Galactose-phosphonates as mimetics of the sialyltransfer by trypanosomal sialidases

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Galactose‐Phosphonates as Mimetics of the

Sialyltransfer by Trypanosomal Sialidases

Heike Busse ^a, Mitsuru Hakoda ^b, Matthew Stanley ^b& Hansjörg Streicher ^b

^aDepartment of Chemistry, University of Konstanz, Konstanz, Germany

^bChemistry Department, University of Sussex, Brighton, United Kingdom

Published online: 01 Jun 2007.

To cite this article: Heike Busse , Mitsuru Hakoda , Matthew Stanley & Hansjörg Streicher (2007):

Galactose‐Phosphonates as Mimetics of the Sialyltransfer by Trypanosomal Sialidases, Journal of Carbohydrate

Chemistry, 26:3, 159-194

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Journal of Carbohydrate Chemistry, 26:159–194, 2007

Copyright # Taylor & Francis Group, LLC

ISSN: 0732-8303 print 1532-2327 online

DOI: 10.1080/07328300701351532

Galactose-Phosphonates as

Mimetics of the

Sialyltransfer by

Trypanosomal Sialidases

Heike Busse

Department of Chemistry, University of Konstanz, Konstanz, Germany

Mitsuru Hakoda

Chemistry Department, University of Sussex, Brighton, United Kingdom

Matthew Stanley

Chemistry Department, University of Sussex, Brighton, United Kingdom

Hansjo¨rg Streicher

Department of Chemistry, University of Konstanz, Konstanz, Germany and Chemistry

Department, University of Sussex, Brighton, United Kingdom

In an attempt to ﬁnd competitive inhibitors of the trans-sialidase of the pathogen

Trypanosoma cruzi, we have synthesized conjugates of carbocyclic sialylmimetics

(e.g., cyclohexenephosphonates) and galactose derivatives. A trans-sialidase inhibition

assay revealed an interesting preference for ethylidene-spacered conjugates involving

the 3-position of the sugar.

Received January 23, 2007; accepted February 20, 2007.

¨

Address correspondence to Hansjorg Streicher, Chemistry Department, University of

Sussex, Brighton BN1 9QG, United Kingdom. E-mail: h.streicher@sussex.ac.uk

159

H. Busse et al.

160

Keywords Sialidase, Trans-sialidase, Sialylmimetic, Carbasugars, N-acetylneur-

aminic acid, Phosphonates

INTRODUCTION

The sialic acids, derivatives of 3-deoxy-D-glycero-D-galacto-2-nonulosonic

acids, are of great signiﬁcance for recognition processes in higher organisms

(Fig. 1).^[1]The cell wall glycoconjugates of the latter contain sialic acids at

exposed, often terminal, positions, linked to the respective glycan chain via a

2,3-, 2,6-, or a 2,8-glycosidic linkage. At this crucial position, sialic acids

serve as receptors for endogenous or exogenous recognition events such as leu-

cocyte targeting or microbial adhesion.^[2,3]Often, microbial infection processes

involve chemical modiﬁcation, such as acetylation and deacetylation of the host

cell sialic acids and, as a classical example, desialylation. In this case, the gly-

cosidic bond between the sialic acid and the subterminal sugar is hydrolyzed

through the action of microbial sialidases, a class of enzymes that as a result

have been identiﬁed as possible targets to interfere with the respective infec-

tion processes.^[4,5]

Synthetic organic chemists have therefore been seeking to mimic sialosides

effectively while at the same time overcoming their metabolic instability, for

instance, by replacing either the glycosidic or the ring oxygen by other atoms

such as carbon or sulfur.^[5,6]A prominent example for this strategy is the car-

bocyclic sialylmimetic, the inﬂuenza sialidase inhibitor anti-inﬂuenza drug

oseltamivir.^[7]

In many cases, however, mimetics of the sialic acid alone might not be sufﬁ-

cient to achieve effective inhibition. In Scheme 1, the example of the trans-sia-

lidase of the parasite Trypanosoma cruzi is given to illustrate such a case.^[8–11]

Although carrying glycoconjugates on its surface, the parasite T. cruzi

cannot synthesize sialic acid de novo. Instead, it expresses a trans-sialidase

(TcTS), which transfers a sialyl moiety to its own glycans rather than releasing

it to the aqueous environment like “normal” sialidases. This sialyltransfer

proceeds as a two-step reaction via a covalent enzyme-sialic acid intermediate

Figure 1: N-Acetylneuraminic acid (Neu5Ac, R¹-R⁶¼ H), the most prominent member of the

family of sialic acids (R¹–R⁶¼ various modiﬁcations).

Galactose-Phosphonates

161

Scheme 1: Sialyltransfer by T. cruzi trans-sialidase.

involving Tyr 342 (Sch. 1). Detailed investigations have shown that, for efﬁcient

transfer to occur, the acceptor substrate must be bound simultaneously to the

donor substrate. This is supported by the ﬁndings that in the absence of an

acceptor substrate, sialoside hydrolysis is slow and the classical sialidase inhibi-

tor and transition-state analog DANA is only a very weak inhibitor of TcTS.^[12,13]

We have therefore concluded that carbocyclic sialylmimetics containing a

phosphonate would allow attachment of an acceptor mimetic as a monoester

while at the same time retaining a negative charge under physiologic con-

ditions known to be important for recognition by all sialidases.^[14]

Simple cyclohexenephosphonate monoesters developed by us such as 1 and

2 (Fig. 2) exhibited moderate, but encouraging, inhibition of TcTS, and we have

recently succeeded in the synthesis of pseudo-sialosides involving sugars such

as 3.^[15,16]

H. Busse et al.

162

Figure 2: Previously investigated cyclohexenephosphonate monoesters and types of

compounds included in this study.

Our phosphonates of type A (Fig. 2), including 3 itself, do not inhibit TcTS,

so it became clear that a more systematic approach to simulate the TcTS sialyl-

transfer to the acceptor substrate is required. In this contribution, we report on

methodologies to synthesize mimetics involving phosphonate monoesters and

the 3-OH of galactose as the natural acceptor of the sialyl moiety when trans-

ferred by TcTS. Phosphonates of type B include a spacer between the galactose

and the phosphonate, while the even more demanding phosphonates of type C

are linked directly to the sugar hydroxyl group (Fig. 2).

RESULTS AND DISCUSSION

Type A

We have synthesized pseudo-sialoside 3 by alkylation of the protected

L-xylo cyclohexenephosphonate monobenzyl ester 4 with the sugar triﬂate

followed by deprotection (Sch. 2).^[16]Esteriﬁcation of methylphosphonic acid

and galactose 5 under Mitsunobu conditions was straightforward, so we

attempted to apply both strategies with suitable galactose derivatives, that

is, Mitsunobu condensation of 4 with galactoses 8, 9, and 10 as well as alky-

lation of 3 with galactose triﬂates 11, 12, and 13 (Sch. 3). Much to our

Galactose-Phosphonates

163

Scheme 2: Synthesis of type A conjugates.

disappointment, the monobenzyl esters seemed to not be suitable for a general

approach with, for instance, triﬂate elimination being always faster than the

corresponding alkylation reaction.^[17]

Cyclohexenephosphonate-monomethylesters, however, did show a more

promising behavior in Mitsunobu esteriﬁcation.^[16,17]We therefore applied

Scheme 3: Attempted synthesis of type B and C mimetics via the monobenzyl

phosphonate.

H. Busse et al.

164

Scheme 4: Synthesis of type B conjugates via the monomethyl phosphonate.

our established general methodology for the synthesis of xylo-conﬁgured cyclo-

hexenephosphonates^[18]to generate a suitable monomethylester (Sch. 4).

