Section 1
Preparation of CRM
197
conjugate glycodendrimers
1. Introduction
1.1. Streptococcus Pneumoniae 19F
As shown in the Preface, Streptococcus pneumoniae serotypes 19F (SP19F) and 19A (SP19A) are responsible for a large number of infections of the upper respiratory system and meningitis, especially in children and immunodeficient subjects. The repeating units24 of SP19F (1) and SP19A CPS (2, Fig. 1.1) are both made up of a trisaccharide containing an N-acetyl-D-mannosamine unit (A) linked through a β-(14) bond to a D-glucose (B) residue that is linked to an L-rhamnose unit (C) through an α-(12) (SP19F) or an α-(13) bond (SP19A). The repeating units are linked to each other via an α-(14) phosphodiester bridge.
Figure 1.1.
1.2. Literature syntheses of SP19F capsular polysaccaride fragments
Since the elucidation of these structures,24 chemists have been involved with their synthesis, especially with that of SP19F. The reported synthetic approaches involve, in all cases, two different glycosilation reactions, and can be classified into two groups: the first is based on the initial synthesis of the A–B fragment and the successive coupling with the C unit,25 while the second approach involves the coupling of unit A with the B–C fragment (1, Fig. 1.2).26
The most challenging task is the introduction of the N-acetyl-β-D-mannosamine linkage, because of the difficulties in stereoselective formation of β-D-mannosamine glycopyranosides by direct glycosylation with D-mannosamine donors. This represents a specific problem in a most general one in carbohydrate chemistry: the formation of 1,2-cis-glycosidic bond.27 The most employed strategy for this substrate is based on the initial formation of a β-D-glucopyranoside residue, followed by its transformation into a β-D-mannosamine moiety by amination with inversion of configuration at C-2’ through an oxidation–oximation, followed by reduction of the oximino
derivative (Scheme 1.1)25aor through an SN2 displacement with sodium azide on a 2-O-sulfonyl
intermediate followed by reduction (Scheme 1.2).26b
Scheme 1.1
Reagents and conditions. i: a) NaOMe, MeOH; b) Ac2O, DMSO; c) NH2OH.HCl (61%, E+Z, 3
steps); ii: a) LiAlH4, Et2O; b) Ac2O, MeOH; iii: a) Ac2O, TFA; b) HBr (R1=H, 85%; R1=Bn, 15%).
The oximino derivative way ([A-B]+C), developed by Prof. Russo’s group in 1987, has been elaborated from 2’-O-Ac-β-(14) disaccaride 3 (A-B fragment), obtained from monosaccharide glycosylation,25a that was subjected to 2’-O-Ac removal then to an oxidation–oximation sequence. Oxime 4 (isomer mixture E+Z) was selectively reduced to amine, giving, after acetylation, N-acetyl-Man-β-(14)-Glc derivative 5. Anomeric activation was achieved by acetyl group introduction at C-1 using Ac2O/TFA system that removed also benzyl protections at C-6 and C-6’,
then displacement of anomeric acetyl with HBr: in this strong acidic condition Bn at C-3 was also removed. The glycosyl donors 6a and 6b were used in glycosylation reaction with rhamnoside acceptor using Hg(CN)2–HgBr2.25a
Scheme 1.2
Reagents and conditions. i: Tf2O, DCM-Et2O (1:1), -78°C (77%); ii: NaOMe, MeOH (98%); iii:
Im2SO2, NaH, DMF, -30 °C (99%); iv: Bu4NN3, toluene, reflux (97%); v: Zn, CuSO4, THF-Ac2
In the azido derivative way (A+[B-C]), the trisaccaride 9 (Scheme 1.2) was elaborated through amination with inversion at C-2’’, obtaining ManNAc unit containing SP19F CPS protected trisaccharide 13.26b
Other methods described are direct glycosylations with 2-azido-2-deoxy-D-mannopyranose donors, either the glycosyl bromide activated by silver silicate26aor with a C-2 oximino glycosyl donor, followed by stereoselective reduction.25c Still, most of these methods suffer from problems related to low reaction yields and stereoselectivity.
A recently reported method28 for the synthesis of β-D-mannosaminosides and β-D-mannosides is based on the completely stereoselective elaboration of positions 2 (amination with inversion) and 4 (epimerization) of β-D-galactopyranosides. This suggested the possibility to obtain donor β-D -ManNAcp-(14)-D-Glcp disaccharide 18 (Scheme 1.3) from lactose by converting its nonreducing end into a N-acetyl-D-mannosamine moiety. The use of commercial lactose, with a preformed β -(14) linkage permitted to avoid the critical β-mannosylation step and in general the glycosylation reaction that generate the disaccharide substrate.
Scheme 1.3
Reagents and conditions. i: AllBr, KOH, 18-crown-6, wet THF, rt (82%) ii: a) 80% aq AcOH,
80°C; b) Ac2O, AcONa .
3H2O, reflux (82%, 2 steps); iii: a) TMSSPh, ZnI2, 4 Å AW MS, DCE, rt
(82%); b) KOH, EtOH, rt (96%); c) BnBr, KOH, 18-crown-6, wet THF, 0°C (95%).
Thioglycosides as 18 are readily prepared using a variety of methods, but mostly by nucleophilic substitutions at the anomeric center. They are prepared most commonly from anomeric 1,2-trans-acetates by reaction with thiols or with trimethylsilyl ethers of thiols in the presence of Lewis acids such as BF3·Et2O, TMSOTf, SnCl4, and so on. They can also be obtained from the reaction of other
common glycosyl donors (glycosyl halides, trichloroacetimidates, etc.) with thiols, from the opening of the oxirane ring of 1,2-anhydro sugars, or by alkylations of 1-thiosugars. Thioglycosides
show remarkable stability; not only do they have long shelf lives, but they also tolerate very diverse chemical manipulations leaving the thioglycoside function intact. Importantly, most of the common carbohydrate protecting group manipulations can be performed on thioglycosides, a feature making the preparation of highly functionalized thioglycoside donors possible. Attempts to convert 17 into the thioglycoside analogue with the most widely reported methods in the literature (PhSH/BF3.Et2O
or TMSSPh/MeOTf)29led to disappointing results, and the starting material was recovered in any case. Good results for this conversion (82% isolated yield) have been obtained treating 17 with TMSSPh in the presence of zinc iodide,30 and finally converted into perbenzyl thioglycoside 18 in an overall good yield.
Compound 18 was included in a systematic investigation31 on the glycosylation properties of different disaccharide thiophenyl glycosyl donors obtained from lactose, each carrying at the nonreducing end a D-mannosamine, a D-talosamine or a D-galactopyranose unit, with a simple alcoholic acceptor (Figure 1.2).
Figure 1.2
Despite their stability, thioglycosides as 18 can be activated with thiophilic reagents, typically by soft electrophilic reagents, under mild conditions (Scheme 1.4). The formed sulfonium ion 22 is a better leaving group, so loss of the sulfonium ion with the assistance of the ring oxygen or a neighboring group leads to the common intermediates of glycosylation reactions 23, which will react with the O-nucleophile to afford the O-glycosides 24.
Scheme 1.4
The common promoters of thioglycoside activation are a very heterogeneous class of compounds: from the first attempt with mercury salts (HgSO4, HgCl2, Hg(OAc)2), that gave moderate yield and
are not powerful enough to be of general use to triflates, from simple (i.e. TfOMe) to complex ones (DMTST). Activating systems constituted by N-iodosuccinimide (NIS) in the presence of a catalytic amount of triflic acid or other Lewis acid (TMSOTf, AgOTf, BF3.Et2O, etc.) have been
studied as very efficient promoter systems that proceed at low temperature within short time and are capable to activate a wide variety of glycosil donors.32 In this study31 all thioglycoside donors (Fig. 1.2) were glycosylated using isopropanol as acceptor and various reported activating systems. The analysis of the results pointed out that mannosaminic donor 18 presented an “anomeric activation” only with TfOMe. Compound 19 has been glycosylated with i-PrOH (50%, 1:1 α/β) using TfOMe or NIS/TfOH as activating system. Best results in terms of yield and stereoselectivity has been achieved with donor 20 (78% yield, 4:1 α/β) because of the acetamido group absence. In 2009,33 taking into account the completely negative results obtained in the seen preliminary study31 using NIS/TfOH or TfOMe as promoter, the glycosylation properties of β-D -ManNAcp-(14)-D-Glcp thiophenyl glycosyl donor 18 has been explored with other activating systems. Hence, α-glycosylation reactions between donor 18 and rhamnoside acceptor 25, selectively C-2 deprotected (Scheme 1.5) have been carried out with the most widely used activating systems of thioglycosides. In the case of the previously tried NIS/TfOH and MeOTf, also NIS/TMSOTf, PhIO/TMSOTf, TfOMe/collidine or DMTST have been given disappointing results. Again no product has been isolated, and only retrieved starting materials and decomposition products have been collected from workup of the reaction mixture. However relevant results has been obtained using system NIS/AgOTf and running the reaction at -35 to -10 °C gave a 76% yield of trisaccharide 26, corresponding to the repeating unit of Streptococcus pneumoniae 19F, with a 3.4
α/β ratio as the best result.
