2898
J . A m . Chem. SOC.1982, 104, 2898-2910
Structural Studies and Chemistry of Bacterial Capsular Polysaccharides. Investigations of Phosphodiester-Linked Capsular Polysaccharides Isolated from Haemophilus influenzae Types a, b, c, and f NMR Spectroscopic Identification and Chemical Modification of End Groups and the Nature of Base-Catalyzed Hydrolytic Depolymerization’ William Egan,*2nRachel Schneerson,” Kathleen E. Werner,” and Gerald Zon*2b Contribution from the Division of Biochemistry & Biophysics and Division of Bacterial Products, Bureau of Biologics, Food and Drug Administration, Bethesda, Maryland 20205, and the Department of Chemistry, The Catholic University of America, Washington, D.C. 20064. Received February 9, I981
Abstract: The capsular polysaccharide (Figure 1) from Haemophilus influenzae type b organisms was analyzed by a combination of NMR and chemical methods to determine its end-group composition. Phosphorus-containing end groups were present as ~-ribosyl2,3-and D-ribitol4,5-cyclophosphates,as well as D-ribosyl C3-and Dribitol C5-attachedphosphate monoesters; reducing end groups were not found. A similar analysis of the H. infuenzae type- f capsular polysaccharide (Figure 5 ) showed cyclophosphate termini spanning the C3 and C4 positions of D-GalNAc residues and monophosphate end groups bonded to C3of D-GalNAc and C I of 3-(0Ac)-~-GlcNAcresidues. Capsular polysaccharide (Figure 8) derived from H . influenzae type c organisms contained phosphate monoester end groups at C4 in the D-GIcNAcand 3-(0Ac)-~-GlcNAcresidues. In capsular polysaccharide (Figure 10)from H. infuenzae type a organisms, the 31PNMR-detectable monophosphate termini were tentatively assigned to D-ribitol C5 or D-GlcC4or both of these positions. The ”P NMR-derived molar ratio of phosphorus-containingend groups to repeating unit phosphodiester linkages was used to calculate number-average chain length. Comparisons of end-group composition and number-average chain length for capsular polysaccharide samples isolated from various strains of the four serotypesrevealed appreciable differences. The base-catalyzed (pH 10)depolymerization of the capsular polysaccharides (cleavage of the phosphodiester linkages) was monitored by 31PNMR spectroscopy, and pseudo-first-order kinetic analysis led to the respectively. serotype stability order a > c > f > b, with estimated relative rate ratios at 50 OC being 0.0052:0.0066:0.125:1, Types b and f capsular polysaccharide undergo depolymerization with formation of cyclophosphate and phosphate monoester end groups, while depolymerizationof types c and a capsular polysaccharide was accompanied by phosphate monoester end-group formation without accumulation of detectable cyclophosphatetermini. The type a polysaccharide gave evidence of phosphodiester linkage rearrangement. The phosphate monoester end groups in capsular polysaccharide types a and b were selectively derivatized (Scheme V) by a carbodiimide-mediated coupling with adipic acid dihydrazide, while similar nucleophilic trapping reactions free radical and 7-amino-4-methylcoumarin afforded spin-labeled and with 4-amino-2,2,6,6-tetramethylpiperidinyl-l-oxy fluorescence-labeled samples of the type a capsular polysaccharide; average yields were 50 & 20%.
