Eur. J. Biochem. 167, 549-561 (1987) 0 FEBS 1987

Somatic antigens of Pseudomonas aeruginosa

Ins'ri,~; de F '--

%!?T

.a,;~/,-

TILiVl+N

gra 1

If

The structure of 0-specific polysaccharide chains of lipopolysaccharides of P. aerugtnosa 0 2 5 (Wokatsch) and Fisherimmunotypes 3 and 7

6

63 (Lanyi), -z

Yuriy A. KNIREL', Nikolay A. PARAMONOV', Evgeny V. VINOGRADOV', Alexander S. SHASHKOV', Boris A. DMITRIEV', Nikolay K. KOCHETKOV', Elena V. KHOLODKOVA' and Evgeny S. STANISLAVSKY'

'

N. D. Zelinsky Institute of Organic Chemistry, Academy of Sciences of the USSR, Moscow I. I. Mechnikov Institute of Vaccines and Sera, Health Ministry of the USSR, Moscow

(Received November 6, 1986/March 4,1987) - EJB 86 1174

0-specific polysaccharides, obtained on mild acid degradation of lipopolysacchrides of the serologically related strains Pseudomonas aeruginosa 0 3 (Lanyi classification), 0 2 5 (Wokatsch classification) and immunotypes 3 and 7 (Fisher classification), are built up of trisaccharide repeating units involving 2-acetamido-2,6-dideoxy-~-galactose (N-acetyl-D-fucosamine), 2,3-diacetamido-2,3-dideoxy-~-mannuronic acid or 2,3-diacetamido-2,3-dideoxy-~guluronic acid and 3-acetamidino-2-acetam~do-2,3-dideoxy-~-mannuron~c acid or 3-acetamidino-2-acetamido2,3-dideoxy-~-guluronicacid. Lanyi 03(a),3d,3f and Wokatsch 0 2 5 polysaccharides contain also 0-acetyl groups. On the basis of solvolysis with anhydrous hydrogen fluoride, resulting in trisaccharide fragments with N-acetylfucosamine residue at the reducing terminus, chemical modifications of the acetamidino group (alkaline hydrolysis to the acetamido group or reductive deamination to the ethylamino group), as well as analysis by HNMR (including nuclear Overhauser effect experiments) and 3C-NMR spectroscopy, and fast-atom bombardment mass spectrometry, it was concluded that the repeating units of the polysaccharides have the following structures : +4)-j-~-ManNAcAmA-(1 +4)-/3-~-Man(NAc),A-(l+3-P-~-FucNAc-(1 t4 OR R = H Lanyi 03a,3b R = Ac Wokatsch 0 2 5 --f

+4)-j-~-ManNAcAmA-(1 +4)-p-~-Man(NAc)~A-( 1 -+3)-a-~-FucNAc-(l+ Lanyi 03a,3d +4)-p-~-ManNAcAmA-(1 +4)-a-~-Gul(NAc)~A-( 1 3)-p-~-FucNAc-( 1+ Lanyi 03(a),3c = Fisher immunotype 3 --f

+4)-p-~-ManNAcArnA-(1 +4)-a-~-Gul(NAc)~A-( 1+3)-a-~-FucNAc-( 1+ Lanyi 03a,3d,3e +4)-a-~-GulNAcAmA-(1 +4)-P-~-Man(NAc)~A-(1+3)-a-~-FucNAc-( 1+ Fisher immunotype 7 +4)-HexNAcAmA-( 1+4)-~-~-Man(NAc)~A-(1+3)-a-~-FucNAc-(l+ 14 OAc Lanyi 03(a),3d,3f where HexNAcAmA = a-L-GulNAcAmA ( z 70%) or P-D-ManNAcAmA (z30%). Linyi 03(a),3d,3f polysaccharide involves two types of repeating units, which differ from each othcr o n l y in the configuration at C-5 of the 3-acetamidino-2-acetamido-2,3-dideoxyuronic acid residue. Llinyi 03(a),3c, 03a,3d,3e and Fisher immunotypes 3 and 7 polysaccharides contain, together with the major repeating units shown above, a small proportion of units in which the derivative of a-L-guluronic acid is replaced by the corresponding j-D-manno isomer. The data obtained provide the opportunity to substantiate the serological interrelations between these strains of P. aeruginosa by the presence in the 0-specific polysaccharides of common monosaccharides or disaccharide fragments. The distinctions between them stem from the presence or absence of the 0-acetyl group, a different configuration of the glycosidic linkage of the N-acetylfucosamine residue and/or a different configuration at C-5 of one or both derivatives of diaminouronic acids. Correspondence to Yu. A. Knirel, Institut Organicheskoj Khimii imeni N. D. Zelinskogo, Akademiya Nauk S.S.S.R., Leninskij prospekt 47, Moskva, USSR 117913 Abbreviations. D-FucNAc, 2-acetamido-2,6-dideoxy-~-galactose (N-acetyl-D-fucosamine); D-Man(NAc)'A and L-GUI(NAC)~A, 2,3-

diacetamido-2,3-dideoxy-~-mannuronic acid and the corresponding derivative of L-guluronic acid; D-ManNAcAmA and L-GulNAcAmA, 3-acetamidino-2-acetam~do-2,3-dideoxy-~-mannuron~c acid and the corresponding derivative of L-guluronic acid.

550 An opportunistic pathogen Pseudomonas aeruginosa is ( 5 :5 : 1 :3, by vol.) using ninhydrin as the detection reagent. serologically heterogeneous and, according to different Amino sugars were detected by using a BC-200 amino acid classification schemes, subdivides into several (up to 13) 0 analyzer as described previously [12]. Gel filtration was serogroups, some of which include several 0 serotypes [l]. performed on columns of Sephadex G-50 (70 x 3.5 cm) in The most complicated of them is the 0 3 serogroup in the pyridinelacetate buffer (4 ml pyridine and 10 ml acetic acid in Lanyi classification [2] (or the respective 0 2 serogroup in the 1 1 water) or TSK HW 40 (80 x 1.5 cm) in water; elution Lanyi-Bergan classification [lI) which involves five 0 profiles were recorded by using a Technicon sugar analyzer serotypes. According to the data [3] this serogroup should be or a Knauer differential refractometer, respectively. Highenlarged to include the Wokatsch 0 2 5 serotype [4], which is performance ion-exchange chromatography was carried out serologically related to the Lanyi 0 3 serogroup but identical on an Altex instrument with a Knauer variable-wavelength to none of the individual serotypes. P. aeruginosa monitor, using a column of DEAE-3SW (LKB) and a 0.02 M immunotypes 3 and 7 (Fisher classification [5]) also relate by sodium dihydrogen phosphate buffer with linear sodium chlotheir serological properties to exactly the same serogroup [l], ride gradient (0 - 0.5 M) as the mobile phase. however their exact correlation with the known serotypes has not been established conclusively. Isolation of lipopolysaccharides and polysaccharides Chemical and immunochemical studies of 0 antigens of Bacterial cultures of P. aeruginosa 03(a),3d,3f (Lanyi gram-negative bacteria enable one to relate their immunospecificity with the structure of polysaccharide chains of classification) and Fisher immunotype 7, museum strains lipopolysaccharides and to substantiate serological differ- 170007 and 170047, respectively, were kindly provided by Dr entiation of strains at the molecular level. In the course of Lanyi from the Hungarian National Collection of Medical studying P. aeruginosa 0 antigens we have established the Bacteria. P. aeruginosa 0 2 5 (Wokatsch classification) was structures of Lanyi 03a,3b, 0(3a),3c, 03a,3d, and 03a,3d,3e supplied by the L. A. Tarasevich Institute for Standardization 0-specific polysaccharides [6, 71. They were shown to con- and Control of Biological Medical Preparations (Moscow). tain di-N-acetyl derivatives of 2,3-diamino-2,3-dideoxy-~-Cultures were grown as described previously [13]. Acetonemannuronic acid and the corresponding derivative of dried cells (30 g each strain) were extracted with 45% aqueous L-guluronic acid, together with the acetamidino derivative phenol [14], dialyzed against water without separation of of the former, to which the structure involving a l-acetyl-2- aqueous and phenol layers, after separation of cells by cenmethyl-2-imidazoline ring was assigned [6]. Later the structure trifugation and precipitation of nucleic acids with Cetavlon of this derivative was revised in favor of an acyclic structure the lipopolysaccharides were recovered by the addition of of an acetamidino group (preliminary communication [S]), ethanol [14] followed by dialysis against distilled water. The whereas Fisher immunotype 7 0-specific polysaccharide was yield of lipopolysaccharides was 4 - 8% of the cell dry weight. Lipopolysaccharides (800 mg each) were heated with 1?Ao found to contain the a-gulo isomer of this sugar (preliminary communication [9]). A part of this work is devoted to the acetic acid (100 ml, 100°C, 2-4 h) until a well-developed detailed description of the identification of these two unusual precipitate was formed, which was then removed by centrifusugars. In addition this report gives the data on the structural gation. Gel filtration of the supernatant on Sephadex G-50 analysis of Lanyi 03(a),3d,3f 0-specific polysaccharide, afforded 0-specific polysaccharides (180 -200 mg) and which has been hitherto characterized as irregular [7], as well oligosaccharide fractions (200 - 250 mg); the latter were not as Wokatsch 0 2 5 and Fisher immunotype 7 polysaccharides. studied further. ~~