Type B

Starting from 5-silylated 1,2-O-isopropylidene-a-L-xylofuranose 14, we

inverted the absolute conﬁguration at C-3 and benzylated ribose 15 to give

16 according to well-known procedures (Sch. 4).^[18–22]In previous syntheses

of ethyl^[15,18,19]and benzyl esters,^[16,17]we introduced azide as an acetamide

precursor at this stage. In this case, however, the azide was not compatible

with the conditions of methylenediphosphonate introduction. Desilylation of

16 to give 5-deprotected ribose 17 allowed activation as the triﬂate, which

was readily substituted with the tetramethyl methylenediphosphonate anion

to give 6-carbon sugar 18. Removal of the isopropylidene group under acidic

conditions liberated the aldehyde/hemiacetal, which was cyclized by treatment

with strong base to furnish ^L-xylo-conﬁgured cyclohexenephosphonate 19 in

40% yield. Acetylation of 19 to give 20 was carried out under standard con-

ditions to facilitate characterization, but unfortunately the acetyl groups

tended to migrate under debenzylation conditions. To avoid this, we introduced

Galactose-Phosphonates

165

methoxymethyl groups at hydroxy groups 3 and 5 of 19 to give 21 and

subsequently removed the benzyl group by transfer hydrogenation with 1,4-

cyclohexadiene and palladium on charcoal, which gave 22 in good yield.

Initially, we introduced the azide via a Mitsunobu procedure in only

moderate yield of 45%, which we were able to signiﬁcantly improve employing

the two-step procedure via the triﬂate giving azide 23 in 82% yield. Treatment

of 23 with 50% triﬂuoroacetic acid removed the methoxymethyl groups quanti-

tatively to yield 24, which was treated with trimethylphosphine and acetic

anhydride to simultaneously achieve azide conversion into the acetamide

and O-acetylation to give 25. Quantitative cleavage of one methyl ester

group with the thiophenol/triethylamine system resulted in the corresponding

monoester salt 26, which could be converted into the free acid 27 with acidic ion

exchange resin.

Much to our delight, monomethylester 27 readily underwent Mitsunobu

condensation with protected galactose-spacer conjugate 10 to give protected

pseudo-sialoside 28 as a mixture of both diastereomeric diesters. Selective

cleavage of the methyl ester with thiophenol/triethylamine resulted in quanti-

tative formation of monoester 29, which was readily deacetylated to give 30.

Finally, removal of the isopropylidene group resulted in type B target

molecule 31, which had previously been inaccessible employing benzyl

esters. For comparison, we synthesized both the monomethyl ester 32 by sapo-

niﬁcation of diester 27 and the methylphosphonic acid conjugate 33 by Mitsu-

nobu condensation with galactose 10. The latter was ﬁnally deacetylated to

give 34, the second target molecule of type B (Sch. 4).

Type C

As mentioned above, esteriﬁcation or alkylation of our modiﬁed phospho-

nates, to obtain the corresponding 3-phosphono-galactoses of type C,

failed.^[17]Consequently, we decided to introduce the phosphonate via the phos-

phite triester followed by partial hydrolysis and alkylation. To achieve this, a

protected methyl b-galactopyranoside with a free hydroxyl group in the

3-position was required. We chose the stannylidene acetal-supported selective

alkylation of the 3-OH group of methyl b-galactopyranoside with benzyl

bromide followed by acetylation to give 35, which was readily debenzylated

to afford 3-OH free galactose 36 (Sch. 5). The labile triester 37 was not

isolated but directly subjected to mild acid hydrolysis with silica gel to give

phosphonate 38, ready for alkylation. To investigate its behavior toward

TcTS, we deprotected 38 to obtain type C target 39. With the phosphonic

acid triester at hand, synthesis of the corresponding phosphate was straight-

forward enough to include it in our study. Peroxide treatment of 37 gave the

stable trialkyl phosphate 40, which could be deprotected in two steps to give

the 3-phosphate galactopyranoside 41 in high yield.

H. Busse et al.

166

Scheme 5: Synthesis of type C conjugates via alkylation of H-phosphonates.

Phosphonic acid 38 proved to be the ideal starting material for the intro-

duction of alkyl and aryl residues by various methods, two of which we

included in this investigation so far. Firstly, aldehydes such as acetamido ben-

zaldehyde as simple sialylmimetics react readily with phosphonates under

basic conditions to form the respective substituted hydroxymethyl phospho-

nates, here 42. This methodology has been extensively applied to the synthesis

of sialyltransferase inhibitors by others.^[23]We did not separate the resulting

diasteromers, but it is clear that the new hydroxylated stereocenter opens

the way to a wealth of straightforward further modiﬁcations. We deprotected

42 to give the next type C molecule as the diasteromeric mixture 43, which

was tested as such.

Galactose-Phosphonates

167

Alkylation of the phosphonate with alkyl halides does not come with stereo-

center formation. To validate this approach, we alkylated phosphonate 38 with

benzyl bromide and p-acetamido benzyl bromide to give 44 and 46, respectively.

Both molecules were deprotected to the type C targets under standard deben-

zylation and deacetylation conditions to yield 45 and 47 (Sch. 5).

This approach via 3-phosphono galactoses provides many options for

library synthesis of potential TcTS inhibitors, some of which are currently

being developed in our laboratory.

Synthesis of Galactose Derivatives

Generally, standard protecting group methodologies^[24]were applied to the

synthesis of galactose derivatives 9–13, which were used in successful and

attempted syntheses of pseudo-disaccharides (Sch. 6). In brief, methyl b-D-

galactopyranoside was selectively allylated at the 3-hydroxy group via the

stannylidene acetal and then isopropylidenated to give 48. Conversion of the

allyl group into the hydroxyethyl spacer via ozonolysis and reduction furnished

the protected acceptor 9, which, for alkylation attempts, could be activated as

the triﬂate (11). To obtain a fully protected galactose-spacer conjugate, benzy-

lidene galactose^[25,26]was isopropylidenated and then allylated to give 49.

Scheme 6: Synthesis of galactose derivatives.

H. Busse et al.

168

Conversion of the allyl group was carried out as described above to form 10,

which, if required, was triﬂated (12).

The galactose derivatives lacking a spacer were synthesized from methyl

b-D-galactopyranoside by ﬁrst introducing the 4,6-benzylidene acetal and

selectively acylating the 3-position with monochloroacetyl chloride to give 50.

Benzoylation to yield 51, followed by deacetylation with hydrazinium

acetate, furnished 3-OH-unprotected galactose 52 for use in Mitsunobu ester-

iﬁcations. Inversion of conﬁguration for alkylation was achieved by activation

as the triﬂate followed by substitution with nitrite and hydrolysis of the nitrous

acid ester to give gulose 53, the triﬂate of which (13) was readily formed with

triﬂic anhydride and pyridine (Sch. 6).

Trans-sialidase Assay

To assess the inhibitory activity of our compounds against TcTS, we used a

trans-sialidase assay introduced by Schrader et al.^[27]In brief, the reaction to

be inhibited is the transfer of a sialic acid by TcTS from the commercially avail-

able donor 3⁰-sialyllactose to the 3-OH of the acceptor 4-methylumbelliferyl

b-D-galactopyranoside. Sialylated derivatives are separated from uncharged

acceptor by serial ion exchange chromatography and the product formed is

quantiﬁed by total hydrolysis and measurement of 4-methylumbelliferone-

ﬂuorescence under basic conditions.

Methyl b-D-galactopyranoside, as an example for the minimal structural

requirements an acceptor has to fulﬁll, and lactitol, as an example for a

rather active substrate-analogous inhibitor,^[28]were included in the assay.

While results with the former have to be viewed with caution as sialylated

methyl b-D-galactopyranoside can compete with the donor in the assay, sialy-

lated lactitol, in contrast, has been shown to not act as a donor.^[28]

The results obtained are summarized in Table 1. The weak binding of type

A compound 7 did not come as a surprise as we had previously found pseudo-

sialoside 3 to not inhibit,^[16]thus indicating that a negatively charged group

linked to the 6-OH of a galactose does not lead to a substantial increase in

binding (e.g., by electrostatic interaction with the enzyme’s arginine triade).

Linking a phosphonate to the 3-position of methyl b-D-galactopyranosidevia

an ethylene spacer, however, enhances binding compared to the reference

methyl galactoside. The effect obtained with type B inhibitors 31 and 34,

respectively, is not as pronounced as we had expected, but it is strong enough

to shed light on how to proceed. We had previously published weak inhibition

by ^L-xylo cyclohexenephosphonate monoethylesters^[15](i.e., 31 without the

galactose), indicating also a positive effect of an ethyl group. These data

taken together enable us now to establish, albeit still with caution, a ﬁrst struc-

ture activity relationship based on an ethylidene linkage between a phospho-

nate-containing sialylmimetic and a methyl b-D-galactopyranoside.