2. Results and discussion.
For the preparation of conjugate glycodendrimers loaded with SP19F repeating units has been (a) prepared for first the protected trisaccharide A with an azido propyl spacer on anomeric position. The synthesis has been achieved following the [A-B]+C approach shown in Par. 1.2, using the glycosylation condition reported33 for analogue 26. (b) The trisaccharide so obtained has been loaded on PAMAM dendrons using different synthetic methods as thioureidic bridge and amide bond formation. (c) On Novartis Vaccines Labs in Siena, deprotected SP19F trisaccharide and SP19F glycodendrimers have been conjugated to CRM197 as protein carrier. (d) Finally one of the
glycoconjugates has been tested “in vivo”.
2.1. Preparation of trisaccharide 38
Following the [A-B]+C approach, thioglycoside donor 33 and rhamnoside acceptor 37 have been separately prepared then coupled to form desired trisaccharide 38.
The preparation of the thioglycoside donor 33 (Scheme 2.1) has been achieved starting from talosamine derivative 14, readily obtained from lactose.34 This key intermediate has been subjected to C-4’ epimerization following an improved method developed in our Lab, that simplifies a previous reported one to MaNAc derivative 15 (Scheme 2.1).35 This published methodology based on the synthesis of the enol-ether 29 and its regio and stereoselective hydroboration-oxidation required 5 reactions and 4 chromatographic purification with 44% overall yield. With this new approach enolether 30 was prepared using the well-known eliminations of cyclic acetal functions under strongly basic conditions in carbohydrates.36 Butyl-lithium is the base used most frequently and several different types of cyclic and acyclic products have been obtained via the formation of carbanions at one of the dioxolane rings or adjacent carbons. The facile base-catalyzed elimination of acetone from 3,4-O-isopropylidene derivatives of galactopyranosiduronic acid to give hex-4-enopyranosiduronic acids has been observed, but the β-elimination was facilitated by conjugation in the products.37 Derivative 14 has been treated with t-BuOK in THF at 80°C. The reaction was
stopped after 30’ and the crude product directly submitted to benzylation reaction (KOH, BnBr, humid THF, 18-crown-6). Compound 30 was obtained in excellent yield over two steps (91%) and its structure was confirmed through NMR analysis (C-4’ at 99.4 ppm and C-5’ at 150.7 ppm).
Scheme 2.1
Reagents and conditions. i: a) 80% aq AcOH, 80°C; b) 2-methoxypropene, PyHOTs, DCM (82%
from 14); ii: Bu2SnO, toluene reflux then BnBr, TBAB, reflux (95%); iii: a) Im2SO2, NaH, DMF,
-30°C to rt; iv: BH3 .
Me2S, Et2O then NaOH, H2O2 (64% from 28); v: a) t-BuOH, THF, reflux; b)
BnBr, KOH, 18-crown-6, humid THF, 0°C (91% from 14); vi: BH3.Me2S, Et2O then NaOH, H2O2
(80%).
The reaction here reported has been evidently involved an E-2 type elimination of acetone (Fig. 2.1), initiated by attack of base at C-5’ and favoured strongly by the approximately antiperiplanar disposition of the C-5’-H and C-4’-O bonds.
Figure 2.1
The base attack has been selectively directed to H-5’ probably because the H-3’ is linked to a sugar “zone” that has been distorted by the tense 3’,4’-O-dioxolane ring and, for that reason, has not resulted so antiperiplanar to C-4’-O bond as H-5’.
The final hydroboration-oxidation reaction has been again completely regio- and stereoselective and afforded 15 in 71% yield. This new route is easier compared to the other one and needs just 3 reactions and 2 chromatographic purifications with 73% overall yield which represent an good increment face to the reported method. Compound 15 has been subjected to acetal protection removal using 80% aq AcOH at 40°C followed by peracetylation using the system AcONa.3H2O in
Ac2O at reflux (Scheme 2.2). This acetylation method has been chosen instead of standard Ac2O in
pyridine method because it permitted us to obtain more β anomer than α: the velocity of anomeric acetylation is lower than the equilibration one, so β anomer, less stable, reacts faster.38 Compound
31 has been prepared in excellent yield from 15 with a nearly 4:1 anomeric ratio (β:α). The necessity of a major β acetate quantity comes from the fact that in thioglycosylation reactions anomeric β acetate easiest reacts: using the system TMSSPh/ZnI2 in dichloroethane,30 the acetate
mixture 31 has been converted in the thioglycoside 32, that has been finally subjected to protecting groups exchange from acetyl to benzyl ethers. Acetyl protections have been removed using Zémplen conditions (MeONa in MeOH) at 0°C, that avoided hydrolisis of C-2’ acetamido group to amine. Deprotected thiophenyl has been finally transformed to donor 33 using the classic alkylation method BnBr/KOH/crown ether in humid THF: the reaction was stirred at 0°C to avoid N-alkylation. Scheme 2.2 O AcO BnO NHAc OBn O OAcO OAc SPh OAc 32 O AcO BnO NHAc OBn O OAcO OAc OAc OAc 31 O HO BnO NHAc OBn O O O O (MeO)2HC O 15 O BnO BnO NHAc OBn O OBnO OBn SPh OBn 33 i ii iii
Reagents and conditions. i: a) 80% aq AcOH, 80°C; b) Ac2O, AcONa .
3H2O, reflux (94% from
15); ii: TMSSPh, ZnI2, 4 Å AW MS, DCE (65%); iii: a) MeONa, MeOH, 0°C; b) BnBr, KOH,
18-crown-6, humid THF, 0°C (81% from 32).
With the glycosyl donor in hand, the preparation of selectively C-2 deprotected rhamnoside 37 (Scheme 2.3) has been achieved starting from peracetyl rhamnose that has been glycosylated with 3-O-tosylpropandiol (easily synthetized from 1,3-propandiol and tosyl chloride) as acceptor and SnCl4 as activating system39 in dry CH3CN. The glycoside 34 has been obtained in good yield with
complete α-stereoselectivity thanks to the acetyl group at C-2. Applying SN2 reaction condition to
this derivative (NaN3, TBAI, DMF) azide 35 has been prepared (88% yield) and used in a synthetic
sequence that gave protected rhamnoside 36 without purification of the intermediates. Azide 35 has been deacetylated using Zémplen conditions (MeONa in MeOH) and the crude triol subjected to
direct 2,3-O-acetonation40 with 2,2-dimethoxypropane in presence of catalytic amount of TsOH. The acetonation has been regioselective without formation of regioisomer bringing a tense 3,4-diossolane cycle, less stable. The completely protected rhamnose 36 was finally obtained, after C-4 benzylation, in a 90% overall yield from 35. Final acceptor has been prepared from 36 through a sequence including isopropylidene protection removal (50% aq AcOH, 70 °C) followed by regioselective C-3 benzylation, achieved by cyclic stannylidene acetal formation.41
Scheme 2.3
Reagents and conditions. i: 3-tosylpropandiol, SnCl4, CH3CN (86%); ii: NaN3, TBAI, DMF,
reflux (88%); iii: a) MeONa, MeOH, 0 °C ; b) DMP, TsOH; c) BnBr, NaH, DMF (90% from 35);
iv: a) 50% aq AcOH, 70°C; b) Bu2SnO, toluene, reflux then BnBr, TBAB (88% from 36).
Applying the glycosylation conditions found in literature33 on the donor 33 and rhamnoside 37, desired SP19F repeating unit mimic 38 (Scheme 2.4) has been obtained in a good 86% yield with an acceptable α-stereoselectivity (α/β≈2:1).
Scheme 2.4
An alternative way to 38 has been planned following a different synthetic approach: starting from talosamine containing disaccharide 19,35 trisaccharide 39 has been assembled for first then
subjected to C-4’’ epimerization (Scheme 2.5). Applying the same glycosylation conditions seen for the preparation of trisaccharide 32, compound 39 (Scheme 2.5) has been obtained in a good 73% yield with an acceptable α-stereoselectivity (α/β≈2:1). For the C-4’’ epimerization sequence
in EtOH–MeOH solution gave the alcohol 41 in quite good yield (76%). Many problems have been occured in this “symple” reaction as long reaction times, frequent PdCl2 adds and not complete
reaction of the starting material to product 41.