The presence of extracellular capsular polysaccharides on Gram-negative and Gram-positive bacteria is related to their ability to cause invasive disease in human^.^,^ That the details of the structures of these polymers are important is evidenced by the observation that encapsulation is a necessary but not a sufficient condition for pathogenic it^.^^^ Thus, generically related, nonidentically encapsulated organisms can display markedly different abilities to cause disease; for example, of the six encapsulated Haemophilus influenzae serotypes, designated by the letters ”a” through “f“, only type b is associated with disease in humans.s Immunity to encapsulated organisms is mediated by serum antibodies specific for the polysaccharide capsule^.^-^ Since the initial finding that capsule-derived polysaccharides could be immunogenic: efforts have been made to actively immunize various (1) Portions of this work have been presented at the International Symposium on Bacterial Vaccines, Sept 15-18, 1980, National Institutes of Health, Bethesda, MD. (2) (a) Food and Drug Administration; (b) The Catholic University of America. (3) Robbins, J. B. Immunochemistry 1978, 15, 839 and references therein. (4) Robbins, J. B.; Schneerson, R.; Egan, W.; Vann, W.; Liu, D. T. “The Molecular Basis of Microbial Pathogenicity”; Smith, H., Skehel, J. J., Turner, M. J., Eds.; Verlag Chemie: Weinheim, Germany, 1980; pp 115-132 and references therein. ( 5 ) Robbins, J. B.; Schneerson, R.; Parke, J. C.; Liu, T.-Y.; Handzel, Z . T.; Orskov, I.; Orskov, F. “The Role of Immunological Factors in Infectious, Allergic, and Autoimmune Processes”; Beers, R. F., Bassett, E., Eds.; Raven Press: New York, 1976; pp 103-120 and references therein.
populations againt a variety of pathogens by using purified polysaccharide preparations. The first unequivocal demonstration of the efficacy of a polysaccharide vaccine was provided by MacLeod and associates’ in 1945; recently, capsular polysaccharide vaccines for the prevention of meningococcal and pneumococcal diseases have been licensed by the Food and Drug Administration. Unfortunately, success with polysaccharide vaccines has not been total. For example, polysaccharide vaccines are not, in general, highly effective in infants, the group for which they are often the most needed. This finding could reflect a preference, in infants, for T-cell-dependent vaccine antigens, although other factors affecting polysaccharide efficacy (e.g., molecular size) may be operative; in several instances, T-celldependent antigens have been formulated by conjugation of the polysaccharide with a protein carrier.’ The relationship between capsule structure and bacterial virulence, as well as the use of capsule-derived polysaccharides as immunogens, has prompted a number of structural studies of capsular polysaccharides, including those from Streptococcus pneumoniae, Neisseria meningitidis, and H . influenzae and their commonly encountered, cross-reactive, nonpathogenic counterparts In contrast to the well-characterized (usually Escherichia c0li).~3’~ (6) Francis, T., Jr.; Tillett, W. S. J . Exp. Med. 1930, 52, 573. (7) MacLeod, C. M.; Hodges, R. G.; Heidelberger, M.; Bernhardt, W. G. J . Exp. Med. 1945, 82, 445. (8) Schneerson, R.; Robbins, J. B.; Hanson, L.; Kaisjer, B.; Sutton, A.; Vann, W.; Ahlstedt, S.;Egan, W.; Zon, G. Semin. Infect. Dis., in press.
0002-7863/82/1504-2898$01.25/00 1982 American Chemical Society
J . Am. Chem. SOC..Vol. 104, No. 10, 1982 2899
Bacterial Capsular Polysaccharides
r
Table I. 31P NMR-Derived' End-Group Composition for Samples of Capsular Polysaccharide from H. influenzae Type b (Strain 1482)
1
HOH2C
0
OH
1
1"
L - ' - O 0H
Figure 1. Repeating unit structure of H . influenzae type b capsular polysaccharide shown in its protonated form: -3)-@-~-Ribf-( l-l)-Dribitol-5-(P04-.
sampleb
end group
composition: % ratio
before EDAC
D-ribosyl 2,30cyclophosphate D-ribitol4,5-cyclophosphate phosphate monoester
0.33 3,3,1 0.10 0.22 -
after EDAC
0.65 D-ribosyl-2,3-cyclophosphate 0.6 0 D-ribitol-4,5-cyclophosphate 0.17 d phosphate monoester 0.77
-
' See Experimental Section for spectral acquisition parameters.
Ii
See text. Relative molar percentages; calculated from integrated signal intensities relative to the total value for all end groups Not detached. and the repeating unit phosphodiester linkage.
