Section 2
Preparation of SP14 repeating units to be loaded
on PAMAM dendrons
1. Introduction
1.1. Streptococcus Pneumoniae 14
Streptococcus pneumoniae type 14 capsular polysaccharide (Pn14PS) consists of biosynthetic repeating unit {6)-[β-D-Galp-(β-(14)-] β-D-GlcpNAc-(β-(13)-β-D-Galp-(β-(14)-β-D -Glcp-(1}n (1a, Fig. 1.1).
1
The immunogenicity of this polysaccharide and its depolymerized oligosaccharide fragments conjugated to a protein have been reported to produce specific anti-Pn14PS antibodies in mice.2 The poor immunogenicity of the Pn14PS compared to other PnPSs3 may be due to structural similarities between antigenic determinants of the Pn14PS and human OS structures as human milk OSs and blood group carbohydrate structures. To avoid cross-reactivity with human tissue and the induction of autoreactive antibodies, the synthetic glycoside 1b (Fig. 1.1), a mimic of tetrasaccharide corresponding to a single structural repeating unit of Pn14PS, was conjugated to the crossreactive material of CRM197 (see Preface) and found to induce
anti-polysaccharide type 14 antibodies.4 Figure 1.1
A recent study5 was set up to investigate how small the minimal structure in Pn14PS can be and still produce specific antibodies to polysaccharide type 14. To do this, a series of oligosaccharide fragments of Pn14PS, varying from tri- to dodecasaccharides, were synthesized. These oligosaccharide fragments were then conjugated to a protein carrier as CRM197 or bovine serum
albumin (BSA). The biological results pointed out that branched tetrasaccharide 1c produced a specific antibody response to Pn14PS, as described by Mawas et al,4 and is a serious candidate for a synthetic oligosaccharide conjugate vaccine against infections caused by SP14.
1.2. Literature syntheses of SP14 capsular polysaccaride fragments
The reported syntheses of tetrasaccharide 1b and/or of its glycosides equipped with appropriate spacers to the protein conjugation, are based on a sole strategy involving 4 steps:6 (a) synthesis of a 4-OH-β-D-glucosamine glycosyl acceptor 2 with a functionalized spacer, if conjugation is the final target; (b) β-(14) galactosylation using 2 as acceptor; (c) deprotection of the lactosamine OH-6
group; (d) β-(16) lactosylation and final deprotection (Scheme 1.1).
Scheme 1.1
The sole alternative to this “general” method is reported and involves a chemoenzimatic approach, developed by Kamerling et al.7 where an easier access to well-defined synthetic oligosaccharide-protein conjugates was investigated. In this approach an enzymatic reaction mediated by commercially available β-1,4-galactosyltransferase had permitted to avoid the β-1,4-galactosylation reaction, a problematic step in the chemical preparation of 4. In biosynthetic pathways β -1,4-galactosyltransferase, commonly regarded as synthesizing terminal N-acetyllactosamine sequences, catalyzes the transfer of β-D-galactopyranosyl groups from UDP-galactose to the 4-position of terminal N-acetyl-β-D-glucosamine residues.8 The transferase has been extensively studied with regard to synthetic applications and substrate specificity and it has been shown that the enzyme allows various modifications at the GlcNAc acceptor residues. Different aglycons, a variety of N-acyl groups and substitutions at the 3- and 6-position are tolerated. With respect to the 6-position, 6-O-acetyl, 6-O-methyl and 6-O-sulfate groups are accepted. An interesting example of this approach is reported in Scheme 1.2: the trisaccharide 7 was first synthetized coupling a lactoside donor with the functionalized glucosamine. The final galactosyltransferase reaction on the deprotected trisaccharide as acceptor and UDP-Gal as donor was performed permitting a near quantitative conversion of the acceptor to tetrasaccharide 8.
Scheme 1.2
Reagents and conditions. i: TMSOTf, 4 Å MS, DCM, 0°C to rt (68%); ii: a) MeONa, MeOH; b)
NH2(CH2)2NH2, n-BuOH, 80°C; c) Ac2O, Py (98%, 3 steps); iii: MeONa, MeOH (94%); iv: β
-1,4-galactosyltransferase. MBz: p-methylbenzoyl.
1.3. A new synthetic approach to SP14 tetrasaccharide
The method that we wanted to develop was based on a completely synthetic process (Scheme 1.3) using lactose as the sole starting material. That approach might permit to avoid the β -galactosylation step required for the lactosamine unit construction because lactose contains a preformed β-(14) linkage. As shown in Scheme 1.3, protected lactal was obtained in high yield
from lactose through introduction of the 1,2-enol ether double bond. Lactal might represent a valid key intermediate to lactosamine derivatives.
Glycals are very versatile glycosyl donors and are often used as precursors for the preparation of 2-amino-2-deoxysugar by way of N-functionalization at C-2 accompanied by C(1)-O bond formation. Over the last few decades, a variety of methods have been developed for the nitrogen transfer to glycals:9 (a) introduction of 2-azido moiety to glycals via radical mechanism (azidonitration); (b)
cycloadditions with formation of pyrano-oxadiazines or triazolines; (c) phosphoramidation; (d) sulfonamidoglycosylation; (e) transition metal-mediated amidation; (f) 2-acetamidoglycosylation.
1.3.1. Azidonitration
In 1971, Trahanovsky and Robbins reported the formation of α-azido-β-nitroalkanes on treating alkenes with sodium azide and ceric ammonium nitrate (CAN) in moist acetonitrile.10 Since the products obtained were consistent with the reaction being initiated by addition of an azide radical (anti-Markovnikov route), Lemieux and Ratcliffe anticipated that the azide radical-induced addition to glycals would provide 2-azido-2-deoxyglycosyl nitrates. This gave rise to the development of the azidonitration protocol.11 As in the original procedure, the azido radical is generated from sodium azide and CAN. High regioselectivity of this process is an important advantage, yet, very commonly, epimeric mixtures of 2-azido-2-deoxy-1-O-nitropyranoses are obtained.
The ratio of the epimers formed is strongly dependent on the structure of the glycal substrate. For example, for the lyxo-glycal 9a (‘galactal’, Scheme 1.4), the 2-equatorial product was highly favored, leading to 2-azido-deoxygalactose nitrates 10a in 85% yield.11 Differently, for the arabino-glycal 9b (‘glucal’), mixtures of 2-azido-2-deoxy-D-gluco 10band D-manno derivatives 11 were obtained.12 This higher stereoselectivity of the galacto series can be explained by the increased steric hindrance of the top face of the D-galactal derivative 9a in comparison to that of the D-glucal derivative 9b.
Scheme 1.4
Reagents and conditions. i: NaN3, CAN, MeCN, -15°C (10a 85%)
Transformation of the 2-azido-2-deoxy-1-nitro-pyranose intermediate into an N-acetyl-D -hexosamine can be achieved in a number of ways: the most common approach is the acetolysis of
the azidonitration products, that gives 1,3,4,6-tetra-O-acetyl-2-azido glycopyranosides followed by azide reduction and N-acetylation. The azido moiety can then be reduced under a variety of reaction conditions amongst which there are catalytic hydrogenation (H2, Pd/C),treatment with
1,3-propanedithiol, Staudinger ligation (Ph3P in THF/H2O)or Birch reduction (Na/liquid NH3). The
free amine can then be converted into acetamido or other NHR or NR2 derivatives by simple
protecting group chemistry. Direct conversion of azido to acetamido moiety has also been accomplished via reductive acetylation using neat thiolacetic acid. Depending on the strategy employed, the azide deprotection can be achieved either prior or after the glycosylation step: on the one hand this approach allows the synthesis of 1,cis glycosides via direct glycosidation of a 2-azido donor; on the other hand, 1,2-trans glycosides can also be obtained via transformation of the azido moiety into a 2-N-participating group, such as 2-phthalimido or 2-N-acetamido.