Galactose-Phosphonates

169

Table 1: Inhibitory activity of the sialoside mimetics synthesized toward T. cruzi trans-

sialidase (IC₅₀values, rounded to + 0.5 mM based on three experiments).

b-Galactosides

Type A

Type B

Type C

A: 5 mM; 7: .8 mM; 34: ꢀ3 mM; 39: .8 mM; 47: .8 mM; B: 0.57 mM²³; 3: n.i.; 31: ꢀ1.5 mM; 41:

.8 mM; 45: ꢀ6 mM; 43: ꢀ8 mM.

With regard to type C compounds, the introduction of a phosphonate,

selected alkylphosphonates, and a phosphate at the 3-position of methyl b-D-

galactopyranoside without linker did not lead to any improvement, rather

the contrary, compared to galactose alone. It is, however, too early to

abandon this type of structure as the “sialylmimetics” we used to establish

the alkylation chemistry were rather basic. More examples of these readily

accessible structures are deﬁnitely required.

CONCLUSIONS

As part of our program to ﬁnd novel inhibitors of sialoside-processing

enzymes, in particular trans-sialidases, we have established methodologies to

synthesize pseudo-sialosides consisting of carbocyclic sialylmimetics containing

a phosphonate linking them to galactoses. We have extended our established

synthetic approach toward xylo-conﬁgured cyclohexenephosphonates to the

L-xylo cyclohexenephosphonate monomethylester, which allows galactose

attachment via Mitsunobu condensation and alkylation. In order to gain

access to the otherwise elusive phosphonate linkage to the 3-position of galac-

tose, we have investigated alkylation of the previously introduced phosphonate

with simple sialylmimetics. This gives us straightforward access to libraries of

potential inhibitors and is the subject of ongoing projects in our laboratory.

A number of pseudo-sialosides containing phosphonate-galactose lin-

kages via the 6-position (type A), the spacered 3-position (type B), and the

3-position (type C) have been deprotected and subjected to a trans-sialidase

inhibition assay. While type A and type C compounds displayed negligible

inhibition, we found an interesting positive, albeit still moderate, effect of

type B compounds with ethylidene spacer. This corresponds well with our

previous data of cyclohexenephosphonate monoethylesters and indicates that

sialylmimetic-ethylene spacer-galactose conjugates might be a common

H. Busse et al.

170

denominator for more potent bisubstrate-type inhibitors of TcTS. Although

these compounds still track lactitol or benzyl b-N-acetyllactosamine^[29]regard-

ing potency, they offer a wealth of possible modiﬁcations, some of which are

being pursued by us.

EXPERIMENTAL

Reaction solvents were purchased anhydrous and used as received. Solvents

for chromatography were distilled before use. Reactions were monitored by

TLC using precoated silica gel 60 F₂₅₄plates. Compounds were detected

by UV absorption and/or by staining with a molybdenum phosphate

reagent (20 g ammonium molybdate and 0.4 g cerium(IV) sulfate in 400 mL

of 10% aq. sulfuric acid) and subsequent heating at 1208C for 5 min. Silica

gel 60 M (particle size 40–63 mm) from Macherey-Nagel, Du¨ren, Germany,

and 60 A Davisil (particle size 35–70 mm) from Fisher Scientiﬁc, UK, was

used for ﬂash chromatography. ¹H NMR, ¹³C NMR, and ³¹P NMR spectra

were recorded on

a

Bruker DRX 600 spectrometer (at 600 MHz,

150.9 MHz, and 242.9 MHz, respectively) and a Bruker DPX-300 (at

300 MHz, 75.4 MHz, and 121.4 MHz, respectively). Chemical shifts in ¹H

NMR and ¹³C NMR spectra were referenced to the residual proton resonance

of the respective deuterated solvent, CDCl₃(7.24 ppm), D₂O (4.63 ppm), and

D₂O in CD₃OD (4.88 ppm). For ³¹P NMR spectra H₃PO₄was used as

external standard (0 ppm). In some cases, ¹³C chemical shifts were

deduced from heteronuclear multiple quantum correlation (HMQC)

spectra. In pseudo-disaccharidic systems, the cyclohexene ring is indicated

by the sufﬁx “a”, the sugar by the sufﬁx “b”. Diastereomeric mixtures of

mixed diesters are indicated by the sufﬁx “h”(higher moving) and “l”(lower

moving), but no attempts of separation were made. HR-ESI MS spectra

were recorded on a Bruker Daltonics Apex III in positive mode with

MeOH/H₂O as solvent. MALDI MS spectra were recorded on a Bruker

Biﬂex III spectrometer in positive, linear mode with a delayed extraction

MALDI source or on a Kratos Analytic Kompact Maldi 2 using 3,5-dihydro-

xybenzoic acid (DHB), a-hydroxy-a-cyano-cinnamic acid (HCCA), or azido-

thymidine (ATT) as matrix.

Silica-based MPLC chromatography was carried out on the Bu¨chi Sepacore

system equipped with glass columns packed with LiChroprep Si 60 (15–25 mm)

from Merck, Darmstadt, Germany.

Gel permeation chromatography was carried out in the 1- to 5-mg scale

on a XK 16/70 column (bed volume 130 mL), from Amersham packed with

Bio-Gel P2 Fine (particle size 45–90 mm) and 0.1 M NH₄HCO₃as buffer.

Detection was achieved with a differential refractometer from Knauer,

Berlin, Germany.

Galactose-Phosphonates

171

The synthesis of compounds 1–4 has been described by us pre-

viously,^{[15,16,18,19]}and compound

literature.^[30]

5 was synthesized according to the

Fine chemicals were purchased from Aldrich-, Sigma-, or Acros-Chemicals

and were of the highest purity available.

Abbreviations: THF (tetrahydrofurane), Ph₃P (triphenyl phosphine), DIAD

(diisopropyl azodicarboxylate), EtOAc (ethyl acetate), Tol (toluene), CHCA

(a-cyano-a-hydroxycinnamic acid), TFA (triﬂuoroacetic acid), DCM (dichloro-

methane), TBAF (tetrabutylammonium ﬂuoride), DHB (dihydroxy benzotria-

zole), TBAI (tetrabutylammonium iodide), DBU (diazabicycloundec-7-ene),

BnBr (benzyl bromide).

Triethylammonium O-[1,2:3,4-di-O-isopropylidene-a-D-

galactopyranos-6-yl] methylphosphonate (6)

Methanephosphonic acid (21 mg, 0.21 mmol) was dissolved in 2 mL of

dry THF, and galactose

5 (100 mg, 0.38 mmol) and Ph₃P (110 mg,

0.38 mmol) were added under argon. DIAD (75 mL, 0.38 mmol) was added

and the mixture was stirred at 608C for 2 days. The solvent was evapor-

ated and the residue was puriﬁed by ﬂash chromatography (EtOAc:Tol

10:1 ! EtOAc:MeOH 4:1, 1% NEt₃) to afford 6 (45 mg, 52%) as a colorless

triethylammonium salt. TLC (EtOAc:MeOH 5:1): R_f¼ 0.07; [a]²_D⁰¼ 240.2

(c ¼ 1, MeOH); ¹H NMR (600 MHz, CD₃OD) d 5.48 (d, 1H, J_1–2¼ 4.8 Hz,

H-1), 4.63 (dd, 1H, J_2–3¼ 2.4, J_3–4¼ 7.8 Hz, H-3), 4.36 (dd, 1H, H-2),

4.30 (dd, 1H, J_4–5¼ 1.2 Hz, H-4), 4.04–3.91 (m, 3H, H-5, H-6, H-6⁰), 3.18

(q, 6H, N(CH₂CH₃)₃), 1.50 (s, 6H, C(CH₃)₂), 1.39 (s, 6H, C(CH₃)₂), 1.36–

1.29 (m, 15H, P-CH₃and N(CH₂CH₃)₃); ¹³C NMR (151 MHz, CD₃OD) d

109.8 (C(CH₃)₂), 97.9 (C-1), 72.2 (C-3), 72.1 (C-4), 71.9 (C-2), 68.8 (C-5),

64.7 (C-6), 47.5 (N(CH₂CH₃)₃), 26.5, 26.4 (each CH₃of C(CH₃)₂), 12.5

(N(CH₂CH₃)₃), 11.6 (PCH₃); ³¹P NMR (162 MHz, CD₃OD)

d

27.8

(s, PCH₃). MALDI-MS (CHCA, THF, pos. mode) Calcd for

(C₁₃H₂₃O₈P þ Na)^þ361.1. Found 360.4.

