H, OCH3), 3.32 (m, 1H, H-5), 3.02 (m, 2H,
CH2NH), 2.70 (br s, exchangeable H, 1H, NH), 1.48 [s, 6H, (CH3)2C], 1.32–1.22 (m, 8H,
(CH3)2, H-6] 13C NMR (50 MHz, CDCl3): 137.3, 128.9, 128.2 (Ar-C), 112.0, 111.8
[(CH3)2C], 105.2, 105.1 (C-1, C-10), 84.5, 82.5, 82.2, 81.8, 81.7, 80.1 (C-2, C-20, C-4,
C-40, C-3, C-30), 72.1 (CH2Ph), 62.5 (C-7), 57.1 (OCH3), 43.8 (CH2NH), 29.8 (C-6),
27.1, 26.6 [C(CH3)2].
Anal. Calcd. for C26H39NO9: C, 61.29; H, 7.66; N, 2.75; Found: C, 61.14; H, 7.26;
N, 2.30.
5-(30-O-Allyl-50-amino-50-deoxy-10,20-O-isopropylidene-a-D-xylofuranos-50-yl)-3-
O-benzyl-5,6-dideoxy-1,2-O-isopropylidene-b-L-ido-heptanol (18). Reduction of amino
ester 7 (1.0g, 1.73mmol) with LiAlH4, (0.14 g, 3.46mmol) and work up as described
above afforded glycosyl amino alcohol 18 (0.32 g, 60%) as colorless oil; Rf, 0.5 (chloro-
form/methanol, 24: 1); [a]2D0 268.08 (c 0.10, chloroform); MS (FAB) ¼ m/z 536
1
(M þ H)þ; IR (Neat) nmax cm21: 3347, 3757; H NMR (200MHz, CDCl3): d 7.32–7.27
(m, 5H, Ar-H), 5.90, 5.87 (m, 3H, H-1, H-10, OCH2CH55CH2), 5.20 (dd, J ¼ 19.0Hz,
1.4 Hz, 2H, OCH2CH55CH2), 4.71–4.41 (m, 4H, OCH2Ph, H-2, H-20), 4.20–4.06 (m, 4H,
H-4, H-40OCH2CH55CH2), 3.83–3.80 (m, 2H, H-3, H-30), 3.73 (t, J ¼ 5.8 Hz, 2H, H-7),
3.30 (m, 1H, H-5), 2.90 (m, 2H, CH2NH), 1.75 (br s, exchangeable 1H, NH), 1.47 [s, 6H,
(CH3)2C], 1.32–1.30 [m, 8H, (CH3)2C, H-6]; 13C NMR (50 MHz, CDCl3): d 137.2 (OCH2-
CH55CH2), 134.4, 128.9, 128.5, 128.4 (Ar-C), 118.4 (OCH2CH55CH2), 111.9, 111.8
[(CH3)2C], 105.2, 105.0 (C-1, C-10), 83.2, 82.6, 82.2, 82.1 81.8, 81.7 (C-2, C-20, C-4, C-40,
C-3, C-30), 72.7 (OCH2Ph), 71.3 (OCH2CH55CH2), 62.5 (C-7), 57.1 (C-5), 45.9 (CH2NH),
29.8 (C-6), 27.1, 26.7 [2 ꢁ C(CH3)2].
Anal. Calcd. for C28H41NO9: C, 62.80; H, 7.66; N, 2.62; Found: C, 62.32; H, 7.26;
N, 2.38.
