Twenty-three monosaccharides, e.g., D- or t-pentoses. D- or L-hexoses, heptose, 2- or 6-deoxyhexoses, 2-deoxy-2-aminohexoses, hexuronic acids, and N-acetylmuramic acid, werecoupled to the azo dye 4’-NJ’-dimethylamino-4-aminoazobenzene by reductive aminationusing sodium cyanoborohydride as reducing agent and in the presence of pentaerythritol. Thestructure of the colored glycamines was established by mass spectrometry. The average yield ofthe reaction was more than 80%. The sugar derivatives were separated either by silica-gel thinlayerchromatography or by high-performance liquid chromatography. Spectrophotometricquantitation was performed in the visible range at the picomole level. The method was appliedto the determination of the sugar composition of the glycosphingolipid globotetraosyl ceramide
10
ANALYTICAL BIOCHEMISTRY 147, 156-165 (1985) Chromogenic Labeling of Monosaccharides Using 4’-N,N-Dimethylamino-4-aminoazobenzene GUNTER ROSENFELDER,’ MATTHIAS M~RGELIN, JUI-YOA CHANG, CORA-ANN SCHONENBERGER,DIETMARG.BRAUN,ANDHARRYTOWB~N Pharmaceuticals Research Laboratories, Ciba-Geigy Limited, CH-4002 Basel, Switzerland Received October 22, 1984 Twenty-three monosaccharides, e.g., D- or t-pentoses. D- or L-hexoses, heptose, 2- or 6- deoxyhexoses, 2-deoxy-2-aminohexoses, hexuronic acids, and N-acetylmuramic acid, were coupled to the azo dye 4’-NJ’-dimethylamino-4-aminoazobenzene by reductive amination using sodium cyanoborohydride as reducing agent and in the presence of pentaerythritol. The structure of the colored glycamines was established by mass spectrometry. The average yield of the reaction was more than 80%. The sugar derivatives were separated either by silica-gel thin- layer chromatography or by high-performance liquid chromatography. Spectrophotometric quantitation was performed in the visible range at the picomole level. The method was applied to the determination of the sugar composition of the glycosphingolipid globotetraosyl ceramide and the human milk oligosaccharide lacto-N-fucopentaose I. o 1985 Academic PKSS, I~C. KEY WORDS: spectrophotometry; chromatography, carbohydrates: HPLC, carbohydrates; thin-layer chromatography, carbohydrates; radioactivity measurement: sugar analysis High-sensitivity sugar analysis of glycocon- jugates has hitherto been performed mainly by gas-liquid chromatography of trimethyl- silylated methyl glycosides or of alditol ace- tates, in combination with mass spectrometry (l-3). Recently, high-resolution proton nu- clear magnetic resonance spectroscopy (4) and fast-atom-bombardment mass spectrom- etry (5) have proved to be powerful tech- niques. Another widely used method involves tritium labeling at the reducing end of oli- gosaccharides and degradative analysis of ra- dioactive alditols (6). These methods require either highly sophisticated analytical equip- ment or laboratories licensed for radioisotope work. Lately, a variety of HPLC methods have been reported to separate nonderivatized sug- ars, or chromogenic or fluorogenic deriva- tives by ion-exchange, phase-partition, or adsorption techniques [for a review, see Honda, (7)]. ’ To whom correspondence should be addressed. Detection of nonderivatized sugars by measurement of their refractive indices or uv absorption spectra at 190-2 10 nm is restricted to the nanomolar to millimolar range; col- orimetric or fluorometric detection of sugar derivatives is highly sensitive at the picomolar level. Postcolumn derivatization after anion-ex- change chromatography of borate complexes, or after cation-exchange chromatography of amino sugars or glycamines, is a frequently used method (8-12) but it requires a high degree of instrumentation and is rather time consuming. Derivatization using aromatic or hetero- cyclic substituents and subsequent separation by HPLC or HPTLC2 has the distinct ad- vantage of rendering carbohydrates hydro- * Abbreviations used: DAAB. 4’-N,N-dimethylami- no-4-aminoazobenzene; DAAB-Gal, I-deoxy-I-[4-(4’di- methylaminophenyl)azophenyl] amino-@galactitol; for abbreviations of sugars see Table I; HPTLC, high- performance thin-layer chromatography. 0003-2697185 $3.00 Copyright 0 1985 by Academic Press. Inc. All rights of reprczduction I” any form reserved. 156
