WO1992014151A1

WO1992014151A1 - Purified lam and synthetic analogs thereof

Info

Publication number: WO1992014151A1
Application number: PCT/US1992/001129
Authority: WO
Inventors: Patrick J. Brennan; Delphi Chatterjee
Original assignee: Dynagen, Inc.; Colorado State University Research Foundation
Priority date: 1991-02-12
Filing date: 1992-02-12
Publication date: 1992-08-20
Also published as: AU1455192A

Abstract

Methods for purifying LAM from whole bacteria in commercially useful quantities are provided. Methods for synthetically producing portions of LAM also are provided. Further, kits, pharmaceutical compositions and devices containing or including purified LAM or synthetic analogs thereof are provided.

Description

PURIFIED LAM AND SYNTHETIC ANALOGS THEREOF

Field of the Invention

This invention relates to purified

lipoarabinomannan (LAM) from Mycobacterium spp., synthetic analogs of LAM and various diagnostic, therapeutic and immunologic applications thereof.

Background of the Invention

LAM is a major constituent of the cell wall that is widely distributed within the Mycobacterium

species. It is believed to constitute the major carbohydrate-containing immunogen recognized by sera from patients with tuberculosis and leprosy.

Recently, native LAM has been partially purified from Mycobacterium tuberculosis and Mycobacterium leprae. This LAM was analysed and found to contain the predominant saccharide units arabinose and mannose, in addition to lactate, succinate and

phosphatidylinositol. The structure of LAM, however, has never been determined.

Understanding the structure of LAM would allow a better understanding of its mechanism of action. For example, it has been postulated that LAM has

biological attributes such as suppression of

T-lymphocyte activation, inhibition of

gamma-interferon activation of macrophages, induction of the release of tumor necrosis factor, and a generalized inhibition of antigen presentation by antigen presenting cells. By understanding the structure of LAM, it may be determined which portions of this complex molecule produce the foregoing effects. Then, therapeutic measures based upon this knowledge could be derived. Likewise, synthetic LAM or portions thereof could be constructed for

therapeutic, diagnostic, and other purposes.

Current attempts to purify LAM have been only partly successful. While it appeared that LAM had been purified in minute quantities using

gel-electrophoresis, it was still uncertain whether there were contaminants underlying the very broad LAM band. Moreover, the electrophoresis procedure did not permit generating sufficient quantities of the purportedly pure LAM for sufficient structural analysis, and certainly not for commercial purposes such as for use in immunoassays, antibody

purification schemes, therapeutics and the like.

Summary of the Invention

It is an object of the invention to purify LAM, particularly in commercially useful quantities.

It is another object of the invention to develop diagnostic, therapeutic, immunologic and other applications involving purified LAM.

Still another object of the invention is to develop diagnostic, immunologic and other

applications involving synthetic constructs including portions of LAM.

According to one aspect of the invention, LAM is obtained in pure form. The method for purifying LAM from whole bacteria is conducted without the use of preparative gel-electrophoresis. In one method, an ion exchange separation step and an HPLC separation step are employed. In another method, a sonication step, a precipitation step and a sizing column separation step are employed.

The purified LAM may be used for diagnostic, immunologic and other applications. For example, the purified LAM may be attached to a substrate, such as a microtiter plate or a column, for the purpose of conducting an immunoassay or a column separation, respectively. The LAM also may be formulated with a pharmaceutically acceptable carrier into a

pharmaceutical composition, such as to deliver therapeutic molecules. The purified LAM likewise may be included in kits for conducting immunoassays.

According to another aspect of the invention, the structure of LAM is provided. LAM comprises three major portions, a phosphatidylinositol membrane anchor, a mannan core, and an arabinan non-reducing antigenic end. The antigenic end is comprised of at least four major structural motifs. One is an internal, linear stretch of 5-linked

α-arabinofuranosyl (Araf) residues. Another is an internal branched 3,5-linked α-D-Araf unit with

5-linked α-D-Araf residues attached at both

branched positions. Another is

ß-D-Araf-(1→2)-α-D-Araf-(1→-5)-α-D-Araf-.

Still another major structural motif is

[ß-D-Araf-(1→2)-α-D-Araf-(1→-]₂→-(3 and

5)-α-D-Araf-. This latter motif also represents

the terminal segments of the peptidoglycan-bound arabinogalactan of Mycobacterium spp.

According to yet another aspect of the

invention, synthetic arabinofuranosyl

oligosaccharides are provided. Preferably, such arabinofuranosyl oligosaccharides include at least one of the foregoing arabinofuranosyl motifs.

Preferably such saccharides are arabinofuranosyl neoglycoconjugates. Such synthetic molecules may be used as a substitute for LAM in diagnostic,

therapeutic, immunologic, serologic and other such applications.

Brief Description of the Drawings

Fig. 1. is a chemical pathway illustrating the effect of certain reactions on hypothetical

four-linked Arap and five-linked Araf.

Fig. 2 is a graph showing the GC/MS analysis of products A and E .

Fig. 3 is a graph showing the GC/MS analysis of products Y and Z.

Fig. 4 shows the results of the partial, acid hydrolysis of per-O-LAM.

Fig. 5A shows the total ion chromatogram of fraction number 51, the fraction which contained compound 12.

Fig. 5B shows the mass spectrum of compound number 12.

Fig. 5C shows the ¹H-NMR of compound number 12.

Fig. 6 shows four major structural motifs of LAM. Fig. 7 is a graph comparing the ¹³C-NMR spectrum of LAM (A) to that of solubilized

arabinogalactan (B).

Fig. 8 shows the D-arabinopentaose epitope of the LAM from M. leprae.

Fig. 9 is a trisaccharide intermediate used in forming the compound of Fig. 8.

Fig. 10 shows a protected form of the compound of Fig. 8.

Fig. 11 schematically illustrates the chemical synthesis of a linear alpha (1 → 5) Araf segment.

Detailed Description of the Preferred Embodiment LAM is purified, characterized and portions synthesized according to the invention.

LAM may be seen schematically as follows:

Purified LAM may be used in a variety of contexts to achieve a variety of purposes according to the invention. Initially, the ability to purify LAM in significant quantity has allowed its structural characterization, as will be described in greater detail below. Such structural characterization, in turn, permits the synthesis of neomolecules

representing portions of LAM and capable of being used in a variety of contexts.

