Tài liệu Báo cáo khóa học: Determination by electrospray mass spectrometry and 1H-NMR spectroscopy of primary structures of variously fucosylated neutral oligosaccharides based on the iso-lacto-N-octaose core doc

The double glycosidic D-type cleavage [13,14] is unique to 3-linked GlcNAc and Glc residues, whereas0,2A-type cleavages [15] only occur with 4-linked GlcNAc and Glc, resulting in fragmen

Determination by electrospray mass spectrometry and 1H-NMR

spectroscopy of primary structures of variously fucosylated neutral

Heide Kogelberg1, Vladimir E Piskarev2, Yibing Zhang1, Alexander M Lawson1and Wengang Chai1

1

MRC Glycosciences Laboratory, Imperial College Faculty of Medicine, Northwick Park Institute for Medical Research, Harrow, Middlesex, UK;2Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences, Moscow, Russia

We have isolated a nonfucosylated and three variously

fucosylated neutral oligosaccharides from human milk that

are based on the iso-lacto-N-octaose core Their structures

were characterized by the combined use of electrospray

mass spectrometry (ES-MS) and NMR spectroscopy The

branching pattern and blood group-related Lewis

deter-minants, together with partial sequences and linkages of

these oligosaccharides, were initially elucidated by

high-sensitivity ES-MS/MS analysis, and then their full structure

assignment was completed by methylation analysis and

1H-NMR Three new structures were identified The

nonfucosylated iso-lacto-N-octaose, Galb1–3GlcNAcb1–

3Galb1–4GlcNAcb1–6[Galb1–3GlcNAcb1–3]Galb1–4Glc, has not previously been reported as an individual oligo-saccharide The monofucosylated and trifucosylated iso-lacto-N-octaose,Galb1–3GlcNAcb1–3Galb1–4(Fuca1–3) GlcNAcb1–6[Galb1–3GlcNAcb1–3]Galb1–4Glc and Galb1– 3(Fuca1–4)GlcNAcb1–3Galb1–4(Fuca1–3)GlcNAcb1– 6[Galb1–3(Fuca1–4)GlcNAcb1–3]Galb1–4Glc, both con-taining an internal Lexepitope, are also novel structures Keywords: electrospray mass spectrometry; human milk; oligosaccharide; NMR

A role for carbohydrates in cellular events has long been

hypothesized, although strong evidence for this has only

emerged over the last two decades Awareness of the

biological function of oligosaccharide chains in

glycopro-teins, glycolipids and proteoglycans has intensified as an

increasing number of examples have been reported that

reveal that carbohydrate structures participate in various

biological events in addition to modifying protein function

One of the early demonstrations of the role of

carbo-hydrates in recognition was binding of the influenza virus to

red blood cells via sialic acid [1], and later by work on the

chemical basis of the antigenicity of polysaccharides and

of the well-known ABO (H) blood-group system [2,3],

in which specificity is determined by oligosaccharide

sequences Carbohydrates are well placed to act in cellular

recognition as many cells are surrounded by an

oligosac-charide layer made from cell-associated glycoconjugates,

which often overshadows protein and lipid components on

the cell surface Specific oligosaccharide sequences, such

as the type 1 (Galb1–3GlcNAc)/type 2 (Galb1–4GlcNAc)

chains and the blood group-related antigens bearing the

H (Fuca1–2Galb1–3/4GlcNAc), Lewisa [Lea, Galb1– 3(Fuca1–4)GlcNAc] and Lewisx [Lex, Galb1–4(Fuca1– 3)GlcNAc] determinants, occur naturally as structural elements of free oligosaccharides or on the carbohydrate chains of glycoproteins and glycolipids and comprise recognition motifs for cell–cell and cell–matrix interactions [4,5]

