[31]

REDUCTIVE CLEAVAGE METHOD

573

to scan selected ions and graph composites of selected individual ions allows the investigator to perform linkage analysis on picomole quantities of material. 8 The mass spectrometer response can be used to quantitate the identified derivatives. Prepare partially methylated derivatives of each individual type of sugar present as described previously. By comparing the flame ionization response to the differently substituted derivatives with the selected-ion composite response for the same derivatives it is possible to generate an adequate quantitative picture from less than microgram quantities of polysaccharides. 8 The increased interest in complex carbohydrates and their biological functions has stimulated equipment development in this area. Since linkage analysis most often is the most revealing method for generating the fine structure of a complex, biologically active epitope, it is desirable that more sensitive detectors be developed for the small mass range needed for identification of all partially methylated alditol derivatives. Acknowledgments The authoris indebtedto his mentorProfessorBengtLindberg;to Dr. MargaretMoreland, Glycomed,Inc., Alameda,CA, for submittingFigs. l0 and I l; to Drs. Laineand McCIoskey for their commentsand patience;to Mr. EdwardByrnefor editorialwork;and to Mrs. Sandy Rivers for the typing.

[31] L i n k a g e A n a l y s i s U s i n g R e d u c t i v e C l e a v a g e M e t h o d By GARY R. GRAY Introduction The relatively recently introduced ~ technique of reductive cleavage allows the simultaneous determination of position(s) of linkage and ring form(s) in polysaccharides. The technique is based on methylation analysis, but departs from it significantly with regard to the types of fragments ultimately analyzed. In standard methylation analysis, the fully methylated polysaccharide is hydrolyzed, and the partially methylated sugars so formed are reduced (NaBH4) and acetylated, and then characterized by gas-liquid chromatography-mass spectrometry. 2 Unfortunately, the I D. Rolf and G. R. Gray, J. Am. Chem. Soc. 104, 3539 (1982). 2 C. G. Hellerqvist, this volume [30].

METHODS IN ENZYMOLOGY, VOL. 193

Copyright © 1990by Academic Press, Inc, All rights of reproduction in any form reserved,

574

GLYCOCONJUGATES

MeOCH2 -

o OOMe

[3 1]

F MeOCH?_ l

CH2CI2

/

IMe3SiO~""~ L OMe Et3SiH

MeOCH2 O~"MM 0)

MeOCH2 I. Ac?_O(insitu)

AcO~"~ 2. oq. NoHCO5 OMe

o~ Me M Me3Si

OMe

SCHEME 1. Reductive cleavage procedure for 4-1inked D-glucopyranosyl residue.

method does not distinguish between 4-1inked aldopyranosides and 5linked aldofuranosides and, moreover, the relative configurations of the resultant partially methylated alditol acetates must be established by comparison of their gas chromatographic retention times with those of authentic standards. Although a procedure based upon standard methylation analysis has been devised3that distinguishes between 4-1inked aldopyranosyl and 5-1inked aldofuranosyl residues, it is quite complex and laborious and thus not well suited for routine analyses. In contrast, the reductive cleavage procedure, as illustrated in Scheme 1 for a 4-1inked o-glucopyranosyl residue, is much easier to perform, and the products, partially methylated anhydroalditols, are readily characterized by GC/MS of their acetates o r IH NMR spectroscopy of their benzoates. 4 The latter method of characterization is less sensitive but useful in that it establishes the identity of the parent sugar as well as its position(s) of linkage and ring form, whereas the mass spectrometric procedure is more sensitive but does not directly establish the identity of the parent sugar. A comparison of the electron ionization (EI) mass spectra of partially methylated anhydroalditol acetates with those of authentic standards, however, can be used to identify the positions of substitution of methyl and acetyl groups and thus the position of linkage and ring form of the parent sugar residue. Recognizing that i)-glucopyranosyl residues and D-mannopyranosylresidues are frequently encountered in complex carbo3 A. G. Darvill, M. McNeil, and P. Albersheim, Carbohydr. Res. 86, 309 0980). a C. K. Lee and G. R. Gray, J. Am. Chem. Soc. 110, 1292 (1988).