5-(60-Amino-60-deoxy-10,20:30,40-di-O-isopropylidene-a-D-galactoctapyranos-60-yl)-
3-O-benzyl-5,6-dideoxy-1,2-O-isopropylidene-b-L-ido-heptanol (19). Reduction of
amino ester 8 (1.5 g, 2.47mmol) with LiAlH4 (0.18 g, 4.49mmol) and work up as described
above afforded glycosyl amino alcohol 19 (0.55 g, 65%) as colorless oil; Rf 0.50 (chloro-
form: methanol, 24: 1), [a]2D0 276.08 (c 1.0, chloroform); MS (FAB) ¼ m/z 566
1
(M þ H)þ; IR (Neat) nmax cm21: 3655, 3343; H NMR (200MHz, CDCl3): d 7.35–7.32
(m, 5H, Ar-H), 5.93 (d, J ¼ 3.9 Hz, 1H, H-10), 5.53 (d, J ¼ 4.8 Hz, 1H, H-10), 4.70–4.53
(m, 3H, OCHAPh, H-2, H-30), 4.40 (d, J ¼ 11.7Hz, 1H, CHBPh), 4.28 (dd, J ¼ 4.8 and
2.4 Hz, 1H, H-20), 4.16 (m, 2H, H-4, H-40), 3.80 (d, J ¼ 2.7 Hz, 1H, H-3), 3.75 (m, 2H,
H-7), 3.31 (m, 1H, H-5), 2.97 (m, 2H, H-6), 1.90 (br s, exchangeable 1H, NH), 1.50–1.24
[m, 20H, 3 ꢁ (CH)3C, H-6]; 13C NMR (50 MHz, CDCl3): d 137, 128.9, 128.5, 128.3 (Ar-
C), 111.9, 109.6, 108.9 [(CH3)2C], 105.1 (C-1), 96.7(C-10), 82.2, 81.8 (C-2, C-4), 72.2,
72.0, 71.2, 70.8 (C-20, C-40, C-30, OCH2Ph), 66.0 (C-3), 62.7 (C-7), 57.2 (C-5), 45.7
(CH2NH), 29.9 (C-6), 28.1, 27.1, 26.6, 26.4, 25.3, 24.9 [3 ꢁ C(CH3)2].
Anal. Calcd. for C29H43NO10: C, 61.59; H, 7.61; N, 2.48; Found: C, 61.04; H, 7.26;
N, 2.38.
5-(60-Amino-60-deoxy-10,20:30,40-di-O-isopropylidene-a-D-galactoctapyranos-60-
yl)-5,6-dideoxy-1,2-O-isopropylidene-3-O-methyl-b-L-ido-heptanol (20). Reduction
of amino ester 9 (0.8 g, 0.37 mmol) with LiAlH4 (0.12 g, 0.74 mmol) and work up as
described above afforded glycosyl amino alcohol 20 (0.23 g, 60%) as colorless oil; RF

506
Verma et al.
0.50 (chloroform : methanol, 24 : 1), [a]2D0 250.08 (c 0.12, chloroform); MS (FAB) ¼ m/z
1
490 (M þ H)þ; IR(Neat)nmax cm21: 3757, 3357; H NMR (200 MHz, CDCl3): d 5.90
(d, J ¼ 4.0 Hz, 1H, H-1), 5.53 (d, J ¼ 5 Hz, 1H, H-10), 4.60–4.56 (m, 2H, H-30, H-2),
4.31 (d, J ¼ 2.0 Hz, 1H, H-20), 4.21 (m, 1H, H-40), 3.90 (m, 1H, H-4), 3.86–3.60
(m, 3H, H-7, H-50), 3.60 (d, J ¼ 2.0 Hz, 1H, H-3), 3.41 (s, 3H, OCH3), 3.10 (m, 1H,
H-5), 2.98 (m, 2H, H-60), 2.40 (br s, exchangeable H, 1H, -OH), 1.70 (br s, exchangeable
H, 1H, –NH), 1.62–1.25 [m, 20H, 3 ꢁ (CH)3C, H-6]; 13C NMR (50 MHz, CDCl3): d
111.8, 109.6, 108.9 [(CH3)2C], 104.8 (C-1), 96.7 (C-10), 84.3 (C-2), 82.2, 81.5 (C-4,
C-3), 72.2 (C-30), 71.2, 70.9 (C-20, C-40), 67.9 (C-5), 62.6 (C-50), 57.8 (–OCH3), 47.8
(CH2NH), 30.2(C-6), 27.1, 26.6, 26.3, 25.3, 24.9 24.8 [C(CH3)2].