Human milk is a unique source of diverse oligosaccha-rides, and more than 80 have been isolated and sequences assigned [6] Many of these structures are closely related to the carbohydrate chains of glycoproteins and glycolipids [7] These diverse oligosaccharide sequences may also serve as cell differentiation and tumour antigens [5] Milk oligosac-charides are considered to play a part in the inhibition of bacterial adhesion to epithelial surfaces, as they are able to mimic the binding epitope of the epithelial receptor [8] Also, milk contains oligosaccharides that resemble structures recognized by the cell–cell adhesion molecules, the selectins, suggesting a role in inflammatory processes [8,9] Human milk has also been used as a rich source of oligosaccharides

to map the fine binding specificity of E-selectin [10]

In contrast with oligonucleotides and peptides, oligosac-charides can be branched, and hence a relatively simple set

of monosaccharides can form a huge number of complex structures A greater degree of structural complexity produced by branching is the norm for naturally occurring carbohydrates, and often a branched sequence carrying two

or more recognition motifs is more potent [11,12] Free oligosaccharides from human (milk, urine and infant faeces) have a common lactose (Galb1–4Glc) core It can be extended, for example, at the 4-position of the Gal as a linear sequence or at its 3,6-positions as a branched sequence The linear and branched chains are often

Correspondence to W Chai, MRC Glycosciences Laboratory,

Imperial College Faculty of Medicine, Northwick Park Institute for

Medical Research, Watford Road, Harrow, Middlesex HA1 3UJ,

UK Fax: + 44 20 8869 3253, Tel.: + 44 20 8869 3252,

E-mail: w.chai@imperial.ac.uk

Abbreviations: CID, collision-induced dissociation; ES-MS,

electro-spray mass spectrometry; iLNO, iso-lacto-N-octaose; Le a , Lewis a;

Lex, Lewis x; PMAA, partially methylated alditol acetate; rOe,

rotating frame nuclear Overhauser enhancement.

(Received 4 December 2003, accepted 3 February 2004)

fucosylated to varying degrees to form several of the blood

group-related antigens

Methods for detailed characterization of these

recogni-tion motifs are important in modern structural cell biology

to derive structure/function relationships, particularly in the

postgenome era, in order to understand post-translational

glycosylation and its function Expansion of our knowledge

on the repertoire of carbohydrate structures, the
glycome,

and investigation of oligosaccharide epitopes involved

in carbohydrate–protein interactions require their detailed

isolation and structural determination With small amounts

of material (e.g a few picomoles), no single analytical

technique is capable of the complete characterization of an

oligosaccharide structure Structure elucidation is therefore

usually achieved by using several different techniques, of

which MSand NMR are two of the most powerful

Previously, we demonstrated the distinction of chain type

and blood-group type (such as Lea/xand Leb/y) of

underi-vatized oligosaccharides by negative-ion electrospray mass

spectrometry (ES-MS) with collision-induced dissociation (CID) and MS/MS scanning with low picomole sensitivity [13,14] Several characteristic fragmentations are useful for obtaining detailed structural information The double glycosidic D-type cleavage [13,14] is unique to 3-linked GlcNAc and Glc residues, whereas0,2A-type cleavages [15] only occur with 4-linked GlcNAc and Glc, resulting in fragment ions, indicating type 1 and type 2 chains or blood group types For a 3-linked GlcNAc or Glc, the D-type cleavage occurs at the glycosidic bonds of both reducing and nonreducing sides of the residue, by combined C-type and Z-type cleavages (see Results for discussion and Figs 1–4 for illustration) For -3GlcNAc- without further substitution, a fragment at m/z 202 is obtained If a Fuc is present at the 4-position of the -3GlcNAc- (e.g in the case of an Lea determinant), a fragment at m/z 348 (202 +146) results, whereas if the 4-position is substituted by Gal (e.g in the case of an Lexdeterminant), a unique fragment at m/z 364 is produced Therefore, a D-fragment at m/z 202 indicates a

Fig 1 Product-ion spectra of iLNO with [M-H]–(A) and [M-2H]2–(B) as precursors The structure of iLNO is shown to indicate the proposed fragmentation The nomenclature used to define the cleavage is based on that introduced previously [15], and ions marked with
h are fragments produced by dehydration of the major ions.