1.3.2. Cycloadditions with formation of pyrano-oxadiazines
The cycloaddition of azadicarboxylates with simple vinyl ethers was first reported in 1969 to give [4+2] or [2+2] adducts depending on the substrate.13Based on the published mechanistic studies of
this reaction with simple substrates, Leblanc and co-workers reasoned that the diastereoselectivity of the cycloadducts would be most readily attainable with a rigid substrate having an appropriately placed substituent as allylsubstituted cyclic vinyl ethers.14Since glycals fit this criterion, Leblanc et al explored the possibility of applying this reaction to the synthesis of 2-aminosugars from glycals. Thus, irradiation with UV light at 350 nm of a solution of tri-O-silylated glucal 12 with dibenzyl azadicarboxylate in cyclohexane led to the single [4+2] cycloadduct 13 in 70% yield (Scheme 1.5).15
Schema 1.5
Reagents and conditions. i: 350 nm, CH2Cl2, ciclohexane (70%); ii: a) p-TsOH, MeOH (88%); b)
This initially surprising diastereoselectivity was attributed to the preference of the trisilylated glucal to adopt a 1C4 or a half-boat conformation. As such, the axial C-3 silyl ether would hinder the
cycloaddition from the top face of the molecule. Treatment of cycloadduct 13 with a catalytic amount of p-TsOH in MeOH gave the corresponding methyl glycoside 14 in 88% yield. Hydrogenolysis of the protected hydrazine 14 followed by acetylation gave methyl 2-acetamido-2-deoxyglycoside 15 in 72% yield.
1.3.3. Phosphoramidation
The elaboration of activated 1,2-aziridines for the synthesis of 2-aminosugars by direct insertion was developed by Lafont and Descotes.16 Thus, the reaction of tri-O-benzyl glucal 16 with
iodoazide, followed by Staudinger reaction led to the formation of the trans-diaxially substituted 2-deoxy-2-iodomannopyranosyl phosphoroamidate 17 in 58% yield. Along with the main product of the D-mannose series, a 2-iodo phosphoroamidate of β-D-glucose series was obtained in 34% yield. The reaction of 17 under basic conditions in MeOH allowed the corresponding β-methyl glucoside in 92% yield with the inversion of configuration at C-1 and C-2. It was proposed that the glycosylations proceeded via an aziridine intermediate. This approach was found to be significantly less efficient for glycoside acceptors instead of simple alcohol substrates, giving corresponding disaccharides in low yield (about 30%).
Scheme 1.6
Reagents and conditions. i: a) NaN3, ICl, CH3CN; b) P(OMe)3, CH2Cl2 (58% from 16); ii:
NaOMe, MeOH (92%).
1.3.4. Sulfonamidoglycosylation
Inspired by the pyrano-oxadiazineand phosphoramidationprocedures described above, Griffith and Danishefsky explored another possibility for the azaglycosylation of glycals.17 It was assumed that substitution of the nitrogen with a more effective electron-withdrawing group would significantly enhance the glycosidating potential of the supposed aziridine intermediate, and thereby allow glycosylation of unreactive glycosyl acceptors, a major limitation of the previously introduced techniques. Thus, the reaction of tri-O-benzyl glucal 16 with iodonium (di-sym-collidine) perchlorate (IDCP) and benzenesulfonamide gave the trans-diaxial iodosulfonamide 19 in 78%
yield (Scheme 1.7). Both oxygen and sulfur nucleophiles were investigated for subsequent glycosidations with 19. For example, the sulfonamide migration reaction in the presence of lithium ethanethiolate produced thioglycoside 20 in 85% yield.
Scheme 1.7
Reagents and conditions. i: NH2SO2Ph, IDCP, CH2Cl2, 4 Å MS, 0°C (78%); ii: EtSH, LHMDS,
DMF, -40°C to rt (85%). IDCP= I(sym-collidine)2 ClO4; LHMDS= lithium
bis(trimethylsilyl)amide.
O-glycosylation reactions of 19 with monosaccharides gave disaccharide derivatives in highest yields (60-65%) than the reported ones for phosphoramidation method. It was also demonstrated that 2-sulfonamidoglucosides can be readily converted into natural 2-acetamido derivatives by the treatment with excess sodium in ammonia followed by N-acetylation.
1.3.5. Transition metal-mediated amidation
In 1997, Carreira and co-workers introduced a transition metal-mediated approach to amidation of glycal substrates that leads to the formation of 2-deoxy-2-trifluoroacetamido derivatives.18 The impetus for this project was provided by previous studies showing that easily accessible nitridomanganese complexes, when reacted with trifluoroacetic anhydride (TFAA), transferred a CF3CON unit to electron-rich silyl enol ethers. Thus, activation of (saltmen)Mn(N)with TFAA and
transfer of the CF3CON group to glycal 21 (Scheme 1.8) gave 2-N-trifluoroacetamido derivatives in
good yields and excellent diastereoselectivity. The stereochemical outcome at C-2 of this reaction is controlled by the proximal stereocenter at C-3. As illustrated in Scheme 1.8, sequential treatment of the formed complex with thiophenol and BF3.OEt2 afforded the desired thioglycoside 24 with
complete β-stereoselectivity.
Scheme 1.8
Reagents and conditions. i: (saltmen)Mn(N), TFAA, CH2Cl2;ii: PhSH, BF3 .
1.3.6. Acetamidoglycosylation
All these reported methods, although effective in the transfer of nitrogen to the glycal donor, must also entail further functional group manipulations in order to install the naturally occurring acetamido group at C-2 as well as to form the glycosidic bond with the desired glycosyl acceptor. In 2000 Gin and co-workers introduced the so-called C-2-acetamidoglycosylation procedure,19a one-pot method for nitrogen transfer that efficiently effects the direct installation of the native C-2-N-acetylamino functionality onto glycal donors and, additionally, allows glycosidic coupling with various glycosyl acceptors. In this approach a new sulfonium reagent is employed for glycal activation (25, Scheme 1.9). This thianthrene bis(triflate) was prepared starting from thianthrene-5-oxide which in combination with Tf2O generates the reagent in situ. Gin’s acetamidoglycosylation
reaction using glycal donors involves an exceedingly simple procedure, exemplified by the glycosylation of 2-propanol with tri-O-benzyl-D-glucal to generate isopropyl 2-N-acetamido-3,4,6-tri-O-benzyl-2-deoxy-β-D-glucopyranoside: triflic anhydride (2 equiv) is added to a solution of perbenzyl glucal (1 equiv) and thianthrene-5-oxide in a mixture of chloroform and dichloromethane (4:1) at -78°C. Following an initial activation period of 10’, the acid scavenger N,N-diethylaniline (4 equiv) and solid N-trimethylsilyl-acetamide (N-TMS-acetamide, 3 equiv) were added sequentially, and the reaction mixture was stirred at rt for 2 h. Amberlyst-15 acidic resin and the glycosyl acceptor (2- propanol, 3 equiv) were then added, leading to the formation of the isopropyl glycoside in 73% yield.
Scheme 1.9
Reagents and conditions. i: 25, PhNEt2,TMSNHAc, CHCl3-CH2Cl2, -78°C;ii: NuH,
As shown in Scheme 1.9, the reaction could proceed by way of the pathway in which the initial step involves low-temperature activation of thianthrene-5-oxide with Tf2O to generate complex 25 in
situ. Electrophilic activation of the enol ether functionality in 12 by 25 would lead to the formation of a pyranoside intermediate incorporating an oxocarbenium triflate functionality at C-1 and a thianthrene sulfonium moiety at C-2. The introduction of the acetamido group proceeds upon addition of the acid scavenger N,diethylaniline and NTMS- acetamide. A key function of the N-trimethylsilyl protecting group in this amide reagent is to provide steric shielding at the nitrogen atom to favor initial addition of the amide oxygen atom onto C-1 of the activated glycal, thereby generating the putative acetimidate intermediate 26. Subsequent intramolecular displacement of the C-2-thianthrene-sulfonium moiety by the imidate nitrogen accompanied by loss of the N-TMS protective group (either as TMSOTf or more likely as it’s N,N-diethylaniline adduct) would generate the oxazoline intermediate 27. Oxazoline intermediates can be often isolated prior to the introduction of the acid and glycosyl acceptor lending support to the proposed reaction pathway in Scheme 1.9. Acid-mediated oxazoline ring-opening in the presence of a glycosyl acceptor would then afford the desired glycoside 28 in the final stage of the acetamidoglycosylation.