Anal. Calcd. for C23H39NO10: C, 56.44; H, 7.97; N, 2.86; Found: C, 56.14; H, 7.26;
N, 2.38.
5-(50-Amino-30-O-benzyl-50-deoxy-10,20-O-isopropylidene-a-D-xylofuranos-50-yl)-
3-O-allyl-5,6-dideoxy-1,2-O-isopropylidene-b-L-ido-heptanol (21). Reduction of
amino ester 10 (1.1 g, 1.90 mmol) with LiAlH4 (0.14 g, 3.80 mmol) and work up as
described above afforded glycosyl amino alcohol 21 (0.35 g, 60%) as colorless oil; Rf
0.50 (chloroform/methanol, 24 : 1), [a]2D0 250.08 (c 0.10, chloroform); MS
1
(FAB) ¼ m/z 536 (M þ H)þ; IR (Neat)nmax cm21: 3368, 3678; H NMR (200 MHz,
CDCl3): d 7.33–7.26 (m, 5H, Ar-H), 5.92–5.81 (m, 3H, H-1, H-10, OCH2CH55CH2),
5.23 (dd, J ¼ 19.0 and 1.4 Hz, 2H, OCH2CH55CH2), 4.75–4.52 (m, 4H, CHAPh,
CHBPh, H-2, H-20), 3.92–3.89 (m, 4H, H-7, H-4, H-40), 3.83–3.76 (m, 6H, H-3, H-30,
H-7, OCH2CH55CH2), 3.20 (m, 1H, H-5), 3.06 (m, 2H, CH2NH), 2.80 (br s, exchangeable
1H, –OH), 1.70 (br s, exchangeable 1H, -NH), 1.57, 1.31 [s, 6H, (CH3)2C], 1.22 [m, 8H,
(CH3)2C, H-6]; 13C NMR (50 MHz, CDCl3): d 137.9 (OCH2CH55CH2), 133.9, 128.8,
128.1, 127.9 (Ar-C), 118.5 (OCH2CH55CH2), 111.9, 111.8 (CH3)2C, 105.2, 105.1 (C-1,
C-10), 82.7, 82.5, 82.3, 82.1, 81.9 (C-2, C-20, C-4, C-40 C-3), 80.3 (C-30), 72.0
(OCH2Ph), 62.3 (C-7), 62.4 (OCH2CH55CH2), 57.4 (C-5), 44.4 (CH2NH), 30.1 (C-6),
32.5, 30.1, 27.1, 26.6 [C(CH3)2].
Anal. Calcd. for C28H41NO9: C, 62.80; H, 7.66; N, 2.61; Found: C, 62.12; H, 7.66;
N, 2.38.