type 1 chain, whereas an0,2A-ion doublet at m/z 281/263

indicates a type 2 chain D-ions at m/z 348 or m/z 364 are

characteristic of either terminal Lea or Lex determinants,

respectively [13] We also established a method for

core-branching pattern analysis using CID MS/MS of singly and

doubly charged molecular ions [14] These spectra give

complementary structural information In the CID spectra

of [M-H]–, fragment ions from the 6-linked branch are

dominant, and those from the 3-linked branch are absent,

whereas fragment ions from both branches occur in the

product-ion spectra of [M-2H]2– This allows us to

distin-guish between fragment ions derived from either the 3- or

the 6-branch and to deduce the branching pattern and also

assign structural details of the 3- and 6-branches

Although MSis the more sensitive method of structural

analysis, NMR spectroscopy is the choice for more

complete assignment of carbohydrate structure when

suffi-cient material is available In this report, we demonstrate

our strategy of the combined use of ES-MS and NMR for

analysis of the core-branching pattern and full sequence assignment of four oligosaccharides isolated from human milk These are variously fucosylated structures based

on the iso-lacto-N-octaose core, of which three are novel sequences

Materials and methods

Isolation and purification of oligosaccharides Oligosaccharides were isolated from human milk obtained from a healthy 25-year-old woman, blood group B secretor, Lebpositive, giving negative reaction in hepatitis

B and HIV tests Written consent was obtained from this volunteer for analysis of the milk sample Fat was removed by centrifugation at 4C (5000 g, 30 min) and proteins by precipitation with cold 50% (v/v) acetone Oligosaccharides were separated from lactose on a Sephadex G-25 column (5· 90 cm), and then neutral

Fig 2 Product-ion spectra of MFiLNO with [M-H] – (A) and [M-2H] 2– (B) as precursors The structure of MFiLNO is shown to indicate the proposed fragmentation The nomenclature used to define the cleavage is based on that introduced previously [15], and ions marked with
h are fragments produced by dehydration of the major ions.

from acidic oligosaccharides on a Dowex 1 (· 2; 100–200

mesh; acetate form) column Further gel-filtration

chro-matography was carried out on a Fractogel HW-40(S)

column (5· 90 cm) Oligosaccharides were eluted from

the gel-filtration and ion-exchange columns with distilled

water The partially resolved octasaccharide to

undeca-saccharide fractions were further fractionated by

normal-phase HPLC on a preparative Separon amino column

(10· 250 mm) by elution with 50% (v/v) acetonitrile to

give octasaccharide (F8), nonasaccharide (F9),

decasac-charide (F10) and undecasacdecasac-charide (F11) fractions Each

subfraction was purified by reverse-phase HPLC on a

Zorbax octadecyl column (10· 250 mm) by elution with

water The octasaccharide iso-lacto-N-octaose (iLNO) was

obtained from F8, monofucosyl iLNO (MFiLNO) from

F9, difucosyl iLNO (DFiLNO) from F10, and trifucosyl

iLNO (TFiLNO) from F11 Repeated normal-phase

HPLC was carried out to ensure the purity of each

oligosaccharide fraction before their analyses by ES-MS

and1H-NMR spectroscopy

Methylation analysis After initial reduction with NaBD4, oligosaccharides were methylated, hydrolysed, reduced, and acetylated as des-cribed previously [16] GC-MSanalysis of the partially methylated alditol acetates was performed on a Thermo-Quest Trace system using a 15-m RTX-5 capillary column The initial column temperature was 50C programmed to

100C at 25 CÆmin)1, to 220C at 5 CÆmin)1 and to

310C at 10 CÆmin)1 ES-MS

Negative-ion ES-MS and CID MS/MS were carried out on

a Micromass (Manchester, UK) Q-Tof mass spectrometer Nitrogen was used as desolvation and nebuliser gas at a flow rate of 250 LÆh)1 and 15 LÆh)1, respectively Source tem-perature was 80C, and the desolvation temperature

150C Typically, a cone voltage of 80 V was used for CID MS/MS of singly charged ions [M-H]–and 50 V for

