[ACS Symposium Series] Chemical Separations with Liquid Membranes Volume 642 || Sugar Separation Using Liquid Membranes and Boronic Acid Carriers

Chapter 14

Sugar Separation Using Liquid Membranes and Boronic Acid Carriers

Bradley D. Smith

Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556

Boronic acids are shown to facilitate the transport of a range of hydrophilic sugars through lipophilic membranes. The results suggest that boronic acids have promise as transport carriers in membrane-based sugar separations. Most of the work involves Bulk Liquid Membranes (BLMs), for which the various chemical and physical factors that control transport are examined in detail. The use of boronic acids in large-scale separations will require changing to more practical liquid membrane systems, such as Supported Liquid Membranes (SLMs) with hollow fiber geometries. Boronic acids can also transport sugars through lipid bilayers. The possibility of developing a sugar separations method based on liposomes is discussed.

This chapter describes a method of transporting sugars through lipophilic membranes using boronic acid carriers. Potential applications of this technology range from industrial-scale separations to drug delivery. The focus of this article is on separations. While the utility of liquid membranes in metal cation separations is well-recognized, there are very few examples of liquid membranes being used in molecular separations (7). Boronic acids are currently the only synthetic compounds known to selectively facilitate the transport of hydrophilic sugars through lipophilic membranes (2). In principle, sugar-selective carriers should be useful in a variety of membrane-based separation problems ranging from sugar refining to the purification of fine chemicals and pharmaceuticals (3). This article summarizes our recent progress in this developing research area.

Sugar Complexation Using Boronic Acids.

Boric acid complexes of sugar compounds have been investigated for well over one hundred years (4). By the end of the nineteenth century it was known that addition of boric acid to aqueous sugar solutions changed the optical rotation of the sugars, increased the acidity of the boric acid, and increased the solution's electrical conductivity (5). Analogous complexation studies with boronic acids were apparently not conducted until the middle of the twentieth century. In 1954, Kuivila reported

0097-6156/96/0642-0194$15.00/0 © 1996 American Chemical Society

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In Chemical Separations with Liquid Membranes; Bartsch, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

14. SMITH Sugar Separation Using Boronic Acid Carriers 195

that phenylboronic acid formed trigonal boronate ester precipitates with sugars such as mannitol (6). In 1959, Lorand and Edwards proved that phenylboronic acid was not a Br0nsted acid but rather a Lewis acid (7). While doing so, these workers determined boronate association constants for a number of diol-containing compounds in aqueous solution.

The covalent associations between boronic acids and diol-containing compounds can be summarized in the following way. In anhydrous aprotic solvents, boronic acids readily condense with diols to form trigonal boronate esters, 1. In aqueous solution, the trigonal boronates are unstable and either hydrolyze back to starting compounds or ionize to form anionic, tetrahedral boronates, 2. Since a boronate ester is more acidic than its parent boronic acid, the predominant complexation product between phenylboronic acid (pKa = 8.9), and a diol at pH 7 is boronate 2. A salient point is that although covalent bonds are formed, the associated activation energy is low, so the process is rapid and reversible. This reversible interaction has been exploited extensively as a diol protecting group strategy in organic synthesis (#), and is the basis of a chromatographic method for separation of polyols (9).

O H Cd loP) A P R O T I C solvent P h — β ' + r^T - 0 0 + 2 H2O

O H H 0 0 H V

I Ph

O H ^ T ~ T ^ aqueous solvent p h _ B f H

+ C g > . 0 0 + H 3 0 + O H H O O H Β -

Ph' O H

Boronic Acids As Sugar Transport Carriers.

To date, most of the boronic acid transport experiments have used Bulk Liquid Membranes (BLMs) in a standard U tube apparatus. In this configuration an aqueous source phase is separated from an aqueous receiving phase by a dense organic layer {e.g., dichloroethane) that is stirred using a magnetic stir bar (2,10). The main advantages of BLMs, compared to other membrane systems, are the ease of operation, low cost, and the less stringent requirement for the carriers to be highly lipophilic (11). Thus, BLMs are often used as the initial screen of transport ability for carrier candidates. However, BLMs are not practical on an industrial scale. For commercial applications, Supported Liquid Membranes (SLMs), where the organic layer is immobilized within the pores of a thin (-100 μπι) polymer support, or Emulsion Liquid Membranes (ELMs), are more attractive (1,11). Since the goal of the separation is to reclaim the transported sugars, the use of SLMs in a hollow fiber geometry is probably the membrane system of practical choice (12). This means the final-generation carriers need to be be compatible with hollow fiber SLMs.