5-(50-Amino-50-deoxy-10,20-O-isopropylidene-30-O-methyl-a-D-xylofuranos-50-yl)-
3-O-allyl-5,6-dideoxy-1,2-O-isopropylidene-b-L-ido-heptanol (22). Reduction of amino
ester 11 (1.3 g, 2.59 mmol) with LiAlH4, (0.19 g, 5.18 mmol) and work up as described
above afforded glycosyl amino alcohol 22 (0.41 g, 70%) as colorless oil; Rf 0.50 (chloro-
form/methanol, 24 : 1), [a]2D0 265.08 (c 0.10, chloroform); MS (FAB) ¼ m/z 460
(M þ H)þ; IR (Neat) nmax cm21: 3658, 3332; 1H NMR (200 MHz, CDCl3): d 5.92
(m, 2H, H-1, H-10), 5.87 (m, 1H, OCH2CH55CH2), 5.23 (dd, J ¼ 19.0 and 1.2 Hz, 2H,
OCH2CH55CH2), 4.54 (two d, J ¼ 3.8 Hz, 2H, H-2, H-20), 4.19–4.13 (m, 3H, H-4, OCH2-
CH55CH2), 3.93–3.82 (m, 4H, H-7,H-40, H-30), 3.78 (d, J ¼ 3.2 Hz, 1H, H-3), 3.39 (s, 3H,
OCH3), 3.20 (m, 1H, H-5), 3.04 (d, J ¼ 6.4 Hz, 2H, H-50), 1.70 (br s, exchangeable 1H, NH),
1.49 [s, 6H, (CH3)2C], 1.30 [m, 8H, (CH3)2, H-6]; 13C NMR (50 MHz, CDCl3): d 133.95
(OCH2CH55CH2), 118.4 (OCH2CH55CH2), 111.9, 111.8 [2 ꢁ (CH3)2C], 105.1, 105.0
(C-1, C-10), 84.5, 82.3, 81.9, 81.8, 81.7, 80.1 (C-4, C-40, C-2, C-20 C-3, C-30), 71.1 (OCH2-
CH55CH2), 62.3 (C-7), 57.9 (OCH3), 57.0 (C-5), 44.1 (CH2NH), 30.1 (C-6), 27.1, 26.3
[C(CH3)2].
Anal. Calcd. for C22H37NO9: C, 57.51; H, 8.06; N, 3.05; Found: C, 57.04; H, 8.26;
N, 2.78.

Reductive Amination of Glycosyl Aldoses
507
6-(50-Amino-30-O-benzyl-50-deoxy-10,20-O-isopropylidene-a-D-xylofuranos-50-yl)-
6,7-dideoxy-1,2 : 3,4-di-O-isopropylidene-b-L-glycero-a-D-galactoctanol (23).
Reduction of amino ester 12 (2.2 g, 3.62 mmol) with LiAlH4, (0.27 g, 7.24 mmol) and work
up as described above afforded glycosyl amino alcohol 23 (1.22 g, 60%) as colorless oil; Rf
0.50 (chloroform/methanol, 24 : 1), [a]2D0 267.18 (c 0.14, chloroform); MS (FAB) ¼ m/z
1
566 (M þ H)þ; IR (Neat) nmax cm21: 3654, 3439; H NMR (200 MHz, CDCl3): d 7.34–
7.26 (m, 5H, Ar-H), 5.93 (d, J ¼ 4.1 Hz, 1H, H-10), 5.49 (d, J ¼ 5.2 Hz, 1H, H-1), 4.68–
4.56 (m, 4H, OCH2Ph, H-20, H-3), 4.32–4.23 (m, 3H, H-2, H-40, H-50), 3.93
(d, J ¼ 3.4 Hz, 1H, H-30), 3.83–3.77 (m, 3H, H-4, H-8), 3.43–3.25 (m, 3H, H-6,
CH2NH), 2.25 (br s, 1H, -OH), 1.78 (br s, exchangeable 1H, NH), 1.46–1.26 [m, 20H,
3 ꢁ (CH3)2 C, H-7]; 13C NMR (50 MHz, CDCl3): d 138, 128.8, 128.7, 128.0 (Ar-C),
111.9, 109.8, 109.1 [3 ꢁ (CH3)2C], 105.3 (C-10), 96.9 (C-1), 82.6, 82.5, 80.0 (C-20, C-40,
C-30), 72.2, 71.4, 71.2, 70.8 (C-2, C-3, C-4, OCH2Ph), 62.4 (C-8), 57.7 (C-6), 43.7
(CH2NH), 28.4 (C-7), 27.1, 26.4, 24.9 [3 ꢁ C(CH3)2].
Anal. Calcd. for C29H43NO10: C, 61.59; H, 7.61; N, 2.48; Found: C, 61.78; H, 7.26;
N, 2.38.
6-(50-Amino-50-deoxy-10,20-O-isopropylidene-30-O-methyl-a-D-xylofuranos-50-yl)-
6,7-dideoxy-1,2 : 3,4-di-O-isopropylidene-b-L-glycero-a-D-galactoctanol (24).