Transcript
ANALYTICAL BIOCHEMISTRY 147, 156-165 (1985)
Chromogenic Labeling of Monosaccharides Using 4’-N,N-Dimethylamino-4-aminoazobenzene
Pharmaceuticals Research Laboratories, Ciba-Geigy Limited, CH-4002 Basel, Switzerland
Received October 22, 1984
Twenty-three monosaccharides, e.g., D- or t-pentoses. D- or L-hexoses, heptose, 2- or 6- deoxyhexoses, 2-deoxy-2-aminohexoses, hexuronic acids, and N-acetylmuramic acid, were coupled to the azo dye 4’-NJ’-dimethylamino-4-aminoazobenzene by reductive amination using sodium cyanoborohydride as reducing agent and in the presence of pentaerythritol. The structure of the colored glycamines was established by mass spectrometry. The average yield of the reaction was more than 80%. The sugar derivatives were separated either by silica-gel thin- layer chromatography or by high-performance liquid chromatography. Spectrophotometric quantitation was performed in the visible range at the picomole level. The method was applied to the determination of the sugar composition of the glycosphingolipid globotetraosyl ceramide and the human milk oligosaccharide lacto-N-fucopentaose I. o 1985 Academic PKSS, I~C.
High-sensitivity sugar analysis of glycocon- jugates has hitherto been performed mainly by gas-liquid chromatography of trimethyl- silylated methyl glycosides or of alditol ace- tates, in combination with mass spectrometry (l-3). Recently, high-resolution proton nu- clear magnetic resonance spectroscopy (4) and fast-atom-bombardment mass spectrom- etry (5) have proved to be powerful tech- niques. Another widely used method involves tritium labeling at the reducing end of oli- gosaccharides and degradative analysis of ra- dioactive alditols (6). These methods require either highly sophisticated analytical equip- ment or laboratories licensed for radioisotope work.
Lately, a variety of HPLC methods have been reported to separate nonderivatized sug- ars, or chromogenic or fluorogenic deriva- tives by ion-exchange, phase-partition, or adsorption techniques [for a review, see Honda, (7)].
’ To whom correspondence should be addressed.
Detection of nonderivatized sugars by measurement of their refractive indices or uv absorption spectra at 190-2 10 nm is restricted to the nanomolar to millimolar range; col- orimetric or fluorometric detection of sugar derivatives is highly sensitive at the picomolar level.
Postcolumn derivatization after anion-ex- change chromatography of borate complexes, or after cation-exchange chromatography of amino sugars or glycamines, is a frequently used method (8-12) but it requires a high degree of instrumentation and is rather time consuming.
Derivatization using aromatic or hetero- cyclic substituents and subsequent separation by HPLC or HPTLC2 has the distinct ad- vantage of rendering carbohydrates hydro-
* Abbreviations used: DAAB. 4’-N,N-dimethylami- no-4-aminoazobenzene; DAAB-Gal, I-deoxy-I-[4-(4’di- methylaminophenyl)azophenyl] amino-@galactitol; for abbreviations of sugars see Table I; HPTLC, high- performance thin-layer chromatography.
0003-2697185 $3.00 Copyright 0 1985 by Academic Press. Inc. All rights of reprczduction I” any form reserved.
156
CHROMOGENIC LABELlNG OF SUGARS 157
phobic and thus permitting the application of phase-partition or adsorption-chromato- graphic procedures.