Ammonium O-[a,b-D-galactopyranoside-6-yl-]

methylphosphonate (7)

7 (10 mg, 0.023 mmol) was dissolved in 2 mL of 50% TFA and stirred at rt

overnight. The mixture was lyophilized, dissolved in 1M NH₄HCO²₃buffer, and

1

lyophilized again to afford 7 (6 mg, 95%) as a white solid. H NMR (600 MHz,

CD₃OD) d 5.15 (d, 1H, J_1–2¼ 4.2 Hz, H-1a), 4.49 (d, 2H, J_1–2¼ 8.4 Hz,

H-1b), 4.02–3.70 (m, 14H, H-4, H-5, H-6, H-6⁰both anomers and H-2a,

H-3a), 3.55 (dd, 2H, J_2–3¼ 3.6, J_3–4¼ 9.0 Hz, H-3b), 3.38 (dd, 2H, H-2b),

H. Busse et al.

172

1.21 (d, 6H, PCH₃); ¹³C NMR (151 MHz, CD₃OD) d 96.4 (C-1 b), 92.3 (C-1a),

72.5 (C-3), 71.7 (C-2), 10.6 (PCH₃) ppm, C4-C6 not resolved; ³¹P NMR

(162 MHz, CD₃OD) d 28.6 (s, PCH₃). MALDI-MS (CHCA, THF, pos. mode)

Calcd for (C₇H₁₅O₈P þ Na)^þ281.1. Found 281.1.

4,6-O-Benzylidene-1,2-O-isopropylidene-a-D-

galactopyranoside (8)

Benzylidene galactose^[21](6.0 g, 0.022 mmol) was suspended in dry acetone

(300 mL), and dimethoxypropane (3.57 mL, 0.0290 mmol) and p-TsOH

(100 mg) were added and the reaction was stirred at rt overnight. The

reaction mixture was neutralized with saturated NaHCO²₃solution, washed

with DCM, and dried over MgSO₄. Flash chromatography (EtOAc:Tol 1:2)

afforded 8 (3.1 g, 46%) as a colorless syrup. TLC (EtOAc:Tol 1:2): R_f¼ 0.4;

1

[a]²_D⁰¼ 238.4 (c ¼ 1, CHCl₃); H NMR (600 MHz, CDCl₃) d 7.52–7.36 (m, 5H,

C₆H₅), 5.73 (s, 1H, CHPh), 5.67 (d, 1H, J_1–2¼ 5.4 Hz, H-1), 4.65 (dd, 1H,

J_2–3¼ 2.4, J_3–4¼ 8.4 Hz, H-3), 4.45 (dd, 1H, H-2), 4.33 (dd, 1H,

0

J_4–5¼ 1.8 Hz, H-4), 3.99 (ddd, 1H, J_5–6¼ 4.8, J_5–6¼ 6.6 Hz, H-5), 3.89 (dd,

1H, J_6–6¼ 11.4 Hz, H-6), 3.78 (dd, 1H, H-6⁰),1.56, 1.34 (s, 6H, C(CH₃)₂); ¹³C

0

NMR (151 MHz, CDCl₃) d 137.8 (C-Ph), 129.3–127.4 (C₆H₅), 105.1 (C(CH₃)₂),

104.1 (CHPh), 96.6 (C-1), 72.1 (C-3), 71.9 (C-4), 70.2 (C-2), 68.2 (C-5), 62.2

(C-6), 26.1, 25.3 (C(CH₃)₂). MALDI-MS (CHCA, THF, pos. mode) Calcd for

(C₁₆H₂₀O₆þ Na)^þ331.1. Found 331.1. Calcd for (C₁₆H₂₀O₆þ K)^þ347.1.

Found 347.1.

Methyl 3-O-hydroxyethyl-4,6-O-isopropylidene-b-D-

galactopyranoside (9)

48 (300 mg, 1.1 mmol) was dissolved in MeOH (10 mL) and one spatula

of solid NaHCO₃was added as buffer. The mixture was cooled to 2788C

and a constant stream of ozone was discharged until the solution turned

blue. The reaction was allowed to warm up to rt and NaBH₄(248 mg,

6.5 mmol) was added. After 2 h the reaction was complete, the solvent

was removed in vacuo, and the residue was puriﬁed by ﬂash chromato-

graphy (EtOAc:MeOH 5:1) to afford 9 (240 mg, 78%) as a colorless syrup.

TLC (EtOAc:MeOH 5:1): R_f¼ 0.27; [a]²_D⁰¼ þ25.8 (c ¼ 1, MeOH); ¹H NMR

(600 MHz, CD₃OD) d 4.38 (pd, 1H, J_3–4¼ 3.0 Hz, H-4), 4.18 (d, 1H,

0

J_1–2¼ 7.2 Hz, H-1), 4.14 (dd, 1H, J_5–6¼ 1.8, J_6–6¼ 12.6 Hz, H-6),

3.83 (dd, 1H, H6⁰), 3.72–3.65 (m, 4H, HOCH₂CH₂O), 3.62 (dd, 1H,

J_2–3¼ 9.6 Hz, H-2), 3.51 (s, 3H, OCH₃), 3.38 (m, 2H, H-3, H-5), 1.46,

1.36 (s, 6H, C(CH₃)₂); ¹³C NMR (151 MHz, CD₃OD) d 105.3 (C-1), 100.3

(C(CH₃)₂), 81.8 (C-3), 71.6 (C-a), 70.9 (C-2), 67.7 (C-5), 66.7 (C-4), 63.9

Galactose-Phosphonates

173

(C-6), 62.4 (C-b), 57.3 (OCH₃), 29.5 (C(CH₃)₂). MALDI-MS (CHCA, THF,

pos. mode) Calcd for (C₁₂H₂₂O₇þ Na)^þ301.1. Found 301.3. Calcd for

(C₁₂H₂₂O₇þ K)^þ317.1. Found 317.3.

4,6-O-Benzylidene-3-O-hydroxyethyl-1,2-O-isopropylidene-

a-^D-galactopyranoside (10)

49 (200 mg, 0.570 mmol) was dissolved in MeOH (12 mL) and the

reaction was carried out as described for 9. Following addition of NaBH₄

(88 mg, 2.9 mmol), the reaction was stirred until TLC indicated complete

reduction. The mixture was evaporated in vacuo and the crude product

was puriﬁed by ﬂash chromatography (EtOAc:Tol 1:2 ! 10:1) to yield 10

(110 mg, 55%) as a colorless syrup. TLC (EtOAc:Tol 10:1): R_f¼ 0.57;

[a]²_D⁰¼ 242.1 (c ¼ 1, CHCl₃); ¹H NMR (600 MHz, CDCl₃) d 7.52–7.25

(m, 5H, C₆H₅), 5.75 (s, 1H, HPh), 5.64 (d, 1H, J_1–2¼ 4.8 Hz, H-1), 4.66

(dd, 1H, J_2–3¼ 2.4, J_3–4¼ 8.4 Hz, H-3), 4.45 (dd, 1H, H-2), 4.35 (dd, 1H,

J_4–5¼ 1.8 Hz, H-4), 4.10 (pt, 1H, H-5), 3.75–3.58 (m, 6H, CH₂CH₂0; H6,

H6⁰), 1.57, 1.35 (s, 6H, C(CH₃)₂); ¹³C NMR (151 MHz, CDCl₃) d 136.0 (C-

Ph), 130.0–126.3 (C₆H₅), 108.9 (C(CH₃)₂), 103.7 (CHPh), 96.3 (C-1), 72.5

(C-a), 72.0 (C-3), 71.5 (C-4), 70.0 (C-2), 69.5 (C-6), 66.6 (C-5), 61.5 (C-b),

26.0, 24.9 (C(CH₃)₂). MALDI-MS (CHCA, THF, pos. mode) Calcd for

(C₁₈H₂₄O₇þ Na)^þ375.1. Found 375.0. Calcd for (C₁₈H₂₄O₇þ K)^þ391.1.