Reduction of amino ester 13 (2.70 g, 5.1 mmol) with LiAlH4 (0.38 g, 10.2 mmol) and work
up as described above afforded glycosyl amino alcohol 24 (1.37 g, 55%) as colorless oil; Rf
0.50 (chloroform/methanol, 24 : 1), [a]2D0 261.28 (c 0.16, chloroform); MS (FAB) ¼ m/z
1
490 (M þ H)þ; IR (Neat) nmax cm21: 3728, 3290; H NMR (200 MHz, CDCl3): d 5.87
(d, J ¼ 3.8 Hz, 1H, H-10), 5.50 (d, J ¼ 5.0 Hz, 1H, H-1), 4.61–4.49 (m, 3H, H-2, H-20,
H-3), 4.28–4.25 (m, 4H, H-4, H-40, H-8), 3.74 (m, 2H, H-30, H-5), 3.40 (s, 3H, OCH3),
3.60 (m, 1H, H-6), 2.94 (m, 2H, CH2NH), 1.63 (br s, exchangeable 1H, -NH), 1.43 [s, 6H,
(CH)3C)], 1.32–1.26 [m, 8H, (CH3)2C, H-7]; 13C NMR (50 MHz, CDCl3): d 111.9,
109.3, 108.8 [3 ꢁ (CH3)2C], 105.2 (C-10), 96.9 (C-1), 84.2 (C-20), 82.0 (C-40), 80.0, 79.9
(C-3, C-30), 71.2, 70.8 (C-2, C-4), 68.7 (C-5), 62.4 (C-8), 58.1 (OCH3), 54.4 (C-6), 44.1
(CH2NH), 28.7 (C-7), 27.1, 26.4, 24.9 [C(CH3)2].
Anal. Calcd. C23H39NO10: C, 56.43; H, 8.03; N, 2.86; Found: C, 56.43; H, 8.03;
N, 2.86.
6-(30-O-Allyl-50-amino-50-deoxy-10,20-O-isopropylidene-a-D-xylofuranos-50-yl)-
6,7-dideoxy-1,2 : 3,4-di-O-isopropylidene-b-L-glycero-a-D-galactoctanol (25).
Reduction of amino ester 14 (1.2 g, 2.15 mmol) with LiAlH4 (0.16 g, 4.30 mmol) and
work up as described above afforded glycosyl amino alcohol 25 (0.66 g, 60%) as colorless
oil; Rf 0.50 (chloroform/methanol, 24 : 1), [a]2D0 265.08 (c 0.10, chloroform); MS
1
(FAB) ¼ m/z 516 (M þ H)þ; IR (Neat) nmax cm21: 3650, 3346; H NMR (200 MHz,
CDCl3): d 5.91–5.80 (m, 2H, H-10, OCH2CH55CH2), 5.55 (d, J ¼ 5.2 Hz, 1H, H-1),
5.25 (dd, J ¼ 19.0 and 1.4 Hz, 2H, OCH2CH55CH2), 4.59–4.53 (m, 2H, H-3, H-20),
4.33 (d, J ¼ 5.2 Hz, 1H, H-2), 4.21–4.10 (m, 3H, OCH2CH55CH2 H-40), 3.90 (m, 1H,
H-30), 3.86 (m, 3H, H-8, H-4), 3.11–3.05 (m, 3H, CH2NH, H-6), 2.91 (m, 1H, H-5),
2.40 (br s, exchangeable 1H, -OH), 1.70 (br s, exchangeable 1H, -NH), 1.62–1.25 [m,
20H, [3 ꢁ (CH3)2C, H-7]; 13C NMR (50 MHz, CDCl3): d 134.4 OCH2CH55CH2), 118.2
(OCH2CH55CH2), 111.8, 109.6, 108.8 [3 ꢁ (CH3)2C], 105.2 (C-10), 96.9 (C-1), 82.7,
82.1, 79.9 (C-20, C-40, C-30); 71.4, 71.1, 70.9 (C-3, C-2, C-4, C-8), 68.3 (C-5), 62.4 (OCH2-
CH55CH2), 57.7 (C-6), 43.6 (CH2NH), 28.4 (C-7), 27.1, 26.7, 26.4, 26.3 25.3, 24.9
[3 ꢁ C(CH3)2].