[31]

575

REDUCTIVE CLEAVAGE METHOD

hydrates, this chapter provides the mass spectra of the respective partially methylated 1,5-anhydro-D-glucitol (1) and 1,5-anhydro-D-mannitol (2) acetates derived from all terminal (nonreducing), singly, and doubly linked residues. 6 ROCH 2

4

6 ROCH 2

I

Ro3"- [

4

I

R = Ac

or

Me

Rot-- 2

OR

!

2

Experimental Procedures

Reductive Cleavage Total reductive cleavage is performed with triethylsilane as the reducing agent and with either trimethylsilyl trifluoromethane sulfonate (TMSOTf) or a mixture of trimethylsilylmethane sulfonate (TMSOMs) and boron trifluoride etherate (BF3 • Et20) as the catalyst. Reductive cleavage with EtaSiH and TMSOTf and subsequent in situ acetylation5 are accomplished as previously described in this series: Reductive cleavage with Et3SiH in the presence of TMSOMs and BF 3 • EtzO and separate acetylation are performed as described by Jun and Gray. 7 Briefly, a 5-mg sample of the per-O-methylated polysaccharide and a small stirring bar are added to a Wheaton V-vial (Aldrich, Milwaukee, WI), the inside of which had been previously silanized. 6 The vial and contents are kept under high vacuum for 2 hr, then dichloromethane (0.25 ml, predried with CaHz), Et3SiH (5 Eq/Eq of acetal, Aldrich), TMSOMs (5 Eq/Eq of acetal, prepared as described in Ref. 7), and BF 3 • Et20 (1 Eq/Eq of acetal, Aldrich) are sequentially added. The vial is then capped with a Teflon-lined screw top and the contents stirred for 24 hr at room temperature. Methanol (I .0 ml) is then added, stirring is continued for 30 min, and the mixture deionized by passage through a column (0.5 × 5 cm) of Bio-Rad AG501-X8 (Richmond, CA), analytical-grade, mixed-bed resin. Methanol and dichloromethane are removed by careful evaporation under vacuum, and the product acetylated by treating with 5 Eq each of acetic anhydride and 1methylimidazole in 0.2 ml of dichloromethane for 30 min. Acetylation 5 D. Rolf, J. A. Bennek, and G. R. Gray, Carbohydr. Res. 137, 183 (1985). 6 G. R. Gray, this series, Vol. 138, p. 26. 7 J.-G. Jun and G. R. Gray, Carbohydr. Res. 163, 247 (1987).

576

GLYCOCONJUGATES

[31]

is terminated by the addition of saturated, aqueous sodium hydrogen carbonate (0.5 ml), the aqueous and organic layers are separated, and the dichloromethane layer is washed twice with l-ml portions of water prior to analysis by GLC. Analysis by Gas Chromatography-Mass Spectrometry Gas-liquid chromatography is performed with a Hewlett-Packard 5890A gas-liquid chromatograph equipped with a J & W Scientific (Folsom, CA) DB-5 fused-silica capillary column (0.25 mm x 30 m; 0.25/zm film thickness). The column temperature is held at 110° for 2 min and then programmed to 300° at 6°/min. The column is eluted with helium at a flow rate of 1.3 ml/min. Mass spectra are acquired using either a Finnigan 4000 mass spectrometer equipped with a VG Multispec data system or a VG Analytical Ltd. Model VG 7070E-HF high-resolution, double-focusing mass spectrometer, as designated in Figs. 1 and 2. All spectra are acquired at an ionizing energy of 70 eV and at a source temperature of 250°. The VG instrument is scanned from m/z 600 to 35 at 1 see/decade whereas the Finnigan instrument is scanned from m/z 650 to 20 in 1 sec. The VG instrument is operated at an accelerating voltage of 5 kV. Authentic Standards All the compounds whose spectra are reported herein are synthesized by routes involving standard protection/deprotection strategies. Most are available from previous work,5,8-1~ but eight of the compounds are synthesized for the present study. The structures of all compounds are confirmed by 1H NMR spectroscopy.

Results and Discussion

Mass Spectra Given in Figs. 1 and 2 are the EI mass spectra of methylated/acetylated 1,5-anhydro-D-hexitols having the gluco and manno configurations, respectively. Included in the figures are all possible products arising from terminal (nonreducing), singly, and doubly linked o-glucopyranosyl and o-mannopyranosyl residues. Inspection of the spectra reveals diagnostic s j. u. Bowie and G. R. Gray, Carbohydr. Res. 129, 87 (1984). 9 D. Roll and G. R. Gray, Carbohydr. Res. 152, 343 (1986). 1oS. A. Vodonik and G. R. Gray, Carbohydr. Res. 172, 255 (1988). It S. A. Vodonik and G. R. Gray, Carbohydr. Res. 175, 93 (1988).