'I
i'
Scheme I
I
I
O-
-C-OH
PhOH
phr 4
I I
20
15
10
5
,5 ,l
0
-5
I
PPm
Figure 2. 'H-decoupled 40.25-MHz N M R spectrum of H . influenzae type b capsular polysaccharide (strain 1482, 20 mg/mL); 7r/2 pulse, 30-s repetition time, no NOE, and 5048 accumulations; the middle trace was recorded a t 50 times the amplitude of the lower trace; P refers to the pulse position and X refers to a spinning sideband. The upper trace was similarly obtained with a more concentrated sample (strain Eagen, 100 mg/mL); however, a signal at 3.95 ppm was not detected.
molecular structure of the repeating monomeric units in these polysaccharides, virtually nothing is known about their end-group composition. We have addressed this problem within the phosphodiester-containing H . influenzae capsular polysaccharides (types a, b, c, and f> and, following characterization, have used these end groups in selective chemical transformations. We have also studied the hydrolysis of phosphodiester linkages in these capsular polysaccharides to gain insight into the origin of the terminal groups and to aid in vaccine control and production. Our findings are reported herein.
Results and Discussion Type b. End-Group Studies. H . influenzae type b capsular polysaccharide has the repeating unit structure shown in Figure l.9 The 31PN M R spectrum (Figure 2) of this material (strain 1482) featured an intense signal at 1.09 ppm for the repeating unit phosphodiester linkage as well as very low intensity signals at 3.95,20.83, and 18.85 ppm. The large downfield chemical shifts of the latter pair of signals were suggestive of five-membered ring cyclophosphate end groups, which presumably spanned the C3-C2 positions of D-ribose and the Cs-C4 positions of D-ribitol, on the basis of the connectivity of the phosphodiester linkage in the repeating unit. Isomers of cyclic adenosine monophosphate (CAMP) were used as 31PN M R chemical shift model compounds, and the 20.25-ppm value measured for 2',3'-cAMP was consistent with assignment of a D-ribosyl2,3-cyclophosphateterminus to the 20.83-ppm signal exhibited by the polysaccharide. The 31P chemical shift of 3',5'-cAMP was -0.60 ppm, ruling out the (9) Egan, W. "Magnetic Resonance in Biology"; Cohen, J. S., Ed.; Wiley: New York, 1980; Vol. 1, Chapter 5 and references therein. (10) Larm, 0.;Lindberg, B. Adu. Carbohydr. Chem. Biochem. 1976,33, 295.
-c-0
~
O-
-C-OH
'
I
i
- C -I O , y y P h
I
-c-0
/p\
0-
I
presence of D-ribosyl 3,5-cyclophosphate end groups in the type b polysaccharide. Intramolecular cyclization of the phosphate monoester group in D-ribitol 5-phosphate by reaction with a carbodiimide was investigated as a convenient synthetic route to D-ribitol 4 5 cyclophosphate, which would then serve as a chemical shift model compound for the ribitol segment of the polysaccharide. An aqueous solution of D-ribitol 5-phosphate was reacted at pH 3-5 with a 2-fold molar excess of l-ethyl-3-(3-(dimethylamino)propy1)carbodiimide hydrochloride (EDAC). 