Labeling of reducing sugars and fluoro- metric or uv detection have been done by reductive amination using 2-aminopyridine (13) 7-amino-4-methylcoumarin (14) or an- iline, p-aminoacetophenone, and a-amino- benzoic ethyl ester (15). Dansyl hydrazine has been used to produce hydrazones of neutral sugars ( 16) and dansyl chloride (17) or 1 dimethylaminonaphthalene-5-sul- fonyl chloride ( 18) has been coupled to the primary amino group of deacetylated N- acetylhexosamines. None of these methods readily produces the desired chromophoric derivatives in high yields.
The azo dye 4’-N,N-dimethylamino-4-azo- benzene has been used to synthesize a chromophoric reagent to label amino acids (19). Recently it was reported to reductively aminate mono- or oligosaccharides (20). However, no attempts were made to study the reaction in detail, or to elaborate quan- titative methods of separating the color-la- beled sugars.
We report the preparation of 23 colored DAAB-monosaccharides and the quantitative analysis of these colored sugars by both thin- layer chromatography and high-performance liquid chromatography. This new method offers advantages over existing methods in terms of sensitivity, efficiency, and simplicity.
MATERIAL AND METHODS
Materials. 4’-N,N-Dimethylamino-4-ami- noazobenzene (DAAB) purum was obtained from Fluka (Buchs, Switzerland). It was recrystallized from hot ethanol and stored at -20°C.
Radioactive sugars were purchased from New England Nuclear (Dreieich, FRG): D- [ 1 -‘4C]Glc, D-[ 1 -‘4C]Gal, D-[ 1 -i4C]Fuc, D-[ l- 14C]GalNAc, D-[ 1 -i4C]GlcNAc; the specific radioactivities varied from 30 to 60 Ci/mol. For reductamination they were diluted with nonradioactive sugar and used at a ‘concen- tration of 10 mM in methanol.
Cold sugars were of reagent grade: pen- taerythritol, D-Glc, D-Gal, D-GalNAc, and L- Fuc were purchased from Fluka, and D-GlcU and D-GalU from Sigma Chemical Company (St. Louis, MO.). All others, as well as glo- botetraosyl ceramide, were obtained from Supelco Inc. (Bellefonte, Pa.). Sodium cy- anoborohydride was from Serva (Heidel- berg, FRG).
Lacto-N-fucopentaose I was purified from pooled human breast milk according to the method of Kobata (2 1).
Reductive amination of sugar standards. All components of the reaction mixture were dissolved in methanol and transferred to a Teflon-lined screw-capped vial (Pyrex 13, Pyrex, France). The final reaction volume was 200 ~1. The concentrations in a typical experiment were 0.5 mM sugar, 4 mM DAAB, 37.5 mM NaCNBH3, 37.5 mM pentaerythri- tol, and 4 M acetic acid. The sample was heated for about 10 min at 80°C until a color change from green to orange-yellow had taken place.
Purification cf DAAB-glycamines. Water- soluble constituents of the reaction mixture, i.e., nonreacted sugar, salt, and pentaerythri- tol, were separated from excess DAAB and DAAB sugar derivatives by reversed-phase chromatography on Sep-Pak Cl8 cartridges (Waters Assoc., Milford, Mass.) as follows. Methanol was evaporated using a nitrogen evaporator. The samples were dissolved in water and loaded on the cartridges (pretreated with methanol and water). The cartridges were then flushed with a large excess of water (20 ml) under increased pressure produced by a glass syringe. All colored material was completely bound to the C18-bonded phase. Excess DAAB was largely washed off the cartridge with 5 ml of chloroform:hexane (1: 3, v/v). DAAB-glycamines were eluted with 2-5 ml of methanol. Between these elution steps, the cartridge was flushed with nitrogen.
If necessary, complete separation of DAAB or contaminating colored by-products from DAAB-glycamines was achieved by chroma- tography on glass columns (20 cm length,
158 ROSENFELDER ET AL.
0.5 cm i.d.) packed with spherical silica gel (Iatrobeads, 6RS80.60, Iatron Chemical Products, Tokyo, Japan). The eluting solvent was chloroform:methanol:water 55:45: 10 (v/ v). Substances migrating down the column were easily monitored as distinct colored bands. DAAB was eluted with the solvent front.