Transport via Tetrahedral Boronates. Membrane transport using boronic acid carriers was first reported in 1986 by Shinbo and co-workers who discovered that a mixture of phenylboronic acid (PBA) and trimethyloctylammonium (TOMA) chloride transported reducing monosaccharides through BLMs (13). Transport was observed to be pH sensitive, in that uphill saccharide transport could be achieved from a basic source phase into an acidic receiving phase. The transport mechanism

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196 CHEMICAL SEPARATIONS WITH LIQUID MEMBRANES

invoked to explain these observations is described in Figure 1. Subsequent studies by Czarnik (14,15) and our group (16-20) showed that other hydrophilic diol-containing compounds, namely nucleosides and aryl glycosides, could be transported by this ion-pair process. Under low extraction conditions the order of transport enhancements reflected the known order of boronic acid affinities for cyclic diols which is cis-α,β-diol > cis-a,y-diol > trans-a,y-diol » trans-a,P-diol. Thus, the order of transport enhancements for nucleosides was ribonucleosides » 2,-deoxyribonucleosides, and for glycopyranosides, galactoside > mannoside > glucoside > xyloside. In the case of monosaccharides, the transport enhancement order was fructose ~ mannose > galactose > glucose (13). In this series, however, the order of sugar selectivity is more difficult to rationalize because reducing sugars are known to isomerize in the presence of boronic acids making the identity of the sugar/boronate complex less certain (18). In particular, there is a strong bias towards complexation of hexoses as their furanose isomers (21).

C d i o i p OH"

HO OH

PH Ar—Β Q + c r

OH

O H " diol

HO OH

c r 2 H 2 0

H2O

Q + Ο Ο B-

Ar' v OH

membrane

2H2O c r

H 2 0

Figure 1. Transport Mediated by Reversible Tetrahedral Boronate Formation. pH < Boronic Acid pKa. Q+ = Quaternary Ammonium Cation Such as Trioctylmethylammonium (TOMA).

Czarnik devised an elegant improvement on PBA-TOMA as a carrier admixture by synthesizing the lipophilic alkylated pyridylboronic acids 3 (15). Functioning as covalent versions of PBA-TOMA, carriers 3 were found to transport nucleosides up to eight-fold betfer (e.g., a BLM containing 0.5 mM of carrier 3 enhanced uridine transport to over 100 times the rate of background diffusion). At pH 7, compounds 3 exist as zwitterions, thus the principal binding equilibrium is that shown in equation

" B ( O H ) 3

V N Î R H O O H

3 R = n - C 1 8 H 3 7 , steryl

Uphill Transport Carriers. Although zwitterionic carriers 3 are attractive because of their improved transport rates, they suffer from a potential drawback; their binding equilibria are pH independent. Unless a highly acidic receiving phase is employed (the pK a for 3 is approximately 4) (75), these carriers cannot be used to

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transport sugars uphill using a pH gradient. The ability to transport uphill is an essential prerequisite if a practical sugar separation method is to be developed. As already stated, uphill transport driven by a pH gradient has been demonstrated for the ion-pair transport pathway shown in Figure 1. In certain separations it may be undesirable to deviate from neutral pH (e.g., sample instability). Therefore, we have explored ways of driving uphill transport using electrochemical gradients other than pH. Our first attempt employed a fluoride ion gradient. Since the ability of F - to form dative bonds with trigonal boron acids is similar to that of OH -, we wondered if F - ions could substitute for OH". In other words, could F" ions promote formation of the anionic tetrahedral fluoroboronate 4 (equation 2), and induce transport via an ion-pair mechanism analogous to that described in Figure 1? This was indeed the case (16) Addition of KF (0.5 M) to a source phase buffered at pH 7 was found to increase the passive nucleoside transport ability of a PBA-TOMA carrier admixture by a factor of three. Moreover, uphill nucleoside transport was achieved in the direction of a F" concentration gradient. Control experiments showed that KC1 and KBr had no effect on transport.