31PN M R monitoring of the mixture showed a gradual disappearance of the starting material signal at 3.12 ppm with formation of a product signal at 19.06 ppm; the reaction was complete after 2 h at 25 "C, and there was no evidence for intermolecular pyrophosphate formation (no signals at ca. -10 ppm). 13CN M R data for &ribitol 5-phosphate and the isolated product were consistent with identification of the latter material as D-ribitol 4,5-~yclophosphate.~' The close chemical shift correspondence for this model compound (19.06 ppm) and the polysaccharide (18.85 ppm) was preliminary evidence for D-ribitol 4,5-cyclophosphate termini in the bacterial polymer. (1 1) The ')C NMR spectrum for D-ribitol 5-phosphate was as follows (tentative resonance assignments in parentheses): 64.86 ppm (C,); 68.71 ppm, Jcp = 4.9 Hz (C4: 73.51 ppm, Jcp = 7.3 Hz (C4);74.61 and 74.19 ppm (C, and C2); for D-ribitol 4,5-cyclophosphate: 65.1 1 ppm (C,); 74.83 ppm (Cz); 73.95 ppm, Jcp = 7.3 Hz (C3); 78.31 ppm (C4): 68.61 ppm (CJ. With D-ribitol 4,5-cyclophosphate, the absence of observable spin couplings of phosphorus to C4 and C5 and the presence of a relatively large spin coupling of phosphorus to C3are similar to the coupling patterns found in 2',3'-cyclic nucleotides wherein small (40. (4) Chain lengths, which were calculated with the assumption of one phosphorus-containing end group per chain, do not correlate with polysaccharide partition coefficients ( K d )measured by size-exclusion chromatography (cf. Table 11). While this observation can be attributed to aggregation of polysaccharide chains to form “networks” having roughly the same overall size characteristics, it should be noted that size-exclusion chromatography of a sample of type b capsular polysaccharide afforded three fractions that were analyzed by 31PN M R and found to have chain lengths that were proportional to weighted Kd values (Figure 3). (13) Marsh, F. J.; Weiner, P.; Douglas, J. E.; Kollman, P. A.; Kenyon, G. L.; Gerlt, J. A. J . Am. Chem. SOC.1980, 102, 1660. (14) Haake, P. C.; Westheimer, F. H. J . Am. Chem. SOC.1961,83, 1102.
J . Am. Chem. SOC., Vol. 104, No. 10, 1982 2901
Bacterial Capsular Polysaccharides 1 oa
Scheme I1
0 I
PhO--P=O
I
0-
OH
0
1 I
0
0-
PhO-P=O
\
/ O
75 -0
0-
The corollary to assuming one phosphorus-containing end group
per chain of type b polysaccharide is that the remaining end groups are D-riboSyl or D-ribitol moieties or both. A CJinked terminal Pribosyl group (cf. Figure 1) allows for tautomerization between its cyclic hemiacetal and acylic aldehydic forms, the latter of which may be trapped and quantified by oxime formation. D-Ribose and type b polysaccharide (strain Eagen) were separately reacted with an excess of 0-methylhydroxylamine hydrochloride (CH3ONH2.HC1). After 24 h at pH 7 f 0.5, the recovered (88%) polymer showed no detectable ‘H N M R doublet for an imine proton of either the major or minor isomer of the D-ribosyl 0methyloxime model compound: E (78%), 7.57 ppm, 3JHH = 6.4 Hz; Z (22%), 6.90 ppm, 3 J =~6.0 ~Hz. This novel method for
90
P
50
25
A 4.96
0
10
20 TIME (hi
E ; X = lone pair, Y = OCH3
I:X = OCH3.
