BIOPSEL: verification

Explanations to the table:

Bacterial strains (column 1) includes the following species abbreviations: P.v. = Proteus vulgaris, P.p. = Proteus penneri, P.m. = Proteus mirabilis.
Program input (column 2) includes monomeric composition and widespread mode either ON or OFF. The experimental spectrum is omitted. The first character in each line is the anomeric configuration, the second is the absolute configuration, rest are the residue name. The question mark stands for unknown configurations. If any substitution constraints were specified, they are recorded before the closing parenthesis like "xx)"
Program output (column 3) includes the list of five (may be from 1 to 100) structures most closely fitting the experimental spectrum. The proper structure is given in bold. Each line contains also schematic representation of topology (e.g. -[x]xxx-) and the deviation of the calculated 13C NMR spectrum from the experimental.
References (column4) are to publications with these structures determined independently.
Additional experiments (column 5) column lists the additional NMR and chemical methods that are required to select the proper structure from the list generated. If a particular experiment is needed to distinguish two certain structures, their numbers are presented in parentheses, e.g. (1 vs. 4).

Bacterial
strain
Program
input
Program output    (the proper structure is highlighted) Ref. Additional
experiments


P.m. G1

 Llys
?Dgaa
?Dgaln
?Dgla
?Dgaln

Widespread
=ON
1: [Llys--6)aDgaa]
              |4
        --6)bDgaln--4)bDgla--3)bDgaln--           (-[xx]xxx-)      Dev.: 0.27
2: [Llys--6)aDgaa-] |4 --6)bDgaln--2)bDgla--3)bDgaln-- (-[xx]xxx-) Dev.: 0.33 3: [Llys--6)aDgaa-] |4 --6)bDgaln--3)bDgla--3)bDgaln-- (-[xx]xxx-) Dev.: 0.33 4: [Llys] |6 [bDgla--3)bDgaln--4)aDgaa] |4 --6)bDgaln-- ([xx[x]x]x) Dev.: 0.34 5: [bDgla--3)bDgaln] [Llys] |6 |6 --4)bDgaln--4)aDgaa-- ([xx]x[x]x) Dev.: 0.34

[1]

HMQC-TOCSY
or methylation
analysis


P.m. O3

bDgal
bDglcn
  aletP
aDgal
bDglc

Widespread
=ON
1: [alet--P]
          |6
   --3)aDgal--6)bDglc--3)bDgal—-3)bDglcn--        (-[xx]xxxx-)     Dev.: 0.13
2: [alet--P] |6 --3)aDgal--6)bDglc--3)bDglcn--3)bDgal-- (-[xx]xxxx-) Dev.: 0.13 3: [alet--P] |6 --3)bDgal--3)bDglcn--3)aDgal--6)bDglc-- (-[xx]xxxx-) Dev.: 0.23 4: [alet--P] |6 --3)bDglcn--3)bDgal--3)aDgal--6)bDglc-- (-[xx]xxxx-) Dev.: 0.23 5: [alet--P] |6 --2)aDgal--6)bDglc--3)bDglcn--3)bDgal-- (-[xx]xxxx-) Dev.: 0.23

[2]

NOESY (ROESY)
(1 vs. 2)
(3), (4), (5) posses
much greater
deviation

P.m. O10
?Dgla
?Dglcn
?Dgaln
aLalta

Widespread
=ON
1:   [aLalta]
        |3
  --4)aDgaln--3)aDgla--3)aDglcn--                 (-[x]xxx-)       Dev.: 0.34
2: [aDglcn] |4 --3)aDgaln--3)aDgla--3)aLalta-- (-[x]xxx-) Dev.: 0.38 3: [aLalta] |3 --4)aDgaln--3)aDgla--4)aDglcn-- (-[x]xxx-) Dev.: 0.41 4: [aLalta] |3 --4)aDgaln--3)aDglcn--3)aDgla-- (-[x]xxx-) Dev.: 0.41 5: [aLalta] |3 --4)aDgaln--4)aDglcn--3)aDgla-- (-[x]xxx-) Dev.: 0.46

[3]

HMQC-TOCSY
or methylation
analysis
(1 vs. 2,3)