As used herein, the term "neoportions" means synthetic portions of LAM derived without use of naturally occurring LAM. Such neoportions may be used alone or conjugated to larger, carrier molecules

The term "neomolecules of LAM" as used herein means neoportions, neoportion conjugates and

fragments of naturally occurring LAM conjugated to carrier molecules.

The term "subject" as used herein means living organisms susceptible to mycobacterial infection. Examples of subjects include humans, dogs, cats, horses and cows.

Purified LAM, as well as portions thereof and neotnolecules may be used as immunogens to stimulate an antibody response in a subject. This may be for the purpose of generating polyclonal antibodies or for generating monoclonal antibodies, such as by generating precursors for hybridoma cell lines.

Other in vivo uses include therapeutic applications, such as in the delivery of therapeutic molecules.

Other therapeutic applications include suppression of T-lymphocyte activation, inhibition of activation of macrophages, and generalized inhibition of antigen presentation by antigen presenting cells.

When used as a therapeutic agent, the purified LAM, portions thereof or neomolecules are

administered in a therapeutically effective amount. An effective amount can be determined on an

individual basis and will be based, at least in part, in consideration of the size of the individual, and therapeutic goal and/or the severity of the symptons to be treated. Thus, an effective amount can be determined by one of ordinary skill in the art employing such factors and using no more than routine experimentation.

Administration of the therapeutic molecules of this invention may be made by any method which allows the therapeutic molecules to reach the target site. Typical methods include oral, rectal, peritoneal, topical, intravenous and subcutaneous applications. If administered orally, the compositions can be in the form of dragees, tablets, syrups and ampules.

When the compounds are administered rectally, the composition can be in the form of a suppository.

When the compounds are administered by topical application, they can be in the form of a gel. Thus, the compounds can be prepared in pharmaceutical preparations containing the compound themselves and a pharmaceutically acceptable carrier. The

pharmaceutically acceptable carrier may be solid or liquid. Examples of liquid carriers include water,

clear aqueous solutions of non-toxic salts, or aqueous solutions containing organic solvents such ethanol. Also suitable are emulsions, such as oil-in-water emulsions. Solid carriers include both nutritive carriers, such as sucrose or gelatin, and non-nutritive carriers, such as cellulose or talc.

Purified LAM, portions thereof or neomolecules also may be used diagnostically, such as in

immunoassays for determining the presence or absence of antibodies to LAM in sera, which could indicate presence or absense of mycobacterial disease.

Typical such assays include direct assays, indirect assays, competitive binding assays and the like, all of which are well known to those of ordinary skill in the art. The purified LAM, portions thereof, or neomolecules may be bound or unbound in such

contexts. If bound, they may be covalently bound to a substrate or passively bound.

Purified LAM, portions thereof or neomolecules also may be used in methods of manufacture, such as for example in screening procedures for identifying and isolating hybridoma cell-lines producing antibody to LAM, or on columns or in gels for isolating antibodies or determining the affinity of antibodies to LAM. Such schemes of using purified antigen are well known to those of ordinary skill in the art.

Portions of LAM or neomolecules may be used to generate or identify antibodies which react only to specific portions of LAM. Such antibodies themselves may be used therapeutically. For example, antibodies

against the core portion of LAM may be used to interfere only with those in vivo functions mediated by the core portion of LAM either solubilized or in its native form on the bacterial cell surface. The same is true for antibodies against the antigenic portions of LAM. Such highly specific antibodies may also be configured as immunotoxins and used

therapeutically. By the same token, target specific neomolecules may be prepared, such neomolecules having the same binding/recognition capability as the corresponding portion of LAM. Such neomolecules may be in the form of conjugates providing for the targeted delivery of conjugated moieties.

The structure of the antigenic end of LAM includes at least four major structural motifs, as follows:

The foregoing motifs may be synthesized in whole or in part and may be used for the purposes described above. In addition, these motifs may be attached to larger carrier-molecules, such as proteins like bovine serum albumin (BSA) to form neoconjugates which may be used in the contexts described above. An advantage to forming a neoconjugate is that a particular motif may be synthesized in great quantity and densely packed on the carrier-molecule, which dense packing would provide for good antigenic presentation as well as favorable kinetics in

immunologic reactions.

EXAMPLE I

Preferred Method of Generation and Purification of LAM from Mycobacterium tuberculosis

Mycobacterium tuberculosis strain TMC 107 (Erdman) is grown for eight weeks in a

glycerol-alanine-salts medium as a shaken culture. The Erdman stain was obtained from the Trudeau

Mycobacterium Culture Collection, Trudeau Institute, Saranac Lake, N.Y. USA, 12983, culture number TMC 107 and is available at the ATCC, No.35801 , Rockville, Md., U.S.A. Other strains may be employed including the rapid growing, attenuated strain H37Ra, obtained from K. Takayama, Madison, WI, described by Takayama, K., et al. (1975), J. Lipid Res., 16, 308-317 and available at the ATCC, No.

LAM may be

isolated from nontuberculosis mycobacteria as well.

The cultures were autoclaved at 80°C for 1 h, cooled and filtered using sterile 0.22 micron

filtration system (Nalge Co., Rochester, NY). The harvested cells were washed several times with distilled water and stored frozen (-20°C) until ready for breakage. Harvested cells (~130 g wet weight) were resuspended in PBS containing 0.5% Triton X100 and 0.02% NaN₃ (200 ml). A thick suspension is desirable in order to achieve complete breakage of cells. LAM, LM and PIM have a great affinity for detergent. Use of Triton X100 when breaking the cells helped to keep most of these amphipathic molecules in solution, thereby giving a maximum yield during acetone precipitation.

The suspension was sonicated while cooling in an ice bath for 10 min with a W-385 Sonicator Ultrasonic

Liquid Processor (Heat Systems-Ultrasonic, Inc., Framingdale, NY) operating at optimal cavitation intensity. The sonicate was passed four times through a French pressure cell (Model SA073; American Instruments Co., Urbana, IL) at 20,000 1b per sq. in. The sonicate pressate was centrifuged at 27,000 × g for 45 min, two times. The pellet was washed twice with the above buffer (50 ml each time) and recentrifuged. The supernatant fluids were combined and recentrifuged (at 27,000 × g) in order to remove most of the cell wall. (The supernatant fluid appeared translucent after centrifugation.)