Fig 3 Product-ion spectra of DFiLNO with [M-H] – (A) and [M-2H] 2– (B) as precursors The structure of DFiLNO is shown to indicate the proposed fragmentation The nomenclature used to define the cleavage is based on that introduced previously [15], and ions marked with
h are fragments produced by dehydration of the major ions.

doubly charged ions [M-2H]2– The capillary voltage was

maintained at 3 kV Product-ion spectra were obtained

from CID using argon as the collision gas at a pressure of

0.17 MPa The collision energy was adjusted to 23–43 V for

optimal fragmentation and, typically, 40–43 V was used for

CID of [M-H]–, and 23–27 V for [M-H]2– A scan rate of

1.5 s per scan was used for both ES -MS and CID MS /MS

experiments, and the acquired spectra were summed for

presentation

For analysis, oligosaccharides were dissolved in

acetonit-rile/water (1 : 1, v/v), typically at a concentration of

5–10 pmolÆlL)1, of which 5 lL was loop-injected Solvent

(acetonitrile/1 mMammonium bicarbonate, 1 : 1, v/v) was

delivered by a Harvard syringe pump (Harvard Apparatus,

Holliston, MA, USA) at a flow rate of 10 lLÆmin)1

Alternatively, 0.5–1 lL sample solution was placed in a

capillary needle for the nanospray experiment

NMR spectroscopy

Oligosaccharides were coevaporated with 2H2O (99.9

atom% 2H2) twice by lyophilization and dissolved in

500 lL high-quality2H2O (100.0 atom%2H2), containing

0.1 lL acetone

NMR spectra were acquired on Varian (Palo Alto, CA,

USA) Unity plus-500 (500.07 MHz 1H), Unity-600

(599.89 MHz1H) and Unity-800 (800.27 MHz 1H)

spec-trometers and processed with standard Varian software

The observed1H chemical shifts were reported relative to

internal acetone (2.225 p.p.m.) The NMR spectra were

recorded at 15C for iLNO, 10 C for MFiLNO, 13 C for

DFiLNO, and 27C for TFiLNO The temperatures were

chosen in order to place the HO signals with minimal

disturbance to carbohydrate protons For MFiLNO, for example, a temperature of 10C placed the H2O signal optimally downfield from the Fuc IX H5 proton For TFiLNO, the same temperature would have resulted in total overlap of the H2O signal with the Fuc X and XI H5 protons Therefore the temperature of 27C was chosen for TFiLNO This placed the H2O signal between Fuc IX H5 and the GlcNAc V and VII H1 protons; nevertheless, the Fuc IX H5 was slightly obscured

2D phase-sensitive TOCSY spectra were recorded at mixing times of 10, 30, 50, 70 and 140 ms A spectral width

of 3500 Hz was used in both dimensions, with eight scans per increment 2D phase-sensitive ROESY experiments were performed with a mixing time of 300 ms, a spectral width of 8000 Hz in both dimensions, and 16 scans per increment The spectrum offset was set 1.5 p.p.m to lower field of the most downfield shifted proton, Glc1a,

to minimize TOCSY transfer The raw data sets of the homonuclear 2D experiments typically consisted of 4K· 256 complex data points

Results

Determination of branching pattern and blood group determinants by ES-MS/MS

Negative-ion CID ES-MS/MS product ion scanning with the strategy established previously [13,14] was used first for analysis of the branching patterns and blood group determinants with low (picomole) amounts of the underi-vatized oligosaccharides The significant aspects of this strategy are the combined use of CID MS/MS of singly and doubly charged molecular ions as precursors to deduce the Fig 4 Product-ion spectrum of D 2b-5 ion m/z 1183 of DFiLNO as precursor (A) and the D 2b-5 ion m/z 1037 of a contaminant in DFiLNO as the precursor (B).