508
Verma et al.
Anal. Calcd. for C25H41NO10: C, 58.25; H, 7.96; N, 2.48; Found: C, 58.04; H, 7.26;
N, 2.38.
6-(60-Amino-60-deoxy-10,20:30,40-di-O-isopropylidene-a-D-galactoctapyranos-60-
yl)-6,7-dideoxy-1,2 : 3,4-di-O-isopropylidene-b-L-glycero-a-D-galactoctanol (26).
Reduction of amino ester 15 (1.72 g, 2.93 mmol) with LiAlH4 (0.22 g, 5.86 mmol) and work
up as described above afforded glycosyl amino alcohol 26 (0.79 g, 50%) as colorless oil; Rf
0.50 (chloroform/methanol, 24 : 1), [a]2D0 248.08 (c 0.12, chloroform), MS (FAB) ¼ m/z
1
546 (M þ H)þ; IR (Neat) nmax cm21: 3679, 3445; H NMR (200 MHz, CDCl3): d 5.52
(two d, J ¼ 5.0 Hz, 2H, H-1, H-10), 4.56 (two d, J ¼ 6.0 Hz, 2H, H-3, H-30), 4.33–4.28
(m, 4H, H-2, H-20, H-8), 3.88–3.78 (m, 4H, H-4, H-40, H-5, H-50), 2.6 (m, 1H, H-6),
2.50–2.40 (m, 2H, CH2NH), 1.53 (m, 2H, H-7), 1.54–1.25 [m, 20H, 4 ꢁ [(CH3)2C]; 13
C
NMR (50 MHz, CDCl3): d 109.7, 109.0 [2 ꢁ (CH3)2C], 96.8, 96.7 (C-1, C-10), 72.5,
72.3, 71.6, 71.4, 70.9 (C-3, C-30, C-2, C-20, C-4, C-40), 68.1, 67.9 (C-5, C-50), 62.8 (C-8),
57.4 (C-6), 44.2 (CH2NH), 28.4 (C-7), 26.4, 25.3, 24.9, 24.8 [(CH3)2C].
Anal. Calcd. for C26H43NO11: C, 57.23; H, 7.94; N, 2.57; Found: C, 57.04; H, 8.26;
N, 2.38.
Glucose-6-phosphatase (D-glucose-6-phosphate phosphorylase; EC 3.1.3.9)
activity determination.[12] The liver of a Wistar rat that was fasted overnight was
excised and a 10% homogenate was prepared in 150 mM KCl (w/v) using a Potter Elvej-
hem glass homogenizer fitted with Teflon pestle. The homogenate was centrifuged at
1000 ꢁ g for 15 min at 48C, and the supernatant was decanted and used as enzyme source.
The effect of the test compound was studied by preincubating 100 mM of the com-
pound in 1.0 mL reaction system for 10 min and then determining the residual glucose-
6-phosphatase activity according to the method of Hubscher and West (1965). The
assay system contained 0.3 M citrate buffer (pH6.0), 28 mM EDTA, 14 mM NaF,
200 mM glucose-6-phosphate, and appropriate amount of enzyme protein. The mixture
was incubated at 378C for 30 min, after which reaction was stopped by the addition of
1.0 mL of 10% TCA. Estimation of inorganic phosphate (Pi) in protein-free supernatant
was done according to the method of Taussky and Shorr (1953). Glucose-6-phosphatase
activity was defined as mM of Pi release per min per mg protein.