NOESY (ROESY)
(1 vs. 4)

(5) posses
much greater
deviation

P.m. O13
3?)?Dglcn
34)?Dgal
   ?Dgla
    Lalys

Correction
+1.6 ppm
Widespread =ON
1: [Lalys--6)bDgla]
               |3
         --4)aDgal--3)bDglcn--                    (-[xx]xx-)       Dev.: 1.23

2:   [aDgla] [Lalys]
        |4       |2
  --3)aDgal--3)bDglcn--                           (-[x]x[x]x-)     Dev.: 1.23

3: [Lalys--6)bDgla]
               |4
         --3)aDgal--3)bDglcn--                    (-[xx]xx-)       Dev.: 1.23
		 
4: [Lalys--6)aDgla]
               |4
         --3)aDgal--3)bDglcn--                    (-[xx]xx-)       Dev.: 1.24
5: [Lalys--6)aDgla] |4 --3)bDgal--3)bDglcn-- (-[xx]xx-) Dev.: 1.28

[4]

HMQC-TOCSY
or methylation
analysis
(4 vs. 2)

NOESY (ROESY)
(4 vs. 1)

13C Gated
(4 vs. 3,5)

P.m. O14

De-OAc

bDgaln
aDgal
bDgaln
  alet
  P
aDgal

Widespread
=ON
1: [alet--P]
          |6
    --4)aDgal--4)bDgaln--3)aDgal--3)bDgaln--      (-[xx]xxxx-)     Dev.: 0.20
2: [alet--P] |6 --4)aDgal--3)bDgaln--3)aDgal--4)bDgaln-- (-[xx]xxxx-) Dev.: 0.20 3: [alet--P] |6 --4)aDgal--3)bDgaln--4)aDgal--4)bDgaln-- (-[xx]xxxx-) Dev.: 0.23 4: [alet--P] |6 --4)aDgal--4)bDgaln--4)aDgal--3)bDgaln-- (-[xx]xxxx-) Dev.: 0.23 5: [alet--P] |6 --4)aDgal--3)bDgaln--2)aDgal--4)bDgaln-- (-[xx]xxxx-) Dev.: 0.24

[5]

NOESY (ROESY)
or HMBC


P.m. O16

?Dgaln
?Dgaln
  etnP
?Dglcn
 ?ribP

Widespread
=ON
1:  [etnP]
        |6
  --3)aDglcn--4)DribP--6)bDgaln--4)aDgaln--       (-[x]xxxx-)      Dev.: 0.28
2: [etnP] [bDgaln] |6 |6 --4)aDgaln--4)DribP--4)aDglcn-- ([x]xx[x]x) Dev.: 0.31 3: [etnP] |6 --4)aDglcn--4)DribP--6)bDgaln--4)aDgaln-- (-[x]xxxx-) Dev.: 0.32 4: [etnP] |3 --6)aDglcn--4)aDgaln--4)DribP--6)bDgaln-- (-[x]xxxx-) Dev.: 0.32 5: [etnP--6)bDgaln] |6 --3)aDglcn--4)aDgaln--4)DribP-- (-[xx]xxx-) Dev.: 0.32

[6]

{1H-31P} HMQC
(1 vs. 2,5)

NOESY (ROESY)
(1 vs. 3,4)

P.m. O26

De-OAc

 Llys
?Dgla
?Dgal
?Dgla
?Dglcn

Widespread
=ON
1:    [Llys]           [aDgal]
        |6                |4
  --4)aDgla--3)bDgla--3)bDglcn--                  (-[x]xx[x]x-)    Dev.: 0.27
2:    [Llys]
        |6
  --4)aDgla--4)aDgal--3)bDgla--3)bDglcn--         (-[x]xxxx-)      Dev.: 0.36
3: [Llys] [aDgal] |6 |3 --4)aDgla--3)bDgla--4)bDglcn-- (-[x]xx[x]x-) Dev.: 0.43 4: [Llys] [aDgal] |6 |4 --3)aDgla--3)bDgla--3)bDglcn-- (-[x]xx[x]x-) Dev.: 0.45 5: [Llys] 6| --4)aDgla--3)bDgla--2)aDgal--3)bDglcn-- (-[x]xxxx-) Dev.: 0.47