branching pattern, sequence, and partial linkages, and

assign structural details of each branch (e.g the 3- and

6-branches) Blood group H, Lea/Lex and Leb/Ley

deter-minants, together with type 1 and type 2 chains, can be

determined from their unique fragment ions

iLNO The singly charged molecular ion [M-H]– at m/z

1436 (Fig 1A) and doubly charged [M-2H]2–at m/z 717.8

(Fig 1B) are consistent with an octasaccharide of

compo-sition Hex5HexNAc3 The approximate relative proportions

of partially methylated alditol acetates (PMAAs) from

methylation analysis (Table 1) are in agreement with this

and indicate that the monosaccharide residues include one

reducing terminal 4-linked Glc, four Gal (two terminal, one

internal 3-substituted and one 3,6-disubstituted) and three

GlcNAc (one 4-linked and two 3-linked) As established

previously, the product-ion spectrum of [M-H]–only shows

the fragment ions on the 6-linked branch The C-type ions

C1a, C2a, C3a, C4aand C5(see the fragmentation scheme in

Fig 1) are in agreement with a 6-linked branch

Gal-GlcNAc-Gal-GlcNAc-, while ions D2b-5and 0,3A5at m/z

891 and 819, respectively, define a 3,6-branched core Gal

[14] The mass difference between the ion C5(m/z 1274) and

[M-H]–(m/z 1436) indicates a Hex at the reducing terminus

The characteristic0,2A6doublet (m/z 1376/1358) and2,4A6

(m/z 1316) ions confirm this to be a -4Glc [13,14] Thus, a

reducing terminal core 3,6-disubstituted -Gal-4Glc can be

proposed, in agreement with methylation analysis

Further-more, 3-substituted GlcNAc next to the nonreducing

terminal Gal can be deduced from a D1a-2aion [13] at m/z

202 while the HexNAc linked to the core Gal is deduced to

be a -4GlcNAc from the unique0,2A fragmentation (ions at

m/z 646/628) Together with the monosaccharide linkage

analysis data (Table 1), this further defines the sequence to

be

Gal1–3GlcNAc1–3Gal-4GlcNAc-6(Gal-GlcNAc-3)Gal-4Glc From the knowledge that the product ion spectrum of

[M-2H]2–shows fragment ions from both branches, and as

no additional fragment ions are apparent, it can be deduced that the disaccharide sequence on the 3-branch shares the same terminal sequence Gal1–3GlcNAc- Taken together, these features indicate iLNO to be:

MFiLNO The singly charged molecular ion [M-H]– at m/z 1582 (Fig 2A) and doubly charged [M-2H]2– at m/z 790.8 (Fig 2B) are consistent with a nonasaccharide of composition dHex1Hex5HexNAc3 The approximate rel-ative proportions of PMAAs from methylation analysis (Table 1) are in agreement with this and indicate that the monosaccharide residues include Fuc, in addition to one reducing terminal 4-linked Glc, four Gal (two terminal, one internal 3-linked and one 3,6-disubstituted) and three GlcNAc (one 3,4-disubstituted and two 3-linked) The location of the Fuc residue can be deduced from the CID MS/MS fragmentation In comparison with the spectrum

of iLNO (Fig 1A), the D2b-5 ion has shifted 146 Da to m/z1037 corresponding to the presence of a fucose on the 6-branch The fragment ions at m/z 161, 382, 544 and 202

as B1a, C2a, C3aand D1a-2a, respectively, are the same as those in the spectrum of iLNO (Fig 1A) However, C4a has shifted from m/z 747 for iLNO to m/z 893, indicating the Fuc is at the internal 3,4-disubstituted GlcNAc The absence of a 0,2A4a doublet and the presence of a D4f-4a ion at m/z 729 (see the fragmentation scheme in Fig 2) are in agreement with a Fuc at the 3-position of a GlcNAc, indicating a Lex determinant Again, no addi-tional fragment ions were revealed in the product ion spectrum of the doubly charged precursor (Fig 2B), confirming that the 3-linked disaccharide branch is the

Table 1 Linkage and monosaccharide composition assignment from methylation analysis of milk oligosaccharides PMAA, partially methylated alditol acetate Molar ratios are relative to 1,5-di-O-acetyl-2,3,4,6-tetra-O-methylgalactitol –, Not detected.