Found 391.0.

Methyl 4,6-O-isopropylidene-3-O-(2⁰-

triﬂuoromethanesulfonyloxyethyl)-b-^D-galacto-

pyranoside (11)

9 (50 mg, 0.18 mmol) was dissolved in dry DCM (2 mL) and dry pyridine

(29 mL, 0.36 mmol) was added. The solution was cooled to 2308C and triﬂic

anhydride (39 mL, 0.23 mmol, in 500 mL of dry DCM) was added dropwise

under argon. The reaction was complete after 1 h. The organic phase was

washed with saturated NaHCO₃²solution, 1 M KH₂PO²₄solution, and NaCl²

solution and dried over MgSO₄. The solvents were evaporated and the yellow

syrup (30 mg, 43%) was used in the next step without further puriﬁcation.

TLC (EA:MeOH 5:1): R_f¼ 0.6.

4,6-O-Benzylidene-1,2-O-isopropylidene-3-O-(2⁰-

triﬂuoromethanesulfonyloxyethyl)-a-^D-

galactopyranoside (12)

To a solution of 10 (40 mg, 0.11 mmol) in dry DCM (3 mL) was added dry

2,6-lutidine (18 mL, 0.22 mmol). The solution was cooled to 2308C and triﬂic

H. Busse et al.

174

anhydride (24 mL, 0.12 mmol in 500 mL of dry DCM) was added dropwise under

argon. The reaction was complete after 30 min. The organic phase was washed

with saturated NaHCO²₃solution, 1M KH₂PO₄²solution, and NaCl²solution

and dried over MgSO₄. The solvents were evaporated in vacuo and the

residue was coevaporated three times with dry toluene. The orange residue

(30 mg, 62%) was used in the next step without further puriﬁcation. TLC

(EtOAc:Tol 5:1): R_f¼ 0.83.

Methyl 2-O-benzoyl-4,6-O-benzylidene-3-O-

triﬂuoromethanesulfonyl-b-^D-gulopyranoside (13)

Gulose 50 (50 mg, 0.13 mmol) was dissolved in dry DCM (1 mL), and dry

pyridine (83 mL, 1.0 mmol) was added. The reaction mixture was cooled to

08C and triﬂic anhydride (43 mL, 0.26 mmol) was added dropwise via canula.

The reaction was allowed to warm up to rt and stirred for further 6 h. The

reaction mixture was washed with sat. NaHCO²₃solution and 1 M KH₂PO²₄

solution, the organic phase was dried over MgSO₄, and the solvents were evap-

orated. The crude product was puriﬁed by ﬁltration over a short column with

DCM as eluent to give 13 (70 mg, qu) as a yellow syrup. TLC (EtOAc:Tol 1:2):

1

R_f¼ 0.75; H NMR (250 MHz, CDCl₃) d 8.10–7.00 (m, 10H, C₆H₅), 5.6 (s, 1H,

CHPh), 5.52 (m, 1H, H-3), 5.42 (pt, 1H, H-2), 4.97 (d, 1H, J_1–2¼ 8.4 Hz, H-1),

0

4.45 (dd, 1H, J_5–6¼ 1.3, J_6–6¼ 12.6 Hz, H-6), 4.28 (bs, 1H, H-4), 4.15 (dd,

1H, J_5–6¼ 1.7 Hz, H6⁰), 3.90 (bs, 1H, H-5), 3.53 (s, 3H, OMe).

0

1,2-O-Isopropylidene-5-O-tert-butyldimethylsilyl-a-L-

xylofuranose (14) and 1,2-O-isopropylidene-5-O-tert-

butyldimethylsilyl-a-^L-ribofuranose (15)

Compounds 14 and 15 were synthesized as described by others (for

D-xylose) and us previously.^[18–22]

3-O-Benzyl-1,2-O-isopropylidene-5-O-tert-butyldimethylsilyl-

a-^L-ribofuranose (16)

L-Ribose 15 (15.0 g, 49.3 mmol) and BnBr (8.8 mL, 74 mmol) were dissolved

in dry DMF (280 mL). The solution was cooled to 08C, NaH (1.77 g, 73.9 mmol)

was added in portions under argon, and the temperature was allowed to rise to

rt overnight under stirring. The reaction was quenched with MeOH (5 mL), the

solvent was removed in vacuo, and the residue was dissolved in EtOAc, washed

with water, dried over MgSO₄, and evaporated in vacuo. The crude product was

puriﬁed by ﬂash chromatography (Tol:EtOAc 20:1) to yield 16 (16.5 g, 85%) as a

colorless syrup. TLC (EtOAc:Tol 1:10): R_f¼ 0.5; [a]²_D⁰¼ 255.7 (c ¼ 1, CHCl₃);

¹H NMR (600 MHz, CDCl₃) d 7.38–7.25 (m, 5H, C₆H₅), 5.68 (d, 1H, J_1–

Galactose-Phosphonates

175

¼ 3.6 Hz, H-1), 4.74, 4.60 (d, 2H, J ¼ 12.6 Hz, CH₂Ph), 4.53 (pt, 1H, H-2), 4.06

2

0

(m, 1H, H-4), 3.89–3.87 (m, 2H, H-3, H-5), 3.72 (dd, 1H, J_4–5¼ 3.0,

J_5–5¼ 12 Hz, H-5⁰), 1.57, 1.32 (2s, 6H, C(CH₃)₂), 0.88 (s, 9H, C(CH₃)₃),

0

0.04 (s, 6H, Si(CH₃)₂); ¹³C NMR (151 MHz, CDCl₃) d 137.8 (C-Ph), 128.5–

127.5 (C₆H₅), 112.8 (C(CH₃)₂), 103.9 (C-1), 79.4 (C-4), 77.7 (C-2), 76.4 (C-

3), 72.5 (CH₂Ph), 61.1 (C-5), 26.9, 26.6 ((CH₃)₂), 25.9 (C(CH₃)₃), 18.3

(C(CH₃)₃), 25.0 (Si(CH₃)₂). MALDI-MS (CHCA, THF, pos. mode) Calcd for

(C₂₁H₃₄O₅Si þ Na)^þ417.2. Found 417.8. Calcd for (C₂₁H₃₄O₅Si þ K)^þ433.2.

Found 433.7.

3-O-Benzyl-1,2-O-isopropylidene-a-L-ribofuranose (17)

16 (16.5 g, 42.0 mmol) was dissolved in dry THF (150 mL), the solution was

cooled to 08C, and TBAF-solution (1M, 46.5 mL in dry THF) was added. The

reaction was stirred at rt for 2 h, the solvent was removed in vacuo, and the

residue was puriﬁed by ﬂash chromatography (Tol:EtOAc 2:1) to afford 17

(10.0 g, 85%) as a colorless syrup. TLC (EtOAc:Tol 1:10): R_f¼ 0.25;

[a]²_D⁰¼ 282.7 (c ¼ 1, CHCl₃); H NMR (600 MHz, CDCl₃) d 7.39–7.26 (m, 5H,

1

C₆H₅), 5.73 (d, 1H, J_1–2¼ 3.6 Hz, H-1), 4.75 (d, 1H, J ¼ 12.0 Hz, CH₂Ph),

4.59 (d, 1H, J ¼ 12.0 Hz, CH₂Ph), 4.57 (dd, 1H, J_2–3¼ 8.4 Hz, H-2), 4.12

0

(ddd, 1H, J_3–4¼ 4.2, J_4–5¼ 2.4 Hz, J_4–5¼ 9.0 Hz, H-4), 3.92 (dd, 1H,

J_4–5¼ 2.4 Hz, J_5–5¼ 12.0 Hz, H-5), 3.84 (dd, 1H, H-3), 3.63 (dd, 1H, H-5⁰),

0

1.59, 1.34 (2s, 6H, C(CH₃)₂); ¹³C NMR (151 MHz, CDCl₃) d 137.5 (C-Ph),

128.5–127.9 (C₆H₅), 113.1 (C(CH₃)₂), 104.0 (C-1), 78.8 (C-4), 77.7 (C-2), 76.5

(C-3), 72.3 (CH₂Ph), 60.6 (C-5), 26.8, 26.5 ((CH₃)₂). MALDI-MS (CHCA, THF,

pos. mode) Calcd for (C₁₅H₂₀O₅þ Na)^þ303.1. Found 303.2. Calcd for

(C₁₅H₂₀O₅þ K)^þ319.1. Found 319.2.