Using this procedure several glycosylations were performed with a series of selectively protected glycal donors.19 It is worth highlighting that the yield in each of these one-pot operations is the result of glycal activation, nitrogen transfer by oxazoline formation, oxazoline ring-opening, and glycosidic bond formation. Thus, the acetamidoglycosylation of simple alkyl alcohols as 2-propanol, benzyl alcohol, dihydrocholesterol as well as of the carbohydrate acceptor methyl 2,3,4-tri-O-benzyl-α-D-glucopyranoside led to the formation of the corresponding C-2 acetamidoglycosides and C-2 acetamidodisaccharides directly from protected glycal donors in good yields (45-70%). In addition, glycal protective groups such as benzyl, triisopropylsilyl (TIPS), and allyl ethers as well as the acid-labile isopropylidene ketal can be used in this glycosylation process. The Gin’s method involves a novel process for nitrogen transfer to glycals that: a) employs thianthrene-5-oxide and triflic anhydride as a new reagent combination for glycal activation; and b) effects, for the first time, the direct installation of the naturally occurring C-2 N-acetamido functionality onto glycal donors in conjunction with glycosidic coupling to various glycosyl acceptors in an overall one-pot procedure.
1.4. The Heyns rearrangement revisited
In 1999 as an alternative approach to lactosamine derivatives Stütz et al developed a synthetic method starting from a commercial product with a preformed β-(14) linkage, the lactulose 37.20
constipation and hepatic encephalopathy, a complication of liver disease. It is a disaccharide where galactose is β-(14) linked with D-fructose. This approach is presented to be an “exceptionally simple” two step chemical synthesis of lactosamine in a multigram scale based on Heyns rearrangement revisited. In the 1950s, Heyns and Koch reported the formation of D-glucosamine 35 from the reaction of D-fructose 29 with ammonia (Scheme 1.10). Yields of isolated product for this reaction were found to be around 10% with rare exceptions of 30% depending on the reaction conditions employed.21 Scheme 1.10 OH OH HO O OH OH OH OH 29 O HO OH OH HO O OH OH NHR OH 30 NHR HO OH OH OH OH NHR HO OH OH OH OH 32 31 HO OH OH O OH NHR HO OH OH O OH RHN OH O HO NHR OH HO 35 OH O HO NHR OH HO 36 34 33
From the reaction of D-fructose with primary or secondary aliphatic amines, N-substituted D -glucosamine derivatives were subsequently obtained by Carson22 as well as the Heyns group. The latter workers also examined the reactivities of the other ketohexoses and the condensation products of D-fructose with aromatic amines and amino acids. In all reported cases, yields rarely exceeded the 20% mark. In the Stütz approach20 the Heyns rearrangement proceeded, presumably because of destabilizing interactions of the glycosyl moiety at O-4 with the other polar groups, considerably more efficiently than with unsubstituted D-fructose, allowing for yields of 60 to 70% of the N-benzyl-D-lactosamine 38 (Scheme 1.11), with the formation of a small portion of the corresponding D-mannosamine derivative. Lactulose 37 was reacted with commercial grade benzylamine to give a crystalline mixture of the ketosylamine and unreacted starting material. This mixture underwent rearrangement in methanolic solution containing 10% glacial acetic acid as the catalyst to furnish crystalline N-benzyllactosamine (38) together with small amounts of side products. The corresponding 2-aminodeoxy disaccharide was obtained simply adding Pd(OH)2 and
stirring the suspension under a H2 atmosphere. Subsequent acylation (excess Ac2O and solid
NaHCO3) of the free amino group gave desired N-acetyllactosamine 39 in a overall yields between
8:4:1). This sequence, which can be performed conveniently as a one-pot procedure, does not require any protecting group manipulations or elaborate chromatographic separation and is well suited for scaling-up. A wide range of N-modified derivatives suitable as intermediates for sophisticated oligosaccharides are available in good yields by employing different acylating reagents.20
Scheme 1.11
Reagents and conditions. i: a) BnNH2, 40°C; b) MeOH-AcOH (25:1); ii. for 39a: a) H2, Pd(OH)2
-C; b) Ac2O, NaHCO3 (38-45% from 37). For 39b: DTPM reagent, TEA (65% from 37).
In 2003 an optimization of this method has been achieved by the same group:23 (a) ketosyl amine product formed in the first reaction of 37 with benzylamine and unreacted 37 were precipitated by treatment of the reaction mixtures with Et2O; (b) hydrogenolysis was performed in presence of HCl
(pH 1-2) for an “indirect” partial purification of the mixture (rearrangement side products do not survive to this condition); (c) N-protection was achieved in methanol employing Et3N and
DTPM-reagent (41), easily prepared from 1,3-dimethylbarbituric acid 40 (Scheme 1.12).24
Scheme 1.12
Reagents and conditions. i: N,N-DMF dimethylacetal, CHCl3 (67%).
DTPM protecting group is stable during most reaction conditions commonly used in carbohydrate chemistry such as acetylation, Zemplén conditions, alkylation, hydrogenolysis, acetal formation, silylation and can be easily removed with ammonia, hydrazine or primary amines at rt in a few minutes. Employing this protecting group, the N-DTPM protected 39b precipitates from the reaction mixture, whereas unreacted lactulose 37 remains in solution. Peracetylation of this lactosamine derivative (Ac2O, Py) allowed, after recristallisation from diisopropyl ether, access to
2. Results and discussion
As the first step towards the SP14 tetrasaccharide preparation, synthesis of a lactosamine derivative has been attempted using commercial lactose as starting material that permits to avoid the β-(14)
galactosylation, a problematic step in monosaccharide assembly of lactosamine. LacNAc preparation has been attempted using: (a) Gin’s acetamidoglycosylation19 starting from lactal; (b) Heyns rearrangement revisited by Stütz20 starting from commercial lactulose. These approaches have presented many limits, low yields and difficulties in product purification: those problems will be discussed in each section. At the end the best way to synthesize a properly substituted lactosamine has been the monosaccharide assembly starting from commercial glucosamine hydrochloride.
2.1. Gin’s acetamidoglycosylation: lactosamine from lactal
As shown in the introduction, glycals have been widely employed in the synthesis of various types of glycosides and oligosaccharides. Glycals also serve as the key building blocks in the synthesis of optically active natural products. Therefore, their efficient, cost-effective large scale preparation would be of great value. Typically, the preparation involves three steps: (a) peracetylation of a carbohydrate; (b) replacement of anomeric acetate with a halide or sulfide; (c) reductive amination to generate the 1,2-double bond. The mechanism of reductive elimination (Scheme 2.1) is believed to involve a two-electron reduction process localized at the anomeric centre, followed by elimination of an acetoxonium anion from the vicinal position.25
Scheme 2.1
The classic method for (b) and (c) steps is the reaction of acetobromosugar with zinc dust alone or in combination with salts of other metals in aqueous acetic acid, but the yield are variable and solvolysis products, among others, are formed in side reactions.26 In 1993 an improvement of this method has been introduced by Somsàk et al,27 with the use of a base as 4-methylpyridine or 1-methylimidazole in the reductive elimination step. This variation in the reaction condition had permitted to obtain many types of glycals from good to excellent yields, often without side products.
A one-pot preparation of peracetylated glycosyl bromide from reducing sugars has been reported28 using acetic anhydride and a catalytic amount of HBr/AcOH to effect peracetylation, followed by the addition of several more equivalents of HBr/AcOH to complete the formation of the anomeric peracetylglycosyl bromides. However it was noted that quite frequently the products from this treatment have been described as being accompanied with furanosyl acetate type of byproducts: this problem could be effectively circumvented by mantaining the reaction temperature at or slightly below room temperature. That procedure was successfully used in a one-pot preparation of peracetylated glycals from reducing sugars without anomeric bromide isolation.29
Applying that procedure to commercial lactose, the unprotected sugar has been efficiently converted into bromide 46 (Scheme 2.2). After complete formation of 46, analyzed by TLC, the excess of HBr was neutralized with sodium acetate, followed by pouring the resulting solution into a suspension of activated Zn/CuSO4 in water and acetic acid buffered with sodium acetate. Finally,
exa-O-acetyl lactal 47 has been isolated in 76% yield, higher than reported one (61%).