Scheme 2.5
Alcohol 41 was subjected to a two-step epimerization protocol,35 starting with enolether 43 formation through a one-pot process (Scheme 2.6). After initial deprotonation of the alcohol with NaH in DMF, alkoxyde 41 has been treated at low temperature (-30 °C) with N,N’-sulfuryldiimidazole (Im2SO2), and then allowed to reach room temperature.
Scheme 2.6
The overall procedure is supposed to involve (Scheme 2.6) the formation of an imidazylsulfonate intermediate (42) from alkoxyde 41, spontaneously evolving through an imidazolate promoted elimination to enolether 43 in a nearly quantitative yield. Applying standard hydroboration-oxidation condition35 on 42, derivative 40 has been obtained in good yield (87%) in a regio- and
2.2. SP19F glycodendrimer preparation
After the preparation of the desired saccharide 38 with a functionalizable spacer on anomeric position, we decided to make early loading attempts on smallest PAMAM-(NH2)2 using the
thioureidic bridge formation approach.17 Reduction of azide group to amine has been the first step for the introduction of an isothiocyanate moiety on the propyl spacer of 38 (Scheme 2.7). Different azide reduction methods have been applied on the saccharide: using NiCl2·6H2O/NaBH4,43
problems in work up and quite low yield (60%) have been occured; problems as long time reactions, frequent catalyst adds and difficulties in base dosage have been occured also using hydrogenolysis with base poisoned 10% Pd-C: nitrogen containing bases as ammonia or Et3N
might dramatically suppress removal of O-benzyl protecting groups.44 Those reduction problems have been overcame with the use of PPh3 supported on resin in aq THF,45 a simple, clean, quite
rapid and quantitative reaction. The only subproduct of this reaction, triphenylphosphine oxide (still linked with resin), has been simply filtered off the reaction solution.
Scheme 2.7
Reagents and conditions. i: for 44: PPh3 on resin, THF-H2O (quant.); for 45: H2, 10% Pd-C,
(Boc)2O, EtOAc-EtOH then Ac2O, Py (89%); ii: for 46: DPT, DCM (64% from 38); for 47:
DCM-TFA-H2O then DPT, DCM (73% from 45); iii: PAMAM-(NH2)2, DMF-DCM, 60°C (no reaction
for R=Bn; 55% yield for R=Ac).
In amine to isothiocyanate conversion, the most well known method is based on the thiophosgene use,46 an extremely toxic reagent. Recently dipyridil-thionocarbonate (DPT, Scheme 2.8),47 a
“thiocarbonyl transfer” reagent, has been shown as a good alternative to thiophosgene and has been used also in this synthetic sequence.
Scheme 2.8 N O O S N DPT R NH2 R N C S N OH 2
Applying standard procedure (DPT in DCM) to 44, isothiocyanate 46 was obtained in 64% yield and used in the coupling reaction with the simplest PAMAM structure (51, Scheme 2.9), with two amino groups on its surface. The chosen dendron had a core with a protected amino group that permits higher dendron assembly or conjugation with different molecules as carrier proteins. PAMAM-(NH2)2 51 has been prepared using a divergent approach starting from
N-Boc-ethylenediamine 49 (EDA), easily obtained in high yields from the reaction between (Boc)2O and a
great excess of EDA, that avoided dicarbamate formation.48 Amine 49 has been subjected to a (a) Michael addition (methyl acrylate in MeOH) followed by (b) aminolysis using great excess of EDA. Repeating (a)-(b) sequence PAMAM-(NH2)4 52 was also prepared in nearly quantitative
yield. Scheme 2.9 Me3CO N H NH2 O i BocHN N OMe OMe O O 49 50 BocHN N NH NH O O NH2 NH2 ii 51 i; ii N NH H N O O NH2 NH2 BocHN N NH NH O O N NH N H O O NH2 NH2 52
Coupling reaction between 46 (1.2 eq excess for each NH2) and dendron 51 has been performed in
an optimized solvent mixture (DMF-DCM) that well solved both reagents at room temperature. Unfortunately no coupling product has been detected or isolated probably due to the steric hindrance of benzyl groups that didn’t permit the thioureidic bridge formation: unreacted 46 was recovered after flash chromatografy. The problem has been overcame, as shown in Scheme 2.8, through a reaction sequence that permitted azide reduction to protected amine and exchange from benzyl ether protections to acetyl ones.49 Trisaccharide 45 has been synthetized through a catalytic hydrogenation (H2, Pd-C in presence of Boc2O) that at the same time afforded to debenzylation,
azide reduction and N-Boc protection, followed by complete acetylation. The completely acetylated
45 has been transformed to 47 after standard Boc removal (TFA, DCM) and conversion of the
amino group into the isothiocyanate one using DPT in DCM. Saccharide 47 has been finally loaded on the dendron 51 using the same conditions seen for 48 (R=Bn) and the desired glycodendrimer 48 (R=Ac) has been isolated in 55% yield (Fig. 2.2).
Figure 2.2 O N H O N HN O H N N H O O AcO AcO NHAc O AcO O AcO OA c OAc AcO O O AcO O O AcO AcO AcHN O AcO O AcO AcO OAc AcO O O AcO O NH H N S N H S 48
In literature has not been reported yet the potential immunogenicity of thioureidic bridge in glycoconjugate system. This lack of informations might alter the comprehension of immunogenic tests on conjugate glycodendrimer 48. After a research in literature about different conjugation methods toward synthetic vaccines, it has been decided to use an homobifunctional linker as N,N’-bis-hydroxysuccinimide ester of adipic acid, often utilized in glycoconjugate preparation. This linker was used for example to prepare conjugates of synthetic saccharide fragments of Streptococcus pneumoniae type 14 with CRM197.50 In this reported study it was also pointed out
that SP14 conjugates prepared using the widely utilized squaric acid dietyl ester as linker decreased immune response relative to those that contained an adipic acid linker. This probably may be an effect of differences in the distribution of glycan chains on CRM197 due to the different coupling
conditions with conjugation reactions at pH 7.2 for adipic acid and pH 9 for squarate and/or differences in the distance or linkage between the oligosaccharide and protein carrier. On this basis, it has been planned to use adipic linker also in saccharide-PAMAM coupling to mantain the final structure homogeneity.
The first step to PAMAM loading has been the deprotection of trisaccharide 38 to polar compound
53 (Scheme 2.10). A two step approach to amine 53 has been made for first: protected saccharide
has been subjected to hydrogenolisis with base (Et3N) poisoned 10% Pd-C (azide reduction to
amine) then hydrogenolisis in acid MeOH. The same problems seen for 44 have been occurred as long time reactions, frequent catalyst adds and difficulties in base and acid dosage.
Scheme 2.10
Reagents and conditions. i: H2, 10% Pd-C, 25% aq NH3, tBuOH-H2O then H2, 10% Pd-C, AcOH,
tBuOH-H2O (74%); ii: SIDEA, dioxane-H2O (15-20%); iii: PAMAM-(NH2)4, H2O.
Desired deprotected key intermediate 53 (Scheme 2.10) has been finally prepared applying a deprotection “one-pot” sequence developed by Kamerling et al.51 Hydrogenolysis has been performed first in polar solvent in presence of ammonia: after azide reduction to amine (TLC analysis), ammonia was removed by argon bubbling in the reaction suspension and drops of AcOH
and more catalyst were added. Finally mixture was loaded on a short Dowex 50x8 (H+) column, eluted with 10% aq NH4OH and lyophilized. Deprotected 53 has been obtained NMR and
ESI-Q-Tof MS-analysis pure in good yield (74%, Fig. 2.3). Activation of 53 as adipoyl N-hydroxy-succinimidyl active ester 54 has been first attempted following a reported method by Panza et al,52
using adipoyl disuccinimidil ester (SIDEA) as bifunctional crosslinking reagents.53 A critical point has been represented by the necessity of water exclusion during the storage avoiding the hydrolysis of the half-ester intermediate: this sensitivity of the succinimide moiety to hydrolysis also precludes chromatographic purification of the intermediate. Treatment of 53 with 10 molar equivalents of SIDEA in dioxane-H2O gave monosubstituted compound 54 which was separated from the excess
of SIDEA by washing the crude reaction mixture with water. The activated ester remained undissolved while 54 should be recovered pure from the water solution by lyophilization and stored at -20°C. The NMR analysis of the crude pointed out that this purification method was not efficient to remove all the unreacted SIDEA, which might react itself with PAMAM during loading reaction. Activated 54 with an higher purity grade has been prepared by product precipitation from different reaction mixture (Et3N/DMSO) using NaCl-dioxane solution and washing the precipitates many
times with dioxane. The washed and lyophilized product has been finally analyzed with ESI-QTof and used in the protein coupling. A better result in terms of active ester quantity has been achieved by washing the reaction crude with EtOAc, that removed the excess of unreacted material (not activated trisaccharide and SIDEA) and permitted a better active ester recovery. A complex mixture of products has been obtained coupling the activation crude with PAMAM-(NH2)4 using 1.2 eq
excess of 54 for every NH2 group of dendron. The crude has been purified using C-4 column,
eluting with increasing ratio of MeOH in H2O and the collected fractions analyzed through
ESI-QTof method: traces of full loaded glycodendrimer were detected in presence of partially loaded products and activated trisaccharide dimers. After the evaluation of this “deprotected” method with its problems in SIDEA-free activated 54 preparation and coupling crude purification, we decided to plan a different approach to SP19F glycodendrimers preparation, using protected intermediates. A preliminary study about this “protected” approach has been made using rhamnoside 56, an easy-to-prepare mimic of trisaccharide 38.