[7]

HMQC-TOCSY
or methylation
analysis
or NOESY (ROESY)
(2 vs. 1,3)

(4), (5) posses
much greater
deviation

P.m. O30

bDgla
aDgaln
bDglcn
bDglcn

Widespread
=OFF
1: --4)bDgla--3)bDglcn--6)aDgaln--6)bDglcn--      (-xxxx-)         Dev.: 0.35
2: --4)bDgla--6)aDgaln--6)bDglcn--3)bDglcn--      (-xxxx-)         Dev.: 0.37
3: --2)bDgla--3)bDglcn--6)aDgaln--6)bDglcn--      (-xxxx-)         Dev.: 0.39

4: [bDglcn--4)bDgla]
                 |3
          --6)bDglcn--6)aDgaln--                  (-[xx]xx-)       Dev.: 0.39

5: [bDglcn--6)aDgaln]
                 |6
          --3)bDglcn--4)bDgla--                   (-[xx]xx-)       Dev.: 0.39

[8]

HMQC-TOCSY
or methylation
analysis
(1,2 vs. 3,4,5)
 
+NOESY (ROESY)
(1 vs. 2)


P.m. O33
(P.m. D52)

 DribP
?Dgal
?Dglcn
  etnP
?Dglc
?Dglcn

Widespread
=ON
1:  [DribP]             [etnP]
         |3                |6
   --2)bDgal--3)aDglcn--3)bDglc--3)bDglcn--       (-[x]xx[x]xx-)   Dev.: 0.33
2: [DribP] [etnP] |6 |3 --3)bDglc--3)bDglc--2)bDgal--3)aDglcn-- (-[x]xx[x]xx-) Dev.: 0.33 3: [etnP] [DribP] |3 |6 --2)bDgal--3)bDglc--3)aDglcn--3)bDglcn-- (-[x]xxx[x]x-) Dev.: 0.33 4: [DribP] [etnP] |3 |6 --2)bDgal--3)bDglc--3)aDglcn--3)bDglcn-- (-[x]xxx[x]x-) Dev.: 0.33 5: [DribP] [etnP] |3 |6 --2)bDgal--3)aDglcn--3)bDglc--3)bDglcn-- (-[x]xxx[x]x-) Dev.: 0.33

[9]

{1H-31P} HMQC
 
+ NOESY (ROESY)
(1 vs. 2)

P.p. 1

bDglc
bDgaln
bDgaln
aDgal

Widespread
=OFF
1:   [bDgaln]
         |3
   --4)aDgal--6)bDglc--3)bDgaln--                 (-[x]xxx-)       Dev.: 0.18
2:   [bDgaln]
         |4
   --3)aDgal--6)bDglc--3)bDgaln--                 (-[x]xxx-)       Dev.: 0.18

3: [bDglc--3)bDgaln]
               |4
         --3)aDgal--6)bDgaln--                    (-[xx]xx-)       Dev.: 0.23

4: [bDglc--3)bDgaln]
               |3
         --4)aDgal--6)bDgaln--                    (-[xx]xx-)       Dev.: 0.23

5: --6)bDglc--3)bDgaln--4)bDgaln--3)aDgal--       (-xxxx-)         Dev.: 0.34

[10]

NOESY (ROESY)

P.p. 2

?Dr23n
?Dglc
??ta6d
?Dglcn

Widespread
=ON
1:   [bDr23n]
        |3
  --4)aDglc--3)aLta6d--3)bDglcn--                 (-[x]xxx-)       Dev.: 0.41
2: [bDr23n] |4 --2)aDglc--3)aLta6d--4)bDglcn-- (-[x]xxx-) Dev.: 0.44 3: [bDr23n] |4 --3)aDglc--3)aLta6d--3)bDglcn-- (-[x]xxx-) Dev.: 0.44 4: -4)bDr23n--4)bDglcn--3)aDglc--3)bLta6d-- (-xxxx-) Dev.: 0.44 5: [bDr23n] |3 --4)aDglc--3)aLta6d--4)bDglcn-- (-[x]xxx-) Dev.: 0.44