Scheme 2.2
Reagents and conditions. i: HBr-AcOH, Ac2O; ii: Zn, CuSO4.5H2O, AcONa.3H2O, H2O-AcOH
(76% from lactose); iii: a) Na2CO3, MeOH; b) BnBr, NaH, TBAI, DMF (89% from 47).
As a final step lactal 47 has been converted into the perbenzyl analogue 48 with excellent yield (89% from 47), following a reported sequence of acetyl protection removal using Na2CO3 in dry
MeOH and perbenzylation under standard conditions (BnBr, NaH).30
With the lactal 48 in hand, the preparation of lactosamine derivatives bringing a functionalized spacer on anomeric position has been approached using the Gin’s method.
A preliminary study has been made to find the optimal reaction conditions using alcohols 49 and 50 (Fig. 2.1) as glycosyl acceptors and glucal 16 as a symplified model of 48 (Scheme 2.3).
Glucal 16 has been transformed into oxazoline 51 following the reported method (Scheme 2.3):19 triflic anhydride has been added to a solution of 16 and thianthrene-5-oxide in a mixture of CHCl3
-CH2Cl2 (4:1) at -78°C. After 10’ the solution has been treated with N,diethylaniline and solid
N-TMS-acetamide and allowed to warm at room temperature. Formation of oxazoline 51 has been detected by TLC analysis and analyzed after chromatographic purification (58% yield). The physico-chemical properties of 51, including 1H- and 13C-NMR spectra, have been identical to those reported in the literature.19
Scheme 2.3 BnO O OBn BnO i 16 O O BnO N BnO BnO ii OR O BnO NHAc OBn BnO 51 52: R=(CH2)2NHBoc 53: R=(CH2)2OTs OH O BnO AcHN OBn BnO 54
Reagents and conditions. i: thiantrene-5-oxide, Tf2O, PhNEt2,TMSNHAc, CHCl3-DCM, -78°C to
rt (58%); ii. for 52: 50, amberlist-15, DCM then Ac2O, Py (13%); for 53: 49, amberlist-15, AW-300 MS, DCM then Ac2O, Py (14%).
Oxazoline 51 has been regioselectively opened to obtain β-glycosydes 52 and 53 using amberlist-15 as acid catalyst in presence of alcohols 50 and 49. The use of 3-N-t-butoxyaminopropanol, easily prepared through treatment of 3-aminopropanol with Boc2O in DCM,
31
as acceptor gave, after cromatography, a main fraction of impure β-glycosides 52 and a fraction containing hydrolized product 54, probably formed because of not complete anhydricity. After standard acetylation of the mixture with β-glycosyde, desired 52 has been isolated in poor yield (13%). Also with the use of acceptor 49, prepared through a simple reaction between 1,3-propandiol and TsCl, and applying the same reaction conditions, β-glycoside 53 has been obtained pure in low yield (14%). The structure of each glycosides has been demonstrated by 1H-NMR experiment: the signals
of C-2 acetamido group as NH (2 doublets, 6.34 and 5.60) and CH3CO (2 singlets, 1.42 and 1.85),
the J1,2 (8.4 and 8.3 Hz, axial-axial interactions) and signals related to aglicons have confirmed the
glycosides 52 and 53 formations. The low yields of oxazoline opening are probably caused by low reactivity of acceptors face to the reaction conditions. Formation of hydrolyzed compound 54 has not been avoided also using flamed glassware, cooled under Ar, in presence of activated AW molecular sieves.
Using perbenzyl lactal 48 as starting material, related oxazoline 57 has been prepared applying the same conditions used for glucal 16. After chromatographic purification of the crude, a prevalent portion containing desired 57 has been obtained in 28% yield. Also a little portion containing a mixture of gluco- and manno-oxazoline has been isolated. The NMR analysis of this mixture has
permitted to obtain some signals related to manno diastereoisomer 56 (Scheme 2.4) as singlet at δ 2.16 (oxazoline CH3) and the doublet at δ 5.64 (H-1) that presents a J1,2 6.0 Hz, lower than the
vicinal coupling constant of the gluco one, confirming a different configuration of H-1 and H-2.
Scheme 2.4
In Scheme 2.5 is represented a possible rationalization of the formation of oxazoline 56 and 57: manno-oxazoline has been formed probably for a starting approach of the thiantrene electrophile on the α-side of lactal 48 instead of the usual β-side approach, because of the bulky C-4 galactopyranosyl substituent. The formation of intermediate 55 gave 56 after the attack of N-TMS substituent on C-2. With the glucocompound 57 in hand, the regioselective opening of oxazoline ring has been achieved applying the same reaction conditions seen for the model 51, using MeOH and 49 as nucleophile. The formation of the methyl β-glycoside 58 has been obtained in a 34% yield from oxazoline 57 (Scheme 2.5) as confirmed by NMR analysis: doublet at δ 6.43 (NH) and singlet at δ 1.84 (acetamido CH3) related to C-2 acetamido group and singlet at δ 3.39
related to anomeric OMe. The β-stereoselectivity of the reaction has been confirmed by high value of J1,2 (8.3 Hz), related to axial-axial interaction between H-1 and H-2. Trying to apply the one-pot
procedure without isolation of oxazoline intermediate on lactal 48, glycoside 59 has been isolated in very low yield (8% from lactal 48) after acetylation of the crude and chromatographic purification. An improved yield has been obtained applying a reported method of β-glycosylations with oxazoline donors and camphorsulfonic acid (CSA) as acid promoter.32
Scheme 2.5
Reagents and conditions. i: for 58: MeOH, amberlist-15, DCM (34%); for 59: 49, CSA, DCE,
80°C (44%); for 60: THF-AcOH-H2O (44%).
Oxazoline 57 has been opened in presence of acceptor 49 in anhydrous DCE at 80°C following this method. Derivative 57 has been trasformed in the desired glycoside 59 in quite good yield (44%). Also a fraction with hydrolyzed product 60 has been also isolated but in low yield (15%). The direct preparation of 60 has been also approached to obtain a possible precursor of highly reactive donor as trichloroacetimidate. Oxazoline 57 has been hydrolized using a solution of THF-glacial AcOH-H2O (1:1:1 v/v) rapidly obtaining α-derivative 60 in 44% yield. The preparation of 60 has
been also achieved starting from lactal 48 without isolation of oxazoline intermediate: treatment of the oxazoline solution with H2O in presence of amberlist-15 resulted in a mixture of desired
derivative 60 and the mannosamine analogue (C-2 epimer) in a 9:1 ratio, not separable by flash chromatography and in low yield.
Analyzing all these results and planning the preparation of multi-gram quantities of lactosamine glycosylated with functionalized spacer, some problems have arised during the scale-up of this synthetic approach: (a) in the preparation of lactal from lactose decreasing in yields was observed due to difficulties in the work-up of peracetyl intermediate 47; (b) the Gin’s approach to lactosamine has presented a difficult control of the various variables on reaction conditions; (c) the Gin’s approach to β-lactosamine has shown low yields (30%-40% as the best result) also with nearly gram quantities of oxazoline 57.
2.2. Heinz rearrangement of lactulose
As an alternative way to Gin’s approach, Stütz method20 from lactulose to lactosamine has been approached for some reasons: (a) starts from lactulose, a precursor with a preformed β-(14)
linkage; (b) is presented as a good way for multi-gram production of lactosamine; (c) target compound 39 is “easily” prepared with no chromatographic purification in quite good yield (38-45%).
However, following the reported procedure, many problems occurred in every step of the synthetic sequence: (a) difficulties in TLC detection of intermediates, (b) uncomplete precipitation of N-Bn derivatives in Et2O and (c) following the DMTP approach,
23
only a dark mixture of products has been obtained.
Alternatives have been attempted to overcome the observed problems but peracetylation trials on deprotected crude containing N-benzyl lactosamine 38 failed in the final chromatographic purification (many products, not defined TLC spots) and functionalization of the final crude with different protecting groups, as 4’,6’-O-benzylidene, failed in the crystallization of the final products.