MATERIALS AND METHODS

Acid hydrolysis

Miscellaneous methods

Lanyi 03(a),3d,3f and Fisher immunotype 7 polysaccharides (40 mg each) were hydrolysed with 4 M hydrochloric acid (3 ml, 100°C, 4 h), the hydrolysates were evaporated, the residues repeatedly evaporated with water, D-fucosamine was isolated by preparative paper chromatography and evaporated with 0.01 M hydrochloric acid and then with water to convert it into the hydrochloride (3 - 3.5 mg), [aID 78" or +88", respectively, ( c = 0.3), cf. [15] [aID +93" (water).

'H nuclear magnetic resonance (NMR) spectra were run on an AM-300 (Bruker) instrument in D 2 0 at 30°C using acetone, 6, 2.23 ppm, as an internal standard. Nuclear Overhauser effect (NOE) data were obtained by the truncated driven nuclear Overhauser effect method [lo] and performed in the difference mode; the time constants used were D1 = 4 s, relaxation delay; D2 = 0.5 s, build-up NOE. 13C-NMR spectra were recorded with the same instrument in D 2 0 at 60 "C for polysaccharides or 25 "C for oligosaccharides using methanol, & 50.15 ppm, as an internal standard. Fast-atom bombardment mass spectra were obtained on a VG 70170 HS instrument with an ION TECH argon fast-atom source; beam intensity 1 mA, energy 8 kV, target temperature about 30°C; glycerol (Serva) was employed as the matrix. Optical rotations were measured with a Perkin-Elmer polarimeter, model 141, in water at 20°C. Protein content was measured by the method described in [113. Solutions were freeze-dried or evaporated in vucuo at 40°C.

+

Treatment with aqueous triethylamine Polysaccharides (70- 100 mg each) were dissolved in 5% aqueous triethylamine, heated for 3.5-4 h at 70°C, evaporated, and the corresponding modified polysaccharides were isolated in yields of 80-90% by gel filtration on TSK HW 40. Oligosaccharides 3, 9 and a mixture of 7 and 13 were treated with aqueous triethylamine in a similar way.

Solvolysis with hydrogen fluoride Chromatography Ascending paper chromatography was performed on FN-11 paper in the system pyridine/butanol/acetic acidlwater

Polysaccharides (100 - 120 mg) were dried in vucuo over phosphorus pentoxide (7OoC, 3 h), then treated in a Teflon vessel with anhydrous hydrogen fluoride (10 - 15 ml, 20"C,

551 3 - 3.5 h) freshly distilled over cobalt trifluoride [16]; hydrogen fluoride was removed in vacuo by absorption with solid sodium hydroxide; the product was dissolved in water, evaporated and the respective trisaccharides were isolated in yields of 60 - 70% by gel filtration on TSK HW 40. Reduction with complex borohydrides

a) Trisaccharide 1 (60 mg) was reduced with sodium borohydride (50mg) in water (2m1, 20"C, 16 h), acidified with 2 M hydrochloric acid, and oligosaccharide 3 (50 mg) was isolated by gel filtration on TSK HW 40. Oligosaccharide 6 and a mixture of oligosaccharides 6 and 12 were reduced in a similar way. b) Trisaccharide 6 (40 mg) was reduced at 20 "C by adding a solution of sodium borohydride (100 mg) in saturated aqueous boric acid (2 ml) in two portions with an 8-h interval, then treated as described above to give oligosaccharide 9 (32 mg). c) Oligosaccharide 3 (40 mg) in 60% aqueous 2-propanol (1.25 ml) was treated with lithium borohydride (30 mg, 60 "C, 2 h), acidified with 2 M hydrochloric acid and deinonized by gel filtration on TSK HW 40. Oligosaccharide 5 (lOmg), contaminated, according to the 'H-NMR and 13C-NMR data, by an unidentified by-product (w30%), as well as oligosaccharide 4 (3 mg) was isolated by high-performance ion-exchange chromatography on DEAE-3SW followed by deionization on TSK HW 40. RESULTS Isolation of lipopolysaccharides and 0-spec@ polysaccharides

Dry bacterial cells of P . aeruginosa 03(a),3d,3f (Lanyi), 0 2 5 (Wokatsch) and immunotypes 3 and 7 (Fisher) were extracted by Westphal's method [14] with 45% aqueous phenol with the only modification that the combined phenol/ aqueous layer was dialyzed without preliminary separation; the insoluble precipitate was separated after dialysis by centrifugation, nucleic acids were precipitated with Cetavlon [14] and lipopolysaccharides were finally recovered by the addition of ethanol. The preparations obtained in this way contained somewhat greater amounts of proteins (up to go/) in comparison with lipopolysaccharides isolated from the aqueous layer (up to 2%). Nevertheless, since proteins can be removed together with the lipid precipitate in the course of mild acid degradation of lipopolysaccharides, their elevated content does not cause trouble in the structural analysis of 0-specific polysaccharide chains. In fact the protein content in polysaccharides, obtained by the cleavage of lipopolysaccharides with 1% acetic acid followed by gel filtration on Sephadex G-50 of the soluble fraction, did not exceed 1%. Acid hydrolysis of polysaccharides

Lanyi 03(a),3d,3f and Fisher immunotype 7 polysaccharides were hydrolyzed, and fucosamine was identified in the hydrolysate by using an amino acid analyzer. This sugar was isolated by preparative paper chromatography and the optical rotation value showed it to have the D configuration. No other components of the polysaccharides were detected in the hydrolysate. This phenomenon has already been observed in the case of Lanyi 0 3 polysaccharides containing, together with fucosamine, the derivatives of diaminouronic acids, and was accounted for by the stability of glycosidic linkages of

these monosaccharides towards cleavage with acids and the lability of the sugars in drastic acidic conditions [6, 71. Identification of 3-acetamidino-2-acetamido-2,3-dideoxy-~ mannuronic acid; structure of trisaccharide 1 (Scheme I)

Fisher immunotype 3 0-specific polysaccharide was solvolysed with anhydrous hydrogen fluoride to give oligosaccharide I . The 13C-NMR data showed it to be identical to the trisaccharide obtained in the similar solvolysis of Lanyi 03(a),3c and 03a,3d,3e polysaccharides. This trisaccharide contained two derivatives of 2,3-diamino-2,3-dideoxyuronic acids (unit A with the D-manno and unit B with the L-gulo configuration) and N-acetylfucosamine (unit C) [7]. Unit B represents the di-N-acetyl derivative, whereas unit A involves an acetamidino group (6, 19.9, CH3, 167.3, N = C - N). The trisaccharide was studied by fast-atom bombardment mass spectrometry. The mass spectrum contained the only intensive peak corresponding to the ion [M + HI+ with mjz 721 and indicating the molecular mass of this trisaccharide to be 720 Da. These data are inconsistent with the formerly proposed [7] structure 2 involving the cyclic acetamidino group, while being in conformity with the structure I containing this group in the acyclic form. Trisaccharide 6, obtained on solvolysis of Fisher immunotype 7 polysaccharide and containing an acetamidino derivative of L-guluronic acid (see below), has the identical mass spectrum. Reduction of trisaccharide I with sodium borohydride followed by treatment of the resulting oligosaccharide 3 with aqueous triethylamine, afforded oligosaccharide 4. The '3CNMR spectrum (Table 1) and the fast-atom bombardment mass spectrum, which contained an intensive peak of the ion [M HI+ with m / z 724, confirmed its structure, characterized by N-acetyl substituents at all five amino groups of the amino sugars. Treatment of oligosaccharide 3 with lithium borohydride in 60% aqueous 2-propanol led to oligosaccharide 5 as a result of the reductive deamination of the acetamidino group. The structure of 5 was established on the basis of the 'H-NMR and I3C-NMR spectra, in which, instead of the signals for the acetamidino group (6,2.28, CH3;dC20.4, CH3, 167.3, N = C-N), there appeared those for the ethylamino group (~5~1.28, t, Jl,z7 Hz, CH3, 3.14 and 3.40, both dq, J 1 , l , 12 Hz, CHZ; 6c 11.6, CH3,42.5, CHZ). The formation of oligosaccharides 4 and 5 confirmed the presence of the acetamidino group in oligosaccharide 3, and, hence, in trisaccharide 1. The location of the acetamidino group at position 3 of the derivative of D-mannuronic acid (unit A) followed from a considerable displacement of the signal for C-3 of this monosaccharide from 57.7 ppm to 54.6 ppm or 61.4 ppm upon alkaline hydrolysis (conversion of 3 into 4 ) or reductive deamination (conversion of 3 into 5), respectively, while the positions of the remaining signals in the 13C-NMR spectrum were practically unaffected. Thus, trisaccharide I and, as a consequence, Fisher immunotype 3 polysaccharide involve 3-acetamidino-2-acetamido2,3-dideoxy-~-mannuronicacid, and trisaccharide I has the structure shown in Scheme 1.

