β-D-glucosyl and α-D-galactosyl yariv reagents: Syntheses from p-nitrophenyl-D-glycosides by transfer reduction using ammonium formate

Article abstract of DOI:10.1021/jf0401571

Yariv β-D-glucosyl (4a) and Yariv α-D-galactosyl (4b) reagents are multivalent phenylglycosides. The β-D-glucosyl reagent is considered diagnostic for arabinogalactan proteins (AGPs) to which it can reversibly bind, stain, and precipitate. The α-D-galacto

Full text of DOI:10.1021/jf0401571

J. Agric. Food Chem. 2004, 52, 7453−7456 7453
â
-
D
-Glucosyl and
r
-
D
-Galactosyl Yariv Reagents: Syntheses
from p-Nitrophenyl-
D-glycosides by Transfer Reduction Using
Ammonium Formate
DOMINICK V. BASILE* AND IRAJ GANJIAN
Departments of Biological Sciences and Chemistry, Lehman College of the City University of
New York, Bronx, New York 10468
Yariv â-D-glucosyl (4a) and Yariv R-D-galactosyl (4b) reagents are multivalent phenylglycosides. The
â-D-glucosyl reagent is considered diagnostic for arabinogalactan proteins (AGPs) to which it can
reversibly bind, stain, and precipitate. The R-D-galactosyl reagent does not bind AGPs and is used
as a control. In a new strategy, we accomplished the large scale synthesis of the Yariv reagents in
one continuous step by a transfer reduction method and without a need for any specialized apparatus.
As the starting material, p-nitrophenyl-D-glycosides (1) were reduced to p-aminophenyl-D-glycosides
(2) using ammonium formate as the hydrogen donor. The excess formate was converted to formic
acid and ammonia, which then were removed from the reaction by simple distillation. Without isolation,
p-aminophenyl-D-glycosides were diazotized (3) and coupled to phloroglucinol to give the Yariv
reagents in ∼40% yield. AGPs are a major component of gum arabic, an emulsifying agent widely
used in the food and pharmaceutical industries. Increasing interest in AGPs prompted the development
of a relatively easy and inexpensive method for the synthesis of these reagents.
KEYWORDS: Yariv reagents; arabinogalactan proteins; ammonium formate; synthesis; transfer reduction
INTRODUCTION
foodstuffs serve as a good example of the value of Yariv â-D-
glucosyl reagent in food and agricultural industries. Yariv â-D-
glucosyl reagent is used for quality assurance to differentiate
between bona fide gum arabic and gums of lesser quality for
use in foodstuffs (17).
Yariv â-D-glucosyl reagent [1,3,5-tris(4-â-D-glucopyranosyl-
oxyphenylazo)-2,4,6-trihydroxybenzene] (4a) is a multivalent
phenylglucoside (1). This reagent is variously used to detect
(stain) (2-6), selectively precipitate (7-9), and quantify (10)
macromolecules belonging to a large family of proteoglycans
and glycoproteins associated with virtually all plant cell surfaces.
Although most of the weight of the glycoproteins is derived
from its complex carbohydrate moiety, all members of this
family are commonly referred to as arabinogalactan proteins or
AGPs. Their ubiquitous presence at plant cell surfaces suggests
one or more basic functions. AGPs appear to perform a wide
variety of functions in plants including regulation of growth
and development and chemoprotection. They are sufficiently
variable in composition to serve as determinants of identity at
every level of plant organization from cell type to species.
Consequently, how AGPs may be functioning to influence the
structure and biology of plants has become an intriguing question
for an ever-increasing number of investigators (11-15). How
the seemingly infinite variation in composition of individual
AGP molecules influences the processing of plant-derived foods
and drugs is an area that has been of only limited interest thus
far. Gum arabic, derived from Acacia senegal Wild., is a
commercially important plant product that is known to be rich
in AGPs (16). The procurement and use of gum arabic in
Yariv â-D-glucosyl reagent (4a) is considered the most
definitive test for detecting the presence of AGPs in plant tissues
or plant products (18). Because the binding of this reagent is
both highly selective and completely reversible, it is proving
to be a valuable tool for identifying, quantifying, and affinity
selecting/isolating AGPs from aqueous plant extracts (loc. cit.).