Glycogen phosphorylase (a-1,4 D-Glucan: Orthophosphate a- glucosyl Transfer-
ase, EC 2.4.1.1) activity determination.[13] Livers of Wistar strain of albino rats were
excised. Ten percent homogenate (w/v) was prepared in 150 mM KCl using Potter Elvejhem
glass homogenizer fitted with Teflon pestle. The homogenate was centrifuged at 1000 ꢁ g for
15min at 48C; supernatant was decanted and used as an enzyme source.
The effect of the test compound was studied by preincubating 100 mM of the com-
pound in 1.0 mL reaction system for 10 min and then determining the residual glycogen
phosphorylase activity according to the method of Rall et al. (1957). The assay mixture
contained 0.2 mL mixture A (glycogen 57 mg, G-1-P 188 mg, NaF 42 mg and 50 AMP
(4 mM) in 10 mL distilled water) and 0.1 mL mixture B (enzyme protein). It was incubated
at 378C for 30 min, after which reaction was stopped by the addition of 0.1 mL of 10% TCA
and then 0.4 mL sodium acetate (100 mM) was added to prevent the spontaneous hydroly-
sis of G-1-P present in the reaction mixture. The estimation of inorganic phosphate in the
protein-free supernatant was done according to the method of Taussky and Shorr (1953).
Glycogen phosphorylase activity was defined as mM of Pi release per min per mg protein.
a-Glucosidase (EC 3.2.1.20) activity determination.[14] The intestine of a male
albino rat (CF strain) was excised and opened, and the mucosa was collected and

Reductive Amination of Glycosyl Aldoses
509
pooled. A 10% homogenate was prepared in 150 mM KCl using a Potter Elvejhem glass
homogenizer fitted with Teflon pestle. The homogenate was centrifuged at 1000 ꢁ g for
15 min, and the supernatant was decanted and stored at 48C. The supernatant was dialyzed
at 48C against 50 mM Tris–HCl buffer pH 7.0 with two or three changes of buffer. The
dialyzed supernatant was saturated with ammonium sulphate to the final concentration
of 30%. The sample was kept at 48C overnight and then centrifuged to collect the super-
natant and precipitate separately. Thirty percent ammonium sulphate saturated supernatant
was further saturated to 60% with ammonium sulphate. Again the precipitate and super-
natant were separated by centrifugation. Finally the 60% ammonium sulphate saturated
supernatant was further saturated to 100% with further addition of ammonium sulphate.
The precipitate and supernatant was once again separated, and all the samples were
analyzed for a-glucosidase activity using p-nitrophenyl-a-D-glucopyranoid (PNPG) as
substrate. The enzyme activity was found maximum in 60–100% ammonium sulphate
precipitate, and this fraction was used as a source of enzyme for studying the effect of
the test compounds.
Added were 100 mL of purified a-glucosidase (0.1 mg/mL) and 25 mL of glutathione
(1.0 mg/mL), and the total volume was made up to 1 mL by adding 0.67 mM phosphate
buffer (pH 6.8). The reaction mixture was incubated at room temperature for 10 min
with the desired test compound (10 mM) dissolved in 100% DMSO. Reaction was
started by the addition of 50 mL p-nitrophenyl-a-D-glucopyranoside (3 mg/mL), and
increase in absorbance was recorded at 400 nm for a period of 5 min at the interval of
30 sec (Lebovitz, 1997).
Protein estimation.[15] The proteins of liver homogenate was precipitated with an
equal volume of 10% TCA (w/v), washed twice with 5% TCA, dissolved in 0.1 N
NaOH, and estimated according to the method of Lowry et al. (1951) using bovine
serum albumin as standard.
ACKNOWLEDGMENTS
Authors are thankful to the Director of CDRI for his encouragement and to the Heads
of microbiology and new target discovery and development divisions for antimicrobial
screening. Financial assistance from ICMR New Delhi in the form of a Project BMS-II
58/6/2000 is also acknowledged.
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