Tetramethyl [3-O-benzyl-1,2-O-isopropylidene-

3,5,6-trideoxy-a-L-ribofuranos-6,6⁰-diyl]

bisphosphonate (18)

17 (2.00 g, 7.14 mmol) was dissolved in dry DCM (24 mL), dry 2,6-lutidine

(1.65 mL, 14.2 mmol) was added, and the solution was cooled to 2308C. Triﬂic

anhydride (1.44 mL, 8.56 mmol) in dry DCM (12 mL) was added via canula and

the mixture was stirred at this temperature for 2 h. The mixture was extracted

with saturated NaHCO²₃solution and KH₂PO₄²solution and dried over MgSO₄.

The organic phases were evaporated in vacuo and the triﬂate was puriﬁed

by column ﬁltration with DCM. This afforded the corresponding triﬂate

as an orange syrup (2.8 g, 95%). Tetramethyl methylenediphosphonate^[32]

(1.62 mL, 86.9 mmol) was dissolved in 50 mL of dry DMF under argon, and

NaH (163 mg, 6.79 mmol) and 125 mL crown ether (15-crown-5) were added

at 08C.

H. Busse et al.

After stirring for 30 min the freshly prepared triﬂate of 17 (1.80 g,

176

4.36 mmol) in dry DMF (50 mL) was added to the solution via a syringe.

The mixture was stirred in the melting ice bath for 4 h and the reaction was

quenched by addition of solid NH₄Cl. Following removal of DMF in vacuo, the

residue was dissolved in DCM and washed several times with water. The

combined organic phases were dried over MgSO₄and evaporated.

The residue was puriﬁed by ﬂash chromatography (EtOAc:Tol

10:1 ! MeOH:EtOAc 1:20) to give 18 (1.20 g, 56%) as a colorless syrup. TLC

(EtOAc:Tol 10:1): R_f¼ 0.3; [a]²_D⁰¼ 224.3 (c ¼ 1, CHCl₃); H NMR (600 MHz,

1

CDCl₃) d 7.36–7.25 (m, 5H, C₆H₅), 5.68 (d, 1H, J_1–2¼ 3.7 Hz, H-1), 4.75

(d, 2H, J ¼ 11.94 Hz, CH₂Ph), 4.53 (dd, 1H, J_2–3¼ 8.8 Hz, H-2), 4.29 (ddd,

0

1H, J_3–4¼ 4.2, J_4–5¼ 9.2, J_4–5¼ 12.9 Hz, H-4), 3.79 (12H, 4 OCH₃), 3.40

0

(dd, 1H, H-3), 2.83 (dd, 1H, J_{5 -6}¼ 3.8, J_5–6¼ 8.8 Hz, H-6), 2.29 (ddd, 1H, J_5–

¼ 14.3 Hz, H-5), 2.03 (m, 1H, H-5⁰), 1.57, 1.36 (2s, 6H, C(CH₃)₂); ¹³C NMR

5⁰

(151 MHz, CDCl₃) d 137.3 (C-Ph), 128.5–127.9 (C₆H₅), 103.7 (C-1), 82.6 (C-3),

78.0 (C-2), 75.4 (C-4), 72.1 (CH₂Ph), 53.4 (POOCH₃), 31.7 (C-6), 28.6 (C-5),

26.6 (C(CH₃)₂); ³¹P NMR (151 MHz, CDCl₃) d 27.86, 27.51 (2s, PO₃Me₂).

MALDI-MS (CHCA, THF, pos. mode) Calcd for (C₂₀H₃₂O₁₀P₂þ Na)^þ517.2.

Found 516.8. Calcd for (C₂₀H₃₂O₁₀P₂þ K)^þ533.2. Found 532.8.

Tetramethyl [3-O-benzyl-3,5,6-trideoxy-a,b-L-ribofuranos-

6,6⁰-diyl] bisphosphonate and Dimethyl (3R, 4S, 5S)-4-

benzyloxy-3,5-dihydroxy-1-cyclohexenephosphonate

(19)

Synthesis and Characterization of the Hemiacetal Intermediate

18 (530 mg, 1.07 mmol) was dissolved in 30 mL dioxane/5% H₂SO₄²

solution (1:1, 30 mL) and the mixture was stirred at 808C until monitoring

by TLC indicated the absence of starting material. After neutralization

with saturated NaHCO²₃solution, the mixture was extracted several

times with DCM. The organic phases were combined, dried over MgSO₄,

ﬁltered, evaporated, and puriﬁed by ﬂash chromatography (EtOAc:MeOH

10:1) to yield the hemiacetal (315 mg, 65%) as a colorless syrup. TLC

(EtOAc:MeOH 3:1): R_f¼ 0.4; ¹H NMR (600 MHz, CDCl₃, major anomer) d

7.37–7.33 (m, 5H, C₆H₅), 5.23 (d, 1H, J_1–2¼ 3.4 Hz, H-1), 4.65 (d, 2H,

J ¼ 14.1 Hz, CH₂Ph), 4.35 (m, 1H, H-4), 4.02 (m, 1H, H-2), 3.80 (m, 12H,

POOCH₃), 3.65 (m, 1H, H-3), 2.61 (m, 1H, H-6), 2.14 (m, 1H, H-5), 1.96

(m, 1H, H-5⁰); ¹³C NMR (151 MHz, CDCl₃) d 136.7 (C-Ph), 128.7–127.9

(C₆H₅), 96.7 (C-1), 82.9 (C-2), 81.0 (C-3), 77.2 (C-4), 72.8 (CH₂Ph), 53.2

(POOCH₃), 32.3 (C-6), 31.6 (C-5); ³¹P NMR (151 MHz, CDCl₃) d 28.9,

28.17, 27.69, 27.56 (4s, PO₃Me₂). MALDI-MS (CHCA, THF, pos. mode)

Galactose-Phosphonates

177

Calcd for (C₁₇H₂₈O₁₀P₂þ Na)^þ477.1. Found 477.0. Calcd for

(C₁₇H₂₈O₁₀P₂þ K)^þ493.1. Found 493.0.

Cyclization of the Hemiacetal

To a solution of the hemiacetal (500 mg, 1.10 mmol) in dry dioxane (20 mL)

was added lithium bis(trimethylsilyl)amide (1M in dry THF, 1.65 mL,

1.65 mmol) at 08C. The mixture was stirred for 4 h and then allowed to warm

up to rt. The reaction mixture was neutralized with Dowex 50- H^þ, ﬁltered,

and evaporated in vacuo. Puriﬁcation by ﬂash chromatography (EtOAc:MeOH

10:1) furnished 19 (207 mg, 62% from the hemiacetal) as a colorless syrup. TLC

(EtOAc:MeOH 3:1): R_f¼ 0.6; [a]²_D⁰¼ 277.0 (c ¼ 1, CHCl₃); ¹H NMR (600 MHz,

CDCl₃) d 7.36–7.32 (m, 5H, C₆H₅), 6.67 (d, 1H, J_2-P¼ 21.4 Hz, H-2), 4.80

(d, 2H, J ¼ 11.9 Hz, CH₂-Ph), 4.34 (bs, 1H, H-3), 4.13 (bs, 1H, H-5), 3.73–

3.71 (m, 7H, H-4, POOCH₃), 2.50 (m, 1H, H-6), 2.40 (m, 1H, H-6⁰); ¹³C NMR

(151 MHz, CDCl₃) d 137.8 (C-2), 128.6–128.1 (C₆H₅), 76.9 (C-4), 72.6

(CH₂Ph), 68.5 (C-5), 66.4 (C-3), 52.6 (POOCH₃), 31.3 (C-6); ³¹P NMR

(151 MHz, CDCl₃) d 22.26 (s, PO₃Me₂). MALDI-MS (CHCA, THF, pos. mode)

Calcd for (C₁₅H₂₁O₆P þ Na)^þ351.1. Found 351.5. Calcd for (C₁₅H₂₁O₆P þ K)^þ

367.1. Found 367.6.

Dimethyl (3R,4S,5S)-4-benzyloxy-3,5-diacetoxy-1-

cyclohexenephosphonate (20)

19 (45 mg, 0.14 mmol) was dissolved in Ac₂O/pyridine (1:9, 8 mL) and

stirred at rt overnight. The solvents were evaporated and the syrup was

puriﬁed by ﬂash chromatography (Tol:EtOAc 1:10) to give 20 (48 mg, 90%).