As the awareness grows that AGPs are always present in plant
material but distinctly different in different plant parts and
species, there should be an increase in demand for this reagent.
There is at present only one commercial source for Yariv-
D-glycosyl reagent (i.e., Biosupplies, Pty. Ltd., Parksville,
Victoria, Australia) (19). Because of its relatively high cost,
however, many workers seek to obtain it as gifts from those
who can synthesize it. In an earlier publication, we endeavored
to make it easier to prepare â-D-glucosyl Yariv reagent for those
investigators with either limited funds or who lacked the training
and/or the facilities for organic synthesis (20). The procedure
described the reduction of p-nitrophenyl-â-D-glucoside followed
by the diazotization of the corresponding amine and subsequent
coupling of the diazonium ion to phloroglucinol. Two methods
for the reduction of p-nitrophenyl-â-D-glucoside were described
as follows: heterogeneous catalytic hydrogenation and catalytic
transfer hydrogenation (transfer reduction). The catalytic hy-
* To whom correspondence should be addressed. Tel: 718-960-8800.
Fax: 718-960-8236. E-mail: basile@lehman.cuny.edu.
10.1021/jf0401571 CCC: $27.50
© 2004 American Chemical Society
Published on Web 11/02/2004

7454 J. Agric. Food Chem., Vol. 52, No. 25, 2004
Basile and Ganjian
Caution! It is important to add the reagents according to the sequence
that they were added. There is a chance of igniting the hydrogen gas
(produced in situ) if the catalyst is added after the ammonium formate.
Removal of the Excess Ammonium Formate and Diazotization.
Because of high solubility of the amino derivative (2a) along with
ammonium formate, it was not possible to separate the product from
the excess ammonium formate from an aqueous solution by extracting
with an organic solvent. Therefore, to remove the excess of the
ammonium formate from the reaction, it was converted to formic acid
and ammonia by the following process. The residue was dissolved in
water (50 mL), and the solution (pH 8.0) was first acidified with 5%
v/v sulfuric acid (∼4.1 mL) to pH 3.0. About 25 mL of water was
distilled off at 29 °C, under vacuum, to remove the formic acid. It is
crucial to keep the distilling temperature below 50 °C, using either a
high vacuum distillation set up or a rotary evaporator equipped with
vacuum pump. The vacuum generated by a water aspirator is not
efficient enough. To remove the ammonia formed in the solution, the
volume of the solution was brought back to 50 mL by the addition of
water and it was then basified with 5% w/v sodium hydroxide solution
(∼6.9 mL) to pH 9.2; then, about 25 mL of the water was distilled off
at 29 °C under the high vacuum. Because of the presence of gaseous
ammonia, the connection of the flask to the vacuum should be done
very gradually and at room temperature using extreme caution in order
to avoid an initial splash of the solution that would result from applying
a sudden vacuum. After the complete removal of ammonia from the
solution of p-aminophenyl-â-D-glucoside (2a), the volume of the
solution was brought back again to 50 mL by the addition of water in
preparation for the diazotization process. The flask was cooled in an
ice bath (ice water, rock salt) and 0.5 M hydrochloric acid (55 mL)
was added to the solution. While the solution was kept at a temperature
of 0-5 °C, a cold solution of 0.643 g (9.3 mmol) sodium nitrite in
water (35 mL) was added dropwise from a separatory funnel over a
period of 30 min. To maintain the temperature of the reaction at the
0-5 °C range, small pieces of ice were introduced directly into the
reaction flask during the diazotization process, while stirring with a
magnetic stirrer.
