A universal approach to the synthesis of carbohydrate ... - Springer Link

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2006, Vol. 32, No. 6, pp. 568–577. © Pleiades Publishing, Inc., 2006. Original Russian Text © A.V. Orlova, N.N. Kondakov, A.I. Zinin, B.G. Kimel’, L.O. Kononov, I.B. Sivaev, V.I. Bregadze, 2006, published in Bioorganicheskaya Khimiya, 2006, Vol. 32, No. 6, pp. 632–642.

A Universal Approach to the Synthesis of Carbohydrate Conjugates of Polyhedral Boron Compounds as Potential Agents for Boron Neutron Capture Therapy A. V. Orlovaa, N. N. Kondakova, A. I. Zinina, B. G. Kimel’a, L. O. Kononova,1 I. B. Sivaevb, and V. I. Bregadzeb a Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences,

Leninskii pr. 47, Moscow, 119991 Russia b Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences, ul. Vavilova 28, Moscow, 117813 Russia Received February 13, 2006; in final form, February 28, 2006

Abstract—A universal approach to the synthesis of carbohydrate conjugates with polyhedral boron compounds (PBCs) was developed. Oligosaccharide derivatives with amino group in aglycone moiety can be conjugated with PBC carboxy derivatives using N-methyl-N-(4,6-dimethoxy-1,3,5-triazin-2-yl)morpholinium chloride as a condensing agent. Both N- and O-glycosides differing in the conformational mobility around the glycoside bond were shown to be useful as oligosaccharides with a functional group in the aglycone moiety. This allows the application of this approach to the synthesis of PBC conjugates with a wide range of oligosaccharides. For example, not only oligosaccharides obtained by chemical synthesis but also reducing oligosaccharides isolated from natural sources can be transformed into N-glycosides with a functional group in aglycone. The approach was tested by conjugation of the carboxy derivatives of ortho-carborane and dodecaborate anion with lactose as a model oligosaccharide. Lactose, an easily available disaccharide, is a ligand of lectins expressed on the surface of melanoma cells. The approach suggested is the first example of the synthesis of such conjugates that does not require protective groups for the carbohydrate residue. It is especially important for obtaining dodecaborate–carbohydrate conjugates for which the removal of protective groups is often a non-trivial task.

DOI: 10.1134/S1068162006060100 Key words: boron neutron capture therapy, conjugates of polyhedral boron compounds with oligosaccharides, dodecaborate–carbohydrate conjugates, ortho-carborane–carbohydrate conjugates

INTRODUCTION Boron neutron capture therapy is a binary (chemoradiation) method for the treatment of cancer diseases based on the introduction of a stable 10B isotope into tumor.2 The subsequent tumor irradiation with a flux of thermal neutron beam results in the generation of high 1

Corresponding author; phone: +7 (495) 135-7570; fax: +7 (495) 135-5328; e-mail: [email protected]. 2 Abbreviations: BNCT, boron neutron capture therapy; DMT-MM, N-methyl-N-(4,6-dimethoxy-1,3,5-triazin-2-yl)morpholinium chloride; ESI, electrospray ionization; HONSu, N-hydroxysuccinimide; and PBC, polyhedral boron compounds.

energy fission products characterized by a short path length comparable with the cell dimensions. Ideally, this allows the selective destruction of tumor cells, without affecting the surrounding healthy tissue [1]. The second generation preparations for BNCT used in clinical practice [1] do not possess the required selectivity for the accumulation in tumor tissues. The use of directed delivery of boron-containing compounds to tumor cells due to the specific carbohydrate–protein interactions can be one of the ways for the increasing selectivity. The endogenous lectins (protein receptors) located on the surface of many normal and tumor cells function as the specific receptors and are the messen-

568

A UNIVERSAL APPROACH TO THE SYNTHESIS OF CARBOHYDRATE CONJUGATES

Boron-containing fragment

Oligosaccharide

N-Glycosides with a functional group in aglycone

569

O-Glycosides with a functional group in aglycone

Functionalized polyhedralÂ boron compounds

Oligosaccharides obtained by chemical synthesis

Reducing oligosaccharides isolated from natural sources‚

Scheme 1. A universal approach to synthesis of the carbohydrate conjugates with polyhedral boron compounds.

gers in the carbohydrate-specific endocytosis of (neo)glycoconjugates [2]. The PBC [1] conjugates with

carbohydrate ligands of lectins expressed on the surface of tumor cells can be the promising agents for BNCT.

3–

O

O O

HO

3Bu4N+

(CH2)4 O

(I)

(III)

O

, CH ,C , BH ,B

Cl

(II)

Nowadays, there is no unified approach to the synthesis of the oligosaccharide conjugates with various PBC. Only few examples of successful syntheses of conjugates of carborane anion with monosaccharides [3–6] and ortho-carborane with mono- and disaccharides (see [7] and references therein) are reported. This is due to both the complexity of the synthesis of PBC functional derivatives (first of all, dodecaborate ion derivatives) and the fact that the known conditions for functionalization and coupling of carbohydrate fragments to hydrophobic carboranes and hydrophilic dodecaborate anions significantly differ and cannot be used for complex oligosaccharides [3–7]. At the same time, there is a necessity for the development of the approach to various PBC that should allow the coupling of similarly functionalized oligosaccharide derivatives of various types and any complexity, including those isolated from biological sources. RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY

RESULTS AND DISCUSSION We herein3 suggest a uniform approach that allows the coupling of various PBC to oligosaccharide derivatives using lactose, a disaccharide known as a ligand for lectins expressed on the surface of melanoma cells [11], as a model. We have chosen closo-dodecaborate anion and 1,2-dicarba-closo-dodecaborane(12) (ortho-carborane) as PBC most widely used for BNCT. The universal approach to the synthesis of lactose conjugates with PBC requires: (1) lactose-based precursors of the same type containing the aglycone with a functional group, (2) ortho-carborane or closo-dodecaborate anion derivatives with similar functionalization, and (3) the effective method for the coupling of these fragments (Scheme 1). Both N- and O-glycosides can be used as carbohydrate components with a functional group in the aglycone. For example, N-glyco3

Some parts of this study have earlier been reported [8–10].