Y = lone pair
end-group analysis, which indicated < 10 aldehyde-forming Dribose termini per 100 phosphorus-containing end groups, was further evaluated with type b polysaccharide that had been partially depolymerized in 1 M HOAc at 37 “C. The recovered material showed a low intensity doublet ( J = 6.4 Hz,7.61 ppm) for the (E)-oxime derivative of C3-linked terminal D-ribosyl residues; 31PN M R analysis indicated 5% hydrolysis of phosphodiester repeating-unit linkages (vide infra). Attempts to carry out periodate o x i d a t i ~ n of ’ ~ the type b capsular polysaccharide for quantitative colorimetric15 determination of formaldehyde (from D-ribitol termini) were foiled by excessive interference due to color-forming side reactions. The biosynthesis of this capsular material may involve a chain-elongation step which proceeds by D-ribosyl C3 hydroxyl displacement of lipid monophosphate from a lipid pyrophosphate carrier of the repeating unit;I6however, other mechanisms akin to those proposed16 for teichoic acids in Grampositive bacteria are also possible. Hydrolysis. A solution of the type b polysaccharide in 0.1 M glycine-NaOH buffer (pH 10) containing 0.1 M CaC12 was monitored at 50 O C by 31PN M R spectroscopy, and kinetic profiles for the various phosphorus species were obtained from changes in their relative signal intensities as a function of time. The individual profiles during 85% hydrolysis of the repeating unit phosphodiester linkage are presented in Figure 4. It can be seen that as the phosphodiester repeating unit linkage (1.35 ppm) undergoes cleavage, there is a steady production of three phosphate monoester end groups (5.49, 4.96, and 4.66 ppm). The D-ribosyl 2,3-cyclophosphate end group (20.96 ppm) reaches a maximum concentration after ca. 4 h and then diminishes in intensity, while the D-ribitol 4,5-cyclophosphate end-group (18.98 ppm) concentration attains an apparent plateau. Figure 4 also shows a pseudo-first-order kinetic plot for disappearance of the repeating unit phosphodiester linkages. Deviation from linearity is apparent after ca. 5 h, which corresponds to approximately 60% hydrolysis. The initial linear portion of this plot affords a value of r1,2= 4.3 h (k’ = 4.47 X s-I) for phosphodiester cleavage. (15) Speck, J. C., Jr. “Methods in Carbohydrate Chemistry”; Whistler, R. L., Wolfrom, M. L., Eds.; Academic Press: New York, 1962; Vol. 1, pp 44 1-445. (16) Baddiley, J. Acc. Chem. Res. 1970, 3, 98.
2.0
r
In‘
OY
I
5
10
15
20
25
TIME ih)
Figure 4. (Top) Plot of relative concentration (% P, relative NMR signal intensity) vs. time for phosphorus-containing moieties in H.influenzae type b capsular polysaccharide (strain 1482,4 mg/l.5 mL) at 50 OC during depolymerization at pH 10 in 0.1 M glycine-NaOH buffer containing 0.1 M CaCI,; repeating unit phosphodiester linkages (squares) at 1.35 ppm, phosphate monoester end groups (circles) at 4.66, 4.96, and 5.49 ppm, ribosyl 2,3-cyclophosphate end groups (triangles) at 20.96 ppm, and D-ribitol 4,5-cyclophosphate end groups (triangles) at 18.98 ppm. (Bottom) Pseudo-first-order kinetic plot for the disappearance of repeating unit phosphodiester linkages; concentration values C, and C correspond to % P initial and at time 1, respectively.
The kinetics for hydrolysis of the phenyl ester of cis-4hydroxytetrahydrofuran 3-phosphate have been studied” in detail as models for the elementary steps of ribonuclease action, and it has been suggested” that the mechanism involves an “in-line” SN2-typedisplacement of phenoxide ion by the neighboring alkoxide to form the cyclophosphate shown in Scheme 11. For comparison of the relative reactivity of this compound and the type b polysaccharide, the reported” pseudo-first-order hydrolytic s-l) can be corrected rate constant at pH 9.7/50 “C (1.58 X for replacement of PhO with HOCH2CH20.’* This gives a value ~~~~~
(17) Usher, D. A.; Richardson, D. I., Jr.; Oakenfull, D. G . J . Am. Chem. SOC.1970, 92, 4699. (18) Hydrolysis of esters of 2-hydroxypropyl phosphate occurs by neighboring group participation to give 1 -methylglycerol cyclophosphate (Brown, D. M.; Usher, D. A. J. Chem. SOC.1965, 6547). For hydrolysis of the PhO and HOCH2CH2esters in 1 N NaOH at 80 ‘C,k’= 7.40 X lod and 2.35 X lo-* s-’, respectively (Brown, D. M.; Usher, D. A. J . Chem. SOC.1965, 6558).
2902 J. Am. Chem. SOC.,Vol. 104, No. 10, 1982
Egan et al.
Scheme I11 [HO
AcO
Figure 5. Repeating unit structure of H . influenzae type f capsular 1+ polysaccharide shown in its protonated form: --3)-@-~-GalNAc-( 4)-a-~-GalNAc-l-(POp.