Scheme 2.11
Reagents and conditions. i: a) PPh3 on resin, THF-H2O; b) methyladipoyl chloride, Py, DCM
The first phase of this new route has been represented by the azido-propyl spacer “elongation” to the derivatives 58 and 59 (Scheme 2.11) followed by coupling condition study with PAMAM-(NH2)2 and PAMAM-(NH2)4 asthe second phase. Final complete removal of benzyl protections
gave desired glycodendrimers.
Starting compound 56, prepared from peracetyl analogue 35, has been subjected to azide reduction using PPh3 on resin43 followed by a condensation with commercial methyladipoyl chloride, that
permits the introduction of an adipic spacer with a methyl ester at its end. Thanks to the versatility of this group, acid derivative 58 has been prepared through basic hydrolysis (KOH in MeOH) of
57 and amine 59 after aminolysis using large EDA excess. With these two intermediate in hands, a
methodological study of coupling reagent condition has been started to find the best way to desired glycodendrimers (Scheme 2.12).
Generally coupling reagents for peptide synthesis (also for glycodendrimer coupling) are a very heterogeneous family of chemical compounds:phosphonium, uronium, immonium, carbodiimide, imidazolium, organophosphorous, acid halogenating, cloroformate and pyridinium reagents.54 We had performed an extensive screening of coupling conditions focusing our attention on the activating systems EDC/DMAP,54 EDC/HOBt/TEA55 and HBTU/HOBt/DIPEA56 (Fig. 2.3).
Figure 2.3
EDC (ethyl-(N’,N’-dimethylamino) propylcarbodiimide hydrochloride) is a water-soluble and stable carbodiimide and it is used as a versatile coupling agent to form amide, ester or thioester bond and thus to cross-link proteins and nucleic acids. HBTU (O-(1H-Benzotriazole-1-yl)-N,N,N’,N’-tetramethylaminium hexafluorophosphate) with its tetrafluoroborate equivalent TBTU is a benzotriazole uronium reagent and one of the most popular in situ activation reagents in solid and solution phase peptide synthesis.
The use of additives, as N,N-dimethylaminopyridine (DMAP), 1-hydroxy-1,2,3-benzotriazole (HOBt) or N-hydroxysuccinimide (NHS), was required to prevent side reactions in the acid activation or in the coupling step: N-acylurea formation, using EDC or guanidylation of the amine, using HBTU.54
Scheme 2.12
Reagents and conditions. i: see Table 2.1; ii: H2, 10 % Pd-C, AcOH, MeOH (quant.).
At this point compound with acid moiety 58 (1.3 eq. for NH2 group) has been loaded on the
PA-(NH2)2 using EDC/HOBt coupling system, as a first trial on the smallest PAMAM (Table 2.1, entry
1). As clear from TLC analysis, reaction has been brought to the formation of fully loaded product (50% after chromatographic purification). Then rhamnose 58 has been also coupled with PA-(NH2)4 using the same system EDC/HOBt/TEA (entry 2). The reaction had required longer reaction
time than entry 1 and difficulties in TLC analysis and chromatographic purification, leading to fully loaded glycodendrimers isolated in a 30%-40% yield.
Table 2.1
Entry Acid Amine Coupling reagent Yield
1 58 PA-(NH2)2 EDC/HOBt 50% (full loaded)
2 58 PA-(NH2)4 EDC/HOBt 30-40% (f.l.)
3 58 PA-(NH2)4 EDC/DMAP
Only partially loaded products
4 58 PA-(NH2)4 HBTU/HOBt/DIPEA
Only partially loaded products
5 PA-(COOH)4 59 EDC/HOBt
3- loaded PAMAM (main product)
Using EDC/DMAP (entry 3), same analysis and purification problems have been occurred isolating partially loaded glycodendrimers (1H- and 13C-NMR) in presence of N-acylurea derivatives of 58. The formation of these sub-products should be slower than carboxylate activation as benzotriazole ester (Scheme 2.13).
Scheme 2.13
slow
Using HBTU/HOBt/DIPEA (entry 3), no loaded product has been isolated probably due to the formation of guanidine moiety on amino groups of PA-(NH2)4 as shown in the Scheme 2.14. This
study on PAMAM-glycodendrimer formation has been completed with the conjugation between the amino group of rhamnose 59 and PA-(COOH)4 using EDC/HOBt (entry 4). PA-(COOH)4 has been
prepared by saponification (KOH/EtOH) of PA-(COOMe)4, precursor of PA-(NH2)4 (Scheme 2.9).
In entry 4 partially loaded glycodendrimers (main product was three-loaded PAMAM at 1H- and
13
C-NMR) have been isolated. Probably the formation of activated esters on PA-(COOH)4 has
been required longer reaction times and the partial formation of benzotriazole esters (bulky groups) has been prevent an exhaustive activation.
As the final step of this explorative study, glycodendrimer 60 has been subjected to hydrogenolysis using Pd-C in acid MeOH to avoid catalyst poisoning by amine groups. After one night stirring deprotected compound has been obtained in quantitative yield.
The spacer elaboration conditions seen for rhamnoside 56 have been applied on trisaccharide 38 as shown in Scheme 2.15. Scheme 2.15 O BnO BnO NHAc O BnO O BnO BnO OBn BnO O O BnO O N3 O BnO BnO NHAc O BnO O BnO BnO OBn BnO O O BnO O NH O 4 O O O BnO BnO NHAc O BnO O BnO OBn OBn BnO O O BnO O NH O 4 H N O PAMAM 2 O HO HO NHAc O HO O HO OH OH HO O O HO O NH O 4 H N O PAMAM 2 i ii iii 38 63 64 65
Reagents and conditions. i: a) PPh3 on resin, THF-H2O; b) methyladipoyl chloride, Py, DCM
(76% from 38); ii: a) KOH, EtOH; b) PA-(NH2)2, EDC, HOBt, TEA, DMF (49% from 63); iii: H2,
10% Pd-C, AcOH, MeOH (quant.).
For the coupling key step the best reaction conditions found for rhamnoside 56 have been used also for 38: system EDC/HOBt/TEA as coupling reagents between acid analogue of saccharide 63 and amino groups of PAMAM-(NH2)2 and PAMAM-(NH2)4. As expected, problems have been occured
in TLC analysis of reaction (many spots) and especially during chromatographic purifications of the crudes because of particular eluting mixtures (CHCl3:MeOH:ammonia). However glycodendron
64 has been finally isolated in quite good yield (49% from methyl ester 63) and subjected to
complete benzyl protection removal (Fig. 2.4). In glycodendron 55 preparation problems have been amplified by the complexity of the final product. Finally both SP19F clusters 65 and 55 have been subjected to MALDI-Tof MS, ESI-QTof MS and high-field 1H-NMR analysis.
Figure 2.4 NH O O O N H O N N H HN O H N N H O NH O O O HO HO NHAc O HO O HO OH OH HO O O HO O O HO HO AcHN O HO O HO OH OH HO O O HO O 65 (Exact Mass: 1780,87) O HO HO AcHN O HO O HO OH OH HO O O HO O NH O O O NH N H N N H N O O O NH H N O H N NH O N H N NH O HN N H O O HOHO A cHN O HO O HO HO OH HO O O HO O NH O O O HO H O AcHN O H O O HO H O OH OH O O HO O NH O O O OH HO AcH N O HO O HO HO OH OH O O HO O NH O O 55 (Exact Mass: 3629,78)
For the MALDI (Matrix-Assisted Laser Desorption/Ionization) spectra clusters were mixed with different matrices: α-cyano, DHB and super-DHB. α-cyano or CHCA (α-cyano-4-hydroxycinnamic acid) is a cinnamic acid derivative, a member of the phenylpropanoid family and it is used as a matrix for peptides and nucleotides (max. 10 kDa); DHB (2,5-dihydroxybenzoic acid) an aromatic carboxylic acid is used for protein digests, carbohydrates, oligosacharides, glycopeptides and both proteins and peptides below 10 kDa; super-DHB improved the detection of glypeptide and glycocluster signals compared to normal DHB matrix. Matrix CHCA was suspended in 30% aq ACN in presence of 0.1% aq TFA; DHB and super DHB in 50% aq MeOH. Finally clusters have been added to matrices and analyzed. In Fig. 2.5 MALDI spectra of 65 (exact mass 1780.87 Da) using different matrices are shown.