Thin-layer chromatography. Aliquots taken from the reaction mixture or from purified samples were applied to silica-gel 60 HPTLC plates (10 X 10 cm, 0.24-mm silica layer, Merck, Darmstadt, FRG) using an automated sample applicator (Linomat III, Camag, Muttenz, Switzerland). Solvent systems were (A) chloroform:methanol:0.4 M aqueous so- dium tetraborate adjusted to pH 3.5 with glacial acetic acid (55:45: 10, v/v) or (B) chlo- roform:methanol:water (55:45:10, v/v). After they were dried the plates were sprayed with 0.25 N sulfuric acid. Following this treatment, all bands appear bright red.
Densitometric detection was done at 520 nm on a TLC scanner, Model 76502 (Camag, Muttenz, Switzerland) connected to a SP 4100 computing integrator (Spectra Physics, Darmstadt, FRG).
Radioactivity scanning on thin-layer plates was performed with an Automatic TLC Model LB2832 linear analyzer (Berthold, Wildbad, FRG) in combination with a 7500/ B BS 27/N multichannel analyzer system (Silena, S.p.A., Milan, Italy).
High-performance liquid chromatography. The components of the system were a Su- pelcosil 5-pm column (25 cm length, 4.6 mm i.d., Supelco), a Model U6K injector (Waters Assoc.), two Model 1lOA pumps (Altex, Berkeley, Calif.), an Altex Model 420 programmer, a Model 160 fixed-wavelength absorbance detector (Beckman Instruments, Berkeley, Calif,) equipped with a 436-nm filter, a SP4020 interface, and a SP4000 integrator (both Spectraphysics).
Sample volumes were 5-25 ~1. The pro- gram was as follows: (i) Solvent A (chloro- form:methanol:O. 1 M sodium tetraborate ad- justed to pH 3.5 with glacial acetic acid (65:
25:4, v/v) for 15 min, flow rate 0.75 ml/min; (ii) linear increase to Solvent B (chloroform: methano1:O.l M sodium acetate adjusted to pH 3.5 with glacial acetic acid (65:25:4, v/v) in 2 min, flow rate 1.5 ml/min; and (iii) Solvent B for 13 min, flow rate 1.5 ml/min. The sensitivity range was set at 0.005 AUFS.
Hydrolysis of globotetraosyl ceramide and facto-N-fucopentaose I. A combined acetoly- sis/hydrolysis procedure was used (22). Briefly, the substances were dissolved in 300 ~1 of 0.5 N H2S04 in 90% acetic acid and heated for 16 h at 80°C in Pyrex 13 screw- capped vials with Teflon liners (Pyrex, France). Then 300 ~1 of water was added and heating was continued for another 5 h. The samples were neutralized with Dowex 1 X 8 HC03 ion-exchange resin. After re-N- acetylation of amino sugars, the samples were derivatized.
Mass spectrometry.3 A ZAB fast-atom- bombardment mass spectrometer (Vacuum Generators, Altrincham, U. K.) was used. Thioglycerol served as matrix. Ionization was induced by a IO-keV xenon source.
Safety precautions. All procedures, includ- ing HPLC separation, involving toxic or pos- sibly carcinogenic solvents were performed under a well-ventilated hood. DAAB has been found to generate liver tumors in rats (23). Therefore, precaution measures have to be taken to avoid breathing the powdered reagent or any contact of solutions with the skin. However, due to its intense color, even very small amounts of spilled reagent can be noticed.