Molar ratio

Fucitol

Glucitol

Galactitol

N-Acetylglucosaminatol

same as the terminal disaccharide sequence of the

6-linked branch Hence, this monofucosylated

octasaccha-ride, MFiLNO, can be assigned as:

DFiLNO The singly charged molecular ion at m/z 1729

and the doubly charged ion at m/z 863.8 in the spectrum

of DFiLNO (Fig 3) are consistent with a difucosylated

octasaccharide (dHex2Hex5HexNAc3) in agreement with

methylation analysis (Table 1), which also shows a

mono-saccharide composition corresponding to MFiLNO with an

additional Fuc It is apparent that both fucose residues are

located in the 6-linked branch, as evidenced by the unique

and intense D2b-5ion at m/z 1183 (see the fragmentation

scheme in Fig 3), from cleavage of the core residue Gal

(Fig 3A), and by0,3A5at m/z 1111 The C2aion at m/z 528

shows that one Fuc is at the subterminal GlcNAc, and the

characteristic D1a-2aion [13] at m/z 348 is consistent with a

Fuc 4-linked to the GlcNAc of a terminal Leadeterminant

As the C3aion is at m/z 690, this excludes the possibility of

the other Fuc being at the Gal next to the subterminal

GlcNAc The position of the second Fuc 3-linked to the

internal GlcNAc forming an internal Lex determinant is

deduced from the characteristic double cleavage D4f-4aion

at m/z 875 The ions at m/z 1037, m/z 729 and m/z 544 are

similar to those in the spectrum of MFiLNO (Fig 2), in

which only one Fuc is in the 6-branch, and believed to be

from a contaminant (see below), having the same molecular

mass with one Fuc in each of the 3- and 6-branches

Comparison of the product-ion spectra of the singly and

doubly charged molecular ion precursor (Fig 3A,B)

show the major additional ions in the doubly charged ion

spectrum to be m/z 202 and m/z 382 The former derives

from a D1b-2bcleavage consistent with a 3-linked GlcNAc,

and the latter from a glycosidic C2b cleavage, which

supports assignment of the unsubstituted disaccharide

sequence on the 3-branch The ion at m/z 803 is the doubly

charged2,4A6fragment, which appears as a singly charged

ion (m/z 1608) in the product-ion spectrum of the singly

charged precursor (Fig 3A) The product-ion spectra of the

doubly charged precursors generally give more intense

fragment ion peaks

The ions at m/z 1037, m/z 729 and m/z 544 appearing in

both spectra are similar to those in the spectra of MFiLNO

(Fig 2), in which only one Fuc is in the 6-branch, and, as

indicated above, are believed to be from a contaminant with

the same molecular mass with one Fuc in each of the 3- and

6-branches This is confirmed by MS/MS scanning of the

D2b-5 ions m/z 1183 (Fig 4A) and m/z 1037 (Fig 4B) as

precursors, produced by cone voltage fragmentation [14]

The fragment ions of m/z 729 and m/z 544 are both from the

monofucosylated D2b-5 ion m/z 1037 and not from the

difucosylated D2b-5ion m/z 1183

Thus the major component, the difucosylated branched

octasaccharide, can be assigned as:

and the minor component as:

TFiLNO The singly charged molecular ion [M-H]–at m/z

1874 (Fig 5A) and doubly charged [M-2H]2–at m/z 937.0 (Fig 5B) are consistent with a trifucosylated octasaccharide with composition Fuc3Hex5HexNAc3 The approximate relative proportions of PMAAs from methylation analysis (Table 1) are in agreement with this and indicate that the monosaccharide residues include three terminal Fuc1-, two terminal Gal1-, one reducing terminal -4Glc, together with internal monosubstituted and disubstituted residues: one -3Gal1-, one -3,6Gal1-, three -3,4GlcNAc1- The branching pattern and the locations of the three fucose residues can

be deduced by similar reasoning to that for the other fucosylated analogues The product-ion spectrum of the singly charged precursor (Fig 5A) is very similar to that of DFiLNO (Fig 3A), indicating a similarly difucosylated tetrasaccharide sequence on the 6-branch The remaining Fuc is on the 3-branch, as indicated by C5at m/z 1712 and