[11]

HMQC-TOCSY
or methylation
analysis
(1 vs. 2,4,5)
 
+NOESY (ROESY)
(1 vs. 3)

P.p. 4

bDglc
bDgaln
bDgaln
aDgal
aDglc

Widespread
=ON
1: [aDglc--6)bDgaln]
               |4
         --3)aDgal--6)bDglc--3)bDgaln--           (-[xx]xxx-)      Dev.: 0.21

2: [aDglc--6)bDgaln]
               |3
         --4)aDgal--6)bDglc--3)bDgaln--           (-[xx]xxx-)      Dev.: 0.21

3:    [bDgaln]          [aDglc]
         |4                |6
   --3)aDgal--6)bDglc--3)bDgaln--                 (-[x]xx[x]x-)    Dev.: 0.22
4:    [bDgaln]          [aDglc]
         |3                |6
   --4)aDgal--6)bDglc--3)bDgaln--                 (-[x]xx[x]x-)    Dev.: 0.22
5:    [bDgaln]
         |4
   --3)aDgal--6)bDglc--2)aDglc--6)bDgaln--        (-[x]xxxx-)      Dev.: 0.28

[10]

HMQC-TOCSY
or methylation
analysis
(1,2,5 vs. 3,4)
 
+NOESY (ROESY)
(3 vs. 4)

P.p.15

De-Pyr

aDgal
bDgal
bDglcn

Widespread
=OFF
Linear:
1a: --3)aDgal--4)bDgal--3)bDglcn--                (-xxx-)          Dev.: 0.23
2a: --2)aDgal--4)bDgal--3)bDglcn--                (-xxx-)          Dev.: 0.30
3a: --4)aDgal--4)bDgal--3)bDglcn--                (-xxx-)          Dev.: 0.31

Widespread branched (with 1 or 2 sugars in side chains):
1b: [bDgal--3)bDglcn]
                |2
          --4)aDgal--                             (-[xx]x-)        Dev.: 0.28

2b: [bDgal--3)bDglcn]
                |3
          --4)aDgal--                             (-[xx]x-)        Dev.: 0.28

3b:   [bDgal]
         |3
   --4)bDglcn--3)aDgal--                          (-[x]xx-)        Dev.: 0.38

Rarely occurring branched (with more than 2 sugars in side chains): absent

[12]

HMQC-TOCSY
or methylation
analysis

P.p.17
(common)

bDmaan
aDglcn
aDfu4n

Widespread
=OFF
1: --4)bDmaan--4)aDglcn--3)aDfu4n--               (-xxx-)          Dev.: 0.27
2: [aDglcn--3)aDfu4n] |3 --4)bDmaan-- (-[xx]x-) Dev.: 0.55 3: --4)bDmaan--3)aDglcn--3)aDfu4n-- (-xxx-) Dev.: 0.56 4: [aDglcn] |3 --2)aDfu4n--4)bDmaan-- (-[x]xx-) Dev.: 0.56 5: [bDmaan-] |3 --4)aDglcn--3)aDfu4n-- (-[x]xx-) Dev.: 0.68

[*]

(2), (3), (4), (5) posses
much greater
deviation

P.p. 63

?Dglcn
?Dglc
?Lfucn
  etnP
?Dglcn
?Dglc

Widespread
=ON
1:  [etnP]                        [bDglc]
        |6                           |4
  --3)bDglcn--2)bDglc--6)aDglcn--3)aLfucn--         (-[x]xxx[x]x-) Dev.: 0.10
2: [etnP--2)bDglc] |4 --3)aLfucn--3)bDglcn--2)bDglc--6)aDglcn-- (-[xx]xxxx-) Dev.: 0.18 3: [etnP] [bDglc] |6 |4 --2)bDglc--6)aDglcn--3)aLfucn--3)bDglcn-- (-[x]xx[x]xx-) Dev.: 0.21 4: [etnP] [aDglcn] |6 |3 --3)bDglcn--2)bDglc--6)bDglc--4)aLfucn-- (-[x]xxx[x]x-) Dev.: 0.21 5: [etnP--6)aDglcn] |4 --3)aLfucn--3)bDglcn--2)bDglc--6)bDglc-- (-[xx]xxxx-) Dev.: 0.26