To the precooled supernatant fluid, distilled acetone was added (to a final concentration of 90% acetone) to precipitate mainly polysaccharides. Some proteins were also precipitated during this

procedure. (Considering that very large volumes of solvents were used, it was more efficient when the supernatants were divided into two 1000 ml Erlenmeyer flasks.) The acetone precipitate was stored at 4°C for 48 h.

The precipitate was collected by centrifugation at 10,000 × g and air dried. Dry precipitate (1 g) was suspended in 6 ml of 6 M guanidine HCl in lOmM Tris HCl, pH 7.4 by pansonication. (The insoluble material remaining is removed by low speed

centrifugation (2000×g) prior to application to the column.) The soluble material is applied to a sephacryl S-400 column (1.5 × 150 cm) in the same buffer. Fractions (2 ml) are collected and monitored by PAGE. Fractions are pooled according to

enrichment with LAM, LM and PIM and dialyzed

extensively against water (5 to 6 changes of water). There is no resolution between LAM, LM or PIM after this preliminary column fractionation. The dialyzed fractions were freeze dried to yield approximately 500 mg of impure material.

Final purification of LAM was achieved by applying the LAM, LM, PIM enriched fraction obtained from the S-400 column above to a Sephacryl S-200 column with a buffer containing 10mM Tris, 0.2 M NaCl, ImM EDTA 0.02% sodium azide and 0.25%

deoxycholate. About 150-180 mg of crude material was applied to a column size of (2.5 × 120 cm) and 4 ml fractions were collected and monitored by PAGE. Use of deoxycholate as a detergent on a simple sizing column keeps LAM, LM and PIM from aggregating;

therefore, they purify rapidly as separate entities. This method of purification replaced several

laborious and tedious ion exchange chromatography steps, as well as purification. Fractions containing pure LAM, LM and PIM (resolved at this stage) were pooled, dialyzed at 37°C for two days (48 hrs.) against the buffer without deoxycholate in order to remove detergent followed by dialysis against water at 4°C for two days (48 hrs.). Pure LAM, LM or PIM was stored as freeze-dried powder.

LAM has many LPS-like biological activities. To ensure that LPS contamination was not present in preparations, lyophilized LAM was redissolved in

pyrogen-free water, filtered through 0.45 μm PTFE filtration unit and passed through 2.0 ml of

Detoxi-Gel column (Pierce Chemical, Rockford, IL), refiltered through a second 0.20 μm sterile filter and the filtrate collected into a sterile, pyrogen free vial using sterile pyrogen-free water to elute it off the gel.

As a means of quality control, all final preparations are subjected to 1) SDS-PAGE and silver stained with a periodate step to visualize the carbohydrates, 2) Western blot using the monoclonal antibody against LAM to verify its LAM content, and 3) Alditol acetate and GC analysis versus neutral sugar standards to estimate arabinose and mannose content.

EXAMPLE II

Alternate Purification of LAM

The isolation of LAM-containing fractions from M tuberculosis H37Ra and primary resolution on columns of DEAE-Sephacel in detergent-containing buffer, have been described (1, 2). In addition to these steps, preparations of LAM, recovered from columns of

DEAE-Sephacel and which were highly pure according to PAGE (1), were dialyzed, concentrated on an Amicon flow cell (10 kDa molecular weight cut-off membrane, Amicon model 8200; Danvers, MA), precipitated with 85% ethanol, and redissolved in 0.01M Tris HCl (pH 7.4) containing 0.1% Triton X-100 and applied to a HYDROPORE Ax HPLC column (21.4 mm × 25 cm, Rainin, Woburn, MA) equilibrated in the same buffer. The column was eluted with the same buffer followed by a shallow gradient of 0 to 0.1M NaCl. Fractions (10 ml) were collected, analyzed for carbohydrate (2) and positive fractions re-examined by PAGE. Pure LM eluted with 0.01M NaCl, followed by PIM which eluted with 0.02M NaCl, and LAM which eluted with 0.05M NaCl. Fractions were pooled and subjected to a folch wash by treating with 6 parts Chloroform:methanol (2:1) and allowed to form a biphase. The aqueous layer was removed and dialyzed, concentrated and dried. LAM was reprecipitated with 85% ethanol at 0°C overnight and then centrifuged at 2000xg. To remove the last traces of detergent, a solution of pure LAM was passed through a column (2 ml) of

Extracti Gel-D (Pierce Chemical Co., Rockford, IL) and eluted with H₂O. Pure LAM (devoid of

impurities) thus obtained from M. turberculosis H37Ra was the subject of the analyses described below.

EXAMPLE III

Procedure for Determining the Structure of the

Antigenic Portion of LAM

A. Procedure for Determination of Ring Form of

Glycosyl Residues in LAM

Pure LAM (10-15 mg) was methylated with CH₃I as described (3, 4). Partial acid hydrolysis with 2M CF₃COOH, reduction with NaB[²H]₄, ethylation

with C₂[²H]₅I, complete hydrolysis, further

reduction with NaB[ ²H]₄ and acetylation, were accomplished as described (4). The resulting partially O-acetylated, partially

O-pentadeuterioethylated, partially O-methylated alditols were analyzed by GC/MS as described (3, 4).

B. Degradation of Per-O-Alkylated LAM, Resolution of Fragments and Analyses

The strategy and exact protocols for the random partial depolymerization of per-O-Me-LAM, followed by reduction with NaB[ ²H]₄ and

per-O-deuterioethylation, have been described in the context of the peptidoglycan-bound arabinogalactan

(3). Exact procedures for initial HPLC separation of the per-O-alkylated oligoarabinosyl arabinitols and subsequent GC/MS resolution and analysis, have also been described (3). Besides GC/MS resolution and analysis of such fractions, other selected fractions were also totally hydrolyzed, reduced, acetylated and analyzed by GC/MS in order to confirm the

substitution arrangements on individual

partially-O-alkylated alditol acetates (3). The per-O-alkylated oligoarabinosyl arabinitols that were the object of H-NMR analysis on intact LAM was done in [²H]₂O as described (3). NMR was

performed on a Bruker ACE-500 or -300 NMR at the Colorado State University Department of Chemistry Central Instrument Facility or at the Regional NMR Center (3). C. Determining Ring Form of Ara Residues in LAM

Pure LAM (15 mg) was per-O-methylated as

described (4) and an aliquot completely hydrolyzed with 2M CF₃COOH at 120°C for 1 hr, reduced,

acetylated and analyzed by GC/MS and GC. Table I shows the glycosyl linkage composition of LAM from M. tuberculosis H37Ra.