D2b-5at m/z 1183 (Fig 5) It can be deduced that the Fuc is 4-linked to the -3GlcNAc-, forming a second terminal Lea

determinant, as no additional fragment ions are apparent

in the spectrum of doubly charged precursor Hence the trifucosylated octasaccharide contains two terminal Leaand one internal Lexdeterminants with the sequence:

Completion of sequence assignment by1H NMR Homonuclear NMR was carried out to verify the MS assignment of the iLNO core structures and its variously fucosylated analogues, and to determine their anomeric configurations and linkages The proton chemical shifts of

individual monosaccharide residues were assigned with the

aid of TOCSY spectra with increasing mixing times (data

not shown) Oligosaccharide sequences were established

from interresidue rotating frame nuclear Overhauser

enhancements (rOes) in combination with chemical shifts

iLNO The monosaccharide composition of iLNO was

shown by methylation analysis to comprise one Glc

(reducing end), four Gal (two terminal) and three GlcNAc

residues (see above) Their1H chemical-shift assignments,

obtained from TOCSY spectra (Fig 6), are given in

Table 2 Anomeric proton chemical shifts of four Gal

residues are present between 4.47 and 4.427 p.p.m., while

those of GlcNAc are between 4.71 and 4.634 p.p.m The

reducing terminal Glc I residue (see structure in Fig 6 and

below) is indicated by the respective chemical shifts of

the a-anomers and b-anomers at 5.22 and 4.665 p.p.m

(Table 2) All residues are in b-anomeric linkages, as

deduced from H1,H2coupling constants between 8.0 and

8.3 Hz (Table 2)

Sequence assignment of iLNO was derived from interresidue rOes (Table 2) and confirmed the MSassign-ment The reducing Glc I is deduced to be substituted at position 4, as the b-anomer of Gal II H1 gives an rOe to this proton Gal II is a branching point and substituted at positions 3 and 6, as GlcNAc III H1 gives an rOe to H6a and H6b of Gal II, and GlcNAc VII H1 gives an rOe to the H3 of this residue The 3-branch is terminated by Gal VIII which is linked to the 3-position of GlcNAc VII, as Gal VIII H1 gives an rOe to GlcNAcVII H3 In addition, the GalVIIIb1–3GlcNAcVII linkage is supported by

an rOe between GalVIII H1 and NAc protons of GlcNAc VII

Extension of the 6-branch is by a GalIVb1–4GlcNAcIII linkage, as GalIV H1 gives an rOe to H4 of GlcNAc III Gal

IV is further extended by a GlcNAcVb1–3GalIV linkage, as GlcNAc V H1 gives an rOe to Gal IV H3 Finally, a terminal Gal VI is linked b1–3 to GlcNAc V, as Gal VI H1 gives an rOe to GlcNAc V H3, and Gal VI H1 gives an rOe

to the NAc protons of GlcNAc V

Fig 5 Product-ion spectra of TFiLNO with [M-H]–(A) and [M-2H]2–(B) as precursors The structure of TFiLNO is shown to indicate the proposed fragmentation The nomenclature used to define the cleavage is based on that introduced previously [15], and ions marked with
h are fragments produced by dehydration of the major ions.

The structure of iLNO is further supported from

comparisons of chemical shifts with those reported

previ-ously for similar oligosaccharides The proton chemical

shifts of the Gal II and Glc I residues and residues on the

3-branch (VII and VIII) are almost identical with those

reported previously for an iso-lacto-N-octaose derivative that is difucosylated on the 6-branch [17] Furthermore the proton chemical shifts of the structural reporter protons of GlcNAc III are almost identical with those reported for the GlcNAcb1–6 residue of lacto-N-hexaose [18]

Fig 6 1D and 2D1H NMR spectra (800 MHz) of iLNO, region 5.5–3.0 p.p.m., at 15 °C Upper trace,1H NMR spectrum; top-left half, 300-ms ROESY spectrum and bottom-right half, 140-ms TOCSY spectrum The structure is shown at the top, depicting the residue labelling.