The only relevant result has been the isolation of an inseparable α- and β-N-acetyllactosamine 39 mixture (Scheme 2.6) after an high polar chromatographic purification (CHCl3-MeOH-NH4OH,
2:3:1), with an overall 10-15% yield from commercial lactulose 37. The separation of the two anomers has also failed after final peracetylation of the mixture containing 39.
Scheme 2.6
Reagents and conditions. i: a) BnNH2, 40°C; b) MeOH-AcOH (25:1); ii: a) H2, Pd(OH)2-C,
AcOH, MeOH; b) Ac2O, MeOH (10-15% from 37); iii. Ac2O, Py (quant).
2.3. Synthesis of SP14 tetrasaccharide: a monosaccharide approach to lactosamine intermediates
The preparation of protected tetrasaccharide related to SP14 CPS has been finally achieved following a method similar to that reported by Yong-Xiang and coworkers for the synthesis of the SP15C pentasaccharide (Scheme 2.7):33 (a) preparation of a 4-OH-β-D-glucosamine glycosyl acceptor with a functionalized spacer, starting from commercial glucosamine hydrochloride; (b) β -(14) glycosylation of the above acceptor with a protected galactosyl donor; (c) deprotection of
the lactosamine OH-6 group; (d) β-(16) glycosylation using a protected lactose as donor. The
preparation of glucosamine oxazoline 63, a good precursor for the selective preparation of 2-acetamido-2-deoxy β-D-glucopyranosides, has been planned. Commercial glucosamine hydrochloride has been selectively transformed into α-D-per-O-acetyl analogue 62 in nearly
quantitative yield through standard acetylation in Ac2O/Py. The most common approach to
oxazoline derivatives as 63 was the treatment of α-D-per-O-acetyl precursors with an acid activator as TMSOTf in dichloroethane at 50°C:34 the oxazoline has been obtained in multigram quantities, in nearly quantitative yield and seemed to be stable if well quenced with Et3N.
Scheme 2.7
The oxazoline donors can undergo nucleophilic addition at C-1: thus, glycosylation of various alcohols and other nucleophiles with the GlcNAc oxazoline peracetate 63 leads to the exclusive formation of β-glycosides, using an acid catalyst as p-TsOH, 35 CSA36 or BF3.Et2O.37 From 63 β
-glycosides have been prepared using different alcohol as acceptors: MeOH, 49 and 50, seen in the Gin’s approach application.
The preparation of the methyl glycoside 75 has been first attempted following a reported method 37 that use BF3.Et2O as activator and MeOH as solvent and acceptor (Scheme 2.8): desired glycoside
has been isolated in 50% yield as the best results because of the formation of hydrolized oxazoline and other not analyzed byproducts.
Trying to optimize the reaction conditions to be applied with all chosen acceptors, oxazoline selective opening to β-glycosides has been performed in anhydrous dichloroethane at 80°C, using camphorsulfonic acid as acid activator.32 As shown in Scheme 2.8, methyl glycoside 75 and
compound 69 have been isolated up to 50% yield but 68 has been obtained as a minor product after 4 days stirring, probably because the less reactivity of the acceptor. The problem has been overcame using TMSOTf instead of CSA as activator: desired glycoside 68 has been isoled in quite good yield (58%).
To prepare the C-4 deprotected acceptors, with orthogonal groups at C-3 and C-6, the intermediates
71 and 76 have been synthesized from the corresponding glycosides 69 and 75. Methyl glycoside 75 has been subjected to a sequence of deacetylation followed by selective C-4, C-6 protection as
isopropylidene acetal: applying Zemplen conditions (MeONa, MeOH) on 75, crude triol has been rapidly obtained and directly subjected to 4,6-O-isopropylidenation using freshly distilled 2-methoxypropene38 in presence of an acid catalyst as camphorsulfonic acid or p-toluensulfonic acid. Glycoside 69 has been first converted into the azide 70 through an SN2 reaction (NaN3, TBAI,
DMF) in a nearly quantitative yield, then subjected to the sequence seen for 75.
Scheme 2.8 O AcO AcO N OAc O O AcO AcO NHAc OAc O R ii O O RO NHAc O O N3 O AcO AcO NHAc OAc OMe iv O O RO NHAc O OMe i iii 68: R=NHBoc 69: R=OTs 70: R=N3 63 71: R=H 72: R=Ac 73: R=Bz 74: R=Bn v vi vii viii 75 76: R=H 77: R=Bz ix
Reagents and conditions. i. for 68: 50, TMSOTf, 4 Å MS, DCE (58%); for 69: 49, CSA, 4 Å MS,
DCE, 80°C (69%); ii: NaN3, TBAI, DMF, 50°C (98%); iii: a) NaOMe, MeOH, 0°C; b) 2-MP,
CSA, DMF (88% from 70); iv: Ac2O, Py (97%); v: BzCl, Py, 0°C (96%); vi: BnBr, KOH,
18-crown-6, humid THF, 0°C (75%); vii: MeOH, CSA, 4 Å MS, DCE, 80°C (58%); viii: a) NaOMe, MeOH, 0°C; b) 2-MP, CSA, DMF; ix: BzCl, Py, 0°C (60% from 75).
The intermediates 71 and 76 have been used as common starting point for the synthesis of various glycosyl acceptors, allowing a methodological study on the optimal condition for β-galactosylation. On C-3 position different protecting groups have been introduced as acetyl or benzoyl esters and benzyl ethers to evaluate their influence on OH-4 reactivity towards glycosylation reaction. As shown in Scheme 2.9, alcohol 71, easy transformed into 3-O-acetyl analogue 72, has been deprotected to diol 78 using acid condition (70% aq AcOH, 40°C) and preparation of a 6-O-orthogonal protected acceptor has been first attempted (Scheme 2.9).
6-O-p-methoxybenzyl protection has been initially chosen: PMB group is orthogonal to acetyl esters and benzyl ethers and is often compatible with some glycosylation conditions.
In literature many methods were reported to achieve selective 6-O-protection in analogue substrates with many different groups in presence of unprotected hydroxyl groups. In an analogue synthetic sequence, one of the most common method is the introduction of 4,6-O-benzylidene, p-methoxybenzylidene or allylidene protection instead of isopropylidene one with final selective reduction that gave selectively alkyl ethers and a free hydroxyl group.39 The reagents used are a hydride reagent in combination with a Lewis acid (or a proton acid). First, combinations of LiAlH4/AlCl3 were employed. From 4,6-O-benzylidene acetals, this yields the 4-O-benzyl
derivative with high selectivity, especially from precursors with bulky substituents in the C-3 position. A drawback of this methodology is that it is not compatible with various other functionalities as ester protecting groups. Later, NaCNBH3/HCl mixtures were introduced. For
4,6-O-benzylidene acetals, this gave the opposite selectivity with formation of the 6-O-benzyl ethers. This reagent is compatible with esters and also with allyl groups, which allowed the regioselective opening of acrolein acetals with the same regioselectivity. By changing only the solvent, the same flexibility was shown for benzylidene acetals and the Me3NBH3/AlCl3 reagent. THF as solvent
gave the 6-O-benzyl ether in a slow and mild reaction, whereas toluene (or diethyl ether/CH2Cl2
mixtures) gave the 4-O-benzyl ether in a fast reaction accompanied by some acetal hydrolysis. This method has sometimes presented many problems in final yields, selectivity and compatibility with some protecting groups.
Scheme 2.9 O O RO NHAc O O N3 72: R=Ac 73: R=Bz 74: R=Bn O HO RO NHAc OH O N3 O HO RO NHAc OR1 O N3 i ii 78: R=Ac 79: R=Bz 80: R=Bn 81: R=Ac; R1=TBDMS 82: R=Bz; R1=TBDMS 83: R=Bn; R1=PMB 84: R=Bn; R1=TBDMS
Reagents and conditions. i: 70% aq AcOH, 40°C (78 68%, 79 92%, 80 99%); ii: TBDMSCl, Py
(81 48%, 82 93%, 84 89%) or Bu2SnO, toluene, reflux then PMBCl, TBAB (83 65%).