ΊϋοΓ C d i o T ) + P h B ( 0 H ) 2 + ρ - ^ 0 N ρ + 2 H 2 O (2)

H O O H B -R i F

We have also developed uphill transport systems that are driven by metal cation gradients. We reasoned that an alternative way of producing a lipophilic cation to ion-pair with a sugar-boronate anion would be to complex a metal cation inside a lipophilic ionophore (17). This idea lead to the design and synthesis of compound 5 as a functionally biomimetic sodium-saccharide cotransporter (20). A B L M containing 1 mM of 5 transported an aryl glucoside five times faster than the background rate, whereas an equimolar mixture of PB A and benzo-15-crown-5 produced negligible transport enhancement. Carrier 5, however, was less than half as effective at glucoside transport as PBA-TOMA, reflecting among other things the inherent difficulty for a heterotopic receptor like 5 to simultaneously bind and cotransport two different solutes (equation 3). The putative binding equilibrium shown in equation 3 suggests that glycoside transport should be sensitive to Na + ion concentrations in the aqueous phases. Moreover, uphill transport in the direction of a Na+ ion gradient was predicted to occur and subsequently found to be the case. Carrier 5 represents the first artificial sodium-saccharide cotransporter to mimic, functionally, the way nature uses the ubiquitous inward-directed Na + gradient to actively transport sugars into cells (17).

I \ Ι~Λ

C ° 2 C « £ > 0 0 ο ™a ο

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Transport via Trigonal Boronates. Under certain experimental conditions, significant BLM transport can be achieved using boronic acids alone. For example, we found that boronic acids can mediate glycopyranoside transport by forming a reversible trigonal boronate ester with a glycopyranoside diol (Figure 2) (18). The order of diol selectivity for this trigonal boronate transport pathway was observed to be cis-a,y-diol > cis-a,p-diol « trans-a,y-diol » trans-a,p-diol, which differs slightly from the selectivity of the tetrahedral boronate pathway. As noted above, trigonal boronate esters are usually unstable in an aqueous environment, and thus are a minor presence. However, at an aqueous/organic interface, a lipophilic boronate ester is able to partition into the organic phase, where it is protected from hydrolysis. Other factors that strongly affect the trigonal boronate transport pathway are the lipophilicity of the boronic acid carrier and the aqueous phase pH. Shinkai and coworkers reported that highly lipophilic boronic acids are able to efficiently extract reducing monosaccharides into an organic layer (22). Although liquid membrane transport was not the goal of their work, it seems likely that it would have occurred. The effect of pH on the trigonal boronate pathway is varied. We have found that PBA alone is unable to transport ribonucleosides at neutral pH, while significant transport is observed at pH 4 (23). On the other hand, galactopyranoside transport mediated by PBA was a maximum at pH 7 (18). Most recently, Rotello has reported that a B L M containing 3 mM of PBA is able to enhance riboflavin transport by more than two hundred times over the background rate (24). There is little doubt that the trigonal boronate transport mechanism is operating in this case, although the stoichiometry and regiochemistry of binding are still to be established.

diol

HO OH

2 H 2 0

HaO

Figure 2. Transport Mediated by Transient Trigonal Boronate Ester Formation.

Dopamine Transport

Boronic acids have been shown to transport hydrophilic diol-containing compounds other than sugars. For example, the crowned boronic acid 6 was designed as a selective carrier for dopamine transport (25). A BLM containing 1 mM 6 transported dopamine 160 times faster than background diffusion (Table I). An interesting design feature with carrier 6 is illustrated in equation 4. Not only is it dopamine shape-selective, but the resulting complex 7 is charge balanced and does not need an accompanying counter-ion for transport. This provided carrier 6 with a novel selectivity mechanism for dopamine transport which is reflected by the data shown in Table I. Since the association of dopamine and carrier 6 was an acid-producing equilibrium, it was possible to use a pH gradient to drive the dopamine uphill into an acidic receiving phase.