[31]

REDUCTIVE CLEAVAGE METHOD

577

1 ,S-AN HYDRO-2,3,4,6-TETRA-O-M ETHYL- D-GLUCITOL (F)

45

,:5, I.-

> x5 175

101

100-

(a)

71

75-

Z LIJ

_>

5088

laJ tY

25-

.... I

143 158 ,,

115

,J,,l

J.I .

'

'

'

IL

'

50

220

,I,

'

'

'

'

1 O0

I

.

.

.

.

'

I

150

'

200

I

'

'

'

'

250

I

300

2-O-ACETYL- 1,5-ANHYDRO-3,4,6-TRI-O- METHYL- D-GLUClTOL (VG) 71

100-

z w I.Z bJ

75-

(b)

> x5

45 lOl

50-

203

i

bJ tic

25 -

59 i

.. J, 5O i

,

,

i

i7 ,.UI J, i

i

1oo

143 ,,~, ,..L ,

,

,

J..L I1, ,

i

150

,

,h,,21,6

185 '

m/z

,

,

I

200

'

'

24-9 '

'

'1

250

'

'

'

'

I

300

FIG. 1. (a-k) Electron ionization mass spectra of some methylated/acetylated 1,5-anhydro-D-glucitol derivatives (1). Spectra denoted with (F) were acquired on a Finnigan 4000 and those denoted with (VG) were acquired on a VG 7070E-HF GC/MS instrument. differences for the positional isomers in terms of the presence or absence of certain ions and their intensity relative to the base peak (usually m/z 43, C H 3 C ~ O + ) . In addition, the molecular ion [or pressure-induced (M + 1) ion] is detected in low intensity in some spectra. The presence of this ion is useful in that it establishes the molecular weight of the component and thus its identity as an anhydrohexitol and its content of O-acetyl and O-methyl groups. The molecular weights of these components are more apparent, however, in chemical ionization (ammonia) mass spectra, wherein (M + H) ÷ and (M + NH4) ÷ ions are detected. Inspection of Figs. 1 and 2 also reveals small differences in ion intensities and, in a few cases, ion differences, in spectra of positional isomers

578

[31]

GLYCOCONJUGATES

3 - O - A C E r Y L - 1,5-ANHYDRO-2,4,6-TRI-O-METHYL- D-GLUCITOL (F) 1O0 03 z hi I--" Z

(c)

4-3

~. x5

75 ¸

~>

50'

'" ""

25

158

75 101 58 I,

,J[

115125

143

I

,,,,.... ,i ,,, ,,[ 100

5O

171 188

,L

I 2~217

150

249

200

i l l l

I 300

250

4--O-ACErYL- 1,5-AN HYDRO-2,3,6-TRI-O-METHYL- D-GLUCITOL (F) 100"

~ ~A Hz

75.

w _>

50"

43

~

bA n,-

(d)

> x5

58 25.

71

8797 103

.I,I] .[I, 50

111

129143

203 18,91

,,,I ..... I,. I,

1O0

150

200

I

250

300

6 - O-ACETYL- 1,5-ANHYDRO- 2 , 3 , 4 - T R I - O - M E T H Y L - D-GLUCITOL (F) 87

t

75

43 u) z bJ p,"7

(e)

> x5

100'

75. lOl

W __> 50"

58

tM

.,,l1130

25.

156 i

.iJ i

50

d,.l ...

188

143.

I~5 IL

2161

,

I O0

150

200 m/z

F I o . l c , d , a n d e. S e e l e g e n d o n p. 577.

'

'

t

250

'

'

I 3OO

REDUCTIVECLEAVAGEMETHOD

[31]

579

2 , 6 - DI-O-ACEI'YL- 1,5-ANHYDRO- 3 , 4 - D I - O - M ETHYL- D-GLUCITOL (F)

43

(f)

]> x5

100"

87 z w Iz

7571 50

hJ ,..y

130

25.