RESULTS
Reductive Amination of Monosaccharides with DAAB and Thin-Layer Separation of the Products
Initial experiments suggested that the re- ductive amination occurs within 10 min. The completion of the reaction is indicated by a
3 We thank Mr. Fritz Raschdorf, Ciba-Geigy Limited, for performing the mass spcctrometric analysis.
CHROMOGENIC LABELING OF SUGARS
A B C D E F
FIG. 1. Quantitation of reductive amination. [ 1 -“‘C]Gal was derivatized and separated on HPTLC as described under Materials and Methods. The same sample track was analyzed by various methods (A, C, D, F). Solvent system: chloroform:methanol:0.4 M sodium tetraborate, adjusted to pH 3.5 with acetic acid (55:45:10, v/v). (A) Linear analyzer scan of radioactive reaction mixture. (B) Migration of purified [ 1-‘4C]Gal (autoradiography). (C) Autoradiogram of reaction mixture. (D) Photography of reaction mixture. (E) Migration of pure DAAB. (F) Densitogram of reaction mixture.
color change from green (acid) to yellow (neutral to slightly alkaline). This is due to acid consumption caused by the destruction of NaCNBH3. Decreasing the concentration of NaCNBH3 or acetic acid results in a low product yield.
Figure 1 shows the experimental setup used to determine the optimal conditions of reductamination. After HPTLC separation of the reaction mixture and acidification of
/
6 .
. . 5
.
/
4
. 3 .
/A . 2
the silica-gel plates, 14C-labeled DAAB-gly- camine and nonreacted Gal were measured on the same sample, both by scanning for radioactivity and by densitometry at 5 10 nm.
At an eightfold excess of reagent, about 86% of Gal is converted to DAAB-Gal, mi- grating as a single band. The residual 14C radioactivity is present as nonreacted Gal, as judged from the migration behavior. Non- reacted DAAB migrates with the solvent front
FIG. 2. Linearity of DAAB derivatization at constant DAAB concentration (4 mM) and varying sugar concentrations. (A) 0, Fuc; c1, GalNac; A, Gal. (B) 0, Man; A, Glc; n G~cNAc. Regression lines were calculated from seven experiments.
160 ROSENFELDER ET AL.
and minor colored degradation products de- rived from the reagent can be recognized. At pH 2, both DAAB and DAAB-glycamines are bright red in color.
If only scant information on the approxi- mate sugar content of a sample is available, a large excess of reagent is a prerequisite of quantitative analysis. Under these conditions there must be strict linearity between the sugar concentration and the color yield of reductive amination. Figure 2 shows DAAB- glycamine formation at a constant DAAB concentration of 4 mM and sugar concentra- tions of lo-500 j&M. Linearity was observed over the whole range. The lower limit of densitometric detection is about 20 pmol. Experiments using a constant 500 PM sugar concentration and DAAB concentrations varying from 0.1 to 10 mM indicated that at a four- to eightfold molar excess of reagent vs sugar a plateau is reached (data not shown). Color yields depend on the sugar species, Fuc giving the highest and GlcNAc the lowest yield.
To obtain the absolute yields, we measured the radioactivities present on thin-layer plates.
L-Fuc D-Man D-Gal D-Glc IXGalNAc D-GkNAc
FIG. 3. Yield of reductive amination in the presence or absence of pentaerythritol. DAAB concentration: 4 mM; sugar: 500 FM; pentaerythritol: 37.5 mM. Stippled columns, without pentaerythritol; striped columns, with pentaerythritol.
FIG. 4. Structure of DAAB-Gal.
Hexoses and Fuc were derivatized at high yields: 8 l-96% of the total sugar-associated radioactivity was present in a single colored band (Fig. 3, stippled columns). The yields of GalNAc and GlcNAc were 56 and 42%, respectively. The yields of the corresponding amino sugar hydrochloride derivatives were much lower, ranging from 10% downward (not shown).
Since sodium borate, arising from Na- CNBH3, might prevent the completion of the reaction due to the formation of a com- plex, we added pentaerythritol to capture the borate. This increased the yields of GalNAc and GlcNAc (now 84 and 7 l%, Fig. 3, striped columns). The effect on the other sugars was less distinct.