(5.02 X lod s-I) only 9 times less than that measured for the type b polysaccharide under comparable reaction conditions. The close correspondence in hydrolytic reactivity and the appearance of cyclophosphate end groups during depolymerization of the polysaccharide are persuasive evidence for analogous mechanisms of phosphodiester bond cleavage. Scheme I11 is a representation of neighboring group participation by D-riboSyl C2-alkoxide at an interchain cross-link in the CaZ+form of type b polysaccharide. The dual role envisioned for CaZ+as a stereochemical template and Lewis acid catalyst is analogous to various M2+-dependent enzymatic hydrolyses of phosphoric acid esters.I9 Since divalent metal ion catalyzed hydrolysis of carboxylic esters having relatively poor leaving groups, such as alkoxides, may involve weak metal ion binding to the reactant and strong binding in the transition state,20which facilitates departure of the nucleofuge, the essential features of Scheme I11 could equally apply to the activated reaction complex. N M R spectroscopy was thus used to probe the extent of binding between the metal ion and polysaccharide in the reactant stage. The chemical shifts of corresponding resonances in the 13C N M R spectra of the CaZ+and Na+ salts of the type b polysaccharide were, to within 0.1 ppm, the same, as were the 31P chemical shifts of the cyclophosphate end groups and the phosphodiester repeating unit resonances. These spectroscopic observations indicate that there is no substantial difference in the mode of cation association with the polymer. By inference, the association is not an intimate one; Le., it does not appear that there is a substantial fraction of condensed Na+ or Ca2+ ions, as is often found with other polyelectrolytes.21 The line width at half-height, W,,,, for the 23Na resonance of the sodium salt of the type b polysaccharide was ca. 8 Hz, which is the same value found for an equimolar NaCl solution; if a significant fraction of Na+ was intimately associated with the polysaccharide, the z3Naresonance would be broad, relative to the 23Nasignal of a pure NaCl solution (as found, for example, with solutions of the sodium salts of DNA or poly(methy1 methacrylate)21). Finally, we note that W,,, for corresponding 13C resonances of the Na+ and Ca2+salts of the type b polysaccharide were, to within experimental error, the same and, moreover, were equivalent to those found for the ( ~ - B u ) ~ N + salt. If Ca2+ions were to cross-link and partially immobilize the type b polymer, we would not expect W,,, for the 13Cresonances to be unaltered. Nonetheless, replacement of Ca2+with Na+ led to a ca. 25-fold diminution in the rate of phosphodiester cleavage ( T ~= / 98.3 ~ h a t pH 10/50 "C), and when a stoichiometric amount of Ca2+ was added to a solution of type b polysaccharide in its (19) Gray, C. J. 'Enzyme-Catalyzed Reactions"; Van Nostrand Reinhold London, 1971; Chapter 5. (20) Fife, T. H.; Przystas, T. J. J . Am. Chem. SOC.1980, 102, 7297. (21) Manning, G. S. Acc. Chem. Res. 1979, 12, 443 and references cited therein.
I
I
I
1
I
I
I
25
20
15
10
5
0
-5
PDm
Figure 6. 'H-decoupled 40.25-MHz 31PN M R spectrum of H . influenzae type f (strain 644) capsular polysaccharide (25 mg/1.5 mL); r / 4 pulse, 3-s repetition time, and 22 128 accumulations. The upper trace is an expanded and offset display of the repeating unit phosphcdiester linkages centered at -1.18 ppm. The middle trace was recorded at 50 times the amplitude of the lower trace; P refers to the pulse position and X refers to a spinning sideband.