Coupling of p-Diazonium-phenyl-â-D-glucoside Ion to the Phlo-
roglucinol. An ice-cold solution of 0.372 g (3.0 mmol) of phloroglu-
cinol in water (120 mL) was added slowly, from a separatory funnel,
to the solution of diazonium salt (3a), at 0-5 °C, over a period of 30
min while stirring vigorously and maintaining the low temperature.
After half an hour, the flask was removed from the ice bath and allowed
to warm to room temperature. To the flask containing the deep red
solution (pH 2.2), a solution of 0.5% sodium hydroxide (∼44 mL) was
added slowly until the pH reached 9.0 and the pH became stable at 9.0
for 1.5 h. The solution (∼300 mL) was diluted with an equal volume
of ethanol and was kept overnight in the refrigerator. The precipitate
that formed was filtered, and after drying, it resulted in 1.15 g (40%)
of the â-D-glucosyl Yariv reagent (4a).
Preparation of r-D-Galactosyl Yariv Reagent. This compound was
synthesized by the same procedure used for the synthesis of â-D-
glucosyl Yariv reagent described above, with the exception that
p-nitrophenyl-R-D-galactoside (1b) was used as the starting material.
The amount of the precipitate, R-D-galactosyl Yariv reagent (4b), was
1.10 g (40%).
Figure 1. Continuous syntheses of â-D-glucosyl (4a) and R-D-Galactosyl
(4b) Yariv reagents from p-nitrophenylsugars by transfer reduction.
drogenation method was capable of a lager scale synthesis, of
the two methods, but it required the availability of a pressure
hydrogenation apparatus. The transfer reduction method de-
scribed did not require this specialized equipment, but it was
limited to small-scale synthesis, due to problems associated with
separating the product from an excess of ammonium formate
used as the hydrogen donor (21). The product, p-aminophenyl-
â-D-glucoside, is soluble in water and could not be extracted
with organic solvents. This created a serious problem in
removing the excess ammonium formate after the reduction of
p-nitrophenyl-â-D-glucoside (20-22).
In this paper, we describe a modified transfer reduction
method (Figure 1) that facilitates a fast removal of ammonium
formate, thereby permitting a larger scale synthesis without a
need for highly specialized apparatus.
METHODS AND MATERIALS
Chemicals. All reagents used in this study were purchased from
Sigma Chemical Co. (St. Louis, MO). The water used was purified
using a Modulab Analytical Research Grade RO/Polishing water
purification system (Continental Water Systems, San Antonio, TX).
Transfer Reduction of p-Nitrophenyl-â-D-glucoside. To a 500 mL
Erlenmeyer flask set on a magnetic stirrer, 250 mL of anhydrous
methanol (dried on molecular sieves 3 Å) and 2.5 g (8.3 mmol) of
p-nitrophenyl-â-D-glucoside (1a) were introduced. The flask was closed
with a rubber septum, and the mixture was stirred for 5 min. The septum
was removed, and 0.5 g of 10% Pd/C was added with stirring until the
catalyst was fully suspended, followed by the addition of 2.4 g (38.2
mmol) of ammonium formate. The flask was closed, this time with
the septum penetrated with a 20-gauge syringe needle (to allow for
the release of hydrogen gas) and placed in a water bath set at a
temperature of 50 °C, with continuous magnetic stirring. After 20 min,
thin-layer chromatography (TLC) of a sample showed a complete
conversion of the nitro compound (Rf ) 0.60) to the amino derivative
(2a) (Rf ) 0.34). The reaction mixture was cooled to room temperature
and was filtered through a pad of infusorial earth (23) (2.5 cm in a
150 mL sintered glass Buchner funnel, packed in methanol). The pad
was washed with methanol (100 mL), and the filtrates were combined
and evaporated under reduced pressure at 40 °C using a rotary
evaporator.
Caution! In an attempt to collect more product, in both cases, after
separation of the first batch, the volume of the supernatant was reduced
to 150 mL by evaporation on a rotary evaporator. Further dilution with
ethanol (150 mL) and cooling in the refrigerator gave an additional
0.50-0.77 g of a product. The nuclear magnetic resonance (NMR)
spectroscopy proved that the second batch was not the Yariv reagent,
and it was discarded. After this compound was dissolved in dimethyl
sulfoxide (DMSO) (for the NMR), it resulted in a light reddish solution,
as compared to the color of a Yariv reagent in DMSO, which was deep
red.