Figure 2.5. MALDI spectrum of 65 with super DHB 50% aq MeOH.
As shown in Fig. 2.5, the base peak (the peak with the greatest intensity in the spectrum) has been assigned to fully loaded cluster 65 (exact mass 1780.87 Da, sodium adduct exact mass 1803.86 Da) in presence of smaller peaks that might be also MALDI-Tof artifacts. Using a different matrix (DHB) the same peaks have been found in the spectra with similar relative intensity. In the analysis using CHCA no sample ionization has been detected. In Fig 2.6 MALDI spectra of 55 (exact mass 3629.78 Da, sodium adduct exact mass 3652.77 Da) using different matrices are shown. The MALDI spectra seemed to be more complicate than previous ones, with base peak represented by 3038.32 Da peak probably relative to partial derivatization of cluster. As shown in Fig. 2.6, many
[M+Na]+ Cluster PA2+2x19F
peaks have been related to non exhaustive loading products due probably to different space disposition of PA-(NH2)4 (globular structure) compared to PA-(NH2)2 that did not permit complete
coupling with perbenzylated (bulky groups) saccharides.
Figure 2.6. MALDI spectrum of 55 with DHB 50% aq MeOH.
ESI-MS analysis on clusters 65 and 55 have been performed by direct infusion and also after C-4 Jupiter 300 Å chromatography. Direct infusion analysis has been more complicated by adducts formed with Na and K salts. However the results of the two methods of MS-analyses applied to the compounds have been comparable. Every analysis has been performed with sample cone at 15 V as a control and 30V that gave more sugar fragmentation.
In the coupling compound with PA-(NH2)2, complete loaded cluster 65 has been detected (peak
eluted at 7.26-7.28 min) in presence of SP19Fsugar fragmentations (Fig. 2.7a-b). Other spectra signals were not related to partial loaded clusters: many signals between 713 and 985 Da not related to clusters, presence of 1189 Da molecule seen in MALDI spectra (PA-(NH2)2+1xSP19F+104 Da)
with elution time 9.24 min (data not show).
In the coupling compound with PA-(NH2)4, complete loaded cluster 55 has been detected (peak
eluted at 6.17-6.19 min) in presence of SP19Fsugar fragmentations (Fig. 2.8a-b). Other assigned spectra signals are PA-(NH2)4+2xSP19F-60 Da probably related to a cluster with two cyclized
branches and the peak related to PA-(NH2)4+3xSP19F+42 Da.
Cluster PA4+3x19F+105 Da [M+H]+
Cluster PA4+4x19F [M+H]+ Cluster PA4+2x19F+103 Da+44Da
[M+H]+ Cluster PA4+1x19F+44 Da
[M+H]+ PA4+169 Da
Figure 2.7a. A portion of MS-spectra of 65 (7.28 min elution time peak, 15V)
Figure 2.7b. A portion of MS-spectra of 65 (7.27 min elution time peak, 30V). Cluster PA2+2x19F [M+3H] +3 Cluster PA2+2x19F [M+2H] +2 Cluster PA2+2x19F [M+H]+1 Cluster PA2+2x19F [M+2H] +2 Cluster PA2+2x19F [M+H]+1
Figure 2.8a. A portion of MS-spectra of 55 (6.17 min elution time peak, 15V).
Figure 2.8b. A portion of MS-spectra of 55 (6.17 min elution time peak, 30V).
Cluster PA4+4x19F [M+2H]+2 Cluster PA4+4x19F [M+3H]+3 Cluster PA4+4x19F [M+4H]+4 Cluster PA4+3x19F+42 Da [M+2H]+2 Cluster PA4+2x19F+cycl. [M+3H]+3 Cluster PA4+4x19F [M+2H]+2 Cluster PA4+4x19F [M+4H]+4 Cluster PA4+4x19F [M+3H]+3 Cluster PA4+3x19F+42 Da [M+2H]+2 Cluster PA4+3x19F+42 Da [M+3H]+3
Monodimensional 1H-NMR experiments on 65 and 55 (Fig. 2.9a-b) has been performed in D2O and
all signals related to trisaccharide anomeric protons, to its spacer and to PAMAM have been assigned in comparison with spectra of not loaded dendrimers.
The “loading ratio” has been an average value not related to distribution of each loading product. Analyzing 65 spectrum (Fig. 2.9a), the values 2.5, 2.5 and 2.4 have been obtained for trisaccharide anomeric protons through integration of CH3 signals of Boc group (9 protons) which have been
calibrated to 9. Those values indicated a complete loading and probably the presence of few “impurities” (low intensity signals near the baseline) in the range of 10-20%. Integrating also CH2
signals (not O or N-linked) of the spacer (2 protons from “propyl portion” and 4 from “adipic portion”), the previous observations have been confirmed.
Analyzing 55 spectrum (Fig. 2.9b), the values 2.4, 2.3 and 2.3 have been obtained for trisaccharide anomeric protons through integration of CH3 signals of Boc group (9 protons) which have been
calibrated to 9. Those values indicated a 57% loading ratio and also with this product the presence of few “impurities” (low intensity signals near the baseline).
Integrating also CH2 signals (not O or N-linked) of the spacer as seen for 65, the previous
observations have been confirmed. The results obtained from MALDI-Tof, ESI-QTof and NMR pointed out that:
a) PA-(NH2)2-SP19F cluster 65 has been completely loaded with two units of SP19F
trisaccharide. Few “impurities” (from trisaccharide or from coupling reaction) in the range of 10-20% have been detected.
b) PA-(NH2)4-SP19F cluster 55 has been loaded with 2.4 SP19F trisaccharide molecules:
this average value confirmed the presence of PA-(NH2)4 loaded with 4, 3 and 2 SP19F
trisaccharide units. Minimal quantity of “impurities” (from trisaccharide or from coupling reaction) have been detected.
With these observations in hands, only cluster 65 and active trisaccharide ester have been coupled with the carrier protein.
Figure 2.9a. 1H-NMR of 65 (D2O, 400 MHz).
Figure 2.9b. 1H-NMR of 55 (D
2.3. Coupling with CRM197
Cluster 65 has been subjected to removal of Boc protection (20% aq TFA) then, as shown in Fig. 2.10 step a, transformed into active ester 67 using large excess of SIDEA.
Figure 2.10
The preparation of active ester 67 has been achieved following the method applied on 53, with many acetone washings of the reaction precipitate to remove SIDEA excess. Deprotection of 66, activation and SIDEA removal have been confirmed by ESI-QTof MS analysis (data not shown). Activated trisaccharide 54 and cluster 67 were conjugated to the carrier protein CRM197.4 For
conjugation to CRM, a 20-fold molar excess of activated saccharide and cluster was reacted with the protein, at room temperature, in 200 mM phosphate buffer (pH 7.2). The conjugation reactions were analyzed through an SDS-PAGE (Sodium Dodecyl Sulphate - PolyAcrylamide Gel Electrophoresis), that permitted to separate proteins according to their electrophoretic mobility (a function of length of polypeptide chain or molecular weight). In Fig. 2.11a there is a gel related to the first conjugation trials: on the left, in a separate gel lane, a molecolar weight marker (prestained SDS-PAGE
standards Broad Range, MW) was allowed to run, composed by proteins of known molecular weight in order to calibrate the gel and determine the weight of unknown proteins by comparing the distance traveled relative to the marker itself; near MW there is the control of CRM197 protein, then
conjugation mixtures of activated trisaccharide 54-CRM197 (TRIS1) and activated cluster 68
(TRISPA2). In TRIS1, conjugation with the protein was not obtained because of not well activated trisaccharide. In TRISPA2 a total conjugation with protein has been detected (no more CRM197)
with two main “families” of conjugates. As a second trial a better activated trisaccharide (TRIS2, Fig. 2.11b) has been conjugated with the protein and was allowed to run in SDS-PAGE using the same “marker” and conditions: on the TRIS2 lane a total conjugation with protein has been detected (no more CRM197) with the formation of a lower running band
(main band) probably related to conjugated trisaccharide 54-CRM197 and uppermost running band
probably related to conjugated with SIDEA crosslinked domains (not exhaustive removal of SIDEA from activation reaction). The conjugates
have been purified by ammonium sulfate precipitation; this method is used to purify proteins by altering their solubility (known as “salting out”). The solubility of protein varies according to the ionic strength of the solution, and hence according to the salt concentration. The commonly used salt is ammonium sulfate, as it is very soluble in water; it has been used as a saturated aqueous solution (500 mg/mL). This purification method allows the removal of the saccharide excess, since the saccharide doesn’t precipitate with the conjugate and remains in solution. The “salting out” precipitates have been constituted by pure conjugates with 54-CRM197 and cluster 68. The
54-CRM197 and 68-CRM197 conjugates have been characterized by protein and saccharide content. The
protein content has been estimated with a MicroBCA Protein assay (Bio-Rad Laboratories), following the manufacturer’s specifications. According to the spectrophotometer results, 54-CRM197 and 68 had a 50 % and 87% of protein yield, respectively.