[13]


(2), (3), (4), (5) posses
much greater
deviation

P.p. 71

bDglcn
bDglcn
aDgal

Widespread
=OFF
1: --3)bDglcn--4)bDglcn--3)aDgal--                (-xxx-)          Dev.: 0.34
2: --4)bDglcn--3)aDgal--3)bDglcn--                (-xxx-)          Dev.: 0.34

3:    [aDgal]
         |3
   --4)bDglcn--4)bDglcn--                         (-[x]xx-)        Dev.: 0.42

4: --3)bDglcn--4)bDglcn--2)aDgal--                (-xxx-)          Dev.: 0.43

5: --4)bDglcn--2)aDgal--3)bDglcn--                (-xxx-)          Dev.: 0.43

[14]

NOESY (ROESY)
or HMBC
(1 vs. 2)

(3), (4), (5) posses
much greater
deviation

P.p. 103

?Dglc
?Dgal
?Dgaln
 ?ribP
  etnP

Widespread
=ON
1:  [DetnP]
         |6
   --4)bDglc--3)bDgal--3)bDgaln--2)DribP--        (-[x]xxxx-)      Dev.: 0.42
2: [DetnP] |4 --6)bDglc--3)bDgal--3)bDgaln--2)DribP-- (-[x]xxxx-) Dev.: 0.42 3: [DetnP] |6 --3)bDgal--3)bDgaln--2)DribP--4)bDglc-- (-[x]xxxx-) Dev.: 0.42 4: [DetnP-4)bDglc] |3 --6)bDgal--3)bDgaln--2)DribP-- (-[xx]xxx-) Dev.: 0.42 4: [DetnP-6)bDglc] |3 --2)bDgal--3)bDgaln--2)DribP-- (-[xx]xxx-) Dev.: 0.42

[15]

HMQC-TOCSY
or methylation
analysis
(1,2) vs. (3,4,5)
 +
{1H-31P} HMQC
(1 vs. 2)

P.v. O17

De-P

?Dglc
?Dglcn
??fu3n
?DglcA
 Dbut

Widespread
=ON
1:    [Dbut]
        |2
   --6)aDglcn--2)bDfu3n--3)aDglc--4)bDgla--       (-[x]xxxx-)      Dev.: 0.34

2:     [Dbut]
         |3
   --2)bDfu3n--6)aDglc--4)bDgla--3)aDglcn--       (-[x]xxxx-)      Dev.: 0.35
3: [Dbut--3)bLfu3n] |3 --2)aDglc--4)bDgla--6)aDglcn-- (-[xx]xxx-) Dev.: 0.36 4: [Dbut--3)bDfu3n] |3 --2)aDglc--4)bDgla--6)aDglcn-- (-[xx]xxx-) Dev.: 0.37 5: [Dbut--3)bLfu3n] |4 --3)aDglcn--6)aDglc--4)bDgla-- (-[xx]xxx-) Dev.: 0.37

[16]

HMQC-TOCSY
(Fuc3N C-3
displacement)

(2) vs. (1)
 
+methylation
analysis or
HMQC-TOCSY
(2 vs. 3,4,5)

P.v. O22

De-OAc

?Dqu3n
??rha
??rha
?Dgla
?Dglcn

Widespread
=ON
1:  [aDqu3n]
        |2
  --3)bLrha--4)aLrha--4)bDgla--3)bDglcn--         (-[x]xxxx-)      Dev.: 0.24
2: [aDqu3n] |3 --4)aLrha--4)bDgla--3)bDglcn--4)bDrha-- (-[x]xxxx-) Dev.: 0.24 3: [aDqu3n] |3 --2)bLrha--4)aLrha--4)bDgla--3)bDglcn-- (-[x]xxxx-) Dev.: 0.25 4: [aDqu3n--3)aLrha] |2 --4)bDrha--3)bDglcn--4)bDgla-- (-[xx]xxx-) Dev.: 0.25 5: [aDqu3n--3)aLrha] |2 --3)bLrha--3)bDglcn--4)bDgla-- (-[xx]xxx-) Dev.: 0.29