ANALYSIS
Verification of products was accomplished by TLC, high-
performance liquid chromatography (HPLC), or NMR or some
combination of these. TLC was performed using Polygram Sil/G

â
-
D
-Glucosyl and
R
-
D
-Galactosyl Yariv Reagents
J. Agric. Food Chem., Vol. 52, No. 25, 2004 7455
ABBREVIATIONS USED
UV254 plastic sheets (Brinkmann) and a methanol/chloroform
30/70 solvent system. HPLC was carried out using an ISCO
model 2360 gradient programmer, a Waters model M 45 solvent
delivery system pump, an Altex sample injection valve 210, a
Waters model 2487 dual λ UV/vis absorbance detector, and a
Kipp and Zonen model BD-41 recorder. The compounds were
eluted on a prepacked Beckman Ultrasphere ODS (particle size
5 µm) stainless steel column 25 cm × 4.6 mm i.d. and were
detected at 254 nm. The solvent system was methanol/water
(50/50, v/v), and the flow rate was 0.5 mL/min. NMR spectra
were recorded on a 60 MHz LM 360 Varian instrument.
Chemical shifts were reported on the δ scale. The aromatic
region (δ 6.0-9.0) of the spectra was used for a diagnostic
purpose and verification of the conversion of the nitro com-
pounds to Yariv reagents.
â-D-Glucosyl Yariv Reagent. TLC: p-nitrophenyl-â-D-
glucoside, Rf ) 0.60 (1a); p-aminophenyl-â-D-glucoside (2a),
Rf ) 0.34. HPLC: The retention time for p-nitrophenyl-â-D-
glucoside was 13.5 min, and the retention time for â-D-glucosyl
Yariv reagent was 12.0 min. Proton NMR: p-nitrophenyl-â-D-
glucoside (DMSO) δ 8.1 (d, 2H, J ) 12.0 Hz), δ 7.1 (d, 2H, J
) 9.0 Hz); â-D-glucosyl Yariv reagent (4a) (DMSO) δ 7.5 (d,
6H, J ) 12.0 Hz), 7.1 (d, 6H, J ) 12.0 Hz).
AGPs, arabinogalactan proteins; TLC, thin-layer chromatog-
raphy; HPLC, high-performance liquid chromatography; NMR,
nuclear magnetic resonance.
ACKNOWLEDGMENT
We are grateful to John Klonowsky for his technical assistance.
We also thank Yolanda Henry for performing one of the
reactions and double checking the procedure.
LITERATURE CITED
(1) Yariv, J.; Rapport, M. M.; Graf, L. The interaction of glycosides
and saccharides with antibody to the corresponding phenylazo
glycoside. Biochem. J. 1962, 85, 383-388.
(2) Basile, D. V.; Kushner, B. K.; Basile, M. R. A new method for
separating and comparing arabinogalactan proteins for the
chemosystematics of the hepaticae. Bryologist 1989, 92, 164-
169.
(3) Basile, D. V.; Basile, M. R. The occurrence of cell wall-
associated arabinogalactan proteins in the hepaticae. Bryologist
1987, 90, 401-404.
(4) Bosch, M.; Knudsen, J. S.; Derksen, J.; Mariani, C. Class III
pistil-specific extensin-like proteins from tobacco have charac-
teristics of arabinogalactan proteins. Plant Physiol. 2001, 125,
2180-2188.
(5) Rauh, R. A.; Basil, D. V. Phenovariation induced in streptocarpus
prolixus (Gesneriaceae) by beta-glucosyl Yariv reagent. Can.
J. Bot. 2003, 81 (4), 338-344.
(6) Triplett, B. A.; Timpa, J. D. â-Glucosyl and R-galactosyl Yariv
reagents bind to cellulose and other glucans. J. Agric. Food
Chem. 1997, 45, 4650-4654.