Taken together these data allow assignment of the iLNO

structure as follows:

TFiLNO From methylation analysis, it was deduced that

TFiLNO comprises four Gal residues, three GlcNAc

residues, one reducing Glc residue, and three fucose residues

(see above).1H chemical-shift assignments of these residues

(Table 3) were made from TOCSY spectra (see Fig 8) with

increasing mixing times (data not shown) The Gal and

GlcNAc residues are all in b-anomeric linkages as deduced

from H1,H2 coupling constants between 7.8 and 8.0 Hz,

while the fucose residues are a-linked to their neighbouring

residues apparent from H1,H2coupling constants of 3.7 and

3.8 Hz (Table 3)

The sequence of the oligosaccharide was derived from

interresidue rOes (Table 3, Figs 7 and 8) which confirmed

the MSassignment The reducing Glc residue is substituted

at position 4; rOes are observed between Gal II H1 and

Glc I H4 in the b-anomer Gal II is a branching point and

substituted at positions 3 and 6, as GlcNAc III H1 gives an

rOe to H6b of this residue, while GlcNAc VII H1 gives an

rOe to H3 of Gal II The 3-branch is further extended by a

terminal Gal VIII residue, as Gal VIII H1 gives an rOe to

GlcNAc VII H3 The Gal VIIIb1–3GlcNAc VII linkage is

further supported by the rOe between Gal VIII H1 and the

NAc protons of GlcNAc VII GlcNAc VII is also

substi-tuted by Fuc XI, which is linked to the 4-position of this

residue, as Fuc XI H1 gives an rOe to GlcNAc VII H4 The

terminal trisaccharide, Gal VIIIb1–3(Fuc XIa1–4)GlcNAc

VII, the Leaepitope, also shows remote interresidue rOes of

Fuc XI H5 and Fuc XI CH3 to Gal VIII H2 This is a

characteristic rOe pattern for this epitope, resulting from

stacking interactions of the Gal VIII and Fuc XI residues

[19,20]

The GlcNAc III on the 6-branch is substituted at

positions 3 and 4, as Gal IV H1 gives an rOe to H4 of

GlcNAc III, and Fuc IX H1 gives an rOe to H3 and to the NAc protons of this residue The trisaccharide, Gal IVb1– 4(Fuc IXa1–3)GlcNAc III, the Lex epitope, is further characterized by remote rOes between Fuc IX H5 and Gal

IV H2 and between Fuc IX CH3 and Gal IV H2 The latter rOes are similar to those seen for the Lea epitope (see above), as the Lexepitope also shows stacking interaction between the Fuc IX and Gal IV residues, resulting from very similar conformational features ([21] and references therein) Gal IV on the 6-branch is further extended at the 3-position,

as GlcNAc V shows an rOe to H3 of Gal IV GlcNAc V is substituted at the 3- and 4-position, as Gal VI H1 gives an rOe to GlcNAc V H3 and to the NAc protons of GlcNAc

V, while Fuc X H1 gives an rOe to GlcNAc V H4 This second Lea epitope (see also 3-branch above) also shows remote rOes between H5 and CH3 of Fuc X and H2 of Gal VI

The TFiLNO structural assignment is further supported

by similar chemical shifts observed for the protons of residues on the 6-branch to those previously reported for an iso-lacto-N-octaose that contains this difucosylation on the 6-branch [17]

Thus, TFiLNO has the following structure:

MFiLNO The monosaccharide composition of MFiLNO was shown by methylation analysis to consist of four Gal residues, three GlcNAc residues, one reducing Glc residue, and one fucose residue (see above) 1H chemical-shift assignments of these residues (Table 4) were made from TOCSY spectra The Gal and GlcNAc residues are involved

Table 2.1H chemical shifts, H 1 ,H 2 coupling constants, and intermolecular rOes from NMR spectra of iLNO Chemical shifts from a 1D1H 800-MHz spectrum recorded at 15 C are given to three decimals Other chemical shifts were taken from 2D spectra.

Chemical shifts in p.p.m and (H1,H2 coupling constants in Hz)

rOes (from H1)

a Intramolecular rOes originating from Gal IV H1.