Identification of Sugar Reaction Products Fast-atom-bombardment mass spectrom-
etry identified the DAAB sugar derivatives as secondary amines of the corresponding l- deoxysugar alcohol and DAAB. Figure 4 shows the structure of 1 -deoxy- 1-[4-(4’-di- methylaminophenyl) - azophenyl] -amino - D - galactitol as determined by the negative mass ion [M-H]- = 403. A second colored product of Gal arising only at a large excess of Gal proved to be the corresponding tertiary amine, as indicated by the negative mass ion [M-H]- = 567 and the positive one at MH+ = 569. This DAAB-sugar derivative migrated at an Rr value of 0.20 in our borate thin- layer system. The production of tertiary amines was also observed with Glc, Man, and Fuc, but not with GalNAc or GlcNAc.
Thin-Layer Chromatography of 23 DAAB- Sugar Derivatives
In addition to L-Fuc, D-Man, D-Gal, D- Glc, D-GalNAc, and D-GlcNAc, all being
CHROMOGENIC LABELING OF SUGARS 161
major constituents of mammalian glycocon- jugates, we extended our study to include a further 17 monosaccharides of vertebrate, plant, bacterial, or synthetic origin. After reductive amination, DAAB-glycamines were purified (see Materials and Methods) and run on highperformance silica-gel thin-layer plates. Table 1 lists the monosaccharides used and their Rr values using two solvent systems, one of them including borate. Figure 5 shows an acidified chromatogram.
The resolving power of the borate sy- stem is sufficient to separate most of the su- gars present in mammalian glycoconjugates. Problems may arise when Xyl and Gal are both present, and in that case the neutral system is more suitable. 2-Deoxy-2-amino- hexose derivatives show strong tailing on silica gel (Fig. 5, lanes Q, R). Derivatization results in several products of unknown struc- ture. Therefore, and because they react at
very low yields, a re-Wacetylation step should follow the hydrolysis of glycoconjugates. N- Acetylated amino hexoses behave differently on thin layer. GalNAc and ManNAc show regular bands whereas GlcNAc complexes strongly with borate and expresses distinct tailing.
D-Sugar derivatives cannot be separated from L-sugar derivatives. Lactones of glyc- uranic acids migrate at Rr 0.61 (GalU) or 0.49 (GlcU) (not shown). Disaccharides mi- grate in the range of GlcNAc (not shown).
High-Pecformance Liquid Chromatography
DAAB derivatives of six sugars were sep- arated on silica gel columns. Using a borate- containing solvent system, DAAB-Fuc, -Man, -Gal. -Glc, -GalNAc, and -GlcNAc were sep- arated under isocratic conditions (Fig. 6). However, DAAB-GlcNAc eluted very late as
FIG. 5. Thin-layer separation of DAAB-glycamines. Solvent system-chlorofoimmethanolzO.4 M sodium tetraborate adjusted to pH 3.5 with acetic acid (55:45: 10, v/v). Approximately 100-200 pmol was applied. (A) 2d-Rib (B) Rib (Cl Ara (D) XY~ 03 LYX
(F) Man (G) Gal (H) Glc (I) 2d-Glc
I I 15 30
(K) Rha (L) Fuc 04 Hep (N) GlcNAc
(0) GalNAc (P) ManNAc (Q) GlcN (R) GalN
(S) MurNAc (T) GalU (U) GlcU (V) DAAB
Retention Time (min)
FIG. 6. HPLC elution profiles of DAAB-glycamines. Samples contained between 5 and 80 pmol. For elution program see Materials and Methods.
(A) DAAB (El Gal (B) Fuc 03 Glc (C) GalNAc (G) GlcNAc (D) Man
a broad and tailing peak. Exchanging borate for acetate and increasing the flow rate re- sulted in a more symmetrical DAAB-GlcNAc peak. Figure 6 shows an elution profile of a mixture of DAAB-sugar derivatives which had previously been purified and run indi- vidually to determine the retention times. The amounts of the individual components vary from 5 to 80 pmol.