(7) Motose, H.; Sugiyama, M.; Fukuda, H. An arabinogalactan
protein(s) is a key component of a fraction that mediates local
intercellular communication involved in tracheary element dif-
ferentiation of zinnia mesophyll cells. Plant Cell Physiol. 2001,
42, 129-137.
(8) Classen, B.; Witthohn, B. W. Characterization of an arabinoga-
lactan-protein isolated from pressed juice of Echinacea purpurea
by precipitation with the â-glucosyl Yariv reagent. Carbohydr.
Res. 2000, 327, 497-504.
Note. In our previous paper (20), the coupling constant (J)
in the aromatic region for â-D-glucosyl compounds was reported
to be 9 Hz and should be corrected to 12 Hz.
r-D-Galactosyl Yariv Reagent. TLC: p-nitrophenyl-R-D-
galacoside (1b), Rf ) 0.66; p-aminophenyl-R-D-galacoside (2b),
Rf ) 0.40. HPLC: The retention time for p-nitrophenyl-R-D-
galacoside was 14.5 min, and the retention time for R-D-
galactosyl Yariv reagent was 11.0 min. Proton NMR: p-nitro-
phenyl-R-D-galacoside (DMSO) δ 8.1 (d, 2H, J ) 12.0 Hz), δ
7.2 (d, 2H, J ) 12.0 Hz); R-D-galactosyl Yariv reagent (4b)
(DMSO) δ 7.2 (d, 6H, J ) 12.0 Hz), 7.6 (d, 6H, J ) 12.0 Hz).
Caution! The aggregation of the most purified sample of â-D-
glucoside Yariv reagent in water has been investigated, using
an ultracentrifuge cell at equilibrium (24). It has been shown
that Yariv compounds undergo a strong self-association in water
with a very wide distribution of species. As a result, the usual
measurement of physical constants such as melting point, optical
rotation, and elemental analysis fail for Yariv compounds. A
mixture of disubstituted and trisubstituted phloroglucinol could
be formed simultaneously, and also, the aggregating system
could readily trap small amounts of the lower homologues,
making measurement of the physical constants invalid.
(9) Classen, B.; Blaschek, W. An arabinogalactan-protein from cell
culture of MalVa sylVestris. Planta Med. 2002, 68 (3), 232-
236.
(10) Chapman, A.; Helleboid, S.; Blervacq, A. S.; Vasseur, J.; Hilbert,
J. L. Removal of the fibrillar network surrounding Cichorium
somatic embryos using cytoskeleton inhibitors: Analysis of
proteic components. Plant Sci. (Shannon, Ireland) 2000, 150,
103-114.
RESULTS AND DISCUSSIONS
The protocol for syntheses of the â-D-glucosyl and R-D-
galactosyl Yariv reagents (Figure 1; 4a,b) described herein is
perceived as an improvement over the two alternate methods
described in a previous publication (20). This is the first case
of using the transfer reduction method for large-scale preparation
of Yariv reagents. The synthesis is performed in one continuous
synthetic procedure (required time, 2-3 days) without isolating
the reaction intermediates, with a good yield (∼40%), using
inexpensive starting material (1a,b). It eliminates the need for
access to a pressure hydrogenation apparatus required for the
heterogeneous catalytic hydrogenation method. The important
change introduced in this revised transfer reduction method was
the addition of a two-step process of acidification followed by
alkalinization that converts the excess ammonium formate to
formic acid and ammonia, respectively. Both of these com-
pounds can be removed easily from the reaction mixture by a
simple distillation.
(11) Evazzadeh, J. A.; Nothnagel, E. A. Cell cycle arrest by
perturbation of arabinogalactan- proteins with Yariv phenyl-
glycoside. Cell DeVelopment in Biology. Arabinogalactan-
Proteins, Proceedings of the Symposium on Plant Physiology,
20th 1999; Kluwer Academic/Plenum: New York, NY, 2000;
pp 71-81.