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OH O

HO OH

O HO

OH O

NH

OH

NH2 O

(IV) RO

OR O

RO OR

O RO

OR O

O

NH2

OR (V) R = H (VI) R = Ac (VII) R = Bzl

sides with a functional group in aglycone can be synthesized not only from oligosaccharides obtained by chemical synthesis, but also from reducing oligosaccharides isolated from natural sources [12]. An assured coupling of lactose derivatives with both ortho-carboranes and closo-dodecaborate anions requires a simple and substrate-insensitive synthetic method. We assumed that the well developed methods of amide synthesis should be sufficiently effective in this case. This coupling method presumes the presence of carboxy group in one of the starting components and amino group (or its precursor) in another component. We decided to use PBC derivatives as synthetic building blocks carrying a carboxyl group, because the carboxyl derivatives of ortho-carborane, e.g., (I) and (II), have long been known [13] and a convenient synthesis

of carboxyl derivative of closo-dodecaborate anion (III) has been reported in 2000 [14]. According to this logic, lactose should bear an amino group in aglycone. Two types of lactose derivatives, the derivatives of 2-aminoethyl lactoside with conformationally mobile O-glycoside bond and the derivatives of N-glycyl-βlactosamine with a more rigid N-glycoside bond, were used in the synthetic scheme. We used the following model compounds: N-glycyl-β-lactosamine (IV) [15] and (2-aminoethyl) lactoside both unprotected (V) [9] and containing é-acetyl (VI) [8] or O-benzyl (VII) [9] protective groups. The synthesis of the carboxyl derivative of closododecaborate anion (III) (Scheme 2) included a modification of the method reported in [14] at the stage of obtaining nitrile (IX) from tetramethyleneoxonium derivative (VIII). The replacement of tetrabutylammonium bromide as the phase transfer catalyst by tetrabutylammonium hydrosulfate with a less nucleophilic counterion resulted in nothing but the target nitrile (IX) rather than its mixture with the corresponding bromide (X), which was generated when reproducing the procedure described in literature. As a result, we succeeded in avoiding the extremely laborious step of the separation of target product from the undesired bromo derivative. The use of oligosaccharide derivatives without éprotective groups and the formation of amide bond in the presence of DMT-MM [16] as a coupling reagent (Scheme 3) appeared to be the most effective variant for the synthesis of the conjugates of carboxyl-bearing PBC with oligosaccharides. Amides (XI), (XII), (XIII),

–

Bu4N+

O

2–

a

O

(VIII)

(CH2)4CN

(IX) 2–

(VIII)

2Bu4N+

b

(IX) +

O

,B , BH

2Bu4N+

(CH2)4Br

(X) 3–

O (IX)

c

O

3Bu4N+

(CH2)4 O (III)

Scheme 2. Synthesis of the carboxyl derivative of dodecaborate anion. Reagents and conditions: (a) NaCN, Bu4NHSO4, CH2Cl2– H2O, 86%; (b) NaCN, Bu4NBr, CH2Cl2–H2O; (c) KOH, MeOH, 82%. RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY

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HO O

NH2 +

X

HOOC

Carbohydrate derivative

Y

PBC derivative DMT-MM MeOH–H2O

HO O

571

X

NH

, B or C , BH or CH , BH X and Y spacers

Y O

Scheme 3. A general scheme of synthesis of unprotected carbohydrate conjugates with polyhedral boron compounds.

a

(I) + (IV) (XI) (50%) a (I) + (V) (XII) (74%) (III) + (IV) a (XIII) (57%) HO

OH O

HO OH

HO

OH O

HO OH

O HO

OH O OH

O HO

OH O

a

(III) + (V) (XIV) (~55%) (XV) b (XII) (80%) (XVI) c (XII) (84%)

O NH

NH

(XI) O O O

– CH –C – BH –B

NH

OH (XII)

HO

OH O

HO OH

HO

O HO

OH O

2–

O NH

OH

O

NH O (XIII)

2–

OH O

HO OH

O HO

OH O

O O

O

NH

OH (XIV)

Scheme 4. Unprotected conjugates of lactose with polyhedral boron compounds. Reagents and conditions: (a) DMT-MM, MeOH–H2O; (b) MeONa, MeOH; (c) H2, Pd/C, MeOH. RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY

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and (XIV) were obtained just in this way (Scheme 4). All the reactions were carried out in MeOH–H2O mixtures, as amines (IV) and (V) are soluble in H2O (rather than in MeOH), carboranylacetic acid (I) and tetrabutylammonium salt of the carboxyl derivative of closododecaborate anion (III), in MeOH (rather than in H2O), whereas the cooupling agent DMT-MM, in both H2O and MeOH. Interestingly, we succeeded in obtaining the target conjugate (XII) in 74% yield, which 1.5-fold exceeds the yields of amines with acetyl (XV) and benzyl (XVI) protective groups (see below). Product (XII) was isolated by reversed-phase chromatography. In aqueous solution, conjugate (XII) slowly undergoes a hydrolytic deboronation to give the corresponding nido-conjugate (XII-nido) (for details, see [9]). The suggested structure of the conjugate was confirmed by 1H, 13C, and 11B NMR spectroscopy and mass spectrometry. The presence of the signal of the CH2CO group neighboring to the carborane fragment (δë 44.0 ppm) in the 13C NMR spectrum along with the signal of the aglycone CH2NH group (δë 40.7 ppm) indicates the formation of amide (XII). The 11B NMR spectrum of amide (XII) exhibits the signals characteristic of the closo-carborane backbone (δB –2.3, –5.4, and –9.5 ppm) [17]. The mass spectrum (ESI, registration of positively charged ions) of an amide (XII) solution gave the signals of the quasimolecular ion (m/z 594.4 [M + Na] and the dimer (m/z 1165.4 [M2 + Na]). The conjugation of N-glycyl-β-lactosylamine (IV) with carboranylacetic acid (I) in the presence of DMT-MM in aqueous MeOH similarly resulted in the formation of amide (XI). The target conjugate (XI) was isolated as a homogeneous substance (in 50% yield) by reversed-phase chromatography. The suggested structure of the conjugate (XI) was confirmed by 1H, 13C, and 11B NMR spectroscopy and mass spectrometry. The presence of signal of the CH2CO group neighboring to the carborane fragment (δë 44.1 ppm) in the 13C NMR spectrum along with the signal of the aglycone CH2NH group (δë 43.5 ppm) indicated the formation of amide (XI). The 11B NMR spectrum of amide (XI) also contained the signals characteristic of the closo-carborane backbone (δB –2.3, –5.4, and −9.5 ppm) [17]. The mass spectrum (ESI, registration of positively charged ions) of a solution of amide (XI) contained not only the signal of the quasimolecular ion (m/z 605.4 [M + Na]), but also the signals of cluster ions, the dimer (m/z 1188.3 [M2 + Na] and trimer (m/z 1770.1 [M3 + Na]). The conjugation of N-glycyl-β-lactosylamine (IV) with the carboxyl derivative of closo-dodecaborate anion (III) proceeded successfully (Scheme 4). However, the purification of the target conjugate (XIII) from the by-product, 4,6-dimethoxy-1,3,5-triazin-2-ol, required significant efforts. The problem was complicated by the fact that 4,6-dimethoxy-1,3,5-triazin-2-ol sodium salt is readily soluble in water like the sodium

salt of dodecaborate–carbohydrate conjugate (XIII). The purification procedure developed included the following sequence of operations: the extraction of 4,6dimethoxy-1,3,5-triazin-2-ol tetrabutylammonium salt from the reaction mixture with chloroform (or dichloromethane), the replacement of tetrabutylammonium cation by sodium cation using an ion-exchange resin in Na+-form, and, in the case of the presence of remaining unreacted starting carboxy derivative of closo-dodecaborate anion (III), its removal by reversed-phase chromatography. The target product (XIII) was isolated in 57% yield and contained a small impurity of sodium hydrocarbonate (δë 156.6 = 156.6) according to 13C NMR. The structure suggested for conjugate (XIII) was confirmed by 1H, 13C, and 11B NMR spectroscopy and mass spectrometry. The presence of the signal of the glycyl aglycone CH2NH group (δë 43.5 ppm) along with the signal of the CH2CO group (δë 36.2 ppm) in the 13C NMR spectrum proved the formation of amide (XIII). The 11B NMR spectrum of amide (XIII) exhibited the signals characteristic of the monosubstituted closo-dodecaborate anion (δB –23.5, –18.6, –16.6, and 6.2 ppm) [14]. The ESI mass spectrum (registration of negatively charged ions) of a solution of amide (XIII) contained the signal of quasimolecular ion (m/z 663.5 [M + Na]). The conjugation of 2-aminoethyl lactoside (V) with carboxy derivative of closo-dodecaborate anion (III) in the presence of DMT-MM in H2O–MeOH proceeded successfully (Scheme 4): the starting compounds were completely exhausted and the analysis of the NMR spectra of the reaction mixture after its treatment confirmed the presence of the target product (XIV). Unfortunately, the purification procedure described above for conjugate (XIII) did not allow the complete purification of product (XIV). The yield of the product in the reaction of amide bond formation can be approximately estimated as 55% on the basis of integration of the signals in the 1H NMR spectrum of the crude product. The final purification of conjugate (XIV) was achieved by gel filtration on Sephadex G-10 column eluted with 1% aqueous ammonium hydrocarbonate. We should note that the isolation of amide (XIV) in pure state was accompanied with a significant decrease in its yield after chromatographic purification. It should be emphasized that the use of 1% aqueous AcOH as the eluent (Fractogel TSK-HW 40 column) results in a partial destruction of the conjugate (11B NMR data) and, therefore, is inappropriate. The structure of conjugate (XIV) was confirmed by 1H, 13C, and 11B NMR spectroscopy and mass spectrometry. The 13C NMR spectrum of (XIV) included the signal of the CH2CO group (δë 36.4 ppm) along with the signal of the aminoethyl aglycone CH2NH group (δë 40.8 ppm), which proved the formation of amide (XIV). The 11B NMR spectrum of amide (XIV) exhibited the signals characteristic of the monosubsti-

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(II) + (VI) (III) + (VII) RO

OR O

RO OR

a a

(XV) (47%) (XVI) (53%)

O RO

RO O

(I) + (VI) (I) + (VII) (I) + (VII)

b b c

573

(XV) (19%) (XVI) (15%) (XVI) (7%)

O O

NH

OR , CH ,C , BH

(XV) R = Ac (XVI) R = Bzl

Scheme 5. Conjugates of polyhedral boron compounds and lactose with acetyl and benzyl protective groups. Reagents and conditions: (a) NaHCO3, H2O–CH2Cl2; (b) DCC, HONSu, THF; (c) DMT–MM, MeOH.

tuted closo-dodecaborate anion (δB −22.3, –17.7, −16.1, and 6.9 ppm) [14]. The ESI mass spectrum (registration of negatively charged ions) of a solution of amide (XIV) gave the signal of the quasimolecular ion (m/z 648.6 [M + Na]). The absence of protective groups in the lactose residue is the distinguishing feature of the conjugation method of PBC with carbohydrates suggested herein from those reported in literature. This enabled us to avoid an additional step of the removal of protective groups, which is especially important for dodecaborate– carbohydrate conjugates, as in this case, the deprotection step appears to be a nontrivial problem according to [6]. The approach we suggested has been tested both on é- and N-glycosides, indicating its applicability to not only synthetic glycosides, but also oligosaccharide glycosides isolated from natural sources. The study of approaches to the synthesis of protected conjugates of oligosaccharides with PBC prompted us to test various procedures. The yield of the conjugate with é-protective groups at the step of amidation appeared to depend on the method used. The highest yields of protected conjugates [47% in the case of the conjugate with acetyl protective groups (XV) and 53% in the case of the conjugate bearing benzyl protective groups (XVI)] were achieved when using carboranylacetic acid chloride (II) [13] as the acetylating agent (Scheme 5). The use of condensing agents appeared to be much less effective. The yield was 19% in the case of acetylated conjugate (XV) and 15% in the case of benzylated conjugate (XVI) when using DCC and HONSu. The use of DMT-MM resulted in the benzylated conjugate only in 7% yield (Scheme 5). The further deprotection of the resulting conjugates proceeded in high yields (Scheme 4). The O-acetyl groups were removed by the treatment with NaOMe in MeOH; the RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY

O-benzyl groups were removed by catalytic hydrogenolysis. NMR spectroscopy (1H, 13C, and 11B) and mass spectrometry for product (XII) led to the results coinciding with those for conjugate (XII) obtained by the direct coupling of amine (V) and carboranylacetic acid (I). Thus, we suggested a uniform approach to the synthesis of PBC–carbohydrate conjugates and tested it on the derivatives of 2-aminoethyl lactoside with the conformationally mobile O-glycoside bond and N-glycylβ-lactosylamine with a more rigid N-glycosylamide bond. The approach suggested can also be used for obtaining the conjugates both with hydrophobic orthocarborane and hydrophilic dodecaborate anion. The compounds synthesized were submitted to the State Scientific Center Institute of Biophysics, for studying the possibility of their use as preparations for BNCT. EXPERIMENTAL Solvents were purified by standard procedures [18]. TLC was carried out on silica gel 60 F254 (Merck) precoated aluminum sheets. The compounds containing carbohydrate residues were visualized by immersion of the sheets into 85% H3PO4 in 96% EtOH (1 : 10) and subsequent heating on a hot plate. Amines were visualized by the treatment with 5% ninhydrin in acetone and subsequent heating on a hot plate. The compounds containing NH fragments (amines and amides) were visualized by successive treatment with gaseous chlorine and o-tolidine (160 mg) in the mixture of acetic acid (30 ml) and water (500 ml). The compounds containing the borohydride fragment were visualized by the treatment with a solution of PdCl2 (1.26 g) in 10% aqueous HCl (25 ml) and methanol (250 ml). The compounds

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with benzyl protective groups were visualized under UV light (at 254 nm). Preparative chromatography was carried out on silica gel L (40–100 µm, Chemapol) and Silasorb 600 (7 µm, Chemapol); the reversed-phase chromatography, on a Superclean LC18 (Supelco) cartridge; gel chromatography on Sephadex G-10 columns (2 × 73 cm, eluent 1% NH4HCO3, elution rate 1 ml/min) or Fractogel TSK-HW 40 (2.5 × 90 cm, eluent 1% AcOH, flow rate 1 ml/min). Detection of eluate was carried out according to the refraction index using a Knauer refractometer. Compounds (I) and (II) were obtained as described in [13]. 1H, 13C, and 11B NMR spectra were registered on a Bruker AC-200 spectrometer at working frequencies of 200.13, 50.32, and 64.21 MHz, respectively. The values of 1H NMR chemical shifts are given in ppm relative to the residual signals of ëHCl3 (δH 7.27 ppm), CHD2OD (δH 3.31 ppm), and HOD (δH 4.80 ppm). The values of 13C{1H} NMR chemical shifts are given relative to ëDCl3 (δC 77.0 ppm), CD3OD (δC 49.0 ppm), or, in the case of solution in D2O, relative to 1,4-dioxane (δC 67.4 ppm, external standard). The values of 11B NMR chemical shifts are given relative to BBF3 · Et2O (δB 0.0, external standard). The signals in 13C NMR spectra were assigned using the DEPT-135 experiment. The values of coupling constants are given in Hz. The mass spectra (ESI) were registered for the 20 µM solutions in methanol on a Finnigan LCQ mass spectrometer. Distributions of intensities of the peaks in mass spectra of boron-containing compounds were compared with the computed values obtained by CS ChemDraw Ultra 9.0 software. In all cases, a close agreement of computed and observed intensities in the groups of peaks corresponding to the isotope distribution of boron atom was observed. The m/z values and relative intensities (I, %) of monoisotope peaks are given. Everywhere, the spectra with registration of the positively charged ions are given, unless otherwise specified. In the case of mass spectra of negatively charged derivatives of dodecaborate anion, å means monoisotope molecular mass of the anion. The values of optical rotation were measured on a Jasco DIP-360 digital polarimeter at 20–25°C. Bis(tetrabutylammonium)4-cyanobutoxyundecahydro-closo-dodecaborate (IX). A solution of tetramethyleneoxonium derivative of closo-dodecaborate anion (VIII) [14] (516 mg, 1.13 mmol) and Bu4NHSO4 (382 mg, 1.13 mmol) was added to a solution of NaCN (110 mg, 2.25 mmol) in 20 ml of 1 M NaOH. The biphasic system was vigorously stirred for 24 h, and the organic layer was separated, washed with water (3 × 25 ml), dried by filtration through a layer of cotton, and evaporated in a vacuum at 40°C. The residue was dried in a vacuum (1 mm Hg) to give the target nitrile (IX) (705 mg, 86%, cf. lit. [4]); Rf 0.54 (2 : 1 toluene–acetone); 1H NMR (CDCl3): 0.98 (24 H, t, J 7.2,