An alternative method for 6-O-selective protection has been approached: reacting the hydroxyl groups of saccharides with tin oxide reagents as Bu2SnO, stannylene ethers and acetals are formed,
which enhances the nucleophilicity of the oxygens in a regioselective way and makes the consecutive regioselective acylation or alkylation of saccharides possible (Scheme 2.10).40
Scheme 2.10
The activation can be performed in various solvents, the most common being methanol or toluene (benzene). In the latter case, a Dean-Stark trap is often used to remove the water formed in the refluxing reaction. Subsequent treatment of the saccharide tin complex with various electrophiles
(acyl chlorides, silyl chlorides, alkyl halides) in various solvents (usually DMF or the toluene/benzene solution of the stannylene formation) yields the corresponding esters or silyl or alkyl ethers. The addition of nucleophiles as bromide, iodide or fluoride ion enhances the rate of the reaction. The regioselectivity associated with stannyl activation is much the same irrespective of which type of alkyltin derivative is used in the activation step. The primary hydroxyl group and the equatorial hydroxyl group in a vicinal cis-dioxygen configuration are activated and alkylated. Following this method diol 78 has been treated with Bu2SnO and overnight refluxed in toluene
using a Dean-Stark apparatus. Unfortunately TLC analysis has revealed degradations of the starting material (stannylene formation is not TLC detectable) because of 3-O-acetyl protection lability in that reaction conditions.
The problem has been overcame changing C-3 protection from ester to benzyl ether: diol 80 has been prepared from azide 70 through a similar three-step sequence of acetyl removal-4,6-O-isopropylidene introduction-C-3 benzylation in heterogeneous conditions (BnBr, 18-crown-6, KOH, humid THF, 0°C). Applying selective C-6 alkylation conditions seen for 78, desired glycosyl acceptor 83 has been isolated in good yield (65% yield).
A different approach has been followed to obtain glycosyl acceptor also with esters on C-3: the use of a bulky reagent as tert-butyldimethylsilyl (TBDMSCl) or tert-butyldiphenylsilyl (TBDPSCl) chloride and various basic conditions gives a high yield of the 6-O-monoprotected hexose derivative starting from deprotected analogue where the anomeric position is already protected. The most common procedure to 6-O-mono silylated derivative preparation have been applied on diols 78, 79, and 80: using TBDMSCl as silylating agent in imidazole catalyzed condition developed by Corey et al,41 no complete conversion from diols to products has been observed. Also with TBDMSCl and imidazole adds, acceptor 81 have been isolated in 48% yield as the best result. Final acceptors 82 and 84 have been finally obtained in yields up to 85% simply by using the same silyl reagent in anhydrous pyridine. Following this procedure methyl acceptor 86 has been synthesized from 77 in an overall good yield (78%, 2 steps).
Scheme 2.11
With various acceptors in hand, a methodological study of β-(14)-galactosylation reaction, a key
synthetic step in the preparation of desired SP14 tetrasaccharide, has been made paying attention to various aspects.
As recently pointed out by Auzanneau et al,42 glycosylations at O-4 of N-acetylglucosamine are an open problem that must be studied case by case. Indeed, the majority of reported β-(1
4)-glycosylation employed acceptors that carried an N-Phth, N-TCP, N-Troc, azido or oxazolidinone at C-2, and only two reports43in addition to these early syntheses use an N-acetylglucosamine acceptor. The first one is that of Roy and co-workersthat describes the regioselective glycosylation of an acceptor free at O-3 and O-4, and the second one is Auzanneau’s recent synthesisof Lex using conditions as 2 equiv BF3
.
OEt2, 5 equiv donor, rt or 40 °C that efficiently promote
glucosylation at O-4 of N-acetylglucosamine. Indeed over the past few years some studies have shown that the success of such glycosylation depended on the nature of the substituent already present at O-3 in the acceptor but also and more surprisingly on the structure of the aglycone carried by the acceptor: for example in the cited Auzanneau work43b a significantly drop in glycosylation yield has been observed from the use of the 6-chlorohexyl glycoside acceptor (72%) to 6-azidoethyl analogue (27%).
Furthermore our substrates carried acid lable groups at O-6 (PMB, TBDMS) and most of the reported conditions for that reaction as activator large excess or high temperatures (Auzanneu’s conditions) cannot be applied on.
In this study it has been decided to use as a starting point the most common glycosyl donor, the tetra-O-acetyl-α-D-galactopyranosyl trichloroacetimidate 87 (Scheme 2.12), easily prepared from commercial peracetyl galactopyranoside through anomeric acetyl removal (hydrazine acetate in refluxing DMF) followed by reaction with CCl3CN in anhydrous CH2Cl2 catalyzed by a weak base
as DBU.44 Glycosyl trichloroacetimidates are relatively stable under basic or neutral conditions, but react readily under acidic conditions. With various acidic nucleophiles, such as carboxylic acids and phosphoric acid derivatives, the corresponding glycosyl esters are formed without any additional catalyst. Reaction with non-acidic O-nucleophiles proceeds in the presence of catalytic amounts of Brønsted or Lewis acids. Originally, p-TsOH and BF3·Et2O were used,45 while the
latter and TMSOTf46 are currently the most frequently employed promoters. Glycosylations with these promoters take place at low temperatures under mild conditions. Neighboring group participation as acetyls results in the formation of 1,2-trans glycosides, whereas with non-participating substituents, the use of BF3·Et2O favors the formation of the reaction product with
As shown in Table 2.1, all acceptors (A) have been coupled with donor 87 using TMSOTf as activating system, leading to disaccharide glycosylation products (G) and related byproducts as orthoesters (OE) or 4-O-acetylated acceptors (AA).
Scheme 2.12
Table 2.1. β-(14)-galactosylation conditions using donor 87
Entry R R1 R2 Activ. syst. React. Condit.
Product, Yield 1 (CH2)2N3 PMB Bn TMSOTf (0.01 eq) 87 (1.77 eq), AW-300, 0°Crt G(88), 13% 2 (CH2)2N3 PMB Bn TMSOTf (0.01 eq) 87 (1.77 eq), 4 Å MS, -30°Crt OE of 88, 47% 3 (CH2)2N3 PMB Bn TMSOTf (0.1 eq) 87 (1.77 eq), 4 Å MS, -30°Crt 6-O-deprotected A
4 (CH2)2N3 TBDMS Ac TMSOTf (0.1 eq) 87 (1.3 eq), 4 Å MS,
-40°C-20°C
OE of 89,
59%
5 (CH2)2N3 TBDMS Bz TMSOTf (0.5 eq) 87 (2 eq), AW-300,
-30°Crt G (90), 26% OE of 90 35% AA 20% 6 (CH2)2N3 TBDMS Bz TMSOTf (0.5 eq) 87 (1.5 eq), AW-300, -30°Crt (overnight) G (90), 47% 7 (CH2)2N3 TBDMS Bn TMSOTf (0.5 eq) 87 (1.5 eq), AW-300, -30°Crt (overnight) G (91), 53% 8 Me TBDMS Bz TMSOTf (0.5 eq) 87 (1.5 eq), AW-300, -30°Crt (overnight) G (92), 62%
Donor and acceptors have been dried using dry toluene that has been removed under high vacuum and finally added with 4 Å molecular sieves or acid washed AW-300, before the solvent adding. Using 6-O-PMB acceptor 83, the most relevant results are reported in Table 2.1 (entries 1, 2, 3): desired glycoside 88 has been obtained in very low yield (13%, entry 1). The use of common 4 Å molecular sieves (little basic) instead of AW-300 (neutral) gave the formation of the corresponding 1,2-orthoester (OE 47%, entry 2) with no disaccharide presence, probably because basicity of MS partially neutralize the acid activator. Trying to increase the TMSOTf quantity, hydrolysis of acceptor with 6-O-protection removal has been observed, leading to the diol 80 accompanied by other degradation products (entry 3).
The results obtained using 6-O-TBDMS acceptors with different 3-O protecting groups (Ac, Bz, Bn) and the same donor 87 are reported in Table 2.11 (entries 4-8).