(12) Chapman, A.; Helleboid, S.; Blervacq, A. S.; Vasseur, J.; Hilbert,
J. L. Arabinogalactan-proteins in Cichorium somatic embryo-
genesis: Effect of â-glucosyl Yariv reagent and epitope locali-
sation during embryo development. Planta 2000, 211, 305-314.
(13) Gao, M.; Showalter, A. M. Yariv reagent treatment induces
programmed cell death in Arabidopsis cell cultures and implicates
arabinogalactan protein involvement. Plant J. 1999, 19, 321-
331.
(14) Thompson, H. J. M.; Knox, J. P. Stage-specific responses of
embryogenic carrot cell suspension cultures to arabinogalactan
protein-binding â-glucosyl Yariv reagent. Planta 1998, 205, 32-
38.

7456 J. Agric. Food Chem., Vol. 52, No. 25, 2004
Basile and Ganjian
(15) Basile, D. V.; Basile, M. R.; Mignone, M. M. Arabinogalactan-
proteins, place-dependent suppression and plant morphogenesis.
Cell DeVelopment in Biology. Arabinogalactan-proteins, Pro-
ceedings of the Symposium on Plant Physiology, 20th 1999;
Kluwer Academic/Plenum: New York, NY, 2000; pp 169-
178.
(16) Ward, F. M. Uses of gum arabic (Acacia sp.) in the food and
pharmaceutical industries. Cell DeVelopment in Biology. Ara-
binogalactan-proteins, Proceedings of the Symposium on Plant
Physiology, 20th 1999; Kluwer Academic/Plenum: New York,
NY, 2000; pp 231-139.
(17) Menzies, A. R.; Osman, M. E.; Malik, A. A.; Baldwin, T. C. A
comparison of the physicochemical and immunological properties
of the plant gum exudates of Acacia senegal (gum arabic) and
Acacia seyal (gum tahla). Food Addit. Contam. 1996, 13, 991-
999.
(18) Nothnagel, E. A. Proteoglycans and related compounds in plant
cells. Int. ReV. Cytol. 1997, 174, 195-291.
(20) Ganjian, I.; Basile, D. V. Reductive syntheses of p-aminophenyl-
â-D-glucoside and its conversion to â-glucosyl Yariv reagent.
Anal. Biochem. 1997, 246, 152-155.
(21) Roy, R.; Tropper, F. D.; Morrison, T.; Boratynski, J. Syntheses
and transformations of glycohydrolase substrates into protein
conjugates based on Michael additions. J. Chem. Soc. Chem.
Commun. 1991, (7), 536-538.
(22) Roy, R.; Tropper, F. D.; Romanowska, A. New strategy in
glycopolymer syntheses. Preparation of antigenic water-soluble
poly(acrylamide-co-p-acrylamidophenyl â-lactoside). Bioconju-
gate Chem. 1992, 3, 256-261.
(23) Sigma Aldrich chemical: http://www.sigmaaldrich.com/Brands/
Aldrich.html; Kieselguhr (Infusorial Earth) product no. 18514,
or Celite 500 fine product no. 22145. It could also be purchased
from Fisher Scientific: www. Fishersci.com; as Diatomaceous
Earth catalog no. S75114.
(24) Woods, E. F.; Lilley, G. G.; Jermyn, M. A. The self-association
of glycosyl phenyl azo dyes (Yariv antigens.). Aust. J. Chem.
1978, 31 (10), 2225-2238.
(19) Biosupplies Australia, Pty. Ltd., P.O. Box 835 Parksville, Victoria
3052,Australia.Fax: +613-93471071.E-mail: enquiries@biosupplies.com.au.
Internet: www.biosupplies.com.au. Check the Internet for a price
list. The current prices for 2 mg are $58 for Yariv â-glucosyl
reagent and $68 for R-galactosyl Yariv reagent.
Received for review March 29, 2004. Revised manuscript received
August 28, 2004. Accepted September 10, 2004.
JF0401571