I

'

'

'

99 ,I,1.. I

'

50

l

184 I

i 142156

I,,, '

216

]

117

59 ,,,,,L .I h ,~

I,. '

I '

,. '

I I

1 O0

'

'

'

203 , '

I

150

3 , 6 - DI-O-ACE'P(L- 1 , 5 - A N H Y D R O - 2 , 4 - D I - O -

75-

z bJ >_

50-

Ld n--

'

'

'

I

'

'

'

'

I

250

300

METHYL- D-GLUCITOL (F)

(g)

43

> x5

100-

z

'

200

156

74

25 58

87

117

....L .IJ.,..,.J... x5

7558

5o-

,.I.

25-

156 ,I 85 103 7 d,ll ..h, , ,,, 129143., ,, , 1 I...1184 2 0; 3 2, 1, 7 23.3

9..7

I

50

'

'

'

'

I

1 O0

'

'

'

'

I

150

'

'

'

'

I

,'

l'

200

m/z

F[G. lf, g, and h. See legend on p. 577.

,

,

277 i

250

,

,

",

,

I

3OO

580

GLYCOCONJUGATES

[31]

2 , 4 - D I - O - A C E T Y L - 1,5-AN H Y D R O - 3 , 6 - D I - O - METHYL-D-GLUCITOL 100-

5

43

(VG) (i)

> x3

75-

57

Z

~

50-

,,lil

97 hi

a:

25. .....I I

'

. ,[ LI. '

~

1

~. . . . . . . . . '

50

I

'

'

~

'

'

100

g

1

. ,

,

217 ,

i

I

150

'

277

245

'

'

'

200

I

i

,

i

I

250

3OO

3 , 4 - DI- O-ACETYL- 1 , 5 - A N H Y D R O - 2 , 6 - D I - O - M E T H Y L - D-GLUCITOL (F) 10o.

~

43

75.

I'Z

~

(J)

> x3

171 99

50"

w

n,-

25.

58

184

128

231

2o,3 ~,?,6 I '

I

,

,

,

,

50

,

,

,

,

100

i'll

,

'

I

150

'

'

'

'

200

I

'

'

~

I

300

2 , 3 - D I - O - A C E T Y L - 1 , 5 - A N H Y D R O - 2 , 6 - D I - O - METHYL-D-GLUCITOL

100.

'

250 (VG)

(k)

43

> x5 111

,,z

75"

FZ

"' >_

50-

'Y

25-

74

li9

9

I

50

'

'

'

'

142156

I

1 O0

'

'

'

'

I

~

174

184

'

150

'

231

'

I

'

'

200

m/z FIG. li, j, and k. See legend on p. 577.

'

'

I

250

'

I

300

[31]

REDUCTIVE CLEAVAGE METHOD

581

1,5-AN HYDRO- 2,3,4,6-TETRA-O- METHYL-D-MAN NITOL (F)

03 Z

75,

(a)

lOl

100

> x5

4-5

Z

71 50-

Ld ,.y

175 88

25-

58 i..,,, I'1 I

'

143 158 all

115129

,LI..~1 '

'

d ..... , .....

.

50

.

.

.

'

1O0

188

, I

I

'

'

'

'

150

i

I

200

250

'

'

'

I

300

2-O-ACEq'YL- 1,5-ANHYDRO-3,4,6-TRI-O-M ETHYL- D - MANNITOL (F) 100.

z laJ I.Z

(b)

#3

x5

75-

71

>___ 50-

a~

1oi

25.

87

59

,.L,, .,I ,

5O

/ TM

171

[ L, 1714,+o

,, i

,

I

100

....

+

r

,

I,L , . . . ,

i

I

150

i

203

1~ J

~/.

i

216 ,

I

h,

200

Ii ,

249 ,

,

II

250

'

'

I

3OO

F z 6 . 2 . ( a - k ) Electron ionization mass spectra of some methylated/acetylated 1,5-anhydro-D-mannitol derivatives (2). Spectra denoted with (F) were acquired on a Finnigan 4000 and those denoted with (VG) were acquired on a VG 7070E-HF G C / M S instrument.

having different configurations (gluco vs. manno). However, the spectra were obtained using different instruments over a period of several years. Since efforts were not made to obtain the spectra under identical conditions, the use of the spectra to assign configuration is not warranted. Indeed, even if reproducible differences did exist in the spectra of comqgurational isomers, the use of such differences for assignment purposes would probably necessitate obtaining a complete set of reference spectra on every instrument used for the analysis. Under these circumstances, a comparison of gas chromatographic retention times would be less laborious.