+

Structure of Lanyi 03a,3b, 0 3 ( a ) ,3c, 03a,3d, 03a,3d,3e, and Fisher immunotype 3 0-specific polysaccharides

As already pointed out, trisaccharide 1 is the chemical repeating unit of Lanyi 03(a),3c, 03a,3d,3e, and Fisher

552 COOH

OH

-0 HNAc immunotype 3 = = Lanyi 03(a).3c

HNAc

2

lHF

HN (0011

HN COOH

NaBH4

C HO HNH

AcNH 3

!

OHNAc COOHAcNH O

O

$

O

OH

A

B

COOH

CH3 A

B

FHzOH

HO

C

C LiBH,

COOH

3

c H HO3 NH ! &AcNH , 0 0 - 0 HNAc ~ { AcNH 1 r

HNAc

1

CHzOH

COOH

I

.$NHAc OH CH3

4

5

Scheme 1. Selective cleavage of Fisher immunotype 3 0-specific polysaccharide and chemical modifications of trisaccharide 1

Table 1. Chemical shifts in I3C-NMR spectra Chemical shifts for CH,CON 22.9-23.7, CH,COO 21.1-21.5, N = C(CH,)N 19.6-20.9, N = C(CH3)N 167, 0-170.2, CH,CO and COOH 173-176, CH3CH2 11.6 and 10.9, CH3CH2 42.5 and 45.1 ppm in oligosaccharides 5 and 7 respectively. PS, polysaccharide; PS*, polysaccharide treated with aqueous triethylamine Compound

c-1

c-2

c-3

c-4

c-5

Unit A PS Lanyi 03a,3b [6] PS Lanyi 03a,3d [6] PS LBnyi 03(a),3c = PS immunotype 3 [7] PS Lanyi 03a,3d,3e [7] PS Wokatsch 0 2 5 PS immunotype 7

100.3 99.8 100.3 100.0 100.3 99.9

50.9 51 .O 51.1 51.3 51.1 45.7 45.4

Oligosaccharide I Oligosaccharide 3 Oligosaccharide 4 Oligosaccharide 5 Oligosaccharide 6

99.7 100.9 100.7 99.8 100.0 100.4 100.2 100.0 101.o 100.4 100.0

50.9 51.7 52.2 45.1 45.1 52.5 51.2 51.1 52.3 52.6 45.8

Oligosaccharide 7 Oligosaccharide 9 Oligosaccharide 10 Oligosaccharide I I Oligosaccharide 12 Oligosaccharide 13 Oligosaccharide 14

100.0 100.1 100.2 100.0 99.5 99.6 100.6

47.3 45.7 45.3 45.3 50.7 50.7 52.1

75.2 71.4 75.1 71.7 75.6 70.7 72.3 70.8 72.2 71.6 75.3 71.7 73.3 72.9 71.9 67.5 67.9 67.2 67.2 67.4 69.3 65.5 67.4 69.2 69.3 67.1 67.2 67.3

77.5 78.7 77.4 78.3 77.6 68.4

99.7

55.7 57.6 55.7 57.8 55.8 50.0 51.1 49.8 50.9 57.4 53.0 54.1 47.5 47.2 54.4 57.7 57.7 54.4 61.4 54.6 55.6 58.0 54.6 51.6 51.4 57.4 57.5 54.6

PS Lanyi 03(a),3d,3f“ PS* Lanyi 03a,3b = PS* Wokatsch 0 2 5 PS* Lanyi 03a,3d [6] PS* immunotype 7 PS* Lanyi 03(a),3d,3fa

67.9 78.5 76.6 77.2 68.4 68.3 77.3 79.0 79.4 77.7 79.3 69.5 69.6 69.4 69.5 69.8 78.4 79.0 78.6

C-6

553 Table 1. (Continued) Compound

c-1

c-2

c-3

c-4

c-5

100.9 100.8 99.9 99.7 99.3 101.0

52.3 52.4 44.9 45.0 52.4 52.5

76.6 76.5 77.0 77.3 77.0 77.4

78.1 77.5 68.1 68.3 78.2 77.9

99.7

52.3

76.9

77.7

99.7 100.9 100.9 101.1 101.0 100.9 100.1 100.0 99.6 99.4 99.6 100.8

52.3 52.0 52.0 52.4 52.6 52.4 45.3

52.8 52.8 50.5 50.8 52.6 53.5 53.4 53.2 53.1 53.0 52.8 52.8 53.3 53.5 53.0 51.1

76.4 75.7 75.6 76.4 76.7 76.2 77.2

77.5 77.0 76.3 77.2 78.4 76.7 68.4

51.0 50.0 51.0

53.5

77.3 77.5 76.9 77.2

68.9 68.3 68.8 78.9

53.4 54.2 53.6 53.7 53.4 53.4 53.2 53.2

78.0 77.2 76.6 76.1 75.8 76.0 75.7

78.1 78.3 78.9 79.6 78.2 78.1 76.5

C-6

PPm Unit B PS Lanyi 03a,3b [6] PS Lanyi 03a,3d [6] PS Lanyi 03(a),3c = immunotype 3 [7] PS Lanyi 03a,3d,3e [7] PS Wokatsch 025 PS immunotype 7

PS Lanyi 03(a),3d,3fa PS* Lanyi 03a,3b = PS* Wokatsch 025 PS* Lanyi 03a,3d [6] PS* immunotype 7 PS* Lanyi 03(a),3d,3P Oligosaccharide 1 Oligosaccharide 3 Oligosaccharide4 Oligosaccharide 5 Oligosaccharide 6 Oligosaccharide 7 Oligosaccharide9 Oligosaccharide 10 Oligosaccharide I 1 Oligosaccharide 12 Oligosaccharide 13 Oligosaccharide 14 Unit C PS Lanyi 03a,3b [6] PS Lanyi 03a,3d [6] PS LBnyi 03(a),3c = immunotype 3 171 PS Lanyi 03a,3d,3e [7] PS Wokatsch 025 PS immunotype 7

PS Lanyi 03(a),3d,3P PS* Lanyi 03a,3b = PS* Wokatsch 025 PS* Lanyi 03a,3d [6] PS* immunotype 7 PS* Lanyi 03(a),3d,3P Oligosaccharide I Oligosaccharide3 Oligosaccharide 4 Oligosaccharide5 Oligosaccharide6 Oligosaccharide 7 Oligosaccharide 9 Oligosaccharide 10 Oligosaccharide I 1 Oligosaccharide 12 Oligosaccharide 13 Oligosaccharide 14 a

45.6 45.2 45.5 52.6

99.7 100.0 100.4 100.8 100.9 100.0 100.3

52.2 52.4 52.5 52.5 52.5 52.2 52.0

102.3 98.3 102.0 98.0 102.0 95.5 95.7 95.4 95.5 98.2 102.3 98.7 96.7 96.2 98.5 92.3 96.2 62.0 62.0 62.0 92.3 96.0 62.1 61.9 62.0 92.3 96.0 92.3 96.1 62.1 62.0