TABLE I.

Glycosyl linkage composition of LAM from M. tuberculosis

Product identified

Glycosyl O-Ac O-Me Deduced linkage Mole % residues positions positions and ring form

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

Ara 1,4 2,3,5 t-Araf 7

Ara 1,2,4 3,5 2-Araf 9

Ara 1,4,5 2,3 5-Araf/4-Arap 43

Ara 1,2,4,5 3 2,5-Araf/2,4-Arap 1

Ara 1,3,4,5 2 3 , 5-Araf/3,4-Arap 12

Man 1,5 2,3,4,6 t-Manp 11

Man 1,5,6 2,3,4 6-Manp 6

Man 1,2,5,6 3,4 2,6-Manp 9

Man 1,2,5 3,4,6 2-Manp 0.6

Such conventional methylation analysis can

establish the ring form of a glycosyl residue only if

a methyl substituent appears on O-4 in the case of sugars in the pyranose form or on O-5 for

glycofuranoses. As shown in Table I, all of the mannosyl residues of LAM after methylation contain a methyl group at O-4 and thus are pyranosyl. In addition, the identification of 2,3,5-tri-O-Me-Ara and 3,5-di-O-Me-Ara clearly demonstrated the presence of t-Araf and 2-linked Araf, respectively. However, methylation analysis (Table I) does not help

establish the nature of the ring form of the majority of arabinosyl residues, those devoid of 5-OCH₃ groups. The product identified as 2,3-di-O-Me-Ara (Table I) was shown to be predominantly 5-linked Araf with a very small amount of 4-linked Arap based upon application of two different sets of acid hydrolysis conditions as discussed below. Evidence that the product identified as 3-O-Me-Ara (Table I) is 2,5-linked Araf rather than 4-linked Arap is presented in Reference 3. Evidence that the product identified as 2-O-Me-Ara (Table I) is 3,5-linked Araf rather than 3,4-linked Arap is shown in Table II, discussed below.

To establish the nature of the ring form of the majority of arabinosyl residues lacking 5-O-Me groups, per-O-methylated LAM was partially

hydrolyzed, reduced with NaB[²H]₄, O-ethylated, fully hydrolyzed, again reduced with NaB[ ²H]₄ and

O-acetylated. The effect of this series of reactions on a hypothetical 4-linked Arap and 5-linked Araf is illustrated in Fig. 1. Thus a hypothetical 4-linked Arap residue cleaved by partial hydrolysis at C-4

(but not at C-1) would be converted to

1,5-di-O-Ac-4-O-C₂[²H]₅-2,3-di-O-Me-1-C-²H- arabinitol (product Z, pathway a), whereas a

hypothetical 5-linked Araf residue when cleaved at

C-5 but not at C-1 would produce

1,4-di-O-Ac-5-O-C₂[²H]₅-2,3-di-O-Me-1-C-[²H]- arabinitol (product E, pathway c). In a similar fashion, a hypothetical 4-linked Arap cleaved at C-1 but not at C-4 would yield

4-O-Ac-1,5-di-O-C₂[²H]₅-2,3-di-O-Me-1-C-[²H]- arabinitol (product Y, pathway b). Finally, a hypothetical 5-1inked Araf cleaved at C-1 but not at

C-5 would produce

5-O-Ac-1,4-di-O-C₂H[²H]₅-2,3-di-O-Me-1-C-[²H]- arabinitol (product A, pathway d).

In order that both furanosyl and pyranosyl residues be identified, if present, it was necessary that different sets of acid hydrolysis conditions be applied. One set had to be appropriate for partial cleavage of the relatively acid-labile furanosyl residues, whereas other chosen conditions should be compatible with the relative acid stability of pyranosides. Thus, referring to Fig. 1, under optimum mild partial acid hydrolysis conditions appropriate for furanosides, neither the 4-linked Arap residues or R', if R' is a pyranosyl residue, would be cleaved, and, thus, neither product Y nor Z would be produced. On the other hand, under the stronger partial hydrolysis conditions appropriate

for pyranosides, 4-linked Arap and R' each should be partially cleaved, and both Y and Z should be generated. Similar reasoning indicated that if R' was furanosyl, product Z would be formed under both sets of conditions. Likewise, products A and/or E would be formed from 5-linked Araf residues,

regardless of the ring form of R, if both conditions of partial acid hydrolysis were applied. The products of all of these possible outcomes are readily distinguishable by MS (5).

Accordingly, two portions of the per-O-Me-LAM were partially hydrolyzed with 2 M CF₃COOH (for 1 h) at either 75°C or 95°C. The lower temperature was shown to facilitate cleavage of about one-third of the furanosyl residues, while the higher temperature allowed hydrolysis of about one-third of the Manp residues and thus presumably one-third of any Arap residues that may be present. Each sample was reduced with NaB[²H]₄, pentadeuterioethylated, completely hydrolyzed, reduced with NaB[ ²H]₄,

acetylated and analyzed by GC/MS (Fig. 2 and Fig. 3). Identification of the partially O-alkylated alditol acetate products arising from

per-O-methylated-LAM and structural conclusions are provided in Table II. TABLE II

Identification of the partially O-alkylated alditol

acetates arising from per-O-Me-LAM of M. tuberculosis

and structural conclusions thereof.