GLYCOCONJUGATES

582

[31]

3-O-ACETYL- 1,5-AN HYDRO-2,4,6-TR1-0- METHYL-D-MANNITOL (F) 43

lO0z

w I.Z

(c)

> x5

7575

-

lOl ~''' 50w ac

d

25-

58 I.I I.

,tl.J, ,.IJ,l,I ,

5O

14,3 /I 1151129 ,,, 15/8 1~1 188 halll

i

I

h

'

'

'

i

100

I

~

'

249

. 12132170.

'

'

'

150

I

'

"

'

'

200

II"

'

I

250

300

4-O-ACETYL- 1,5-ANHYDRO- 2,3,6-TR1-0- METHYL-D-MANNITOL (VG) 97

(d)

100-

z

171

43

75.

++ ++ ti z

129

-

_~

50

58

"~ w

87

,.I

.JL.h

,/[1

1.j43

j h. . . . . . lJ+.. . . . . . ' 50 O0

203

157 '

'

I

150

'

185,. I.. ~

'

'

I

200

'

'

'

'

I

250

'

'

'

'

I

,300

6-O-ACETYL- 1,5-ANHYDRO-2,3,4-TRI-O-M Eq'HYL-D- MANNITOL (F) 100z

L~

43

[

75 +

z

75 87 101

-

"'

(e)

~ x5

50-

l

25-

156

I

1i0143

~1 I

50

.,l '

'

'

.... '

I

1O0

175188 216

,d '

I .,,1 '

'

'

I

150

'

'

'

'

249

I I

200

'

I '

'

FIG. 2c, d, and e. See legend on p. 581.

'

I

250

'

'

'

'

I

300

[31]

CLEAVAGEM E T H O D

R E D U C T I V E

583

2,6-DI-O-ACETYL- 1,5-ANHYDRO-3,4- DI-O-METHYL-D-MAN NITOL (F) 100"

z i,i hz

__

43

75-

50"

87

~ w ""

(f)

> x5

71 117130

]I

25"

216

203..

59 I lOl L.J Ld, 14,.21.1 1,7.118,.4 ,.,I. .I iIIi 1,11 , ,I,I. .

234 245

277 I

,

'

[

'

'

'

'

I

'

'

'

'

100

5O

I

'

'

'

'

150

I

'

'

''

200

'

I

i

i

i

250

'

I

300

3 , 6 - DI-O-ACETYL- 1,5-ANHYDRO- 2 , 4 - D 1 - 0 - ME'THYL- D-MAN NITOL (F) 100"

43

(g)

> x5

75" Z

w

50-

156 97

25-

74

101

58 ,.,.t..U '

50

'

117 ,..,,,I...... 1.~3.h 1.1!0 186 ,, 203217 , 234244

87 ,I. . . . '

'

1

'

'

'

'

1

'

'

100

'

'

1

'

'

'

'

'

150

1

'

'

277

"

200

250

'

I

300

4 , 6 - DI-O-ACE'F'(L- 1 , 5 - A N H Y D R O - 2 , 3 - D I - O - METHYL-D- MANN]TOL (F)

100"

z

w bz

43

(h)

> x5 156

75-

58

__ 50LJ

a:

171

258597103

...... 50

J.ll ..]JJ.Ltl

143

I.,.a 129...l

.[I

203217 [ I 233

[ II,

,, '

100

150

'

I

200

,

, b

,~/z FIG. 2f, g, and h. See legend on p. 581.

I

250

I

300

584

GLYCOCON,TUGATI~$

[31]

2,4-DI-O-ACETYL- 1,5-ANHYDRO-3,6-DI-O- METHYL-D-MANNITOL (VG)

Z

(i)

43

lO0-

i

> x3

231

75-

LI.I I-Z

157

__. 5097

171

w

a:

87 / 111 129139

25l i

...,,

,

IlL L

.,l l , i. . l ~

~........