Using 3-O acetyl acceptor 81 at low temperature (from -40°C to -20°C) and with basic molecular sieves, the most relevant result has been the formation of the orthoester related to the desired disaccharide 89 (59% yield, entry 4).
Conversely, the increasing of reaction temperature to rt and the use of AW-300 in the glycosylation reaction between the same donor 87 and 3-O-Bz acceptor 82 had permitted to obtain the glycoside
90 (26%) in presence of the acetylated acceptor (20%) and the related orthoester (35%, entry 5).
Long time reaction applied on the same substrate and reaction conditions (entry 6) seemed to avoid the formation of undesired products as orthoesters or 4-O-acetylated acceptors. Changing the 3-O acceptor protection from esters to benzyl ether (entry 7) or the aglycone from 3-azidopropyl to methyl (entry 8), an improvement in yield has been observed in both cases.
The analysis of all the results has pointed out that: (a) for those substrates the optimal glycosylation conditions were a 1.5 eq excess of donor 87 and 0.5 eq of TMSOTf added at -30°C and slowly allowed to warm at rt; (b) the presence of AW-300 neutral molecular sieves was necessary; (c) 6-O-PMB protection resulted not optimal for those substrate because of higher sensibility towards acid activators face to TBDMS protection; (d) the major occuring problem has been the formation of related orthoesters (OE, entries 2, 4, 5); (e) long time of the reaction was an important factor that avoid the formation of undesired byproducts.
A possible cause of that undesired compound formation as orthoesters, can be probably found in the quenching timing: low temperature and short time reaction quenching might prevent the glycosylation intermediates to rearrange into desired glycosides, with the isolation of byproducts. As reported in Table 2.1, desired glycosides 90, 91, 92 have been finally obtained in quite good yield (entry 6, 7, 8). There is only little difference in yield between 3-O-Bz disaccaride and 3-O-Bn analogue ponting out that O-4 reactivity has been influenced only by steric hindrance of phenyl ring and not by electronic differences between ester and ether group. As noticed by Auzanneau et al,42 aglycon has been its “weight” on the reactivity of O-4 towards glycosylation: methyl group
(entry 8) instead of 3-azidopropyl spacer (entry 6) has given a yield enhancement from 47% to 62%.
Sugar 1,2-orthoesters are classic glycosyl donors used in the construction of 1,2-trans glycosidic linkages, which, in particular, have been studied by Kochetkov and co-workers.47 On the other
hand, sugar 1,2-orthoesters are often undesired side products in glycosylation with donors carrying C-2 acetyl protecting groups.
Several experiments further demonstrated that the normal glycosidic products in the glycosylation reactions were actually derived from the corresponding orthoester intermediates. The mechanism of the rearrangement of the orthoester to the glycosidic product has been postulated, as shown in Scheme 2.13 (pathway a). The mechanism involves the dissociation of the orthoester to a 1,2-acyloxonium ion and glycosidation of the transient oxocarbenium species.48
Scheme 2.13
Alternative pathway b begins with coordination of the O-2 of orthoester with a Lewis acid followed by the O-2–C(orthoester) bond cleavage, and the resultant reactive intermediate reacts with acceptor to give a 1,2-translinked glycoside with C-2-free hydroxyl group and 4-O-acetylated acceptor as observed in entry 5.
The isolated orthoesters have been tried to open as reported by Yu et al:48 orthoester can be
converted into corresponding glycosidic product under the action of a promoter as TMSOTf or AgOTf (0.25 eq). This method has been given the desired glycoside only in presence of the corresponding acceptor (0.3 eq) and AW-300 in a not satisfactory 50% yield.
To complete this study, other common donors have been used to evaluate their influence on the galactosylation (Scheme 2.14, Table 2.2).
Commercial penta-O-acetyl-β-D-galactopyranoside 93 has been utilized following a reported procedure (entry 1):49 glycosyl esters has long drawn attention as potential glycosyl donors but the acyloxy group is not as good as leaving group as the trichloroacetimidoyl, for example, so their
activation generally requires harsher conditions as higher concentration of activator with removal of acid sensitive groups as PMB or TBDMS as observed.
Also bromide tetra-O-acetyl-α-D-galactopyranoside 94 (entry 2) has been tested as glycosyl donor following a reported procedure:50 glycosyl bromides and chlorides were the first (and for a long time, practically the only) type of glycosyl donors used for the synthesis of complex glycosides. This archetype of glycosyl donors combines high reactivity with reasonable stability. Because of the anomeric effect, glycosyl halides with axial halogens are more stable than the equatorial isomers. Their stability and reactivity is also greatly influenced by the protecting groups on the sugar ring. For example, electron-withdrawing (acyl) groups decrease, whereas electron-donating (ether-type) protecting groups increase their reactivity. Even in this case acceptor 82 degradation has been observed.
Scheme 2.14
Table 2.2. β-(14)-galactosylation conditions using other donors
Ent. X Y R R1 R2 Activ. syst. React. Condit. Yield
1 OAc H (CH2)2N3 PMB Bn TMSOTf (2.2 eq) D (1.1 eq), AW-300, -20°Crt49 6-O-deprotected A 2 H Br (CH2)2N3 TBDMS Bz
AgOTf (2 eq) D (1.6 eq), AW-300, rt50 deprotected
6-O-A 3 SPh H (CH2)2N3 TBDMS Bz NIS (1.8 eq) AgOTf (0.6 eq) D (1.8 eq), AW-300, -30°Crt 29% G (90) 10% GG 4 SPh H (CH2)2N3 TBDMS Bn NIS (1.8 eq) AgOTf (0.6 eq) D (1.8 eq), AW-300, -30°Crt 34% G (91) 5 SPh H Me TBDMS Bz NIS (1.8 eq) AgOTf (0.6 eq) D (1.8 eq), AW-300, -30°Crt 29% G (92)
Finally thiophenyl donor 95 has been coupled with acceptors 82, 84 and 86 (Table 2.2, entry 3, 4,
easy to prepare from related acetates, stable and higly reactive at the same time. Their preparation and activation is discussed in the SP19F trisaccharide preparation (Part 1, Section 1, introduction). As reported in Table 2.2 desired glycosides have been isolated in a quite modest yield related to the ones obtained for TCA donors (Table 2.1).
The formation of byproduct acetyl 2,3,4,6-tetra-O-acetylgalactopyranosyl-β-(12)-3,4,6-tri-O-α -D-galactopyranoside (GG, entry 3) can be rationalized by the C-1 hydrolysis of donor, followed by 21 acetyl shift on the α-anomer with the formation of a C-2 alcohol that reacts with the donor excess (Scheme 2.15).
Scheme 2.15
Disaccharide 90 has been chosen as acceptor precursor to be coupled with lactosyl donor. That intermediate has been subjected to 6-O-silyl protection removal following the most common reported method: tetrabutylammonium fluoride (TBAF) is commonly used as selective silyl removal agent in THF solution.41 During this selective deprotection, nucleophilic attack of the small fluoride anion leads to a pentavalent silicon centre which is permitted due to hybridisation with the vacant d-orbitals of silicon (Scheme 2.16). In addition, the formation of the strong Si-F bond is the driving force for a fast cleavage.
Scheme 2.16
Applying reported conditions,51 problems occured in the reaction probably because fluoride ion is very basic, especially under anhydrous conditions and thus may cause side reactions with base-sensitive substrates as acetyl groups. As a matter of fact de-O-silylated disaccharide 96 has been isolated as a minor product (35%) and the disaccaride 97, C-6 and C-2’ deprotected, as the main
derivative (65%). The problem has been overcame applying acid hydrolysis conditions (70% aq AcOH, 70°C) that gave a complete conversion to 6-O deprotected 96 in a nearly quantitative yield (Scheme 2.17).
In the final step of the synthetic sequence, compound 96 has been used as glycosyl acceptor in the
β-(16)-lactosylation reaction. As donor peracetyllactose trichloroacetimidate has been easily
prepared from commercial lactose through a similar sequence used for donor 87 preparation: peracetylation, anomeric deacetylation and final DBU catalyzed reaction with CCl3CN in DCM.