GC Product identified Deductions peak

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

Glycosyl Positions(s) of Linkage Position residue O-Ac O-Me O-C ₂[²H]₅ position cleaved

and ring by partial form acid hydrolysis

A Ara 5 2,3 1,4 5-Araf 1

B Ara 3 2 1,4,5 3,5-Araf 1 and 5

C Ara 1,4 2,3,5 - t-Araf -

D Ara 1,4 3,5 2 2-Araf 2

E Ara 1,4 2,3 5 5-Araf 5

F Ara 1,4 2 3,5 3,5-Araf 3 and 5

G Ara 3,5 2 1,4 3, 5-Araf 1

H Ara 1,2,4 3,5 - 2-Araf -

I Ara 1,4,5 2,3 - 5-Araf -

J Ara 1,3,4 2 5 3,5-Araf 5

K Ara 1,4,5 2 3 3,5-Araf 3

L Ara 1,4,5 3 2 2,5-Araf 2

M Ara 1,3,4,5 2 - 3,5-Araf -

Y Ara 4 2,3 1,5 4-Arap 1

Z Ara 1,5 2,3 4 4-Arap 4

N Man 1,5 2,3,4,6 - t-Manp -

P Man 1,5,6 2,3,4 - 6-Manp -

Q Man 1,2,5,6 3,4 - 2,6-Manp -

R Man 6 2,3,4 1,5 6-Manp 1

S Man 1,5 2,3,4 6 6-Manp 6

T Man 1,5 3,4 2,6 2, 6-Manp 2 and 6 u Man 1,5,6 3,4 2 2, 6-Manp 2

V Man 1,2,6 3,4 6 2 , 6-Manp 6 w Man 1,5 3,4,6 2 2-Manp 2

X Hex 1,5,6 2,3,6 4 4-Hexp 4

"GC peak" refers to the peaks previously

identified in Figures 2 and 3. At the lower

temperature (75°C), those products expected from

5-linked Araf (A & E, Fig. 1 and Fig. 2) were produced in large amounts. Accordingly, we concluded that 5-linked Araf as opposed to 4-linked Arap

predominates in LAM. Analysis of other products, e.g., F, G and J (Table II), demonstrated the

presence of 3,5-linked Araf rather than 3,4-linked Arap in LAM. Under the 95°C partial hydrolysis conditions, few partially alkylated, partially acetylated aribinitols were detected; the majority of the arabinofuranosides were cleaved to

monosaccharides at this temperature. However, quantitatively minor amounts of two products, Y

(4-O-Ac-1,5-di-O-C₂[²H]₅-2,3,-di-O-CH₃-arabinit ol) and Z

(1,5-di-O-Ac-2,3-di-O-[²H]₅-4-O-Me-arabinitol)

(Fig. 1, Fig. 3 and Table II) were observed,

indicative of the presence of 4-linked Arap.

Nevertheless, the amounts were extremely low,

suggestive to us of the existence of only one or two residues of 4-linked Arap, at most. No products indicative of 3,4-linked Arap were observed (Table II), corroborating results obtained from the

application of the milder temperature. Therefore, the 2-O-Me-Ara formed during methylation analysis (Table I) resulted from a 3,5-linked Araf only and not from 3,4-linked Arap, and the 2,3-di-O-Me-Ara resulted mainly from 5-linked Araf and, perhaps, from one or two residues of 4-linked Arap. No derivatives of 3-O-Me-Ara (Table I), which could establish its ring form, were observed under either set of

hydrolysis conditions, presumably since this residue is present in extremely small amounts. Consequently, its ring form has not been rigorously determined. However, partial acid hydrolysis showed that this residue was cleaved at the same rate as the

unambiguously assigned 3,5-di-O-Me-Araf, suggesting the presence of 2,5-linked Araf rather than

2,4-linked Arap in LAM.

Linkage and ring form cannot be determined for GC peaks B, I, K and M, due to the lack of C₂

[ ²H]₅ groups at C-4 or C-5, or the presence of

C₂[ ²H]₅ groups at both C-4 and C-5. Instead,

the assigned ring form (Table II) is based on the presence of other derivatives listed in Table II.

Compound I may be a product of the small amount of 4-linked Arap present.

D. Structures of the Oligoarabinofuranosyl Fragments of LAM

The per-O-LAM was again hydrolyzed with 2M

CF₃OOOH, this time at 75°C for 45 min, reduced with NaB[ ²H]₄ and penta-O-deuterioethylated; these

partial acid hydrolysis conditions were such that the more acid-stable Manp linkages were not cleaved. The resulting per-O-alkylated oligoarabinosyl-arabinitol mixture was recovered en block from cartridges of

Sep-Pak (Waters, Milford, MA) and applied to reverse

phase HPLC (3, 6). The partially fractionated population of per-O-alkylated

oligoarabinosyl-arabinitols were further resolved and analyzed by GC/MS. The application of this series of degradations, derivatizations, separations and mass spectometric analyses to a portion of the arabinan segment of LAM is illustrated in Fig. 4.

Conclusions as to the location of the

O-C₂[ ²H]₅ groups are important to the

structural interpretations, in that they indicate the original point of attachment of other glycosyl residues. Thus, the O-C₂[ ²H]₅ group at C-2 of glycosyl residue "a" (Fig. 3D) unequivocally

established that this residue was originally

substituted at C-2 by another glycosyl unit. The presence of O-C₂[ ²H]₅ substituents on C-1 and

C-4 of the alditol ("aid") is due to the fact that the arabinosyl residue is furanoid which, when cleaved and reduced at C-1, exposed OH-functions at C-1 and C-4 for penta-O-deuterioethylation. As an example, the results of GC/MS resolution/analysis of HPLC fraction no. 51 from the fractionation of the per-O-alkylated oligoarabinosyl-arabinitols is shown in Figs. 5A, 5B and 5C. The total ion chromatogram (Fig. 5A) demonstrated that fraction no. 51 on HPLC yielded only the pure diarabinosyl alditol (compound no . 12 , Table III) . The mass spectrum of compound 12 (Fig. 5B) showed the presence of a major aA, ion at m/z 175, proving that the terminal residue "a" contained no O-C₂[ ²H]₅ group. The ion at m/z 335 (abA₁) demonstrated that the internal residue had no O-C₂[ ²H]₅ unit, and the ion at m/z 230

(aid J₂) indicated that the alditol group was

endowed with two O-C₂[ ²H]₅ groups. The

presence of both aid J₁ (m/z 290) and aid J₀ (m/z 276) showed that the internal Ara was 2-linked. This was confirmed by alditol acetate analysis and

documented in Table II. The alditol cleavage ions at m/z 428 and m/z 472 also clearly demonstrated that the alditol is linked at C-5. Thus, based on these considerations, the structure of compound no. 12 is 2,3,5-tri-O-CH₃-D-Araf-(1→2)-3,5-di-O-Me-D-Araf-(1→5)-1,4-di-O-C₂[²H]₅-2,3-di-O-CH₃-D-arabinitol, which translates into the sequence t-Araf-(1→2)-Araf-(1→5)-Araf-→ (Table III).