5O

J'

,

IO0

,

199 189 2]6

,

,',

t'

150

,',

'

2OO

I

I

250

3O0

3,4- DI-O-ACE'P(L- 1,5-ANHYDRO-2,6-DI-O- METHYL-D-MANNITOL (VG)

IO0-

43

U)

> x3 171

,z

75-

Z w

>-

50-

97 ,,,,

r,,- 25-

9

58 69 .llh 87

dl

142 h~

I

'

'

'

'

50

]

'

231

185

111 '

'

100

'

I

'

'

'

'

150

I

'

'

'

'

I

'

'

250

200

I

300

2,3- DI-O-ACETYL- 1,5-ANHYDRO-4,6-DI-O- METHYL-D-MANNITOL (VG) IO 0

z

75.

w _>

50

m I--Z

w

r,,.

43

(k)

:::> x5

111

25.

74 87

129 97

231 142156 174185 I

50

100

150

,.,.,/=

200

FIG. 2i, j, and k. See legend on p. 581.

250

300

[31]

REDUCTIVE CLEAVAGE METHOD TABLE

585

I

I N T E N S I T Y R E L A T I V E TO B A S E P E A K O F S E L E C T E D I O N S IN M A S S S P E C T R A O F METHYLATED/ACETYLATED

1,5-ANHYDRO-D-GLUCITOL DERIVATIVES (1)

Relative intensity(%)~ Position of O-acetyl

Molecular weight

M-32

M-45

M-60

M-73

M-77

M- 105

None

220

5.5

20.8

b

--

17.9

! 1.9

2

248

0.7

9.3

0.2

0.2

3.7

3 i. 1

3

248

0.1

0.9

2.1

--

1.9

13.1

4

248

--

2.2

--

0.1

4.7

8.1

6

248

1.2

--

2.7

1.3

--

2.7

2,6

276

--

--

3.3

0.3

--

-0.3

3,6

276

--

--

0. I

0.1

--

4,6

276

--

--

0.4

0.4

--

i.2

2,4

276

--

8.0

--

--

8.4

7.3

3,4

276

--

3.3

0.7

0.2

--

2,3

276

--

3.5

0.2

0.3

0.1

19.7 3.6

Actualintensitiesvarydependingon the instrumentused, but observedvaluesare given to provide an indicationof their relative prominence. b Not observed.

Fragmentation Patterns The fragmentation patterns for derivatives of this type have not been established, but inspection of the spectra reported herein reveals diagnostic differences based upon the presence or absence of only a few ions in the spectra. Some key ions are those that arise from elimination of methanol ( M - 3 2 ) or acetic acid ( M - 6 0 ) from the molecular ion, and those that arise by cleavage between C-5 and C-6 with loss of C-6 and an attached O-methyl (M - CH2OMe; M - 4 5 ) or O-acetyl (M - CH2OAc; M - 7 3 ) group. Fragment ions are also observed at M - 77 and M - 105 due to the further loss of methanol and acetic acid, respectively, from the M - 4 5 ion. The presence of the M - 105 ion can also be explained in terms of loss of methanol from the M - CH2OAc ion. It should be noted, however, that there are other ions in the spectra of as yet unexplained origin that are also diagnostic for identification of a particular positional isomer. Summarized in Tables I and II are the results obtained from a comparison of the spectra of the gluco and manno isomers, respectively, for the relative intensities of the aforementioned fragment ions. Since the spectra were acquired using different instruments, it is recognized that comparisons based upon the actual intensities are not appropriate. The values listed do, however, provide an indication of the relative prominence of a given ion and on this basis they are useful for distinguishing between the

586

GLYCOCONJUGATES TABLE

[31]

II

INTENSITY RELATIVE TO BASE PEAK OF SELECTED IONS IN MASS SPECTRA OF METHYLATED/ACETYLATED 1,5-ANHYDRO-D-MANNITOL DERIVATIVES (2} R e l a t i v e intensity (%)" Position of

Molecular

O-acetyl

weight

M - 32

M - 45

M - 60

M - 73

M - 77

M - 105

None

220

1.7

8.9

b

--

14.0

8.1

2

248

0.4

3.1

0.2

0.1

3.3

6.4

3

248

--

0.9

2.8

0.1

1.8

18.4

4

248

--

8.4

--

--

72.2

26.4

6

248

1.2

--

1.9

1.3

--

3.5

2,6

276

0.1

--

3.1

0.3

--

0.6 0.4

3,6

276

0.4

--

0.4

0.1

--

4,6

276

--

--

--

2.3

--

2,4

276

--

29.4

0.5

--

1.8

3,4

276

--

5.5

--

--

--

26.1

2,3

276

--

4.0

--

0.3

--

0.8

5.0 11.0

A c t u a l intensities v a r y d e p e n d i n g on the i n s t r u m e n t used, but o b s e r v e d v a l u e s are g i v e n to p r o v i d e a n i n d i c a t i o n o f t h e i r r e l a t i v e p r o m i n e n c e . b Not observed.