CH3), 1.47 (16 H, m, CH2CH3), 1.63 (16 H, m, NCH2CH2), 1.99 (4 H, m, OCH2CH2, CH2CH2CN), 2.47 (2 H, t, J 7.1, CH2CN), 3.25 (16 H, t, J 7.5, CH2N), 3.63 (2 H, t, J 5.8, CH2O); 13C NMR (CDCl3): 13.7 (CH3), 16.7 (CH2CN), 19.6 (CH3CH2), 22.8 (CH2CH2CN), 24.0 (CH2CH2N), 29.6 (CH2CH2O), 58.7 (CH2N), 67.4 (CH2O), 120.2 (CN); 11B NMR (CDCl3): –23.0 (1 B), –18.0 (5 B), –16.6 (5 B), 6.6 (1 B). Tris(tetrabutylammonium)4-carboxybutoxyundecahydro-closo-dodecaborate (III). A solution of KOH (10 g, 178.6 mmol) in 25 ml of water was added to a solution of nitrile (IX) (700 mg, 0.96 mmol) in 25 ml of MeOH, and the resulting solution was refluxed for 33 h. The reaction mixture was cooled, concentrated HCl (17 ml) and Bu4NHSO4 (340 mg, 1 mmol) were added successively, and the resulting solution was extracted with CH2Cl2 (2 × 30 ml). The combined extract was washed with water (2 × 40 ml), saturated aqueous NaHCO3 (50 ml), and water (50 ml). The organic layer was evaporated on a rotary evaporator in a vacuum and dried in a vacuum (1 mm Hg) to give 553 mg (58%) of salt (III) (cf. lit. [14]); Rf 0.66 (100 : 10 : 10 : 10 : 3 EtOH–BuOH–Py–AcOH–H2O); 13C NMR (CDCl3): 13.0 (CH3), 18.8 (CH2CH2COO), 23.2 (CH2CH3), 24.5 (CH2CH2N), 27.6 (CH2COO), 34.4 (CH2CH2O), 57.7 (CH2N), 68.9 (CH2O), 176.4 (COO); 11B NMR (CDCl ): –22.2 (1 B), –17.0 (10 B), 5.5 (1 B); 3 MS [registration of negatively charged ions, m/z (I, %)]: 502.4 (28) [M + H + Bu4N]. C21H56B12NO3, calc. 502.5 [M + H + Bu4N]; 775.4 (18) [M + MeOH + 2Bu4N], C38H95B12N2O4, calc. 775.8 [M + MeOH + 2Bu4N]; 1247.1 (9) [M2 + 2H + 3Bu4N], C58H148B24N3O6, calc. 1247.4 [M2 + 2H + 3Bu4N]. N-[(1,2-Dicarba-closo-dodecaboran(12)-1yl)acetyl]aminoacetyl-4-O-(b-D-galactopyranosyl)b-D-glucopyranosylamine (XI). DMT-MM (70.3 mg, 0.254 mmol) was added to a solution of acid (I) [13] (44.4 mg, 0.231 mmol) and amine (IV) ([15] (92 mg, 0.231 mmol) in a mixture of 1 ml MeOH and 0.7 ml of water under stirring, and the reaction mixture was stirred for 45 h at room temperature and evaporated in a vacuum. The residue was purified by reversed-phase chromatography on a Superclean LC18 cartridge (elution with water MeOH gradient) to give 67.4 mg 20 (50%) of homogeneous target product (XI); [ [ α ] D +2.3 (Ò 4.3, H2O); Rf 0.58 (100 : 10 : 10 : 10 : 3 EtOH– BuOH–Py–AcOH–H2O); 1H NMR (characteristic signals, D2O): 2.91 (2 H, s, COCH2C(B10H10)CH), 4.44 (1 H, d, J 7.1, H1 Gal), 4.45 (1 H, br. s, NH), 5.00 (1 H, d, J 9.3, H1 Glc); 13C NMR (D2O): 43.5 (CH2N), 44.1 ([C2HB10H10]CH2CO); 60.7 (C6 Gal), 61.9 (C6 Glc), 69.4 (C4 Gal), 70.6 (OCH2), 71.8 ([CHB10H10C]), 72.4 (C2 Gal), 73.4 (C3 Gal), 75.9 C2 Glc), 76.2 (2C, C5 and C3 Glc), 77.3 (C5 Gal), 78.6 (C4 Glc), 80.1 (C1 Gal), 103.8 (C1 Glc), 169.7, 172.5 (CO); 11B NMR (D2O): –9.5 (8 B, br. s), –5.4 (1 B, br. s), –2.3 (1 B, br.


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s); MS, m/z (I, %) 605.4 (33) [M + Na), C18H38B10N2NaO12, calc. 605.3 [M + Na]; 1188.3 (100) [M2 + Na], C36H76B20N4NaO24, calc. 1 187.7 [M2 + Na]; 1770.1 (16) [M3 + Na], C54H114B30N6NaO36, calc. 1770.0 [M3 + Na]. {2-[(1,2–Dicarba-closo-dodecaboran(12)-1yl)acetylamino]ethyl}4-O-(b-D-galactopyranosyl)b-D-glucopyranoside (XII) and {2-[(1,2–dicarbanido-undecaboran(11)-1-yl)acetylamino]ethyl}4-O(b-D-galactopyranosyl)-b-D-glucopyranoside (XIInido). Method 1. Sodium methoxide (2.4 µl of 0.2 M solution in MeOH) was added to a solution of dry acetylated conjugate (XV) (38 mg, 0.044 mmol) in absolute methanol (1.5 ml). The mixture was kept for 27 h at room temperature. Then 1 ml of Dowex 50Wx8 (H+) was added, the reaction mixture was kept for 1 h, and the cation exchange resin was filtered off. The filtrate was evaporated in a vacuum, and the residue was dried at a residual pressure of 1 mm Hg to give 20 mg (80%) of the target product (XII). Method 2. A 10% Pd/C catalyst (10 mg) was added to a solution of benzylated conjugate (XVI) (68 mg, 0.057 mmol) in 2 ml of MeOH. The suspension was stirred under hydrogen for 18 h at room temperature. The catalyst was filtered off through a Celite layer, and the filtrate was evaporated in a vacuum. The resulting crude residue (80 mg) contained 16% of the by-product (XII-nido) and 84% of the target product (XII) according to the 11B NMR data. Method 3. DMT-MM (111.9 mg, 0.405 mmol) was added to a solution of acid (I) [13] (71.3 mg, 0.370 mmol) and amine (V) [9] (141.8 mg, 0.368 mmol) in 2 : 1 MeOH–H2O under stirring. The mixture was stirred for 22 h at room temperature and evaporated in a vacuum. The residue was purified on a Superclean LC18 cartridge (elution with a water MeOH gradient) to give two fractions. The first fraction Ä (127.5 mg) eluted was a mixture of boric acid (27%), by-product (XII-nido) (8%), and the target product (XII) (65%), whereas the second, a more pure, fraction B (48.8 mg) contained boric acid (1%), by-product (XII-nido) (5%), and the target product (XII) (94%). The composition of each fraction was deduced from integration of the corresponding peaks in the 11B NMR spectra. Calculation of the total yields of conjugates of closo- (XII) and nido-carboranes (XII-nido), taking into account their molecular masses (569.61 and 558.80, respectively), gave the following results: the yield of the conjugate of closo-carborane (XII) was 74.2% and the yield of the conjugate of nido-carborane 20 (XII-nido) was 7.6%. Fraction B: [ α ] D +7.0 (Ò 2.4, H2O). Rf 0.38 (65 : 25 : 4 CHCl3–MeOH–H2O). Conjugate (XII); 1H NMR (characteristic signals, CD3OD): 3.17 (2 H, s, COCH2C(B10H10)CH), 4.33 (1 H, d, J 7.7, H1 Glc), 4.35 (1 H, d, J 7.0, H1 Gal), 4.71 (1 H, m, NH); 13C NMR (CD3OD): 40.7 (CH2N), 44.0 ([C2HB10H10]CH2CO), 61.6 ([CHB10H10C]), 61.8 (C6 RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY

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Glc), 62.4 (C6 Gal), 69.3 (OCH2), 70.2 (C4 Gal), 71.6 ([CHB10H10C]), 72.4 (C2Gal), 74.7 (2C, C3 Gal and C2 Glc), 76.2 (C5 Glc), 76.4 (C3 Glc), 77.0 (C5 Gal), 80.6 (C4 Glc), 104.2 (C1 Gal), 105.0 (C1 Glc).168.6 (CO); 11B NMR (CD OD): –9.5 (8 B, br. s), –5.4 (1 B, br. s), 3 –2.3 (1 B, br. s); 11B NMR (D2O): –10.7 (br. s), –5.1 (shoulder). Additional minor signals in the 11B NMR spectrum (CD3OD): –36.9 and –32.8 (XII-nido), 18.9 (H3BO3). Additional minor signals in the 11B NMR spectrum (D2O): –37.4 and –33.3 (XII-nido), 19.1 (H3BO3). MS, m/z (I, %): 594.4 (30) [M + Na], C18H39B10NNaO12, Calc. 594.3 [M + Na], 1165.4 (19) [M2 + Na]. C36H78B20N2NaO24, Calc. 1165.67 [M2 + Na]. Disodium (5-{2-[4-O;-(b-D-galactopyranosyl)-bD-glucopyranosylamino]-2-oxoethylamino}-5-oxopentyloxy)undecahydro-closo-dodecaborate (XIII). DMT-MM (31 mg, 0.112 mmol) was added to a solution of amine (IV) [15] (40 mg, 0.101 mmol) and acid (III) (100 mg, 0.101 mmol) in 2 : 1 MeOH–water (3 ml). The reaction mixture was stirred for 24 h at room temperature and evaporated in a vacuum. The residue was dissolved in water (3 ml) and extracted with CH2Cl2 (2 × 4 ml). The aqueous phase was treated with Dowex 50Wx8 (Na+-form). The cation exchange resin was filtered off, the filtrate was evaporated in a vacuum and dried in a vacuum (1 mm Hg) to give 40 mg (57%) of the target amide (XIII); Rf 0.66 (100 : 10 : 10 : 10 : 3 EtOH–BuOH–Py–AcOH–H2O); 13C NMR (D2O): 22.7 (COCH2CH2), 30.8 (OCH2CH2), 36.2 (COCH2(CH2)3), 43.5 (COCH2NH), 60.8 (C6 Glc), 61.9 (C6 Gal), 69.5 (C4 Gal), 69.8 (OCH2CH2), 71.8 (C2 Gal), 72.3 (C2 Glc), 73.3 (C3 Gal), 75.9 (C5 Glc), 76.2 (C3 Glc), 77.3 (C5 Gal), 78.7 (C4 Glc), 80.0 (C1 Glc), 103.8 (C1 Gal), 173.7 and 178.7 (NHC(O)CH2); an additional minor signal in the 13C NMR spectrum (D2O): 156.6 (NaHCO3); 1H NMR (characteristic signals, D2O): 5.02 (1 H, d, J 9.1, H1 Glc), 4.46 (1 H, d, J 7.7, H1 Gal); 11B NMR (D2O): –23.5 (1 B, br. s), –18.6 (5 B, br. s), 6.2 (1 B, br. s); MS (registration of negatively charged ions, m/z (I, %): 663.5 (74) [M + Na], C19H44B12N2NaO13, calc. 663.4 [M + Na]. Disodium-(5-{2-[4-O-(b-D-galactopyranosyl)-bD-glucopyranosyloxy]ethylamino}-5-oxopentyloxy)undecahydro-closo-dodecaborate (XIV). DMTMM (40 mg, 0.14 mmol) was added to a solution of amine (V) (50 mg, 0.13 mmol) and acid (III) (128 mg, 0.13 mmol) in 2 : 1 MeOH–water (3 ml). The reaction mixture was stirred for 24 h at room temperature and evaporated in a vacuum. The residue was dissolved in water (3 ml) and extracted with CH2Cl2 (2 × 4 ml). The extract was evaporated in a vacuum. According to the 1ç and 13C NMR, the residue (80 mg) consisted of tetrabutylammonium salts of 4,6-dimethoxy-13,5-triazin2-ol and the target product (XIV) (yield ~55%, estimated from 1H NMR data). The residue was dissolved in water (2 ml) and treated with Dowex 50Wx8