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.
Novartis Instruments
NMR analyses
All the samples were dissolved in deuterium oxide (Aldrich) and the solutions were inserted in 5-mm NMR tubes (Wilmad). Proton NMR experiments were recorded at 25°C on a Bruker Avanc III 400 MHz spectrometer, using a 5 mm broadband probe (Bruker). The TOPSPIN 2.1 software package (Bruker) was used for data acquisition and processing. All the 1H-NMR spectra were collected and standard one-pulse experiments, with 32 k data points, were collected over a 10 ppm spectral width. The transmitter was set at the HDO frequency, which was also used as reference signal (4.79 ppm).
ESI-MS analyses
Analyses by direct sample injection were performed in a Micromass Q-Tof Micro system (Waters MS Technologies, UK). For LC-Mass analyses the Q-tof Micro system was coupled to an UPLC system (ACQUITY UPLC System, Waters, UK). Chromatographic separations of samples were performed on 2.1 mm i.d.×50 mm ACQUITY BEH C18 1.7 µm column (Waters Corp., USA).
MALDI-MS analyses
MALDI-TOF mass spectra of samples were recorded by an UltraFlex III MALDI-TOF/TOF instrument (Bruker Daltonics) in linear mode and with positive ion detection. All the samples for analysis were prepared by mixing 1 µL product and 1 µL of Super DHB matrix, and 1 µL of each mixture was deposited on samples plate, dried at room temperature for 10 min and subjected to the spectrometer.
Spectrometer
Lambda25 UV/VIS Spectrometer (PerkinElmer) 3.1. Preparation of trisaccharide 38
2-Acetamido-3,6-di-O-benzyl-2,4-di-deoxy-L-erithro-hex-4-enopyranosyl-ββββ-(14)- 2,3:5,6-di-
O-isopropylidene-aldehydo-D-glucose dimethyl acetal (30)
A soln of 1434 (5 g, 7.81 mmol) in anhyd THF (74 mL) was warmed to reflux and treated with t-BuOK (9.60 g, 78.4 mmol). After 30’ 14 was completely consumed (TLC, EtOAc) and, cooled at 0°C, 50 mL of satd aq NaHCO3 were added. The aq phase was extracted with CH2Cl2 (3 × 75 mL),
the organic extracts collected, dried and concentrated under diminished pressure. The crude product (4.90 g) was directly solubilized in umid THF (100 mL, 0.5% of water), added first with 18-crown-6 (330 mg, 1.2 mmol) and then, at 0°C, with pulverized KOH (2.318-crown-6 g, 42.14 mmol). After 30’ at 0°C, the suspension was treated with BnBr (2.24 mL, 18.86 mmol). After 7 h the starting material was consumed (TLC, 3:7 hexane-EtOAc) and MeOH (15 mL) was added, the soln stirred for 30’, concentrated under diminished pressure and the residue partitioned between CH2Cl2 (50 mL) and H2O (30 mL). The aq phase was
extracted with CH2Cl2 (3 × 50 mL), the organic extracts collected,
dried 5’ and concentrated under diminished pressure. The crude residue (7.5 g, yellow oil) was subjected to flash chromatography (3:7 hexane-EtOAc + 0.1% Et3N) to give pure 30 (4.77 g, 91% yield) as a colourless syrup, Rf 0.56
(EtOAc), [α]D +6.29 (c 1.2, CHCl3); 1
H-NMR (250 MHz, CD3CN): δ 7.38-7.26 (m, 10H, aromatic
H), 6.46 (d, 1H, J2’,NH 9.3 Hz, NH), 5.41 (d, 1H, J1’,2’ 2.4 Hz, H-1’), 5.04 (d, 1H, J3’,4’ 1.4, H-4’), 4.56
and 4.48 (AB system, 2H, JA,B 11.9 Hz, CH2Ph), 4.52 (s, 2H, CH2Ph), 4.45 (m, 2’), 4.31 (m, 1H,
H-5), 4.28 (dd, 1H, J2,3 8.0 Hz, H-2), 4.25 (d, 1H, J1,2 6.7 Hz H-1), 4.10-3.99 (m, 2H, H-4, H-6b),
3.97-3.92 (m, 4H, H-6a, H-6’a, H-6’b, H-3), 3.35 and 3.34 (2s, each 3H, OCH3), 1.89 (s, 3H, CH3CO),
O NHAc OBn BnO O O O O (MeO)2HC O
1.36-1.28 (3s, 12H, CH3 isoprop); 13C-NMR (63 MHz, CD3CN): δ 170.6 (CO), 150.7 (C-5’), 139.8,
139.5 (Ar-C), 129.3-128.5 (Ar-CH), 110.7, 108.6 (3 × CMe
2), 106.7 (C-1), 99.4 (C-4’), 98.1 (C-1’),
79.3 (C-3), 78.8 (C-2), 76.3 (C-3), 74.9 (C-4), 73.1, 71.9 (CH2Ph), 69.7 6’), 69.0 3’), 65.4
(C-6), 56.6, 54.4 (OCH3), 48.2 (C-2’), 27.6, 26.6, 26.3, 24.4 (2 × CMe2), 23.2 (CH3CO).
Anal. Calcd for C36H49NO11 (671.77): C, 64.36; H, 7.35; N, 2.09. Found: C, 64.38; H, 7.34; N, 2.10.
2-Acetamido-3,6-di-O-benzyl-2-deoxy-ββββ-D-mannopyranosyl-(1
4)-2,3:5,6-di-O-isopropylidene-aldehydo-D-glucose dimethyl acetal (15)
A soln of 30 (4.02 g, 5.98 mmol) in anhyd THF (236 mL) was treated dropwise at 0°C and under argon atmosphere with a solution of borane dimethylsulfide complex in Et2O (5 M, 1.79 mL, 8.97
mmol) with THF (1:10). The solution was stirred at 0°C for 30’, then warmed to room temperature and stirred until the starting material was completely reacted (4 h, TLC, 1:4 hexane-EtOAc). The mixture was cooled to 0°C and treated in the order with H2O (5.8 mL), 10%
aq. NaOH (18 mL) and, finally, 35% aq. H2O2 (46 mL). The biphasic
mixture was stirred for 2 h, diluted with H2O (470 mL), and the organic phase was separated. The
acqueous layer was repeatedly extracted with CH2Cl2 (4 × 40 mL), and the collected organic
phases, after drying, were concentrated at reduced pressure. The crude residue was subjected to a flash chromatographic purification (1:4 hexane-EtOAc) to give pure 15 (3.29 g, 80% yield) whose physico-chemical properties were analogous to those reported in the literature.35a
2-Acetamido-4-O-acetyl-3,6-di-O-benzyl-2-deoxy-ββββ-D-mannopyranosyl-(14)-1,2,3,6-tetra-O-
acetyl-α,βα,βα,βα,β-D-glucopyranoside (31)
A soln of 15 (2.41 g, 3.49 mmol) in 80% aq. AcOH (60 mL) was stirred at 80°C until the starting compound was completely reacted (4 h, TLC, 8:2 CHCl3-MeOH) with formation of slower moving
products (Rf 0.28). The reaction mixture was concentrated at reduced pressure and repeatedly
co-evaporated with toluene (5 × 30 mL). To the crude residue was directly added a soln of AcONa.3H
2O (1.7 g, 12.47
mmol) solubilized in Ac2O (276 ml) at reflux (145 °C). After
1 h at 110 °C, the reaction mixture was repeatedly co-evaporated with toluene (5 × 50 mL) and the residue partitioned between CH2Cl2 (100 mL) and
H2O (60 mL). The aq phase was extracted with CH2Cl2 (3 × 50 mL), the organic extracts collected,
dried and concentrated under diminished pressure. The crude residue (3 g, yellow oil) was
O AcO BnO NHAc OBn O OAcO OAc OAc OAc O HO B nO NHAc OB n O O O O (MeO)2HC O
subjected to flash chromatography (1:1 hexane-EtOAc) to give 31 (2.55 g, 94% yield from 15), an
α/β anomer mixture (25:75, 1H-NMR), as a white foam, Rf 0.54 (EtOAc); 1H-NMR (250 MHz,
CDCl3) δαααα-anomer: δ 6.27 (d, 1H, J1,2 3.6 Hz, H-1), 5.75 (d, 1H, J2’,NH 9.0 Hz, NH), 2.09, 2.04,
2.03, 2.02, 2.00, (5s, each 3H, 5xCH3COO), 1.94 (s, 3H, CH3CON); ββββ-anomer: δ 5.76 (d, 1H, J2’,NH 9.2 Hz, NH), 5.66 (d, 1H, J1,2 8.1 Hz, H-1), 2.04, 2.03, 2.02, 2.01, 2.00, (5s, each 3H,
5xCH3COO), 1.99 (s, 3H, CH3CON). 1C-NMR (63 MHz, CDCl3) αααα-anomer: δ 98.6 (C-1’), 88.7
(C-1), 76.4 (C-3’), 74.2 (C-5’, C-4), 73.4 (C-5), 69.4, 68.5 (C-2, C-3), 68.7 (C-6’); 67.3 (C-4’), 61.6 (C-6), 49.0 (C-2’), 23.1 (CH3CON); ββββ-anomer: δ 98.3 (C-1’), 91.4 (C-1), 76.3 (C-3’), 74.4 (C-5’, C-4), 73.5 (C-5), 72.4 (C-3), 69.9 (C-2), 68.6 (C-6’); 67.4 (C-4’), 61.8 (C-6), 48.8 (C-2’), 23.0 (CH3CON); common signals (α and β): δ 171.5-168.7 (MeCO), 137.5, 137.1 (Ar-C), 128.3-127.5 (Ar-CH), 70.5, 73.2 (2xCH2Ph), 20.6-20.4 (5 x CH3CO).