various positional isomers. As is evident in Tables I and II, compounds containing a 6-O-methyl group are readily identified by the presence of an M - 45 ion, which is always much greater in intensity than the M - 73 ion, if indeed the latter is even present. In contrast, the M - 73 ion is always present in the spectra of 6-O-acetyl derivatives and the M - 4 5 ion is absent. Mono-O-acetyl derivatives (2-, 3-, and 4-O-acetates) that give an M - 45 ion are distinguished by the patterns of elimination of methanol and acetic acid from the molecular ion; i.e., an M - 32 ion at m/z 216 is either weak in intensity or not observed in the spectra of 3-0- and 4-0acetyl derivatives but is observed in the spectra of 2-O-acetates. The 3-0acetates are distinguished from the 2- or 4-O-acetates by the presence of an M - 6 0 ion at m/z 188. The three di-O-acetyl regioisomers (2,4-, 3,4-, and 2,3-diacetates) that give an M - 45 ion are easily distinguished by the intensities of ions at M - 77 and M - 105. In 2,4-di-O-acetyl derivatives, the M - 7 7 ion at m/z 199 is quite prominent, but this ion is either very weak in intensity or not observed in 2,3- and 3,4-diacetates. Mass spectra of the 3,4-diacetates contain a very intense M - 105 ion at m/z 171, however, distinguishing them from 2,3-diacetates. The three di-O-acetyl regioisomers (2,6-, 3,6-, and 4,6-diacetates) that fail to give an M - 4 5 ion are distinguished by the intensities of ions at M - 60 and M - 105. The M - 60 ion at m/z 216 is prominent in the spectra of the 2,6-diacetates, thus

[32]

PERIODATE CLEAVAGE REACTIONS

587

distinguishing the 2,6-diacetyl from the 3,6- and 4,6-diacetyl regioisomers. The 3,6- and 4,6-diacetyl regioisomers are distinguished, in turn, by the intensity of the M - 105 ion at m/z 171 which is prominent only in the spectra of 4,6-diacetates. Whether the patterns of elimination and fragmentation noted above will be observed for other configurational isomers, such as those of the galacto configuration, remains to be established. The similarities noted above for isomers of the gluco and manno configurations suggest, however, that major differences will not be observed in the spectra of other configurational isomers. Acknowledgments The author wishes to thank Samuel Zeller, Angela Ashton, Anello J. D'Ambra, Molly McGlynn, Christine Rozanas, Judith Sherman, and Dinesha Weerasinghe for the synthesis of previously unreported standards, Dr. Edmund Larka for performing GC/MS analyses, and Steven C. Pomerantz (University of Utah) and Kenneth L. Tomer (National Institute for Environmental Health Services) for assistance with plotting of mass spectra. This investi gation was supported by PHS grant number GM34710, awarded by the National Institute of General Medical Sciences, DHHS.

[32] L i n k a g e Positions in G l y c o c o n j u g a t e s b y P e r i o d a t e Oxidation a n d F a s t A t o m B o m b a r d m e n t M a s s S p e c t r o m e t r y By Ar~E-SOPHm ANGEL and Bo NILSSON

The introduction of fast atom bombardment mass spectrometry (FABMS) started a new era in mass spectrometry. Biological compounds like peptides and underivatized glycoconjugates, which cannot be analyzed by the conventional electron ionization mass spectrometry, could be analyzed using this technique. In contrast to electron ionization, the ionization in FAB is not dependent on volatility, since compounds are ionized in solution by a fast atom beam of argon or xenon. FAB-MS of underivatized glycoconjugates gives information about the molecular weight, but fragment ions formed by cleavage are of low abundance. Derivatization, for example, peracetylation or permethylation, enhances the fragmentation. For derivatized glycoconjugates the fragment METHODS IN ENZYMOLOGY,VOL. 193

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