51.5 48.7 52.4 49.7 52.4 48.4 48.5 49.0

81.6 78.7 78.0 75.0 79.1 79.1

70.3 70.9 71.1 71.7 72.1 71.2

71.5 68.2 71.7 68.3 70.5 68.6

16.9 16.4 16.7 16.4 16.9 16.5

74.9

72.9

67.5

16.3

49.4 51.7 48.4 48.7 48.6 48.6 50.4 54.0 53.1 53.2 53.0 49.4 53.1 52.7 52.5 52.7 49.4 53.0 49.3 53.1 52.6 52.6

74.7 81.5 78.8 78.9 79.0 79.0 75.5 78.4 79.0 79.7 78.8 79.0 81.8 77.1 77.2 77.2 78.9 81.8 79.0 81.9 77.0 77.0

72.9 70.6 71.1 71.3 71.2 71.1 72.2 71.6 76.0 75.5 75.9 71.3 70.6 73.8 73.9 73.7 71.3 70.6 71.3 70.7 73.7 73.6

67.3 71.5 68.1 68.1 68.2 68.1 67.3 72.2 67.3 67.7 67.5 67.3 71.7 66.2 66.2 66.3 67.2 71.5 67.1 71.6 66.1 66.2

16.3 16.9 16.4 16.5 16.4 16.4 16.8 16.8 20.4 20.6 20.3 16.7 16.7 19.5 19.3 19.3 16.7 16.7 16.7 16.7 19.4 19.3

First listed are the chemical shifts for the repeating unit with the a-L-gulo configuration of unit A.

554 HN COOH FOOH FH3 immunotype 3 polysaccharides. In consequence of the revision of the structure of this trisaccharide, involving only the C NH H AcNHO o HNAc AcNH & G acetamidino derivative of D-mannUrOniC acid (unit A), the structures of the first two polysaccharides established pre-0 viously [7] should be accordingly altered (Table 4). The 13CNMR spectrum of Fisher immunotype 3 polysaccharide HNAC R = Ac Wokatsch 0 2 5 (Table 1) proved to be identical to that of Lanyi 03(a),3c R = H Lanyi 03a,3b polysaccharide and, hence, these polysaccharides have identical structure. According to the data [6], Lanyi 03a,3b and 03a,3d &o& Q ; COOH polysaccharides differ from Lanyi 03(a),3c and 03a,3d,3e polysaccharides, respectively, only in the configuration at C-5 of the diacetamido derivative of uronic acid (unit B); and, HNAc AcNH HNAc AcNH -0 hence, taking into account the alteration in the structures of two last polysaccharides, it is concluded that Lanyi 03a,3b HNAc and 03a,3d polysaccharides have the structures shown in A B C Table 4. Analysis of the 13C-NMR spectra of Lanyi 03(a),3c and Scheme 2. Modification of Wokatsch 0 2 5 0-specificpolysaccharide Fisher immunotype 3 polysaccharides showed that together with the major series of signals, corresponding to the structure given in Table4, the spectra also contain the minor series which amounts to approximately 10% the intensity of the group is likely to be substituted by the acetyl group in major series and comprises the spectrum of Lanyi 03a,3b Wokatsch 0 2 5 polysaccharide. This suggestion has been conpolysaccharide (Table 1). Hence, together with the repeating firmed by comparison of the 3C-NMR spectra of both intact units of the major structure, Lanyi 03(a),3c and Fisher polysaccharides (Table 1). The signal for C-4 of N-acetylimmunotype 3 polysaccharides contain a small proportion fucosamine in the spectrum of Wokatsch 025 polysaccharide of the units involving the diacetamido derivative of D- is shifted downfield by 1.8 ppm while those for C-3 and C-5 of this monosaccharide are upfield shifted by 2.5 ppm and mannuronic acid in place of the respective L-gulo isomer. Similar analysis of the spectrum of Linyi 03a,3d,3e poly- 1 ppm, respectively, as compared to the position of these saccharide revealed the presence of a minor series of signals signals in the spectrum of Lanyi 03a,3b polysaccharide. These ( ~ 1 0 %in intensity of the major series), comprising the displacements, both in direction and magnitude, are characspectrum of Lanyi 03a,3d polysaccharide (Table l), thus in- teristic of the c1 and effects of 0-acetylation of this monodicating the presence in the former polysaccharide of a small saccharide at position 4 [17], thus allowing us to locate the proportion of the units inherent in the latter. A probable U-acetyl group. It is noteworthy that the signal for C-1 of acid is also reason of such an unusual phenomenon for these poly- 2,3-diacetamido-2,3-dideoxy-~-mannuronic shifted upfield by 1.6 ppm, apparently as a result of the spatial saccharides, as well as for those of Fisher immunotype 7 and proximity between this carbon and the 0-acetyl group. Lanyi 0(3a),3d,3f, is discussed below (see Discussion). Thus, Wokatsch 0 2 5 polysaccharide has the structure shown in Table 4. Structure of Wokatsch 025 polysaccharide

'

The 3C-NMR spectrum of Wokatsch 0 2 5 polysaccharide showed it to be regular polymer built up to trisaccharide repeating units involving the monosaccharides typical of Lanyi 0 3 polysaccharides, namely, two derivatives of 2,3-diamino2,3-dideoxyuronic acids, one of which contains the acetamidino group, and N-acetylfucosamine. It resembled most closely the spectrum of LLinyi 03a,3b polysaccharide [6], but differed from it by the presence of the signals for an 0-acetyl group (CH3 at 21.5ppm and CO in the region of 174176 ppm) and by the displacement of some signals in the region of 70-82 ppm and 99- 103 ppm, apparently as a result of the attachment of the 0-acetyl group. Treatment of Wokatsch 0 2 5 polysaccharide with aqueous triethylamine (Scheme 2) gave a modified polysaccharide, whose 13C-NMR spectrum missed the signal for the 0-acetyl group; and in place of the signal for CH3 of the acetamidino group at 20.2 ppm there appeared that for the CH3 of one more acetamido group at about 23 ppm. The spectrum of this modified polysaccharide was identical to that of the polysaccharide obtained by similar treatment of Lanyi 03a,3b polysaccharide [6] and, hence, the only distinction between Wokatsch 0 2 5 and LLnyi 03a,3b polysaccharides is the presence in the former of the 0-acetyl group. Since LBnyi 03a,3b polysaccharide has only one free hydroxyl group, namely HO-4 of N-acetylfucosamine, this

Identification of 3-acetamidino-2-acetamido-2,3-dideoxy-~guluronic acid; structure of trisaccharide 6

Solvolysis of Fisher immunotype 7 polysaccharide with anhydrous hydrogen fluoride (Scheme 3) yielded oligosaccharide 6, which according to the 'H-NMR and 13C-NMR data represents a trisaccharide similar in composition to trisaccharide I . It involved two derivatives of diaminuronic acids (units A and B), one of which carried the acetamidino group (6, 2.42, S ; 6c 20.6, CH3, 167.8, N = C-N) and N-acetylfucosamine (~5~92.3, C-la, 96.0, C-lp) occupying the reducing terminus. On treatment of 6 with sodium borohydride in water, the residue of N-acetylfucosamine (unit C) was converted into N-acetylfucosaminitol (6, 61.9, C-1, 19.3, C-6), and simultaneously there occurred the reductive deamination of the acetamidino group into the ethylamino group (6,1.37, t, J1,, 7.1 Hz, CH3, 3.25-3.42, m, CH,; aC 10.9, CH3, 45.1, CH2) to yield oligosaccharide 7. The use of sodium borodeuteride as the reducing agent afforded oligosaccharide 8 involving the [l,l-2H2]ethylaminogroup (6,1.35, s, CH3).Exactly the same reaction, carried out with sodium borohydride in a saturated aqueous solution of boric acid, yielded oligosaccharide 9 with the unaffected acetamidino group. On treatment of 9 with aqueous triethylamine the acetamidino group was hydrolysed