[17]

NOESY (ROESY)

P.v. OX19

  ??quin
34)?Dgaln
4x)?Dgal
??quin
3x)?Dglcn
    P

Widespread
=ON
Correction
-0.1ppm
1:   [aLquin]
        |3
  --4)aDgaln--4)aDgal--P--4)aLquin--3)bDglcn--       (-[x]xxxxx-)  Dev.: 0.39
2: [aLquin--3)bDglcn] |4 --3)aDgaln--4)aDgal--P--4)aLquin-- (-[xx]xxxx-) Dev.: 0.39 3: [aLquin] |3 --4)aDgaln--4)aDgal--P--4)aLquin--3)aDglcn-- (-[x]xxxxx-) Dev.: 0.43 4: [aLquin--3)aDglcn] |4 --3)aDgaln--4)aDgal--P--4)aLquin-- (-[xx]xxxx-) Dev.: 0.43 5: [aLquin] [P] |4 |4 --3)aLquin--3)aDglcn--4)aDgal--3)aDgaln-- ([x]xxx[x]x) Dev.: 0.43

[18]

13C Gated
(1) vs. (3)
 +
NOESY (ROESY)
(1) vs. (2,4,5)

E.coli O28
de-OAc

?Dglcn
b?galf
?Dglcn
 ?gro
  P

Widespread
=OFF
1: -4)bDglcn--3)bDgalf--3)aDglcn--2)Lgro--P--        (xxxxx)       Dev.: 0.54
2: -4)bDglcn--3)bDgalf--3)aDglcn--2)Dgro--P-- (xxxxx) Dev.: 0.54 3: [bDglcn--3)bDgalf--2)Dgro--P] |4 --3)aDglcn-- ([xxxx]x) Dev.: 0.55 4: [bDglcn--3)bDgalf--2)Lgro--P] |4 --3)aDglcn-- ([xxxx]x) Dev.: 0.55 5: [bDglcn--3)bDgalf] |4 --3)aDglcn--2)Dgro--P-- ([xx]xxx) Dev.: 0.57

[19]

optical
rotation
measurement
(1 vs. 2)

HMQC-TOCSY
or methylation
analysis
(1 vs. 3,4,5)

The resulting deviation is significantly sensitive to absolute configurations of input residues (which means that in most cases the absolute configurations are predicted with high accuracy), e.g. specifying (bLgal bDglcn Dalet P aDgal bDglc) as a monomeric composition in stead of (bDgal bDglcn Dalet P aDgal bDglc) leads to the following list of five best structures for the polysaccharide of P.mirabilis O3:

as compared to the primary (all monosaccharides have D configuration) list:

As it can be seen from these two lists of structures, the deviation of the best-fitting structure increased from 0.13 to 0.32 after changing the absolute configuration of one of residues to the value not corresponding to the proper structure. Thus BIOPSEL is the reliable way to determine absolute configurations of monosaccharides basing on the thorough differences in glycosylation effects in DD and DL pairs of monomers.


    References:
  1. Z. Sidorczyk, K. Zych, F.V. Toukach, N.P. Arbatsky, A.S. Shashkov, A. Zablotni, Y.A. Knirel - Eur. J. Biochem., 269 (2002) 1406-1412
  2. E.V. Vinogradov, W. Kaca, A.S. Shashkov, D. Kraewska-Pietrasik, Y.A. Knirel, N.K. Kochetkov – Eur. J. Biochem., 188 (1990) 645-651
  3. A.S. Swierzko, A.S. Shashkov, S.N. Senchenkova, F.V. Toukach, A. Ziolkowski, M. Cedzynski, N.A. Paramonov, W. Kaca, Y.A. Knirel - FEBS Lett., 398 (1996) 297-302
  4. A.S. Shashkov, F.V. Toukach, S.N. Senchenkova, A. Ziolkowski, N.A. Paramonov, W. Kaca, Y.A. Knirel, N.K. Kochetkov - Biochem., 62(5) (1997) 509-513
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       *. Authors' unpublished data
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