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Table I. Transport Rates for Catecholamines, Glycosides, and Uridine in the Presence and Absence of Carrier 6.

Initial transport Initial transport rate (10"8 M min"1)15 rate (10"8 M min-1)15 Rate

Entry Transported compound3 no carrier carrier 6 enhancement0

1 dopamined

2 norepinephrined

3 epinephrine^ 4 tyramined

5 aryl P-glucoside^ 6 aryl p-mannoside^ 7 uridine^

2.2 2

3.4 70 1.3 1.6 0.2

356 120 6.8 70 3.9 5.3 0.2

160 60 2 1 3 2 1

aSource phase: sodium phosphate buffer (100 mM, pH 7.4), sodium dithionite (10 mM); Organic phase: carrier 6(1 mM) in chloroform; Receiving phase: sodium phosphate buffer (100 mM, pH 7.4), sodium dithionite (10 mM). b±15 %. transport rate in the presence of carrier 6 divided by the rate in the absence of carrier. dThe source phase initially contained 41 mM catecholamine. eThe starting concentration of transported species was adjusted to give a similar rate of background diffusion. Source initially contained 1.36 mM glycoside. Source initially contained 20 mM uridine.

Dopamine Transport Through SLMs. The goal of this ongoing project is to develop a dopamine carrier that will purify and concentrate body-fluid samples for clinical dopamine analysis (25,26). The most likely membrane system for such an application is a SLM, which means the carrier has to be highly lipophilic. In an attempt to satisfy this requirement, carrier 8 was synthesized and its SLM transport ability determined (27). Transport was measured through a liquid membrane of 2-nitrophenyl octyl ether supported by a flat sheet of Accurel polypropylene (area = 16 cm2). In the absence of carrier, the flux from a source phase containing 50 mM dopamine at pH 7.4 was negligible. In the presence of carrier 8 (2 % wt) a dopamine flux of 5 χ 10"7 mol/irPs was observed. These preliminary results are highly encouraging and strongly suggest a high-flux dopamine transport device can be developed using SLM technology.

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Chemical and Physical Factors That Control Transport Rate.

In an attempt to fully understand the BLM transport process, we undertook a detailed study of the factors that control glycopyranoside transport rates (79). We found that transport was dependent on the extraction ability of the boronic acid carrier. An extraction constant, KeX, was calculated using the following expression:

G + Β

(aq) (org)

Kex GB

(org)

(5)

where: G = uncomplexed glycoside Β = uncomplexed boronic acid GB = glycoside-boronate complex

A plot of transport rate versus log Ke X exhibited an approximate bell-shaped curve with maximal transport occuring when the carrier had an extraction constant, Ke X ( m a x ) ~ 2.2 (Figure 3).

Transport Rate

10'8 M/min

1.5 2.5 3.5 log Kex

Figure 3. Plot of Glycoside Transport Rates vs. log Kex-

We were able to show that of the various transport models which can explain this bell-shaped relationship, a diffusion-controlled process was the most likely. The most convincing evidence was that transport was dependent on the organic phase stirring rate (Figure 4) (19). The diffusion-controlled model, which is often seen in ionophore-mediated transport, assumes the kinetics of carrier complexation are rapid and that the rate-determining step is diffusion of the solutes through the unstirred layers (Nernst layers) of the three-phase system (10). Transport flux through the unstirred layers is in turn determined by the thickness of the layers (hence the dependence on organic phase stirring rate), the diffusion coefficient D from Fick's first law, and the carrier extraction equilibrium constant, Kex- The observed bell-shaped correlation is rationalized in the following way (10). Transport is a multistep process involving extraction of the solute from the source phase, movement of the carrier/solute complex through the organic layer, and subsequent stripping of the complex into the receiving phase. Under conditions of weak extraction, transport is slow due to the low amounts of solute moving from the source phase into the organic layer. Under conditions of high extraction, it is the low solute concentrations moving from the organic layer into the receiving phase that is the rate-determining step. A corrollary of the diffusion-controlled process is that Ke X is the critical variable determining transport rate (10). Therefore, an analysis of the factors that control

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transport can be reduced to an analysis of the factors that change Kex relative to Kex(maxV In the case of glycopyranoside transport, the chemical and environmental factors tnat affect KeX have been described in detail (19).