When this type of reasoning and other rules governing the interpretation of the EI-MS

fragmentation of oligoarabinitol alditols (3, 6) were applied to a total of 25 such fragments of LAM and the results compared to those derived from a similar analysis of the peptidoglycan-bound arabinogalactan (3), the structure shown in Table III emerged.

Table III illustrates the structure of some 25 oligoarabinitol alditol fragments derived from Per-O- Me-Lam. The fragments are listed in chronological order as they emerged from the HPLC column. Part of the structural proof for Compound No. 12 is

described above. The structural proof for Compound 12 (Table III) included the following additional information:

Compound 12: GC R_T: 16.8 min; HPLC R_T: 25.8 min. KI/MS ions at m/x 101, 143 (aA₂), 175 (aA₁), 335 (baA₁), 303 (baA₂), 230 (aldJ₂), 276

(aldJ₀), 290 (aldJ₁), 428 (alditol cleavage) and 472 (alditol cleavage). Glycosyl linkage

composition: 1,4-di-O-Ac-2,3,5-tri-O-Me arabinitol; 1,2,4-tri-O-Ac-3,5-di-O-Me arabinitol and

5-O-AC-2,3-di-O-Me-1,4-di-O-C₂[²H]₃ arabinitol in the ratio of ca. 1:1:1.

The structural proof for compound 2 (Table III) included the following information:

Compound 2: GC R_T: 9.7 min; HPLC R_T: 21.5 min. KI/MS ions at m/x 101, 143 (aA₂). 175 (aA₁), 230 (aldJ₂), 290 (aldJ₁)₂ and 268 and 312

(alditol cleavages).

The structural proof for compound 20 included the following information:

Compound 20: GC R_T: 17.3 min; HPLC R_T: 35.0 min. KI/MS ions at m/x 120, 162 (aA₂), 194

(aA₁), 230 (aldJ₂), 290 (aldJ₁), 354 (baA₁)

and 491 (alditol cleavage). Glycosyl linkage

composition:

1,4-di-O-Ac-3,5-di-O-Me-2-O-C₂[²H]₃ abinitol;

1,4,5-tri-O-Ac-2,3-di-O-Me arabinitol and

5-O-AC-2,3-di-O-Me-1,4-di-O-C₂[²H]₃

arabinitol. The ratio could not be obtained.

The structural proof for compound 23 included the following information:

Compound 23: GC R_T: 22.5 min; HPLC R_τ: 32.5 min. KI/MS ions at m/x 101, 143 (aA₂), 175

(aA₁), 303(baA₂), 335 (baA₁), 230 (aldJ₂),

290 (aldJ₁), 436 (caldJ₀), 450 (caldJ₁) and 610 (bcaldJ₁).

The structural proof for compound 24 included the following information:

Compound 24: GC R_T: 23.1 min; HPLC R_τ: 40.5 min. EI/MS ions at m/x 101, 120, 162 (aA₂), 194 (aA₁), 230 (aldJ₂), 354 (baA₁), 450 (caldJ₁)

and 610 (bcaldJ₁).

In addition to the above determinations, the structural proof for some of the compounds listed in Table III, was based upon structural identity between the compounds reported in Table III and compounds described in Reference 3. For example, the proposed structure for Compound No. 1, t-Araf-(1→-2)-Araf, is based on the structural proof that its structure is identical to compound no. 1 of Reference 3.

Accordingly, the structural proof for compound no. 1 is reported in Table III as "compound no. 1, ref.3", i.e., the structure of compound no. 1 is identical to that of compound no. 1 of Reference 3. The

structural proofs for the remaining compounds of Table III are reported in an analogous manner.

E. The Major Arabinan Structural Motifs of LAM

Examination of the structure of these 25 individual oligosaccharide fragments allowed the recognition of four families of oligoarabinosides and thereby four major structural motifs (Fig. 6).

Structural motifs A, B, C and D are within the peptidoglycan-bound arabinogalactan. Recognition of these particular arrangements is based on the

existence of compounds no. 5, 6, 10, 11, 18, 19 and 21 in the case of motif A; compounds no. 7, 8, 14, 15, 16, 17 and 22 in the case of motif B; and

compounds no. 4, 13 and 25 prove the case of motif D. The presence of structural motif C was determined in part from the existence of arabinosyl alditol no. 3 which demonstrated that at least some of the

2-linked Araf residues are glycosidically linked to a linear (non-branched) 5-linked Araf. A key

component, compound no. 12, confirmed this linkage pattern, i.e., the attachment of a 2-linked Araf at C-5 of a 5-linked Araf and, most importantly, that the unit was part of a terminal motif in that t-Araf was glycosidically linked to C-2 of the 2-linked Araf. The formulation of the full details of motif C (Fig. 6) was allowed by the recognition of compound no. 24 and confirmed by the structures of compounds no. 1, 20 and 23.

F. Assignment of Anomeric Configurations

Table IV shows the results of ¹H-NMR analysis of various compounds.

Both the 2-linked Araf and 5-linked Araf residues appearing in structural motif C (Fig. 6) were shown to be α, based on ¹H-NMR analysis of compound no. 20 (Table IV). Compounds 20 and 13 co-eluted from the HPLC and NMR analysis resulted in a broad singlet ["br.s."] at δ 5.00 and δ 5.04 in a ratio of

about 3:1. Individual assignments cannot be made but all the glycosyl residues must be α-Araf.

The t-Ara of motif C was demonstrated to be β by 1H-NMR analysis of compound no. 12 (Fig. 5 and

Table IV). Assignment of β to the t-Araf and α to the 2-linked Araf of compound no. 12, was based on analysis of compounds 1 and 20 (Table IV).

The →2-Araf residues on both C-3 and C-5 of the branched Araf of structural motif A and the →5-Araf on both C-3 and C-5 of the branched Araf of structural motif B were shown to be in the α

configuration by ¹H-NMR analysis of compounds no.