Molar Ratio of Sugars Present in Globotetraosyl Ceramide and Lacto-N-fucopentaose I
To demonstrate the sensitivity of our sep- aration methods, quantitative analyses were made of two reference substances. Globotet- raosyl ceramide from human erythrocytes (1 nmol) and lactofuco-N-pentaose I from hu- man milk (2.5 nmol) were hydrolyzed and the released sugars reductively aminated. Ta- ble 2 shows the structures of the oligosaccha- rides and the molar ratios of DAAB-glyca- mines. Figure 7 shows the HPLC elution profile of a 4% aliquot of globotetraose-
CHROMOGENIC LABELING OF SUGARS 163
TABLE 2
MOLAR RATIOS OF SUGARS PRESENT IN GLOBOTETRAOSYL CERAMIDE AND LACTO-N-FUCOPENTAOSE I DETERMINED BY HPLC AND TLC-DENSITOMETRY
Molar ratio
Oligosaccharide structure and trivial name Fuc GalNAc Gal Glc GlcNAc
GalNAc~l-3Galcu1-4Gal~I-4Glc (globotetraose)
Fuccu l-2Gal/3 l -3GlcNA@l-3GalP l -4Glc (lacto-N-fucopentaose I)
- 0.90 2.04 1.00 --a
0.91 - 1.83 I .oo 0.85”
a Determined by HPLC. ’ Determined by densitometry of TLC plates.
derived sugars at a 0.005AUFS setting. The lower detection limit is less than 5 pmol.
Densitometric scanning of TLC-separated DAAB-sugar derivatives from lacto-N-fuco- pentaose I (Fig. 8) is less sensitive. Here the lower detection limit is about 20 pmol.
0 5 10 15 : Retention Time bin)
FIG. 7. HPLC separation of DAAB-glycamines obtained from hydrolyzed globotetraosyl ceramide. (A) DAAB, (B) GalNAc, (C) Gal, (D) Glc. An ahquot containing approx 50 pmol of Glc was injected.
In the present study we used the azo compound DAAB to reductively aminate 23 monosaccharides in the presence of sodium cyanoborohydride (24). In preliminary ex- periments we largely adopted the conditions described by Hase and co-workers (13,25), who coupled 2-aminopyridine to sugars at an excess of chromophoric reagent and large excesses of NaCNBH3 and acetic acid over 3 to 7 h at 75°C in methanolic solution. In the
Migration Distance (cm)
FIG. 8. Densitometric scanning of HPTLC-separated DAAB-glycamines obtained from hydrolyzed lacto-lli- fucopentaose I. An aliquot containing 250 pmol was applied.
164 ROSENFELDER ET AL.
DAAB system, however, the reaction was completed after only 10 min. Further addition of NaCNBH3 and/or acetic acid did not substantially increase the reaction yield. The markedly lower yield of N-acetyl aminohex- oses as compared to hexoses or pentoses and the different reactivities of GalNAc vs GlcNAc suggested that in addition to an electronic effect steric hindrance might play a role. The different migration behaviors of DAAB-GalNAc and DAAB-GlcNAc on silica gel TLC in the presence or absence of borate indicated the formation of a strong borate complex with the latter even at low pH (26). Polyols, e.g., pentaerythritol or glycerol (27), added to the reaction mixture at excess con- centrations compete for borate binding sites and thus lead to higher sugar reductamination yields.
In the presence of pentaerythritol, DAAB efficiently labels all the prominent classes of reducing sugars, in contrast to some existing methods which can only be applied to neutral sugars ( 16) or to amino sugars ( 17,18).
The intense color of DAAB-glycamines facilitates sample preparation for TLC or HPLC.
The chromogenic materials can be located visually at any time as migrating bands on columns or thin-layer plates or during phase partition procedures. Thus, the solvent vol- umes can be kept to a minimum. Due to the color-change effect of the DAAB chromo- phore upon protonation/deprotonation (28), the pH of the fractions can be easily moni- tored.