50 -

Transport Flux 1 0 - 8 M/min

30 -

20 -

10-200 400 600 800

Stirring Rate, rpm Figure 4. Glycoside Transport Rate vs. Membrane Stirring Rate.

Since transport is dependent on the rate of diffusion through the membrane, it is anticipated that transport rates should increase as the membrane becomes thinner. This correlation should continue, of course, until the rate of membrane diffusion becomes faster than the kinetics of sugar-boronic acid association. Since the effective membrane thickness associated with a stirred B L M is substantially greater than a SLM, it is expected that transport fluxes will be.greatly increased when the above transport experiments are repeated with SLMs (11,12). Our most recent results with SLMs indicate this to be true (27).

Sugar Transport Through Lipid Bilayer Membranes.

Scattered within the transport literature are a handful of reports on the possibility of using liposomes (lipid bilayer vesicles) in separations (28,29). There are a number of attractive features associated with liposome membrane systems: (i) the bilayer constituents are highly biocompatible phospholipids, organic solvents are essentially eliminated; (ii) liposomes have a small size (100 nm diameter) and a very large surface area, while the walls are very thin (3 nm), thus transport rates can be very rapid; (iii) the technology and economics associated with manufacturing and storing liposomes has improved dramatically over the past ten years (30).

Transport Out of Liposomes. We have begun a study of the ability of boronic acids to transport sugars in and out of liposomes (31). Initially we focused on the experimentally easier problem of determining sugar efflux from liposomes. The experimental set-up for glucose efflux is described in Figure 5. Glucose (typically 300 mM) was encapsulated inside large unilamellar vesicles (LUVs) that were prepared by the rapid extrusion technique (32). It is worth noting that very similar results were obtained with the easier to prepare multilamellar vesicles (MLVs) composed of egg lecithin. A standard hexokinase/glucose-6-phosphate dehydrogenase enzyme assay was used for detection of the escaped glucose (33). The enzymes are unable to penetrate the liposomes, thus an absorbance reading at 340 nm, due to NADPH formation, results only when a glucose molecule is released from the liposome. As shown in Figure 6 and Table II, addition of various boronic acid compounds induced glucose leakage from the liposomes. Glucose efflux from the liposomes continued until the liposomes were completely empty. This active transport effect is attributed to the destructive assay which continually removes glucose from the system. Inspection of the data in Table II suggests efflux is dependent on both the lipophilicity of the boronic acid (as judged by hydrophobicity

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202 C H E M I C A L S E P A R A T I O N S W I T H L I Q U I D M E M B R A N E S

constant, π), as well as the acidity (as judged by pKa). A large number of control experiments have been conducted to prove the glucose efflux is due to a selective transport process, and not a general increase in liposome permeability. In addition, the efflux experiments have been repeated with a range of sugar compounds. To date, the observed order of sugar efflux rates has been sorbitol > fructose > glucose > mannose > sucrose, which reflects the known order of boronate affinity for these sugars (7,34). An illustration of the transport selectivity that can be achieved with this approach is provided in Figure 7 (34). In this experiment, liposomes with equal amounts of sorbitol and sucrose encapsulated inside were teated with boronic acids. An enzymatic sorbitol assay showed that essentially all of the sorbitol was extracted from the liposomes within a couple of hours, whereas a sucrose assay of the same liposomes showed zero sucrose efflux. The selectivity is remarkable. In a single step, an equimolar mixture of these two sugars is completely separated

Figure 5. Liposome Glucose Efflux Experiment. G = glucose, Β = boronic acid, BG = glucose-boronate complex, Ei = hexokinase, E2 = glucose-6-phosphate dehydrogenase, 6-PG = 6-phosphogluconate, NADP = nicotinamide adenine dinucleotide phosphate, NADPH = reduced form of nicotinamide adenine dinucleotide phosphate, ADP = adenosine diphosphate, ATP = adenosine triphosphate.