Anal. Calcd for C28H39NO18 (773.78): C, 49.63; H, 5.80; N, 2.07. Found: C, 49.61; H, 5.82; N,
2.04.
2-Acetamido-4-O-acetyl-3,6-di-O-benzyl-2-deoxy-ββββ-D-mannopyranosyl-(14)-2,3,6-tri-O-
acetyl-1-thiophenyl-ββββ-D-glucopyranoside (32)
A soln of 31 (1.0 g, 1.29 mmol) in anhyd (CH2)2Cl2 (17 mL) was added under argon atmosphere to
rapidly flamed ZnI2 (825 mg, 2.58 mmol) and activated AW molecular sieves 4Å (AW 300, 4 g).
After 30’ in the dark, TMSSPh (0.97 mL, 5.2 mmol) was added at 0°C and the suspension stirred at room temperature until the starting compound was completely reacted (16 h, Rf 0.35, EtOAc). The
suspension was then filtered through Celite, diluted with CH2Cl2, the organic layer washed with
satd aq NaHCO3 and the aq phase extracted with CH2Cl2 (3 × 50 mL). After organic extract drying
and concentration, the crude residue (1.43 g) was subjected to flash chromatography (1:9 hexane-EtOAc) to give 32 (690 mg, 65% yield) as a white foam, Rf
0.35 (EtOAc), [α]D -58.5 (c 1.0, CHCl3); 1
H-NMR (250 MHz, CDCl3): δ 7.50 (m, 2H, H), 7.37-7.24 (13, H, Ar-H,
ArS-H), 6.10 (d, 1H, J2’,NH 9.6 Hz, NH), 5.16 (t, 1H, J2,3,=J3,4, 9.0
Hz, H-3), 5.05 (t, 1H, J3’,4’=J4’,5’=9.1 Hz, H-4’), 4.95, 4.44 (system AB, 2 H, JA,B 11.0 Hz, CH2Ph),
4.90 (dd, 1H, J1,2 9.2 Hz, H-2), 4.81 (d, 1H, H-1), 4.73 (d, 1H, J1’,2’ 1.6 Hz, 1’), 4.71 (m, 1H,
H-2’), 4.43 (s, 2H, CH2Ph), 4.25 (m, 2H, 6a, 6b), 3.70-3.37 (m, 5H, 3’, 4, 5, 6’a,
H-6’b), 3.21 (m, 1H, H-5’), 2.06, 2.05, 2.02, 1.97 (4s, each 3H, 4xCH3CO), 1.89 (s, 3H,CH3CON); 13C-NMR (63 MHz, CDCl
3): δ 173.1, 173.0, 172.4, 172.0, 170.3 (5xCO), 138.9, 138.2 (2xAr-C),
132.2-126.3 (ArCH, ArS-CH), 131.5 (ArS-C), 98.6 (C-1’), 85.3 (C-1), 76.8 (C-3’), 74.6, 74.3, 73.2
O AcO BnO NHAc OBn O OA cO OAc SPh OAc
(C-5’, C-4, C-5), 73.9, 70.5 (2xCH2Ph), 72.3 (C-3), 69.8 (C-2), 68.2 (C-4’), 68.6 (C-6’), 61.9 (C-6),
50.3 (C-2’), 23.6 (CH3CON), 20.6-20.1 (4xCH3CO).
Anal. Calcd for C32H41NO16S (823.90): C, 52.8; H, 5.68; N, 1.92. Found: C, 52.9; H, 5.70; N, 188.
2-Acetamido-3,4,6-tri-O-benzyl-2-deoxy-ββββ-D-mannopyranosyl-(1
4)-2,3,6-tri-O-benzyl-1-thiophenyl-ββββ-D-glucopyranoside (33)
To a soln of 28 (1.02 g, 1.24 mmol) in MeOH (6 mL) was added NaOMe in MeOH (0.33 M, 4.8 mL) at 0°C. After 4 h at 0°C, the reaction mixture was neutralized with Amberlist-15 (washed with MeOH), filtered and concentrated under reduced pressure. The crude product (807 mg, Rf 0.21,
EtOAc) was directly solved in THF+0.5% H2O (25 mL) and treated with the system
BnBr/KOH/crown ether as seen for 25. After standard work-up, flash chromatography purification (3:2 hexane-EtOAc) gave 29 (1.02 g, 81% yield) as a white foam, Rf 0.41 (1:1 hexane-EtOAc), [α]D
-15.5 (c 1.0, CHCl3); 1H-NMR (250 MHz, CD3CN): δ 7.55 (m, 2H, ArSH), 7.48-7.20 (m, 33H,
ArCH+ArCHS), 6.30 (d, 1H, J2’,NH 10.0 Hz, NH), 5.13-4.73 (AB system, 2H, JAB 11.6, CH2Ph),
4.80 (m, 1H, H-2’), 4.77-4.59 (AB system, 2H, JAB 11.0, CH2Ph), 4.76 (d, 1H, J1,2 9.8, H-1), 4.73 (m, 2H, CH2Ph), 4.72-4.38 (AB system, 2H, JAB 10.7, CH2Ph), 4.71-4.44 (AB systemt, 2H, JAB 12.3, CH2Ph), 4.70 (d, 1H, J1’,2’, 1.3, H-1’), 4.63 (AB system, 2H, JAB 12.0, CH2Ph), 3.98 (dd, 1H, J4,5 9.6, H-4), 3.77 (m, 2H, H-6a, H-6b), 3.66 (dd, 1H, J3,4 9.2, H-3), 3.65-3.52 (m, 5H, H-3’, H-5’, H-6’a, H-6’b, H-5), 3.43 (dd, J4’,5’ 9.7, H-4’), 3.33 (dd, 1H, J2,3 8.71, H-2’), 1.82 (s, 3H, CH3CON); 13C-NMR (63 MHz,
CD3CN): δ 170.9 (CO), 140-139.3 (ArC), 135.0 (ArCS), 131.7-128.1 (ArCH+ArCHS), 99.7 (C-1’),
87.5 (C-1), 85.5 (C-3), 81.6 (C-3’), 81.2 (C-5), 78.9 (C-5’), 76.7 (C-4), 76.6 (C-2), 75.8 (CH2Ph),
75.7 (CH2Ph), 75.4 (C-4’), 75.2 (CH2Ph), 73.7 (CH2Ph), 73.5 (CH2Ph), 71.4 (CH2Ph), 70.3 (C-6’),
69.8 (C-6), 50.0 (C-2’), 23.2 (CH3CON).
Anal. Calcd for C62H65NO10S (1016.25): C, 73.28; H, 6.45; N, 1.38. Found: C, 73.25; H, 6.48; N,
1.41.