555

c

HN

H NH

AcNH

~

COOH o

&

z

~

HNAc AcNH

-0

HNA~

HNAc

immunotype 7

I

I

i

HF

COOH

HF

OH OH HNAc 11

A

B

C

6

NaBH, OH

9

A

B

1

C

COOH

CHzOH

7R=H

8R=D

~HNAc AcNH o

~

-

HNAc AcNH

c

-

-

-

~

HO CH3 10

Scheme 3. Selective cleavage of Fisher immunotype 7 0-specific polysaccharide and chemical modifications of trisaccharide 6

into an acetamido group to give oligosaccharide 10 carrying five acetamido groups. The structures of oligosaccharides 6 , 7, 9 and 10 were confirmed by 13C-NMR (Table 1) and fastatom bombardment mass spectra, which contained the intense peaks of ions [ M f H ] ’ and no noticeable peaks corresponding to any fragments of the molecules. All signals in the ‘H-NMR spectrum of oligosaccharide 7 were assigned by using the sequential, selective spin-decoupling experiments (Table 2). On the basis of coupling constants for vicinal protons of the pyranose residues determined from this spectrum, one of the derivatives of the uronic acids (unit A) was shown to have the a-gulo configuration, whereas the other derivative (unit B) was proved to be of the p-manno configuration [18, 191. Further, by using the selective heteronuclear 13C (1H) double resonance the 13C-NMR spectrum of oligosaccharide 7 was fully interpreted (Table 1). The position of signals for C-2,3 of unit A (47.3 ppm and 58.0ppm) and of unit B (52.2ppm and 54.2ppm) showed these carbons to carry nitrogen and, consequently, both monosaccharides are derivatives of 2,3-diamino-2,3-dideoxyuronic acids. The relatively low-field position of the signal for C-4 of unit B at 76.4 ppm is due to the a effect of glycosidation [20], thus indicating this unit to be substituted at position 4. On the other hand the relatively high-field position of the signal for C-4 of unit A at 65.5 ppm is characteristic of the occurrence of this unit at the ‘non-reducing’ terminus. Analysis of nuclear Overhauser effects arising on preirradiation of the anomeric protons in oligosaccharide 7 en-

’

Table 2. Parameters of H - N M R spectrum of oligosaccharide 7 Chemical shifts for C H 3 C 0 2.00, 2.09, 2.14, and 2.17, CH3CH21.37 (t, 5 = 7.1 Hz), CH3CH23.25-3.42 ppm (m). d, doublet; t, triplet; q, quartet; b, broad singlet; m, multiplet Proton

Chemical shift

Observed multiplicity

Coupling constant Hz

Unit A H-1 H-2 H-3 H-4 H-5

5.14 4.58 3.86 4.47 4.55

d dd dd t d

Unit B H-I H-2 H-3 H-4 H-5

5.07 4.63 4.36 3.89 4.18

d dd dd t d

51.2

Unit C H-1,l’ (2 H)

3.77

b

‘I2

3.2 4.5 3.0 3.0

51.2 52.3 53.4

54.5

1.4 4.0 10.1 10.1

52.3 53.4

54.5

+

6.6

(51.2 J1,.2)

H-2 H-3 H-4 H-5 H-6 (3 H)

4.26 4.09 3.37 4.26 1.28

dt dd dd dq d

52.3 53.4

54.5 J5,6

I .7 9.2 1.7 6.6

~

556

100

80

60 Chemical shift (ppml

LO

20

Fig. 1. I3C-NMR spectrum of Fisher immunotype 7 0-specific polysaccharide (with the exception of the CO region)

Thus, trisaccharide 6 involves 3-acetamidino-2-acetabled us to confirm the sequence and types of substitution of acid and has the structure the monosaccharide residues. Thus, pre-irradiation of H-1 amido-2,3-dideoxy-~-guluronic of unit A (at 5.15 ppm) resulted in a considerable ( ~ 7 % ) shown in Scheme 3. enhancement of the signal for H-4 of unit B (at 3.89 pprn), thus showing that the former unit is linked to the latter at position 4. Further, pre-irradiation of H-1 of unit B (at Structure of Fisher immunotype 7polysaccharide 5.07 ppm) caused enhancement ( ~ 4 % of ) the signal for H-3 Comparison of the 13C-NMR spectra of Fisher immunoof the N-acetylfucosaminitol residue (unit C) at 4.09 ppm, type 7 polysaccharide (Fig. 1) and trisaccharide 6 showed that proving the latter unit to be substituted by the former at the latter retains all the structural features of the polysacchaposition 3. In addition, pre-irradation of methyl protons of ride and thus represents its chemical repeating unit. The posithe ethylamino group (at 1.37 ppm) caused a noticeable effect tion of the signal for C-1 of N-acetylfucosamine (unit C) in ( ~ 3 % at ) H-3 of unit A at 3.86 ppm, which proved this the spectrum of the polysaccharide at 95.5 ppm showed that group to be located at position 3 of this monosaccharide. This this monosaccharide is a-linked; this was confirmed by the conclusion was confirmed by the shift of the signal for C-3 of relatively large coupling constant '.IC,"169.2 Hz, determined unit A from 54.6ppm in the 13C-NMR spectrum of for this signal from the gated-decoupling spectrum of the oligosaccharide 9 to 58.0 ppm and 51.6 ppm in the spectra of polysaccharide [22]. The coupling constants lJC,H 171.1 Hz oligosaccharides 7 and 10 as a consequence of the conversion and 162.8 Hz for the signals for C-1 of units A and B of the acetamidino group into the ethylamino or acetamido at 99.9 ppm and 101.0 ppm, respectively, confirmed the group respectively. anomeric configurations of these units [22], whch were estabSince the derivatives of 2,3-diamino-2,3-dideoxyuronic lished formerly in the course of the analysis of the 'H-NMR acids could not be isolated in the free form (see above), the spectrum of oligosaccharide 7. The displacements of the signals for C-3,4,5 of unit A from regularities in the glycosidation effects were used to establish their absolute configurations [21]. These effects were deter- 54.6 ppm, 67.4 ppm and 69.5 ppm in the 13C-NMRspectrum mined for units B and C from the I3C-NMR spectrum of trisaccharide 6 to 50.0 ppm, 70.7 ppm and 68.4 ppm, reof oligosaccharide 6 by comparison with the data for spectively, in the spectrum of the polysaccharide are caused N-acetylfucosamine and oligosaccharide 4, in which 2,3- by the a and p effects of glycosidation [20], thus proving unit diacetamido-2,3-dideoxy-~-~-mannuronic acid occupies the A to be substituted at position 4 in the polysaccharide. The terminal position (Table 1). A small p effect (0.9 ppm) on C-4 relatively small a effect on C-4 (+3.3 ppm) and large (by of unit C (N-acetyl-D-fucosamine), caused by its glycosidation module) p effect on C-3 (-4.6 ppm), caused by the glycosidain oligosaccharide 6 by unit B at position 3, proved the D tion of unit A by the residue of N-acetyl-a-D-fucosamine, are configuration of unit B (2,3-diacetarnido-2,3-dideoxy-p-additional arguments in favour of the L configuration of unit mannuronic acid) since if it were in the L configuration this A, i.e. the guluronic acid derivative. Indeed, in the case of its D configuration these effects would be approximately the same effect would exceed 2 pprn [21]. Next, the p effect on C-3 of unit B, caused by its glycosida- as in the fragment a-~-GulA-( 1-+4)-~-GulAof alginic acid, tion by unit A at position 4, is relatively large (0.9- 1.O ppm). i.e. +9.1 ppm and -2.1 ppm respectively [23]. It is characteristic of the L configuration of this unit (the Hence, Fisher immunotype 7 polysaccharide has the derivative of a-guluronic acid), since in the case of the a-D structure shown in Table 4. configuration of the glycosidating sugar this effect would not It is noteworthy that each of the carbons C-3,4 of unit A exceed 0.3 ppm (cf., for example, the 13C-NMR data for gave two lines (at 50.0 and 51.1,70.7 and 72.3 ppm respectiveunit A in Lhnyi 03a,3d polysaccharide treated with aqueous ly) in a ratio of approximately 2.5:l in the 13C-NMR triethylamine, Table 1). The same regularity is general for all spectrum of the polysaccharide (Table 1). A slight splitting or sugars of the manno configuration [21]. The L configuration broadening was also observed for some other signals in the of the guluronic acid derivative was also confirmed by the spectrum, e.g. for C-1,2 of unit C (95.5 and 95.7, 48.4 and analysis of the effects caused by its glycosidation by the N- 48.5 ppm) and for C-3 of unit B (53.5 and 53.3 ppm). In the acetyl-a-D-fucosamine residue in Fisher immunotype 7 poly- spectrum of trisaccharide 6 there occurred also two lines (54.6 saccharide (see below). and 55.6,67.4 and 69.3 ppm) for exactly the same carbons of

557

100

80

60

LO

20

Chemical shift Ippm)

Fig. 2. I 3C-NMR spectrum of Fisher immunotype 7 polysaccharide treated with aqueous triethylamine (with the exception of the CO region)