80 % Glucose escaped from liposomes 50

40

20 0 20 40 60 80 100

Time (min) Figure 6. Percent of Glucose Escaped at pH 7.5 From Liposomes Containing 300 mM Glucose, After Treatment with: (a) no boronic acid, (b) phenylboronic acid, 1 mM, (c) phenylboronic acid, 5 mM, (d) 3,5-dichloro-phenylboronic acid, 1 mM, (e) 3,5-bis(trifluoromethyl)phenylboronic acid, 1 mM.

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Table II. Relative Rates of Glucose Efflux at pH 7.5, pKa's, and Substituent Hydrophobicity Constants for Various Boronic Acid Derivatives.

Relative rate of Sum of the Boronic acid efflux (± 15 %) pK a π valuesa

Phenylboronic acid 1 8.9 0.0 3,5-Dichlorophenylboronic acid 15 7.4 1.42 3,5-Bis(trifluoromethyl)phenylboronic acid 30 7.2 1.76 4-Methylphenylboronic acid 1.9 9.3 0.56 4-Methoxyphenylboronic acid 0.8 9.3 -0.02 3-Methoxyphenylboronic acid 0.9 8.7 -0.02 4-terf-Butylphenylboronic acid 30 9.3 1.98 4-Carboxyphenylboronic acid 0.02 8.4 -4.36 1-Butaneboronic acid 0.3 10.4 -Boric acid io- 4 9.0 -aSum of hydrophobicity constants for aryl substituents excluding the boronic acid.

Abs (340 nm)

Sorbitol Assay no boronic acid 1 mM t-butylphenyl boronic acid

Abs (340 nm)

1.0 0.8 0.6 0.4 h after Triton-X 0.2 h 0.0

Sucrose Assay

1 mM t-butylphenyl boronic acid

Time (min) 20 40 60 80

Time (min) 100

Figure 7. Liposome Efflux Rates for Liposomes Filled with 150 mM Sorbitol and 150 mM Sucrose. The absorbance at 340 nm corresponds to NADPH production produced by enzymatic assays.

Transport into Liposomes. Most recently, we have begun liposome influx studies using radiolabeled sugars. We have found that lipophilic arylboronic acids can facilitate sugar transport into empty liposomes. Figure 8 shows some typical preliminary results. ^C-labeled liposomes were treated with ^H-labeled glucose in the presence and absence of boronic acids. Every ten minutes an aliquot was removed and the amount of glucose associated with the liposomes (ratio of Glu:PL) was determined (34).

Ratio Glu:PL

0.8 r

0.6 -

0.4 -

0.2, j

o.oh

B * n g 8 f t * x - ι 1 1 1 1 1 1 1 1 1

20 40 60 80 100

0 No Boronic Acid x 4-Carboxyphenylboronic acid

(5mM) • 3,5-Bis(trifluoromethyl)

phenylboronic Acid (2.5 mM)

Time (min) Figure 8. Glucose Delivery into Empty Liposomes Using Radiotracer Techniques.

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Conclusions

Boronic acids can facilitate the transport of a range of hydrophilic sugar derivatives through various lipophilic membranes. Although the research program is still in its infancy, the results to date suggest that boronic acids show great promise as transport carriers in membrane-based sugar separations. Their use in large-scale separations will require moving to more practical membrane systems such as SLMs with hollow fiber geometries. This in turn means the carriers will have to be redesigned to meet the constraints of the membrane system (in the case of SLMs, an important requirement is very high carrier lipophilicity). Research efforts in this general direction are underway.

Acknowledgments

It is with sincere gratitude that I acknowledge the research efforts of Jeffrey T. Bien, Marie-France Paugam, Pamela R. Westmark, Gregory T. Morin, and Martin Patrick Hughes. This work was supported by a grant from the National Science Foundation and a Cottrell Scholar award of Research Corporation.

References

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784. 7. Lorand, J. P.; Edwards, J. O. J. Org. Chem. 1959, 24, 769-774. 8. Ferrier, R. J. Adv. Carbohydr. Chem. 1978, 35, 31-80. 9. Bergold, Α.; Scouten, W. H., In Solid Phase Biochemistry, Analytical and

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