3-O-tosylpropyl 2,3,4-tri-O-acetyl-αααα-L-rhamnopyranoside (34)
A soln of SnCl4 (13 mL) in dry CH3CN (31 mL) was dropped into a soln of peracetyl rhamnose
(3.03 g, 13.2 mmol) in dry CH3CN (123 mL). After 15’ stirring at rt, a soln of 3-O-tosyl-propandiol
(3.00 g, 13.0 mmol) in dry CH3CN (30 mL) was added and, after 2 h at rt, the reaction solution was
concentrated under reduced pressure, then diluted with CH2Cl2, the organic layer washed with satd
O BnO BnO NHAc OBn O OBnO OBn SPh OBn
aq NaHCO3 and the aq phase extracted with CH2Cl2 (4 × 50 mL). After organic extract drying and
concentration, the crude residue (6.20 g) was subjected to flash chromatography (3:2 hexane-EtOAc) to give 34 (5.69 g, 86% yield) as a colourless syrup, Rf 0.41
(1:1 hexane-EtOAc), [α]D -31.4 (c 1.1, CHCl3); 1H-NMR (250 MHz,
CD3CN): δ 7.78, 7.44 (AA’XX’ syst., 4H, Ar-H), 5.07 (dd, 1H, J1,2 1.6
Hz, J2,3 3.3 Hz, H-2), 5.03 (dd, 1H, J3,4 9.6 Hz, H-3), 4.94 (dd, 1H, J4,5 9.3 Hz, 4), 4.65 (d, 1H,
H-1), 4.11 (m, 2H, CH2OTs), 3.77 (dq, 1H, J5,6 6.3 Hz, H-5), 3.67 (dt, 1H, Jvic 6.0 Hz, Jgem 10.1 Hz,
CH2O), 3.40 (dt, 1H, Jvic 5.9 Hz, CH2O), 2.43 (s, 3H, MePh), 2.08, 2.02, 1.93 (3s, each 3H, MeCO),
1.93 (m, 2H, CH2CH2CH2), 1.13 (d, 3H, H-6); 13
C-NMR (63 MHz, CD3CN): δ 170.9 (3xCO),
146.3, 133.7 (2xAr-C), 131.0, 128.7 (4 x Ar-CH), 98.1 (C-1), 71.3 (C-4), 70.2 (C-2), 70.0 (C-3), 68.7 (CH2OTs), 67.2 (C-5), 64.1 (CH2O), 29.2 (CH2), 21.6 (MePh), 21.0, 20.9, 20.8 (3xMeCO),
17.7 (C-6).
Anal. Calcd for C22H30O11S (502.54): C, 52.58; H, 6.02. Found: C, 52.53; H, 5.99.
3-O-azidopropyl 2,3,4-tri-O-acetyl-αααα-L-rhamnopyranoside (35)
To a soln of 34 (5.32 g, 10.6 mmol) in dry DMF (214 mL), NaN3 (2.06 g, 31.8 mmol) and then
Bu4NI (783 mg 21.2 mmol) were added and the resulting suspension was stirred under argon
atmosphere at 50 °C. After 3 h and 30’ TLC analysis (2:3 hexane-EtOAc) revealed the complete disappearance of the starting material, mixture was cooled to rt, concentrated and partitioned between satd aq NaHCO3 (100 mL) and CH2Cl2 (100 mL). The organic phase was separated and
the aq layer extracted with CH2Cl2 (5 x 50 mL). The organic extracts were dried (MgSO4.H2O),
filtered and concentrated under diminished pressure. Purification of the residue by flash cromatography over silica gel (7:3 hexane-EtOAc) gave pure 35 (3.47 g, 88% yield) as a foamy solid, Rf 0.51 (2:3
hexane-EtOAc), [α]D- 57.57 (c 1.21, CHCl3), m.p. 48-52°C (chrom); 1H-NMR (250 MHz, CD3CN): δ
5.15 (dd, 1H, J1,2 1.7 Hz, J2,3 3.4 Hz, H-2), 5.12 (dd, 1H, J3,4 9.7 Hz, H-3), 4.96 (t, 1H, J4,5 9.7 Hz,
H-4), 4.74 (d, 1H, H-1), 3.86 (dq, 1H, J5,6 6.3 Hz, H-5), 3.75 (dt, 1H, Jvic 6.2 Hz, Jgem 10.0 Hz,
CH2O), 3.50 (dt, 1H, Jvic 6.0 Hz, CH2O), 3.41 (t, 2H, J 6.7 Hz, CH2N3), 2.16, 2.08, 2.01 (3s, each
3H, MeCO), 1.85 (m, 2H, CH2CH2CH2), 1.16 (d, 3H, H-6); 13C-NMR (63 MHz, CD3CN): δ 171.0,
170.9, 170.8 (3xCO), 98.1 (C-1), 71.4 (C-4), 70.4 (C-2), 70.1 (C-3), 67.2 (C-5), 65.5 (CH2O), 49.0
(CH2N3), 29.3 (CH2), 21.0, 20.9, 20.8 (3xMeCO), 17.7 (C-6).
Anal. Calcd for C15H23N3O8 (373.37): C, 48.25; H, 6.21; N, 11.25. Found: C, 48.20 H, 6.22; S,
11.29. AcO O O AcO OAc OTs AcO O O AcO OAc N3
3-O-azidopropyl 4-O-benzyl-2,3-O-isopropylidene-αααα-L-rhamnopyranoside (36)
To a soln of 35 (3.42 g, 9.16 mmol) in MeOH (88 mL) was added NaOMe in MeOH (0.33 M, 17.5 mL) at 0°C. After 1 h and 40’ at rt, the reaction mixture was neutralized with Amberlist-15 (washed with MeOH), filtered and concentrated under reduced pressure.
The crude (2.21 g) was solved in anhyd DMF (3.5 mL) and 2,2-dimethoxypropane (10 mL) and added with TsOH (30.2 mg, 0.16 mmol). After 5 h and 30’ TLC analysis (EtOAc) revealed the complete disappearance of the starting material and the soln was diluted with CH2Cl2, partitioned
between satd aq NaHCO3 (100 mL) and CH2Cl2 (100 mL). The organic phase was separated and
the aq layer extracted with CH2Cl2 (5 x 50 mL). The organic extracts were dried (MgSO4.H2O),
filtered and concentrated under diminished pressure.
To a suspension of NaH in mineral oil (60%, 747 mg, 18.7 mmol) pre-washed with hexane under argon atmosphere and cooled at 0°C, a soln of the crude in dry DMF (66 mL) was slowly added. The mixture was stirred at 0°C for 30’ and treated with BnBr (1.33 mL, 11.22 mmol). After 16 h under room temperature stirring, starting compound was completely reacted (1:1 hexane-EtOAc) and the reaction mixture was then cooled to 0°C, excess of NaH was destroyed by addition of MeOH (4 mL) followed by 10’ stirring and MeOH removal under diminished pressure. The solution was partitioned between CH2Cl2 (50 mL) and H2O (50 mL), the organic phase separeted
and the aq layer extracted with CH2Cl2 (3 x 50 mL). The collected organic phases were dried
(MgSO4.H2O), filtered and concentrated under dimished pressure. The flash chromatographic
purification over silica gel of the reaction product (9:1 hexane-EtOAc) gave pure 36 (3.11 g, 90% yield from 35) as a colourless syrup, Rf 0.22 (9:1 hexane-EtOAc), [α]D -40.7 (c 1.18, CHCl3); 1
H-NMR (250 MHz, CD3CN): δ 7.38-7.26 (m, 5H, Ar-H), 4.90 (d, J1,2 0.6
Hz, H-1), 4.82, 4.61 (AB system, 2H, JA,B 11.6 Hz, CH2Ph), 4.16 (ddd, 1H, J2,3 5.7 Hz, J3,4 6.7 Hz, J3,5 0.6 Hz, H-3), 4.11 (dd, 1H, H-2), 3.73
(ddd, 1H, Jvic 5.8 Hz, Jvic 6.4 Hz, Jgem 9.9 Hz, CH2O), 3.64 (ddq, 1H,
J4,5 9.8 Hz, J5,6 6.3 Hz, H-5), 3.46 (ddd, 1H, CH2O), 3.39 (t, 2H, J 6.8 Hz, CH2N3), 3.18 (dd, 1H,
H-4), 1.80 (m, 2H, CH2CH2CH2), 1.46, 1.32 (2s, each 3H, Me2C), 1.21 (d, 3H, H-6); 13C-NMR (63 MHz, CD3CN): δ 139.8 (Ar-C), 129.1, 128.8, 128.4 (Ar-CH), 109.8 (Me2C), 97.7 1), 82.1
(C-4), 79.3 (C-3), 76.8 (C-2), 73.6 (CH2Ph), 65.3 (C-5), 64.8 (CH2O), 49.1 (CH2N3), 29.4 (CH2), 28.2,
26.5 (Me2C), 18.1 (C-6).
Anal. Calcd for C19H27N3O5 (377.43): C, 60.46; H, 7.21; N, 11.13. Found: C, 60.50; H, 7.18; N,
11.10. O O O BnO O N3