100

80

60 Chemical shift (PPm)

LO

20

Fig. 3. 13C-NMR spectrum of Lanyi 0(3a).3d,3f 0-specific polysaccharide (with the exception ofthe CO region)

unit A, the first of which, C-3, carries the acetamidino group and the second, C-4, is the adjacent one, which is most sensitive to any modifications of the substituent at C-3 (cf., e.g. the 3C-NMR data for oligosaccharides 7,9,10, Table 1). At the same time, such splitting was observed in the spectra of neither Fisher immunotype 7 polysaccharide treated with aqueous triethylamine (Fig. 2), nor oligosaccharide I 1 obtained on solvolysis of these modified polysaccharides with hydrogen fluoride, both containing no acetamidino group. In addition, in the low-field region of the spectra of the intact polysaccharide and trisaccharide 6 there were observed two signals (at 168.2 ppm and 170.2 ppm) assigned to the carbon of the group N = C-N, whereas the spectra of Lhnyi 0 3 polysaccharides contained only one signal in this region (near 168 ppm). Hence it can be concluded that the acetamidino group in the Fisher immunotype 7 polysaccharide occurs in the form of two isomers. In addition the 13C-NMRspectrum of Fisher immunotype 7 polysaccharide contains a minor series of signals ( w 10% in intensity as compared to the signals of the major series), which makes up the spectrum of Lanyi 03a,3d polysaccharide

(Table 1). Analogously, the spectrum of oligosaccharide 6 contains a similar minor series belonging to contaminating trisaccharide 12, which was obtained on solvolysis with hydrogen fluoride of Lanyi 03a,3d [6] and Lanyi 03(a),3d,3f polysaccharides (see below). Therefore, Fisher immunotype 7 polysaccharide contains, together with the repeating units of the major structure, a small proportion of the units characteristic of Lanyi 03a,3d polysaccharide.

Structure of Lanyi 0 3 ( a ),3d,3f polysaccharide

The 3C-NMR spectrum of Lanyi 03(a),3d,3f polysaccharide (Fig. 3) contained a large number of signals with different intensities, which is indicative of an irregular structure. In the spectrum could be distinguished the signals of functions typical of the polysaccharides of the group under study, including carbons of pyranose rings carrying nitrogen in the region of 45 - 58 ppm, acetamidino groups (6,19.7 and 20.8, CH3, 167.4, 168.2 and 170.0, N = C-N), N-acetyl groups at 22.9 - 23.0 ppm, as well as an 0-acetyl group at 21.1 ppm.

558

80

100

60

LO

20

Chemical shift ippm)

Fig. 4. 13C-NMR spectrum of Lanyi 0(3a)Jd,3f polysaccharide treated with aqueous triethylamine (with the exception of the CO region) COOH

-0

-0

HNAc

HNAc

Lanyi 03(a),3d,3f

R 1= H or COOH R2= COOH or H

A

lHF

B

C

OH OH HNAc

6 R 1= H, R’ = COOH 12 R 1= COOH, R2= H

14

OH

OH

CH3 7

CH3

13

Scheme 4. Selective cleavage of Lanyi 0(3a),3d,3f 0-specific polysaccharide and chemical modifications of oligosaccharides obtained

Treatment of the polysaccharide with aqueous triethylamine led to de-0-acetylation and, simultaneously, to hydrolysis of the acetamidino groups into the acetamido groups. The 13C-NMRspectrum of the modified polysaccharide thus obtained (Fig. 4) contained two series of signals with a ratio of intensities of about 2: 1. The signals of the major series made up the spectrum of the similarly treated Fisher immunotype 7 polysaccharide, and the spectrum formed by the signals of the minor series was practically identical to that of Lanyi 03a,3d polysaccharide treated with aqueous triethylamine [6] (Table 1). In order to confirm the occurrence of two types of repeating units, Lanyi 03(a),3d,3f polysaccharide was cleaved

with anhydrous hydrogen fluoride (Scheme 4). This treatment was accompanied by de-0-acetylation and resulted in a mixture of trisaccharides 6 and 12, which were obtained formerly upon similar cleavage of Fisher immunotype 7 (Scheme 3) and Lanyi 03a,3d [6] polysaccharides, respectively. These trisaccharides reacted in different ways with sodium borohydride in water in accordance with the different properties of the acetamidino derivatives involved. Therefore, the acetamidino derivative of L-guluronic acid in trisaccharide 6 underwent reductive deamination into the respective ethylamino derivative, whereas that of D-mannUrOniC acid in trisaccharide 12 did not react under these conditions, thus

559 affording oligosaccharides 7 and 13, respectively. Treatment of these oligosaccharides with aqueous triethylamine led to hydrolysis of the acetamidino group in oligosaccharide 13 into the acetamido group to give oligosaccharide 14, while oligosaccharide 7 remained unaltered. The structures of all the modified oligosaccharides thus obtained were established on the basis of 13C-NMR data (Table 1). Thus, Lanyi 03(a),3d,3f polysaccharide is built up of trisaccharide repeating units of two types, the larger portion comprising the 0-acetylated repeating units of Fisher immunotype 7 polysaccharide, while the rest comprises the 0-acetylated repeating units of Lanyi 03a,3d polysaccharide. Since these polysaccharides involve only one free hydroxyl group, namely HO-4 of N-acetylfucosamine, the 0-acetyl group may by unambiguously located at this monosaccharide. The displacements of the signals for C-3,4,5 of N-acetylfucosamine from 78.7 - 78.8 ppm, 70.9 - 71.2 ppm and 68.2-68.5 ppm in the 13C-NMR spectra of Fisher immunotype 7 and Lanyi 03a,3d polysaccharides to 75.0 ppm, 73.0 ppm and 67.6 ppm, respectively, in Lanyi 03(a),3d,3f polysaccharide are consistent with the a and p effects of 0-acetylation of this monosaccharide at position 4 [17]. The signal for C-I of unit B, attached to N-acetylfucosamine, also changed its position by 1.1 - 1.2 ppm upon 0-acetylation, while the chemical shifts of the other signals were practically unaffected. Besides the irregularity connected with the presence of the acetamidino derivatives of the uronic acids with different configuration at C-5, the 13C-NMR spectrum of Lanyi 03(a),3d,3f polysaccharide showed other types of irregularity. One of these is exactly the same as in Fisher immunotype 7 polysaccharide (see above), related to the occurrence of two forms of the acetamidino derivative of L-guluronic acid (to these correspond the pairs of lines for C-3,4 of unit A at 49.6 and 50.9, 70.8 and 72.2 ppm, respectively, for C-3 unit B at 53.2 ppm and 53.1 ppm and for C-1 of unit C at 95.4 ppm and 95.5 ppm, as well as for the N = C-N group at 168.2 ppm and 170.2 ppm). The other type may be due to incomplete 0-acetylation of N-acetylfucosamine (e.g. there occurs the signal at 100.8 ppm belonging to C-1 of unit B, attached to unit C carrying no 0-acetyl group), as well as being due to partial replacement of the acetamidino group at C-3 of the derivative of L-guluronic acid by the acetamido group (e.g. the signals for C-2 of 2,3-diacetamido-2,3dideoxyguluronic acid at 47.2ppm and for C-1 of Nacetylfucosamine, attached to it, at 96.3 ppm are observed). Judging from the intensities of the respective signals in the spectrum, the contribution of each of the last two types of unit does not exceed 10% of the total number of repeating units. Upon treatment of the polysaccharide with aqueous triethylamine all kinds of irregularities mentioned in this paragraph disappeared. Regularities in glycosidation effects in 3C-NMR spectra of gulopyranose derivatives

Analysis of the 13C-NMR data for L-gulopyranose derivatives listed in Table 1 revealed essential differences in the effects of glycosidation of this monosaccharide at position 4. Thus, the a effect at C-4 and the fl effect on C-3 in Lanyi 03a,3d,3e polysaccharide, treated with triethylamine, are +8.1 ppm and -0.8 ppm, while in the similarly treated Fisher immunotype 7 polysaccharide they are 3.4 ppm and -4.8 ppm, respectively, as determined by comparison with the data for oligosaccharide 10, in which this mono-