5, 6, 7 and 8 (Table IV). Compounds 6 and 8

co-eluted from the HPLC and thus it is not known which anomeric signal results from 6 and which from 8. Similarly, compounds 5 and 7 co-eluted from the HPLC and thus it is not known which anomeric signal results from 5 and which from 7. The assignment of α in all four cases, i.e., compounds 5-8, is

unambiguous.

The t-Araf residues present in motifs A and C were shown to be in the β configuration by ¹H-NMR analysis of compound no. 1 (Table IV). The branched 3,5-linked-Araf residues in motifs A and B (Fig. 6)

are both in the α configuration as demonstrated by 1H-NMR analysis of compound no. 9 (Table IV).

Thus, assignment of all of the anomeric

configurations shown in Fig. 6 was accomplished.

G. The Application of ¹³_C-NMR to the Arabinan

Segments of LAM

The application of ¹³C-NMR analysis to

solubilized peptidoglycan-bound arabinogalactan allowed the assignment of C-1 of the t-Araf units appearing in structural motif A to δ 101.9 and δ

101.8; C-1 of the 2-linked Araf residue of the same motif was assigned to δ 106.8 and δ 106.6,

whereas C-2 of 2-linked Araf was assigned to δ 88.2 and 5 87.9 (3). The ¹³C-NMR spectrum of LAM is compared in Fig. 7 to that of solubilized

arabinogalactan (3) in the range of δ 85 to δ

115. The resonances corresponding to C-1 (δ 106.8 and δ 106.6) and C-2 (δ 88.2 and δ 87.9) of the

2-1inked Araf unit of motif A of LAM were determined.

In addition, resonances from C-1 (δ 106.9) and C-2

(broadening signal at δ 87.9) of the 2-linked Araf unit of the closely related structural motif C of LAM was evident. The resonances of the t-Araf units appearing in both motifs A and C of LAM are present between δ 101.7 and δ 101.9, but are somewhat

obscured by resonances arising from the mannosyl residues. Nevertheless, the presence of motifs A and

C in LAM was confirmed by ¹³C-NMR.

EXAMPLE IV

Chemical Synthesis of the Arabinose-Containing

Epitopes of LAM from M. tuberculosis

An epitope of the LAM from M. tuberculosis H37Ra is a D-arabinopentaose shown in Fig. 8. Synthesis of neoglycoproteins containing this unit calls for attention to the fact that it has α, ß linkage at the 2, 3 and 5 positions.

In an oligosaccharide synthesis,

regioselectivity is generally achieved when the glycosylatng component (glycosyl donor) possesses selectively protected hydroxyl groups and an

activating group at the anomeric carbon atom and the other sugar component with a free hydroxyl group (glycosyl acceptor) possesses protecting groups at other -OH positions. In addition, the coupling step should occur diastereoselectively with respect to formation of an α- or ß-linkage.

The reducing end arabinose (Fig. 8) is branched at the 3- and 5- position with α-linked arabinose. For this purpose, we exploit the use of

1,2-orthoester which initially gives a φ-orthoester which favors α-linked glycosides upon

glycosylation. Orthoesters are extremely versatile intermediates in oligosaccharide synthesis. They can be converted to halides, can be glycosylated, and the intermediate also provides a removable protecting group at the 2-OH position.

Thus, 2,3,5-tri-O-benzoyl (^α or ^ß) arabinofuranosyl bromide is prepared by stirring a mixture of D-arabinose in anhydrous methanol

containing HCl and stirring at room temperature until dissolution of the sugar. After reducing power

(Fehling's solution) of the sugar is eliminated, pyridine is added and the solution evaporated to give syrupy methyl α-D-arabinofuranoside which on

further reaction with pyridine and benzoyl chloride (1 h at 0°C) gives methyl

2,3,5-tri-O-benzyol-α-D-arabinofuranoside. The product is crystalline, and NMR should confirm its ring form. To a solution of this compound, glacial acetic acid is added followed by a solution of hydrogen bromide in acetic acid. A faster running product (by TLC), indicating the formation of the halide, can be isolated, and crystallized to give α-glycosyl bromide and the mother liquor contains φ-glycosyl bromide. Either the α-anomer or the φ-anomer can be used to make the orthoester.

There are several methods of preparing a

1,2-orthoester. These involve formation of acyclic acyloxinium ion involving C-1 and C-2 of an aldose moiety in a five-membered ring and can be generated from the tribenzoyl α or φ arabinofuranosyl

halide using anhydrous methanol in the presence of 2,6-lutidine. There also are various other

noteworthy Ag salt catalysts, e.g., silver carbonate, silver oxide, silver nitrate and various scavengers, e.g., 4A molecular sieves, 2,4,6-trimethylpyridine, etc. Additionally, an appropriate

N,N-dimethylformamide dimethylacetal - Me₂N-CH(OR)₂ - can be used in dichloromethane to give the essential

orthoester-3,5,di-O-benzoyl-debenzoylated at 3- and 5-hydroxyl positions using ammonia-saturated methanol and glycosylated using 8-methoxycarbonyloctanol in the presence of mercuric bromide in acetonitrile to give a 1,2-trans related glycoside,

8-methoxy-carbonyloctyl-2-O-benzoyl

α-D-arabinofuranoside, which will act as an

acceptor for glycosyl halides.

Since the branches at 3- and 5-position of the reducing end arabinose have similar araf residues, excess (2 or 3 molar) of the glycosyl donor should substitute both the 3- and 5-position. However, due to the reactivity of hydroxyl group (5- being a primary and 3- being a secondary), if only one position gets substituted, the glycosylation step can be repeated in order to obtain the trisaccharide.

But, for the penultimate araf residues, we use a temporary blocking group at 2, since the terminal araf residues are 2-linked.

For this, we make another 1,2-O-orthoacetate, however, an orthoacetate in place of orthobenzoate. Thus, 2,3,5- tri-O-acetyl-α,φ-arabinofuranosyl bromide can essentially be made where D-arabinose is first converted to its 5-O-trityl derivative, which is acetylated to give the tri-O-acetyl derivative. On treatment with acetic anhydride, in the presence of acetyl bromide, hydrolysis of the trityl group occurs with simultaneous acetylation giving a

per-O-acetyl derivative. Isolation of the product and bromination with hydrogen bromide which can be converted to its 1,2-O-(1-methoxyethylidene)

derivative as described earlier. Deacylation

followed by benzylation will give

3,5-di-O-benzyl-1,2-O-methoxy ethylidene-φ-D-Araf. This orthoester then is converted to a halide

(preferably chloride) which serves as a glycosyl donor and an excess of this halide can be added to the reducing end araf in order to give the

trisaccharide shown in Fig. 9.