Na+ form, the hydrolytic reactivity was restored to ca. 75% of the value initially found for the Ca2+ form. Similar kinetic measurements with added Mg2+ indicated that this divalent cation was approximately one-half as effective as Ca2+in restoring hydrolytic reactivity to the Na+ form, while substitution of (CH3)4N+ for Ca2+in the type b capsular polysaccharide afforded essentially the same kinetic results as did substitution with Na+. The contrast between hydrolytic behavior and spectroscopic characteristics of the polysaccharide is consistent with transition-state stabilization by divalent metal ions during alkaline hydrolysis of the phosphodiester linkages; however, further experiments are needed to test this hypothesis. Type f. End-Group Studies. Previous N M R investigations of H . influenzae type f capsular polysaccharide found a single 31P signal for the phosphodiester linkage in a homopolymer having the repeating unit structure pictured in Figure 5.9 In the present work with purified type f material (strain 644), the 3'P N M R spectrum (Figure 6 ) showed three overlapped phosphodiester signals at -1.05, -1.18, and -1.44 ppm in a relative ratio of ca. 10:80:10, respectively, suggesting structural heterogeneity. The 'H N M R spectral region for the acyl methyl groups (Figure 7) featured two N-Ac signals at 2.18 and 2.1 1 ppm, and an 0-Ac signal at 2.02 ppm, in accord with earlier data reported9 for the GalNAc and 3-(0Ac)GalNAc components in the type f polysaccharide.22 The additional low-intensity singlets seen (Figure
J . Am. Chem. Soc., Vol. 104, No. IO, 1982 2903
Bacterial Capsular Polysaccharides Table 111.
NMR-Deriveda Structural Data for Samples of Capsular Polysaccharide from H. influenzae Type f end group composition,b % sample
644, run 1, fraction Ad 644, run 1, fraction Bf 644, run 2, fraction AS 686 BO95
C,,C,-cyclophosphate
C,-phosphate monoester
C,-phosphate monoester
25 1.10f 98.90 90 c (Ruggerio) 0.25f 99.75 399 c (Ruggerio)g a See Experimental Section for spectral acquisition parameters. Relative molar percentages calculated from integrated signal intensities relative to the total value for all detectable end groups and the repeating unit phosphodiester linkage, except as noted. See text and ref 1 2 . Attached to either D-G~c C, or D-ribitol C, or both of these positions; see text. e Line broadening, possibly due to nucleic acid contamination, led to a relatively high estimated upper limit; no signal was detected. Attached to the C, position in partially 0-acetylated D-GIcNAc;see text. 8 Different preparation from the previous entry.
I
1
I
I
I
I
I
25
20
15
10
5
0
-5
DPm
r
e I
OH '
HO-C-H
1
l
Figure 11. 'H-decoupled 40.25-MHz ,'P NMR spectrum of H . influenzae type a (strain Fin-35) capsular polysaccharide (25 mg/1.5 mL); 7r/4 pulse, 2-5 repetition time, and 22 440 accumulations. The upper trace was recorded at 60 times the amplitude of the lower trace; P refers to the pulse position and X refers to a spinning sideband.
I
HO-C-4
I
CH20H
Figure 10. Repeating unit structure of H. influenzae type a capsular polysaccharide shown in its protonated form: +4)-@-D-Gk-(1-4)-0ribitol-S-(PO,-+.