+

Table 3. Glycosidation effects in 13C-NMR spectra of 1+4 linked disaccharides with the glycosidatedpyranose having the L-gulo confguration Configuration of the glycosidating pyranose general

anomeric and absolute

Glycosidation effects for the glycosidating pyranose on C-1

Reference

for the glycosated pyranose o n C - 3 onC-4

PPm

manno manno manno

p-D p-D b-D

gzdo

a-L a-D

galacto

+7.4 +6.3 6.2 7.5 3.4

+ +

+

-0.8 -1.8 -2.1 -2.1 -4.8

+8.1 +7.4 f9.1 f9.1 +3.4

[7] [24] [23] [23]

this work

saccharide occupies the terminal position. Comparison of these data and the 13C-NMR data for 2-acetamido-2-deoxyL-gulopyranose in the carboxyl-reduced capsular polysaccharide of Neisseria meningitidis group I [24] and L-guluronic acid in alginic acid [23] (Table 3) revealed a clear regularity in the effects of glycosidation of gulopyranose derivatives depending on the anomeric and absolute configuration of the glycosidating sugar. Thus, in the case of the a-L or p-D configuration of the glycosidating pyranosidic sugar, the a effects on its C-1 and C-4 of the glycosidated derivative of L-gulopyranose are large (6.2 - 7.5 and 7.4 - 9.1 ppm respectively), whereas the p effect on C-3 of the glycosidated sugar is relatively small (by module) and does not exceed 2.1 ppm. In contrast, in the case of glycosidation by a-D-pyranoside the a effects are small ( 3.4 ppm), while the p effect is rather large (4.8 ppm). These regularities are analogous to those established by us previously for the glycosidation effects in 1,2-linked disaccharides, involving an a-mannopyranose derivative as the glycosidated sugar [25]. They can be applied also mutadis mutandis to derivatives of D-gulopyranose, and can be used to determine relative absolute configurations of sugars in oligosaccharides and polysaccharides.

+

DISCUSSION A characteristic feature of a large group of the polysaccharides studied in the present work is the presence in the trisaccharide repeating units of two derivatives of 2,3diamino-2,3-dideoxyuronicacids, having the D-manno and Lgulo configurations, the amino group at C-3 of one of the derivatives entering the acetamidino function. Besides these polysaccharides, the only natural carbohydrate known to contain the acetamidino group is P . aeruginosa 0 1 3 (Lanyi) 0-specific polysaccharide involving 2-acetamidino-2,6-dideoxy-L-galactose [26]. Mono-N-substituted amidines are of tautomeric character, and the interconversion of the tautomeric forms occurs so rapidly that physical methods, e.g. NMR spectroscopy, fail to detect the individual forms [27]. At the same time, owing to the sp2 hybridization of both nitrogens E / Z isomerism of amidines can be observed [28]. This seems to be demonstrated by the presence of two series of signals for the acetamidino

Table 4. Structures of 0-specific polysaccharides of P. aeruginosa 0 3 (Ldnyi) and related strains ~~

Strain

Structure of the repeating unit

Lanyi 03a,3b Wokatsch 0 2 5

+4)-p-~-ManNAcAmA-(1+4)-p-~-Man(NAc)~A-( 1 -3)-P-~-FucNAc-(l+

+~)-~-D-M~~NA~A~A-(~+~)-~-D-M~~(NAC)~A-(~~~)-~ f4 OAc

Lanyi 03a,3d Lanyi 03(a),3c = Fisher immunotype 3 Lanyi 03a,3d,3e Fisher immunotype 7 Lanyi 03(a),3d,3f

-+4)-~-~-ManNAcAmA-(l+4)-~-~-Man(NAc)~A-(1+3)-a-~-FucNAc-(l+ +4)-P-~-ManNAcAmA-(1+4)-a-~-Gul(NAc)~A-(l-+3)-~-~-FucNAc-(l+ +4)-~-~-ManNAcAmA-(1+4)-a-~-Gul(NAc)2A-(I +~)-GL-D-FucNAc-(I -+ +4)-a-~-GulNAcAmA-(1+4)-~-~-Man(NAc)~A-(l-+3)-cc-~-FucNAc-(l + +4)-a-~-GulNAcAmA-(l+4)-P-~-Man(NAc)~A-(1-+3)-a-~-FucNAc-(l+ and f4 OAc +4)-a-~-ManNAcAmA-(l+4)-p-~-Man(NAc)~A-( 1 +3)-a-~-FucNAc-(1 + 14 OAc

group and neighboring carbons in the I3C-NMR spectra of oligosaccharides and polysaccharides involving the residue of 3-acetamidino-2-acetamido-2,3-dideoxy-~-guluronic acid. This is probably due to the axial orientation of the acetamidino group in this monosaccharide, since in the case of 3-acetamidino - 2 - acetamido - 2,3 - dideoxy - D -mannuronic acid and 2-acetamidino-2,6-dideoxy-~-galactose [26], with the equatorial acetamidino group, such splitting of the signals in the spectra is not observed. The readiness of the reductive deamination of the acetamidino derivative of guluronic acid into the corresponding ethylamino derivative, to take place even under the action of sodium borohydride in water at room temperature, may also be related to steric factors. Thus, the acetamidino derivatives of fucose [26] and mannuronic acid are not affected under these conditions, and can be converted into the corresponding ethylamino derivatives only on heating with lithium borohydride in aqueous 2-propanol. The amidines are known to possess basic properties, and the supposed role of the acetamidino group is to neutralize one of two carboxyl groups entering the repeating units. In contrast to extracellular polysaccharides, a high concentration of negative charge does not seem to be typical of the polysaccharide chains of lipopolysaccharides of gram-negative bacteria, and for known polymers [29] it does not exceed one carboxyl group per three monosaccharide residues. In those cases when the charge is larger, partial or total compensation of the negative charge takes place, e.g. by the appearance of the acetamidino group. The glycosidic linkages of the derivatives of diaminouronic acids are extremely stable. Under conditions of drastic acid hydrolysis required for their cleavage, the manno isomer largely undergoes epimerization into the gluco isomer [6, 301, while the gulo isomer is practically fully destroyed [7]. Solvolysis with anhydrous hydrogen fluoride does not affect the glycosidic linkages of these acidic diamino sugars and results in depolymerization in a strictly selective way at the glycosidic linkage of the third component of the polysaccharides, N-acetylfucosamine, to give the trisaccharides representing their repeating units. Analysis of these trisaccharides employing chemical modifications, ‘H and I3C NMR spectroscopy, and fast-atom bombardment spectrometry, enabled both the identification the derivatives of the diaminouronic acids without their isolation in the form of free monosaccharides and the establishing of the structures

of the repeating units of these polysaccharides. Such a detailed analysis led to the revision of the structure of the acetamidino derivative of D-mannuronic acid to which the structure involving a 1-acetyl-2-methyl-Zimidazoline ring was erroneously assigned by us previously [6]. Comparison of the polysaccharides studied showed that they have structures similar to each other and posses invariable structural elements, such as the sequence of N-acetylfucosamine residues and the acetamidino and diacetamido derivatives of the uronic acids, as well as the mode of substitution of all the monosaccharides. As a result, the polysaccharides contain common monosaccharide or oligosaccharide fragments, which account for the serological interrelations between 0 antigens and thus provide the chemical basis for combining the corresponding strains into a single 0 serogroup [l - 31. The variable structural elements of the polysaccharides are the configuration of the glycosidic linkage of the Nacetylfucosamine residue, the presence or absence of the 0acetyl group at this monosaccharide, as well as the configuration at C-5 of one or both derivatives of the uronic acids. The change of the configuration at C-5 implies the conversion of the fl-D-manno into the a-L-gulo isomer or vice versa. These distinctions in the structures of the polysaccharide chains of the 0 antigens determine the serological differentiation of the strains studied within the 0 serogroup. Comparison of the structures of the polysaccharides (Table 4) and the most complete serological classification scheme for P . aeruginosa (Lanyi-Bergan-Homma-Akatova-Smirnova [311) showed that the Wokatsch 0 2 5 serotype is quite soundly incorporated into the LQnyi 0 3 serogroup ( 0 2 serogroup in the scheme [l]) as an individual sixth 0 serotype. Moreover, this group should be further extended to include, as a seventh 0 serotype, Fisher immunotype 7 (classification [5]),which 0 antigen is related but not identical to the 0-antigens of the 0 3 serogroup. It is noteworthy that, judging from the 13C-NMR data, the structure of Wokatsch 0 2 5 0-specific polysaccharide is identical to that of the polysaccharide of the mutant strain P . aeruginosa PAO(D3). This strain was obtained by lysogenation with phage D3 of the strain P . aeruginosa PAO1, the 0specific polysaccharide of which has a structure identical to that of Lanyi 03a,3d polysaccharide [32]. On the basis of the I3C-NMR analysis the changes of the structure of the repeating unit caused by lysogenation were established accurately [32]; however, the structures of PA01 and PAO(D3) 0-