¼ 232.0 (c ¼ 0.5, CHCl₃); ¹H NMR

20

TLC (Tol:EtOAc 1:10): R_f¼ 0.5; [a]_D

(600 MHz, CDCl₃) d 7.33–7.27 (m, 5H, C₆H₅), 6.46 (d,1H, J_2-P¼ 21.0 Hz,

H-2), 5.52 (bs, 1H, H-3), 5.05 (bdd, 1H, J ¼ 7.8 Hz, H-5), 4.71 (d, 2H,

J ¼ 12.0 Hz, CH₂-Ph), 4.10 (bs, 1H, H-4), 3.73–3.69 (m, 6H, POOCH₃), 2.65

(m, 2H, H-6, H-6⁰), 2.07, 2.00 (2s, 6H, OAc); ¹³C NMR (151 MHz, CDCl₃) d

170.0 (COCH₃), 138.8 (C-2), 128.3-127.0 (C₆H₅), 74.1 (CH₂Ph), 73.8 (C-4),

70.5 (C-3), 69.8 (C-5), 52.6 (POOCH₃), 26.7 (C-6), 20.9 (COCH₃); ³¹P NMR

(151 MHz, CDCl₃) d 20.95 (s, PO₃Me₂). MALDI-MS (CHCA, THF, pos. mode)

Calcd for (C₁₉H₂₅O₈P þ Na)^þ435.1. Found 435.5. Calcd for (C₁₉H₂₅O₈P þ K)^þ

451.1. Found 451.5.

Dimethyl (3R,4S,5S)-4-benzyloxy-3,5-di(methoxy-

methyloxy)-1-cyclohexenephosphonate (21)

19 (60 mg, 0.18 mmol) was dissolved in 40 mL of a mixture of DCM/

dimethoxymethane (1:1) and 30 mL of triﬂic acid were added under argon.

H. Busse et al.

178

The mixture was stirred at rt for 4 h. The reaction was then washed with satu-

rated NaHCO²₃solution, and the organic phase was dried over MgSO₄and

evaporated in vacuo. Puriﬁcation by MPLC (EtOAc:MeOH 29:1) resulted in

21 as a colorless syrup (55 mg, 71%). TLC (EtOAc: MeOH 10:1): R_f¼ 0.48;

[a]²_D⁰¼ 232.5 (c ¼ 1, CHCl₃); ¹H NMR (600 MHz, CDCl₃) d 7.36–7.21

(m, 5H, C₆H₅), 6.53 (d, 1H, J_2-P¼ 21.6 Hz, H-2), 4.85 (d, 2H, CH₂Ph), 4.65

(dd, 4H, 2CH₂O), 4.34 (bs, 1H, H-3), 4.04 (bs, 1H, H-4), 3.82 (pt, 1H,

J ¼ 7.8 Hz, H-5), 3.68 (2d, 6H, J_P-OMe¼ 10.8 Hz, POOCH₃), 3.35 (2s, 6H,

CH₂OCH₃), 2.49 (m, 2H, H-6, H-6⁰); ¹³C NMR (151 MHz, CDCl₃) d 142 (C-2),

128.6–127.0 (C₆H₅), 95.5 (CH₂OCH₃), 74.5 (C-4), 73.9 (C-5, C-3), 73.4

(CH₂Ph), 55.8 (CH₃OCH₂), 52.7 (POOCH₃), 27.9 (C-6); ³¹P NMR (151 MHz,

CDCl₃) d 21.7 (s, PO₃Me₂). MALDI-MS (CHCA, THF, pos. mode) Calcd for

(C₁₉H₂₉O₈P þ Na)^þ439.2. Found 439.7. Calcd for (C₁₉H₂₉O₈P þ K)^þ455.2.

Found 455.8.

Dimethyl (3R,4S,5S)-4-hydroxy-3,5-di(methoxymethyloxy)-

1-cyclohexenephosphonate (22)

To 21 (65 mg, 0.16 mmol) in EtOH (5 mL) was added Pearlman’s catalyst

(60 mg) and 1,4-cyclohexadiene (3 mL) and the mixture was stirred at 508C

for 4 days. The catalyst was removed by ﬁltration and the solvent was evapor-

ated. Puriﬁcation by column chromatography (EtOAc:MeOH 10:1) afforded 22

(33 mg, 65%) as a colorless syrup. TLC (EA:MeOH 10:1): R_f¼ 0.26;

[a]²_D⁰¼ 210.6 (c ¼ 1, CHCl₃); ¹H NMR (600 MHz, CDCl₃) d 6.50 (d, 1H,

J_2-P¼ 21.6 Hz, H-2), 4.78, 4.73 (dd, 4H, J_a-b¼ 10.2 Hz, CH₂O), 4.32 (m, 1H,

H-3), 4.23 (m, 1H, H-4), 3.82 (bt, 1H, H-5), 3.72 (2d, 6H, J_P-OMe¼ 10.8 Hz,

POOCH₃), 3.44, 3.40 (2s, 6H, CH₂OCH₃), 2.47 (m, 2H, H-6, H-6⁰); ¹³C NMR

(151 MHz, CDCl₃) d 140.0 (C-2), 95.3, 94.8 (CH₂OCH₃), 73.4 (C-3), 72.9 (C-5),

67.6 (C-4), 55.8, 55.4 (CH₃OCH₂), 52.2 (POOCH₃), 26.4 (C-6); ³¹P NMR

(151 MHz, CDCl₃) d 20.7 (s, PO₃Me₂). MALDI-MS (CHCA, THF, pos. mode)

Calcd for (C₁₂H₂₃O₈P þ Na)^þ349.1. Found 349.9. Calcd for (C₁₂H₂₃O₈P þ K)^þ

365.1. Found 365.8.

Dimethyl (3R,4R,5S)-4-azido-3,5-di(methoxymethyloxy)-1-

cyclohexenephosphonate (23)

22 (30 mg, 0.092 mmol) was dissolved in dry DCM (500 mL), dry pyridine

(34 mL, 0.42 mmol) was added, and the solution was cooled to 2308C. Triﬂic

anhydride (30 mL, 0.18 mmol) in dry DCM (300 mL) was added and the

mixture was stirred at 2108C for 2 h. The mixture was washed with saturated

NaHCO²₃solution and KH₂PO₄²solution and dried over MgSO₄. The organic

phases were dried in vacuo and the triﬂate was obtained as an orange syrup

Galactose-Phosphonates

179

(40 mg, 95%), which was used without further puriﬁcation. The triﬂate (30 mg,

0.066 mmol) was dissolved in dry DMF (2 mL), NaN₃(22 mg, 0.33 mmol)