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(Na+-form) cation exchange resin. The resin was filtered off, the filtrate was evaporated and dried in a vacuum (1 mm Hg) to give 40 mg of the mixture of sodium salts of conjugate (XIV) and 4,6-dimethoxy-1,3,5-triazin-2-ol. The residue was dissolved in 1% aqueous NH4HCO3 (1.5 ml), applied on a Sephadex G-10 column, and eluted with 1% aqueous NH4HCO3. The fractions containing the target conjugate (XIV) were combined and evaporated in a vacuum at 50°C to give 8 mg 20 (9%) of (XIV); [ [ α ] D –3.0 (Ò 0.2, H2O); Rf 0.29 (100 : 10 : 10 : 10 : 3 EtOH–BuOH–Py–AcOH–H2); 13C NMR (D2O): 21.7 (CH2CH2CO), 30.7 (CH2CH2OB12H11), 36.4 (CH2C(O)N), 40.8 (OCH2CH2N), 61.0 (C6 Glc), 61.6 (C6 Gal), 69.3 (2C, CH2OB12H1 and OCH2CH2N), 69.6 (C4 Gal), 71.7 (C2 Gal), 73.2 (C3 Gal), 73.6 (C2 Glc), 75.0 (C5 Glc), 75.5 (C3 Glc), 76.0 (C5 Gal), 79.3 (C4 Glc), 102.9 (C1 Gal), 103.7 (C1Glc); 11B NMR (CD3OD): −22.3 (1 B, br. s), –17.7 (5 B, br. s), –16.1 (5 B, br. s), 6.9 (1 B, br. s); MS (registration of negatively charged ions, m/z (I, %): 648.6 (100) [M + Na], C19H45B12NNaO13, calc. 648.4 [M + Na]. {2-[(1,2-Dicarba-closo-dodecaboran(12)-1yl)acetylamino]ethyl}-2,3,6-tri-O-acetyl-4-O(2,3,4,6-tetra-O-acetyl-b-D-galactopyranosyl)-b-Dglucopyranoside (XV). Method 1. DCC (27.2 mg, 0.120 mmol) was added to a mixture of acid (I) [13] (20.6 mg, 0.107 mmol) and HONSu (14.9 mg, 0.129 mmol) in freshly distilled anhydrous THF (2 ml) cooled on ice bath, and the mixture was stirred for 2 h. Then a solution of é-acetylated 2-aminoethyl lactoside (VI) [8] (99.5 mg, 0.141 mmol) and Et3N (0.015 ml, 0.107 mmol) in freshly distilled anhydrous THF (2 ml) was added under stirring. The reaction was monitored by TLC (AcOEt). After 5-h stirring at room temperature, an additional portion of lactoside (VI) (10 mg, 0.014 mmol) was added. After 1 h, the resulting precipitate was filtered off; the filtrate was evaporated; and the residue was dissolved in CH2Cl2 and washed with 1 M H2SO4 (2 × 10 ml), saturated NaHCO3 (2 × 20 ml), and saturated NaCl (30 ml). The organic phase was dried by filtration through a cotton layer and evaporated in a vacuum. The residue was dissolved in dichloromethane, applied onto a Silasorb 600 (7 µm, 250 × 15 mm) col95 : 5 CH2Cl2– umn, and eluted with a CH2Cl2 MeOH gradient. The fraction with Rf 0.44 (95 : 5 CH2Cl2–MeOH) was collected, evaporated, and dried in a vacuum (1 mm Hg) to give 17.6 mg (19%) of pure target (XV). Method 2. Saturated NaHCO3 (2 ml) was added to a solution of amine (VI) (64.2 mg, 0.094 mmol) in 1 ml of dichloromethane. A suspension of chloride (II) [13] (29.5 mg, 0.142 mmol) in 1 ml of dichloromethane was added dropwise under stirring to the resulting mixture. The reaction mixture was stirred for 2 h at room temperature. The organic layer was separated, successively