That glycosylation reaction has been less problematic than seen β-(14)-galactosylation:(a)
primary O-6 is a more accessible and reactive position than O-4; (b) no acid lable groups as TBDMS are present in both donor and acceptor, that permitted the use of an activator excess avoiding the formation of undesired orthoesters. Lactose donor and acceptor 96 have been finally coupled using 1.2 eq of BF3·Et2O and AW-300 giving SP14 tetrasaccharide 98 in a good yield
(74%).
Scheme 2.17
Reagents and conditions. i: 70% aq AcOH, 70°C (99%); ii: lactose TCA, BF3 .
Et2O, AW-300,
DCM, -15°C to rt (74%).
The obtained tetrasaccharide 98 has been analyzed by 1H-NMR experiment: the signals of H-1 protons (4.70, 4.56, 4.55 and 4.53 Hz), the ones related to the acetamido group as the NH doublet (6.46 Hz) and the CH3CO singlet (1.70 Hz), and to aglycone as CH2N3 signals (3.36 Hz) have
2.4. Future developments
Tetrasaccharide 98 will be used in the preparation of glycodendrimers as seen for SP19F trisaccharide (Part 1, Section 1): (a) exchange from acetyls to benzyl ether protections; (b) azide reduction to amine; (c) coupling with an adipic di-functionalized spacer as seen methyl adipoyl chloride; (d) conversion to acid derivative; (e) coupling with PAMAM dendrons using peptide chemistry; (f) final deprotection and (g) coupling with an immunogenic protein (Scheme 2.18). Obtained conjugates will be finally tested towards immune system to evaluate their stimulation of anti SP14 antibodies production.
3. Experimental
General methods
NMR spectra were recorded with a Bruker AC 200 instrument operating at 200.13 MHz (1H) and 50.33 MHz (13C) and with a Varian INOVA600 spectrometer operating at 600 and 150 MHz for 1H and 13C respectively using Me4Si as internal reference. Assignments were made, when possible, with the aid of DEPT, HETCOR, HSQC, by comparison of values for known compounds and applying the additivity rules. In the case of mixtures, assignments were made by referring to the differences in the peak intensities. All reactions were followed by TLC on Kieselgel 60 F254 with
detection by UV light and/or with ethanolic 10% phosphomolybdic or sulphuric acid, and heating. Kieselgel 60 (E. Merck, 70-230 and 230-400 mesh, respectively) was used for column and flash chromatography. Solvents were dried by distillation according to standard procedure, and storage over 4Å molecular sieves activated at least 24 h at 400 °C. MgSO4 was used as the drying agent for
solns.
3.1. The Gin’s approach to lactosamine glycosides: general procedure for oxazoline preparation from glycals
A soln of glycal and thianthrene-5-oxide (2 eq) in toluene was concentrated to dryness under high vacuum to remove H2O traces. After 1 h under vacuum, triflic anhydride (2.0 eq) was added to a
soln of this solid mixture in chloroform and dichloromethane (4:1) at -78°C. The reaction mixture was stirred at this temperature until TLC analysis showed the complete disappearance of the starting material (about 10’). N,N-diethylaniline (5.3 eq) and solid N-TMS-acetamide (5.0 eq) were sequentially added to the reaction mixture that was immediately warmed to rt and stirred at this temperature until TLC analysis showed the formation of oxazolyne. When the oxazoline was isolted, the reaction solution was concentrated under diminished pressure and purified through chromatographic purification.
2-methyl-(1,2-dideoxy-3,4,6-tri-O-benzyl-αααα-D-glucopyrano)-[2,1-d]-2-oxazoline (51)
Following general procedure commercial glucal 16 (200 mg, 0.48 mmol) was converted into oxazoline 51. After standard work-up the crude residue was subjected to flash chromatography (1:1 hexane-EtOAc + 0.1% Et3N) that afforded pure 51 (132 mg, 58% yield) as a colourless oil, Rf 0.36
(3:7 hexane-EtOAc) whose physico-chemical properties were analogous to those reported in the literature:19 [α]D +35.2 (c 1.12, CHCl3); 1H-NMR (250 MHz, CDCl3): δ
7.48-7.14 (m, 15H, Ar-H), 6.01 (d, 1H, J1,2 7.5 Hz, H-1), 4.68, 4.27 (AB system, 2H,
JA,B 12.0 Hz, CH2Ph), 4.63, 4.53 (AB system, 2H, JA,B 11.0 Hz, CH2Ph), 4.51,
4.46 (AB system, 2H, JA,B 12.2 Hz, CH2Ph), 4.22 (m, 1H, H-2), 3.99 (t, 1H,
J2,3= J3,4.2.8 Hz, H-3), 3.68 (dd, 1H, J4,5.8.9 Hz, H-4), 3.47 (m, 1H, H-5), 3.56-3.50 (m, 2H, H-6a, H-6b), 2.04 (s, 3H, CH3); 13 C-NMR (63 MHz, CDCl3): δ 165.8 (C=N), 137.9, 137.7, 137.6 (3 x Ar-C), 128.4-127.5 (Ar-CH), 100.4 (C-1), 76.8 (C-3), 75.0 (C-4), 73.3, 71.9, 71.4 (3 x CH2Ph), 70.3 (C-5), 69.5 (C-6), 65.6 (C-2), 14.1 (CH3). 3-N-(t-butoxycarbonyl)-aminopropyl 2-acetamido-2-deoxy-3,4,6-tri-O-benzyl-ββββ-D -glucopyranoside (52)
To a soln of 51 (44 mg, 0.093 mmol) in anhyd CH2Cl2 (2 mL) was added a soln of acceptor 50 (75
mg, 0.43 mmol) in anhyd CH2Cl2 (1.5 mL) at 0°C. After 10’ stirring at this temperature,
amberlyst-15 (100 mg) was then added. After 17 h at rt, TLC analysis (2:3 hexane-EtOAc) revealed the complete disappearance of the starting material (Rf 0.26) and the formation of slower moving
products (Rf 0.16 and 0.08). The mixture was filtered, diluted with CH2Cl2 (10 mL) and washed
first with satd aq NaHCO3 (10 mL) then with brine (10 mL). The organic extracts were dried
(MgSO4.H2O), filtered and concentrated under dimished pressure. Purification of the residue by
flash cromatography over silica gel (1:4 hexane-EtOAc + 0.1% Et3N then 1:9 hexane-EtOAc +
0.1% Et3N) gave pure 54 (10 mg, 22% yield) and a fraction containing glycoside 52 with traces of
alcohol 54 (35%).
54 is a white solid, Rf 0.08 (2:3 hexane-EtOAc) whose physico-chemical properties were analogous to those reported in the literature:52 m.p. 218-219 °C (MeOH), [α]D+63.0 (c 0.90, pyridine);
1
H-NMR (250 MHz, CDCl3): δ
7.32-7.10 (m, 15H, Ar-H), 5.18 (dd, 1H, J1,2 3.6 Hz, J1,OH 3.0 Hz, H-1), 4.70
(m, 3H, NH, CH2Ph), 4.55-4.38 (m, 5H, OH, 2 x CH2Ph), 3.98 (m, 1H, 2), 3.80-3.30 (m, 5H,
H-3, H-4, H-5, H-6a, H-6b); 13C NMR (63 MHz, CDCl3): δ 168.4 (CO), 138.9, 138.3, 137.8 (3 x
Ar-C), 128.2-127.1 (Ar-CH), 90.8 (C-1), 79.8 (C-3), 78.2 (C-4), 73.9, 72.0, 71.6 (3 x CH2Ph), 72.1
(C-5), 69.1 (C-6), 53.5 (C-2), 23.1 (CH3CO).
The fraction containing the glycoside 52 was subjected to standard acetylation (1:2 Ac2O-Py) and,
after 12 h, the reaction solution was concentrated by toluene coevaporation and the crude purified by silica gel flash chromatography (2:3 hexane-EtOAc + 0.1% Et3N) to give first acetylated
acceptor (10 mg), followed by pure 52 (8 mg, 13% yield).
O BnO BnO N OBn O O BnO BnO AcHN OBn OH