Excess DAAB, salt, free sugar, and pen- taerythritol can be conveniently separated from DAAB-sugar derivatives by a two-step elution from Sep-Pak Cls cartridges. This minimizes sample preparation time, as com- pared with the tedious ion-exchange proce- dures (13,14).
A reproducible reaction is a prerequisite of quantitative analysis. With DAAB, strict linearity of color yield was observed over a broad range of sugar concentrations (lo-500 PM) at excess reagent concentration. The chromophoric sugar derivatives and the re-
agent itself are extremely stable compounds. Thus, purified DAAB-glycamines have been stored in methanol at room temperature in the dark for more than 1 year without any evidence of degradation. The molecular ex- tinction coefficient E,,,,, is 29,500 at 430 nm (28). Upon protonation to a pH of 2.2 there is a shift to 39,000 at 520 nm. This second absorption maximum can be exploited only for densitometric detection on thin-layer plates acidified with mineral acids. HPLC separation at such low pH may cause corro- sion of the pumps.
The fastest and simplest analytical sepa- ration technique is HPTLC. Its major draw- backs are inadequate quantitation methods and the lack of solvent gradient development systems. On silica gel, all sugars of any significance in mammalian glycoconjugates can be separated in a single run as DAAB- glycamines. Densitometric scanning is easy since no background problems arise due to spraying of acid or heating of the plates. The lower detection limit is about 20 pmol or less as compared to 1 nmol of pyridylamino sugar derivatives on paper ( 13). Visual detec- tion limits of 1 pmol or less, as reported in the literature, are of little value for quanti- tative analysis. Using the borate solvent sys- tem at least three modes of separation can be distinguished: (i) according to molecular weight (see pentoses vs hexoses and heptose, Fig. 5); (ii) according to the polarity of func- tional groups (increasing R,- values from amino to carboxyl to hydroxymethyl to iV- acetyl to methyl group); and (iii) according to complex formation with borate (see Gal, Glc, Man, or GalNAc vs GlcNAc. Fig. 5).
Precolumn derivatization and HPLC sep- aration afford the most sensitive means of analyzing sugar mixtures [lower limit 0.1 pmol (25)]. By Cls reversed-phase HPLC, DAAB derivatives of structurally closely re- lated sugars could not be separated completely (G. Rosenfelder, unpublished). Silica gel HPLC of DAAB-sugar-borate complexes, on the other hand, is an excellent method of quantitative sugar analysis, the lower limit
CHROMOGENIC LABELING OF SUGARS 165
being of the order of 5-l pmol at 436 nm. The problems arising with very polar com- pounds such as the DAAB-GlcNAc-borate complex can be overcome by switching to a
i 9
solvent system without borate, which results in an immediate release of DAAB-GlcNAc IO
from the column. During the preparation of this manuscript,
a paper published in 1982 in Chinese came 1 1,
to our notice (20). The authors had used 12 DAAB to reductively aminate the reducing end of mono- and oligosaccharides. They 13.
separated the derivatives by two-dimensional polyamide thin-layer chromatography and 14.
were able to identify 1 nmol to 100 pmol on 15, the plates by visual detection. Their separa- tion system was not able to distinguish be- tween GlcNAc and GalNAc and between 16.
Glc and Gal. 17. The color and the amphiphatic nature of
DAAB derivatives afford much the same 18. advantages in carbohydrate analysis as did related reagents, such as DAB-sulfonyl chlo- 19.
ride (29) DAB-isothiocyanate (30) and DAB- iodoacetamide (31) in peptide and amino
20,
acid analysis. 21. DAAB derivatization is not limited to
monosaccharides. The reaction conditions are sufficiently mild to also allow the reduc- 22.
tive amination of neutral oligosaccharides, as 23, well as of oligosaccharides containing sialic acids (manuscript in preparation). 24.
ACKNOWLEDGMENTS 25.
We thank Mrs. Angelique Bordmann for her skillful 26. technical assistance and Mrs. Dorette Moeschli for her secretarial expertise. 2-l.
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