washed with 2 M H2SO4 (10 ml), saturated NaHCO3 (20 ml), and saturated NaCl (30 ml). The organic phase was dried by filtration through a cotton layer and evaporated in a vacuum. The residue was dissolved in dichloromethane, applied onto a Silasorb 600 (7 µm, 250 × 15 mm) column, and eluted with a CH2Cl2 95 : 5 CH2Cl2–MeOH gradient. The fraction with Rf 0.44 (95 : 5 CH2Cl2–MeOH) was collected, evaporated, and dried in a vacuum (1 mm Hg) to give 38 mg 20 (47%) of pure target (XV); [ [ α ] D +2.2 (Ò 1.2, CHCl3); Rf 0.44 (CH2Cl2–MeOH, 95 : 5). 1H NMR (CDCl3): 0.80–2.30 (10 H, m, BH), 1.98, 2.05, and 2.15 (21 H, all s, 7 OAc), 3.14 (2 H, s, CH2C=O), 3.35–3.50 (2 H, m, OCH2CH2NH), 3.57–3.93 (5 H, m, OCH2CH2NH, H5 and H4 Glc, H5 Gal), 4.03–4.17 (3 H, m, H6a Glc, H6a and H6b Gal), 4.47 (1 H, d, J 7.9, H1 Glc), 4.54 (1 H, d, J 7.7, H1 Gal), 4.68 (1 H, t, J 10.8, H6b Glc), 4.85 (1 H, t, J 8.0, H2 Glc), 4.98 (1 H, dd, J 3.3 and 10.4, H3 Gal), 5.13 (1 H, dd, J 7.6 and 10.4, H2 Gal), 5.18 (1 H, t, J 9.4, H3 Glc); 5.36 (1 H, d, J 3.1 H4 Gal); 13C NMR (CDCl ): 20.5, 20.6, 20.8, 21.0 (CH C=O); 3 3 39.6 (CH2NH); 42.8 ([B10H10]CH2C=O); 58.7 (CH[B10H10]), 60.7 (C6 Gal), 61.2 (C6 Glc), 66.5 (C4 Gal), 69.1 (OCH2CH2NH), 69.1 (C2 Gal), 70.7 (C5 Gal), 70.9 (C3 Gal), 71.5 (C2 Glc), 72.5 (C3 Glc), 73.2 (C5 Glc), 75.6 (C4 Glc), 100.8, 101.0 (C1 Gal, C1 Glc), 166.5 (CH2C=O); 170.1 (CH3C=O ). 11B NMR (CDCl3): –9.9 (8 B, br. s), –5.6 (1 B, br. s), –2.5 (1 B, br. s); IR (film from CDCl3 solution, ν, cm−1: 1752 (ë=é), 2592 (BH), 3380 (NH); MS, m/z (I, %): 888.0 (90) [M + Na], C32H53B10NNaO19, calc. 888.4 [M + Na]. {2-[(1,2-Dicarba-closo-dodecaboran(12)-1yl)acetylamino]ethyl}-2,3,6-tri-O-benzyl-4-O(2,3,4,6-tetra-O-benzyl-b-D-galactopyranosyl)-b-Dglucopyranoside (XVI). Method 1. DCC (50.5 mg, 0.222 mmol) was added to a solution of amine (VII) [9] (101.5 mg, 0.100 mmol), HONSu (13.9 mg, 0.121 mmol), and acid (I) (29.7 mg, 0.102 mmol) in anhydrous THF (2 ml) cooled on ice bath. The mixture was stirred for 48 h, filtered, and the filtrate was evaporated in a vacuum. The residue was dissolved in CH2Cl2 and washed with 2 M H2SO4 (20 ml), saturated NaHCO3 (20 ml), and saturated NaCl (30 ml). The organic phase was dried by filtration through a cotton wool and evaporated in a vacuum. The residue was dissolved in dichloromethane, applied onto a Silasorb 600 (7 µm, 250 × 15 mm) column, and eluted with a petroleum ethe r 7 : 3 petroleum ether–AcOEt gradient. The fraction with Rf 0.32 (7 : 3 petroleum ether– AcOEt) was collected, evaporated, and dried in a vacuum (1 mm Hg) to give 18.6 mg (15%) of the pure target product (XVI). Method 2. DMT-MM (26.5 mg, 0.096 mmol) was added to a solution of amine (VII) (101.5 mg, 0.100 mmol) and acid (I) (17 mg, 0.087 mmol) in MeOH (1.5 ml). The reaction mixture was stirred for


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6 days at room temperature and then evaporated in a vacuum. The residue was dissolved in Et2O, the resulting solution was washed with 2 M H2SO4 (20 ml), saturated NaHCO3 (20 ml), and saturated NaCl (30 ml). The organic phase was filtered through a cotton wool and evaporated in a vacuum. The residue was dissolved in dichloromethane, applied onto a Silasorb 600 (7 µm, 250 × 15 mm) column, and eluted with a petroleum ether 7 : 3 petroleum ether–AcOEt gradient. The fraction with Rf 0.32 (7 : 3 petroleum ether–AcOEt) was collected, evaporated, and dried in a vacuum (1 mm Hg) to give 7.3 mg (7%) of the pure target product (XVI). Method 3. Saturated NaHCO3 (0.8 ml) was added to a solution of amine (VII) (90 mg, 0.087 mmol) in dichloromethane (0.8 ml); a suspension of chloride (II) [13] (55.6 mg, 0.267 mmol) in dichloromethane (1.2 ml) was added dropwise under stirring to the resulting mixture. The reaction mixture was stirred for 1 h at room temperature. The organic layer was separated, successively washed with 2 M H2SO4 (20 ml), saturated NaHCO3 (20 ml), and saturated NaCl (30 ml). The organic phase was dried by filtration through a cotton wool and evaporated in a vacuum. The residue was dissolved in dichloromethane, applied onto a Silasorb 600 (7 µm, 250 × 15 mm) column, and eluted with a petroleum ether 7 : 3 petroleum ether–AcOEt gradient. The fraction with Rf 0.32 (7 : 3 petroleum ether– AcOEt) was collected, evaporated, and dried in a vacuum (1 mm Hg) to give 55.4 mg (53%) of pure target 20 (XVI); [ [ α ] D +4.8 (c 0.84, CHCl3); Rf 0.32 (7 : 3 petroleum ether–AcOEt); 13C NMR (CDCl3): 40.7 41.6 ([C2HB10H10]CH2CO), 58.4 (CH2N), ([CHB10H10C]), 68.1, 69.3, 69.4 (C6 Glc, C6 Gal, OCH2), 70.9 ([CHB10H10C]), 72.5, 73.4 (2 C); 74.7, 75.2 (2 C), 75.4 (OCH2Ph), 73.2, 73.3, 74.5, 77.8, 79.8, 81.8, 82.6, and 82.8 (C2, C3, C4, C5 Glc; C2, C3, C4, and C5 Gal), 103.3, 104.3 (C1 Glc, C1 Gal), 127.5, 127.5, 127.6, 127.8, 127.9, 128.0, 128.2, 128.4, and 128.5 (Ph), 137.3, 138.1, 138.4 (2 C); 138.8, and 139.0 (2 C) (quaternary Ph), 166.5 (CO); 11B NMR (CDCl3): –10.2 (8 B, br. s), –5.6 (1 B, br. s), –2.8 (1 B, br. s); MS, m/z (I, %): 1224.8 (82) [M + Na], C67H81B10NNaO12, calc. 1224.7 [M + Na]. ACKNOWLEDGMENTS We are grateful to L.M. Likhosherstov for valuable advices on the synthesis of N-glycyl-β-lactosylamine. This work was supported by the Russian Foundation for Basic Research (project no. 03-003-32622) and by the Program on the Directed Synthesis of Compounds with Specified Properties and Creation of the FuncRUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY

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