0.1 M CaCl,) were heated at 100 OC for various times. 3*PN M R analysis of the hydrolysates showed that initial depolymerization (90% yield) was achieved by dialysis against NaCl (1 L, 0.2 M, 2 times) and then water (1 L, 2 times) followed by ion exchange with Dowex resin in the Na' form as described above. Reactions with 0-Methylhydroxylamine. D-Ribose. A solution of CH30NH2.HCI (42 mg, 0.50 mmol) in D 2 0 (3 mL) was adjusted to pH 7 with 1 N NaOD in D 2 0 and was then stirred with D-ribose (60 mg, 0.40 mmol) for 2 h at 25 OC, keeping the pH at 7 & 0.5. The reaction mixture was saturated with NaCl and then extracted with EtOAc (3 X 3 mL). Removal of the solvent under reduced pressure gave the expected)' E and Z isomers of D-ribose 0-methyloxime, which were identified by IH N M R spectroscopy (360 MHz); see text for pertinent data. H . htluenzse Type b. A sample of the capsular polysaccharide (strain Eagen, 25 mg, 0.068 mmol) in water (1.5 mL) was reacted with CH,ONH2.HCI (5.1 mg,0.061 mmol) according to the above procedure. The reaction mixture was dialyzed against CaCI, (5 L, 0.2 M) and then water ( 5 L) before lyophilization to afford a sample (88% yield) which had no detectable HC=NOCH3 signals (6-8 ppm) in its 'H N M R (360 MHz) spectrum. Repetition of this experiment with exclusion of the dialysis steps gave a similar result. An identical sample of the capsular polysaccharide starting material was heated at 37 OC in 1 M HOAc for 24 h and then neutralized with 1 N NaOH. )lP N M R signal intensities for the monophosphate end groups (ca. 4 ppm) and remaining phosphodiester linkages (ca. 1 ppm) had a relative ratio of 5:95. The hydrolysate was dialyzed against water (5 L, 3 times), and the retained material was reacted with CH30NH2.HCI (5.5 mg, 0.066 mmol) at p H 7 f 0.5 for 15 h at 25 OC. The reaction mixture was dialyzed against CaCI, (5 L, 0.2 M) and then water (5 L) before lyophilization to give the product (25 = 6.4 Hz. mg, 100% recovery); 'H N M R (360 MHz): 7.61 ppm, d, The extent of D-ribosyl 0-methyloxime formation was not quantified. Reactions with PhOH/NaOAc. A solution of the type b capsular polysaccharide (strain 1482, 28 mg) in water (5 mL) was vigorously shaken for 4 h at 37 "C with an equal volume of PhOH/NaOAc solution (prepared from 454 g of PhOH in 9:l water-saturated aqueous NaOAc, pH 7). After centrifugation, the upper layer was dialyzed against CaCI2 (5 L, 0.1 M, 2 times) and then water (5 L, 2 times). Lyophilization gave recovered material (88%) that had a )IP N M R spectrum identical with that of the starting material (Table 11). Repetition of this experiment using D-ribitol 5-phosphate (15 mg) and excluding the dialysis steps afforded (100%) unchanged starting material ()lP NMR). 0-Deacetylation of Capsular Polysaccharide Types f and c. The following details are representative. A solution of the type f polysaccharide (strain 644, 25 mg) in D 2 0 (2 mL) was adjusted to pH 11 with 30% ND4ODD2O. A 'H N M R (220 MHz) spectrum was recorded after 15 h at 5 "C (see Figure 7), and the pH was increased from 9.5 to 11. After
J. Am. Chem. SOC..Vol. 104, No. IO, 1982 2909 an additional 24 h at 25 O C , the IH N M R spectrum was again recorded (see Figure 7). I I P N M R (40.25 MHz) at pH 10: repeating unit phosphodiester, 0 ppm (relative chemical shift), 62.5% (relative intensity); phosphate monoester, 5.06 ppm, 11.3%, and 6.96 ppm, 5.1%; cyclophosphate, 17.92 ppm, 21.3%. Alkaline Hydrolysis Kinetics. The 0.1 M glycine-NaOH buffer" (pH 10) was stored at 5 "C and was freshly prepared after 30-day periods. Samples contained 4 mg of capsular polysaccharide dissolved in 1.4 mL of buffer and 0.1 mL of aqueous 1.5 M CaCI2. Kinetic runs with the type b and type f materials were carried in tightly capped N M R tubes (10 mm), using the JEOL FX-100 variable temperature controller and the selective 'H-decoupling mode. Aliquots (1.5 mL) of the type a and type c capsular polysaccharide solutions were sealed in standard glass ampules and submerged in a controlled-temperature water bath. After samples (six) were heated for a given kinetic run, they were frozen and stored for batchwise )lP N M R analysis; see text for results. A 7r/2 pulse, 2-s recovery time, and nuclear Overhauser enhancement (NOE) were used in all cases, which gave accurate pseudo-first-order kinetic data (Table IV) but only approximate product ratios, due to NOE and T , differences. Values of k'were derived from linear least-squares fits (