This method is a classical Koenigs-Knorr

condensation, and, with neighboring group

participation, formation of a 1,2 trans glycoside is attained. In case of a problem with correct anomeric configuration, alternatively, a thioglycoside may be prepared, which is a stable intermediate and yet can be activated to reactive donors by powerful promoters like methyltriflate.

The trisaccharide, above, can be deacylated selectively to remove 2-O-acetyl functions with methanolic HCl at 0°C and thus be ready to accept the terminal sugar.

The terminal sugar has a ß-linkage. For

synthesizing a glycoside with 1,2 cis relation, we replace 2,3,5-O-ester function with ether protecting groups in order to prevent the acyloxonium ion formation which invariably leads to a 1,2-trans arrangement. Therefore, a 2,3,5-tri-O-benzyl araf halide can be condensed simultaneously at the 2 × OH positions of the trisaccharide in one step

glycosylation using mercuric cyanide catalyst in dry dichloromethane. Alternatively, we make a

thioglycoside of arabinose, use benzyl protecting groups at 2,3 and 5 positions and then activate the thioglycoside with cupric bromide in the presence of tetrabutylammonium bromide and silver

trifluoromethane-sulfonate to give a finally

protected pentasaccharide (Fig. 10).

Sequential deprotection (deacylation,

debenzylation) followed by conversion of the

carbohylate on the spacer arm to its hydrazide gives a product ready to be coupled to a carrier such as a protein or even to the core 1→5 arabinofuranosyl chain.

For a repeating unit containing a linear α(1→5) araf segment, the synthesis is

straightforward (Fig. 11). Glycosidation of a

3,5-di-O-benzoyl-1,2-O-cyanobenzylidene

φ-D-arabinofuranose with

8-methoxycarbonyloctyl-2,3-di-O-benzoyl-5-O-triphenylmethyl-αD-arabinofuranoside in ahnydrous dichloromethane in the presence of

triphenylcarbanium tetrafluoroborate as catalyst under high vacuum gives a dimeric homolog. The 1,2-cyanoethylidene group is introduced using silver cyanide in boiling xylene or sodium cyanide in acetonitrile and can be used as glycosylating agents directly. The disaccharide formed can be debenzoylated, retritylated rebenzoylated and

condensed to another cyanoethylidene derivative, giving a trimer. In this way a chain elongation can be carried out up to a penta or a hexasaccharide, finally deprotected to remove benzoyl groups, and incorporated to a carrier protein as needed for serological studies.

References

1. Hunter, S. W., Gaylord, H., and Brennan, P. J. (1986) J. Biol. Chem. 261, 12345-12351.

2. Hunter, S. W., and Brennan, P. J. (1990) J. Biol. Chem., 265, 9272-9279.

3. Daffe, M., Brennan, P. J., and McNeil, M. (1990) J. Biol. Chem. 265, 6734-6743.

4. McNeil, M., Wallner, S. J., Hunter, S. W., and Brennan, P. J. (1987) Carbohydr. Res. 166,

299-308.

5. Darvill, A. G., McNeil, M. , and Albersheim, P. (1980) Carbohydr. Res. 1986, 309-315.

6. Valent, B. S., Darvill, A. G., McNeil, M., Robertson, B. K., and Albersheim, P. (1980)

Carbohydr. Res. 79, 165-192.

The foregoing references are hereby incorporated in their entireties herein by reference.

Having now fully described this invention, it will be appreciated by those of ordinary skill in the art that the same can be practiced with a wide range of equivalents, without affecting the spirit or scope of the invention.

What we claim is:

Claims

1. A device for use in conducting a biological procedure comprising

purified LAM attached to a substrate.

2. A device as claimed in claim 1 wherein the purified LAM is attached to a microtiter plate.

3. A device as claimed in claim 1 wherein the purified LAM is attached to a column.

4. In a kit for conducting an immunoassay, the kit including various containers for containing reagents, the improvement comprising at least one of the containers containing purified LAM.

5. A pharmaceutical composition comprising purified LAM and a pharmaceutically acceptable carrier.

6. A method for purifying LAM from whole bacteria in which the LAM is purified without the use of preparative gel-electrophoresis.

7. A method as claimed in claim 6 further comprising

an ion exchange separation step, and

an HPLC separation step.

8. A method as claimed in claim 6 further comprising,

a sonication step,

a precipitation step, and

a sizing column separation step in the presence of detergent.

9. A composition of matter comprising

a synthetic arabinofuranosyl oligosaccharide.

10. A composition of matter as claimed in claim 9 wherein the arabinofuranosyl oligosaccharide includes motif A.

11. A composition of matter as claimed in claim 9 wherein the arabinofuranosyl oligosaccharide includes motif B.

12. A composition of matter as claimed in claim 9 wherein the arabinofuranosyl oligosaccharide includes motif C.

13. A composition of matter as claimed in claim 9 wherein the arabinofuranosyl oligosaccharide includes motif D.

14. A composition of matter as claimed in claims 9, 10, 11, 12 or 13 wherein the synthetic arabinofuranosyl oligosaccharide is an

arabinofuranosyl neoglycoconjugate.

15. A device for conducting a biological procedure comprising a substrate to which is attached the composition of matter as claimed in claim 9.

16. A device for conducting a biological procedure comprising a substrate to which is attached the composition of matter as claimed in claim 10.

17. A device for conducting a biological procedure comprising a substrate to which is attached the composition of matter as claimed in claim 11.

18. A device for conducting a biological procedure comprising a substrate to which is attached the composition of matter as claimed in claim 12.

19. A device for conducting a biological procedure comprising a substrate to which is attached the composition of matter as claimed in claim 13.

20. A device for conducting a biological procedure comprising a substrate to which is attached the composition of matter as claimed in claim 14.

21. A pharmaceutical preparation containing the composition of matter of any one of claims 9-13.

22. A pharmaceutical preparation containing the composition of matter of claim 15.

23. A method for conducting a biological procedure comprising using the composition of any one of claims 9-13.

24. A method for conducting a biological procedure comprising using the composition of claim 14.