561 specific polysaccharides should be refined in accordance with the present revision of the structure of the acetamidino derivative of mannuronic acid. The structure of Lanyi 03(a),3d,3f polysaccharide deserves special comments. This polysaccharide involves comparable a m o u n t s of trisaccharide units of t wo types, differing from one a n o t h e r in the configuration at C-5 of the acetamidino derivative of the uronic acid, o r represents a mixture of two different polysaccharides. Since all the P. aeruginosa type strains used in this work are very well characterized museum ones, we may rule out the possibility of a mixture of microorganisms each making its o w n polysaccharide. Most probably we are dealing with a polysaccharide possessing a masked regularity of the type discovered for the first time in 0 antigens, whose polysaccharide chains are usually either strictly regular polymers or possess irregular structures resulting from the non-stoichiometric 0-acetylation or glycosidation of a regular backbone [33]. At the same time, some other natural polysaccharides (alginic acid, d erm a ta n sulphate, heparin) possess hybrid structure of precisely this type. So alginic acid, a polysaccharide of brown algae [34] a n d a n extracellular polysaccharide of some bacteria (P. aeruginosa and Azotobacter vinelandii [29]), is a block-copolymer of D-mannuronic and L-guluronic acids a n d has no simple repeating unit. This feature is a consequence of epimerization at C-5 of some of the p-D-mannuronic acid residues yielding a-L-guluronic acid residues within the preassembled Dm a n n u r o n a n chain in the course of the biosynthesis [35]. It was also found that Lanyi 03(a),3c, 0 3 a, 3 d , 3e and Fisher immunotype 7 polysaccharides, together with the major repeating units including one D-mannuronic acid a n d one L-guluronic acid derivative, contain a small proportion of units with the D-manno configuration in both the derivatives. Thus, it seems likely, although not proved, that the biosynthesis of the polysaccharides of this group containing derivatives of L-guluronic acid, like the biosynthesis of alginic acid, involves epimerization at C-5 of one of the derivatives of D-mannuronic acid within the glycan chain after polymerization. In the case of Lanyi 03(a),3c, 03a,3d,3e a n d Fisher immunotype 7 polysaccharides, which contain no 0-acetyl groups, the epimerization affected almost all repeating units, while in 0-acetylated Lanyi 03(a),3d,3f polysaccharide a considerable proportion of repeating units remained unaffected. This is consistent with the recently established role of 0-acetyl goups in bacterial alginic acid as inhibitors of epimerization at C-5 of the p-D-mannuronic acid residue, these groups effecting not only the 0-acetylated sugar residue but also adjacent non-

acetylated ones [36]. I n contrast t o alginic acid, where the distribution of epimerized and non-epimerized units can be analyzed, e.g. by using 13C-NMR [24, 371, this cannot be accomplished for Lanyi 03(a),3d,3f polysaccharide since the monosaccharide residues subject to epimerization are separated by t wo residues of other sugars not participating in the modification. Solving this problem as well as proving rigorously t h at the repeating units of t w o different types enter the same glycan chain necessitates the search for the respective specific endoglycosidases in order t o isolate higher fragments of the polysaccharide.

REFERENCES 1. Lanyi, B. & Bergan, T. (1978) Methods Microbiol. 10,93-168. 2. Lanyi, B. (1966/1967) Acta Microbiol. Acad. Sci. Hung. 13,295318.

3. Akatova, N. S. & Smirnova, N. E. (1982) Zh. Microbiol. Epidemiol. Immunol. 7, 87 - 91. 4. Wokatsch, R. (1964) Zentralbl. Bakteriol. Mikrobiol. Hyg. I. Abt. Orig. 192, 468. 5. Fisher, M. W. (1975) Microbiology (Wash. D C ) . 416. 6. Knirel, Yu. A,, Vinogradov, E. V., Shashkov, A. S., Dmitriev, B. A., Kochetkov, N. K., Stanislavsky, E. S. & Mashilova, G. M. (1982) Eur. J . Biochem. 128,81-90. 7. Knirel, Yu. A,, Vinogradov, E. V., Shashkov, A. S., Dmitriev, 9. A,, Kochetkov, N. K., Stanislavsky, E. S. & Mashilova, G. M. (1983) Eur. J . Biochem. 134,289-297. 8. Knirel, Yu. A., Paramonov, N. A., Vinogradov, E. V., Shashkov, A. S., Dmitriev, B. A. &Kochetkov, N. K. (1986) Bioorg. Khim. 12,995-997. 9. Knirel, Yu. A., Paramonov, N. A., Vinogradov, E. V., Shashkov, A. S. & Kochetkov, N. K. (1986) Bioorg. Khim. 12,992-994. 10. Wagner, G. & Wiithrich, K. (1979) J . Magnet. Resonance 33, 675 - 680. 11. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., Randall, R. J. (1951) J. Biol. Chem. 193,265-275. 12. Dmitriev, B. A,, Knirel, Yu. A,, Kochetkov, N. K. & Hofman, I. L. (1976) Eur. J . Biochem. 66, 559-566. 13. Dmitriev, B. A., Knirel, Yu. A,, Kocharova, N. A,, Kochetkov, N. K., Stanislavsky, E. S. & Mashilova, G. M. (1980) Eur. J . Biochem. 106,643 - 651. 14. Westphal, 0. & Jann, K. (1965) Methods Carbohydr. Chem. 5, 83-91. 15. Perry, M. B. & Daoust, V. (1973) Can. J . Chem. 51,974-977. 16. Mort, A. J. & Lamport, D. T. A. (1977) Anal. Biochem. 82,289309. 17. Gagnaire, D., Mancier, D. & Vincendon, M. (1978) Org. Magnet. Resonance 11, 344- 349. 18. Altona, C., Haasnoot, C. A. G. (1980) Org. Magnet. Resonance 13,417-429. 19. Shashkov, A. S. (1983) Izv. Akad. Nauk SSSR, Ser. Khim. 13281336. 20. Gorin, P. A. J. (1981) Adv. Cabohydr. Chem. Biochem. 38, 13104. 21. Kochetkov, N. K., Chizhov, 0. S. & Shashkov, A. S. (1984) Carbohydr. Res. 133, 173- 185. 22. Bock, K. & Pedersen, C. (1974) J . Chem. SOC.Perkin Trans. 2, 293 - 297. 23. Usov, A. I., Kosheleva, E. A. & Yakovlev, A. P. (1985) Bioorg. Khim. 11,830-836. 24. Mishon, F., Brisson, J. R., Roy, R., Ashton, F. E. & Jennings, H. J. (1985) Biochemistry 24, 5592-5598. 25. Knirel, Yu. A,, Vinogradov, E. V., Shashkov, A. S., Dmitriev, B. A,, Kochetkov, N. K., Stanislavsky, E. S. & Mashilova, G. M. (1985) Eur. J . Biochem. 150, 541 - 550. 26. Knirel, Yu. A,, Vinogradov, E. V., Shashkov, A. S., Dmitriev, B. A., Kochetkov, N. K., Stanislavsky, E. S. & Mashilova, G. M. (1987) Eur. J . Biochem. 163,627-637. 27. Hafelinger, G. (1975) in Chemistry of amidines and imidates (Patai, S., ed.) pp. 1 -84, Wiley, London. 28. Osczapowicz, J., Raczynska, E. & Osek, J. (1986) Magnet. Res. Chem. 24,9-14. 29. Kenne, L., Lindberg, B. (1983) in The polysaccharides (Aspinall, G. O., ed.) vol. 2, pp. 287 - 363, Academic Press, New York. 30. Nagaoka, M., Kamisango, K., Fujii, H., Uchikawa, K., Sekikawa, I. & Azuma, I. (1985) J . Biochem. (Tokyo) 97, 1669- 1678. 31. Stanislavsky, E. S., Dmitriev, B. A., Lanyi, B. & JoO, I. (1985) Acta Microbiol. Hung. 32, 3 - 37. 32. Kuzio, K. & Kropinski, A. M. (1983) J . Bacteriol. 155,203-212. 33. Jann, K. & Jann, B. (1985) in Chemistry ofendotoxin (Rietschel, E. T., ed.) vol. 1, pp. 138-186, Elsevier, Amsterdam. 34. Painter, T. J. (1983) in Thepolysaccharides (Aspinall, G. O., ed.) vol. 2, pp. 195-285, Academic Press, New York. 35. Haug, A,, Larsen, B. & Grasdalen, H. (1985) Carbohydr. Res. 145, 169-174. 37. Grasdalen, H., Larsen. B. & Smidsrnd. 0. (1981) Carbohvdr. Res. 89,179-191.