t

and BuNH₄Cl (3 mg) were added, and the mixture was stirred at 508C

overnight. DMF was removed in vacuo and the resulting oil was puriﬁed by

ﬂash chromatography (EtOAc), which afforded 23 (20 mg, 86%) of as a

colorless syrup. TLC (EtOAc): R_f¼ 0.3; [a]²_D⁰¼ 238.6 (c ¼ 1, CHCl₃); ¹H

NMR (600 MHz, CDCl₃) d 6.52 (d, 1H, J_2-P¼ 21.6 Hz, H-2), 4.78, 4.76 (dd,

4H, CH₂O), 4.08 (bd, 1H, H-3), 3.71 (2d, 6H, J_P-OMe¼ 10.8 Hz, POOCH₃),

3.70 (1H, H-5 hidden under MOM), 3.56 (1H, H-4), 3.42, 3.40 (2s, 6H,

CH₂OCH₃), 2.74 (m, 1H, H-6), 2.22 (m, 1H, H-6⁰); ¹³C NMR (151 MHz,

CDCl₃) d 140.8 (C-2), 96.3, 95.9 (CH₂OCH₃), 76.1 (C-3), 74.2 (C-5), 66.9 (C-4),

55.7 (CH₃OCH₂), 52.7 (POOCH₃), 31.2 (C-6); ³¹P NMR (151 MHz, CDCl₃) d

19.9 (s, PO₃Me₂). MALDI-MS (CHCA, THF, pos. mode) Calcd for

(C₁₂H₂₂N₃O₇P þ Na)^þ374.1. Found 374.1. Calcd for (C₁₂H₂₂N₃O₇P þ K)^þ

390.1. Found 390.1.

Dimethyl (3R,4R,5S)-4-azido-3,5-dihydroxy-1-

cyclohexenephosphonate (24)

23 (36 mg, 0.10 mmol) was dissolved in TFA (5 mL, 50%) and stirred at rt

overnight. The solution was lyophilized and puriﬁed by ﬂash chromatography

(EtOAc:MeOH 7:1) to afford 24 (30 mg, qu) as a colorless syrup. TLC (EtOAc:

MeOH 5:1): R_f¼ 0.45; [a]_D²⁰¼ 214.6 (c ¼ 1, CHCl₃); ¹H NMR (600 MHz,

CDCl₃) d 6.36 (d, 1H, J_2-P¼ 22.2 Hz, H-2), 4.16 (m, 1H, H-3), 3.76 (ddd, 1H,

0

J_5–6¼ 5.4, J_4–5¼ 10.2 Hz, J_5–6¼ 15.2 Hz, H-5), 3.64–3.60 (2d, 6H,

J_P-OMe¼ 11.4 Hz, POOCH₃), 3.35 (dd, 1H, J_3–4¼ 9.0 Hz, H-4), 2.57 (m, 1H,

H-6), 2.16 (m, 1H, H-6⁰); ¹³C NMR (151 MHz, CDCl₃) d 144.0 (C-2), 70.6 (C-

3), 69.1 (C-4), 68.1 (C-5), 53.1 (POOCH₃), 32.2 (C-6); ³¹P NMR (151 MHz,

CDCl₃) d 22.7 (s, PO₃Me₂). MALDI-MS (CHCA, THF, pos. mode) Calcd for

(C₈H₁₄N₃O₅P þ Na)^þ286.1. Found 285.7.

Dimethyl (3R,4R,5S)-4-acetamido-3,5-diacetoxy-1-

cyclohexenephosphonate (25)

24 (30 mg, 0.11 mmol) was dissolved in dry THF (1 mL) and added to

a solution of Ac₂O (43 mL, 0.46 mmol) of PMe₃in THF (1M, 680 mL,

0.68 mmol) under argon. The mixture was stirred at rt overnight. The

solution was dried in vacuo and puriﬁed by ﬂash chromatography (EtOAc:Tol

10:1 ! EtOAc:MeOH 5:1) to yield 25 (21 mg, 58%) as a yellow syrup. TLC

(EtOAc:MeOH 10:1): R_f¼ 0.26; [a]²_D⁰¼ 244.8 (c ¼ 1, CHCl₃); ¹H NMR

(600 MHz, CDCl₃) d 6.47 (d, 1H, J_2-P¼ 19.2 Hz, H-2), 5.55 (m, 1H, H-3),

0

5.10 (ddd, 1H, J_4–5¼ 9.0, J_5–6¼ 5.4, J_5–6¼ 10.8 Hz, H-5), 4.25 (dd, 1H,

H. Busse et al.

180

J_3–4¼ 10.8 Hz, H-4), 3.76 (2d, 6H, J_P-OMe¼ 11.4 Hz, POOCH₃), 2.75 (m, 1H,

H-6), 2.34 (m, 1H, H-6⁰), 2.05, 2.03 (2s, 6H, OAc), 1.91 (s, 3H, NHAc); ¹³C

NMR (151 MHz, CDCl₃) d 173.0 (NHCOCH₃), 171.8 (COCH₃), 141.0 (C-2),

72.4 (C-3), 69.6 (C-5), 53.6 (C-4), 53.5 (POOCH₃), 30.8 (C-6), 22.7

(NHCOCH₃), 20.7, 20.6 (COCH₃); ³¹P NMR (151 MHz, CDCl₃) d 19.76

(s, PO₃Me₂). MALDI-MS (CHCA, THF, pos. mode) Calcd for

(C₁₄H₂₂NO₈P þ Na)^þ386.1. Found 385.6. Calcd for (C₁₄H₂₂NO₈P þ K)^þ

402.1. Found 401.6.

Triethylammonium [methyl (3R,4R,5S)-4-acetamido-3,5-

diacetoxy-1-cyclohexenephosphonate] (26)

25 (24 mg, 0.066 mmol) was dissolved in dry THF (750 mL), NEt₃(129 mL,

0.93 mmol) and thiophenol (47 mL, 0.46 mmol) were added, and the mixture

was stirred at rt for 48 h. The solvent was evaporated in vacuo and the

residue was puriﬁed by ﬂash chromatography (EtOAc:MEOH 10:1 ! 4:1þ

1% NEt₃) to give triethylammonium salt 26 (29 mg, qu) in quantitative yield.

TLC (EtOAc:MeOH 5:1): R_f¼ 0.1; [a]²_D⁰¼ 23.2 (c ¼ 0.5, MeOH); ¹H NMR

(600 MHz, CD₃OD) d 6.21 (d, 1H, J_2-P¼ 18.6 Hz, H-2), 5.53 (pd, 1H, H-3),

5.08 (ddd, 1H, J_4–5¼ 9.0, J_5–6¼ 5.4 Hz, H-5), 4.21 (dd, 1H J_3–4¼10.8 Hz,

H-4), 3.48 (1d, 3H, J_P-OMe¼ 10.8 Hz; POOCH₃), 3.18 (q, 6H, CH₂CH₃), 2.75

(m, 1H, H-6), 2.36 (m, 1H, H-6⁰), 2.04, 2.02 (2s, 6H, OAc), 1.90 (s, 3H, NHAc),

1.29 (t, 9H, CH₂CH₃); ¹³C NMR (151 MHz, CD₃OD)

d 173, 173.6

(NHCOCH₃), 172.0 (COCH₃), 171.9 (COCH₃), 133.0 (C-2), 72.1 (C-3), 69.4 (C-

5), 52.7 (C-4), 50.6 (POOCH₃), 46.1 (N(CH₂CH₃)₃), 30.6 (C-6), 22.7–20.7

(COCH₃), 9.2 (N(CH₂CH₃)₃); ³¹P NMR (151 MHz, CD₃OD)

d

13.2

(s, PO₃Me²). MALDI-MS (CHCA, THF, pos. mode) Calcd for

(C₁₃H₂₀NO₈P þ Na)^þ372.1. Found 372.3. Calcd for (C₁₃H₂₀NO₈P þ K)^þ

388.3. Found 388.3.

Methyl [(3R,4R,5S)-4-acetamido-3,5-diacetoxy-1-

cyclohexene] phosphonic acid (27)

26 (15 mg, 0.033 mmol) was dissolved in dioxane/water (1:1, 2 mL). Amber-

lite-IR-120 (H^þ) was added until pH 2, the resin was removed by ﬁltration, and

the product was lyophilized from dioxane to give 27 (12 mg, qu). [a]²_D⁰¼ 28.34

1

(c ¼ 0.5, MeOH); H NMR (600 MHz, CD₃OD) d 6.38 (d, 1H, J_2-P¼ 49.8 Hz,

H-2), 5.52 (m, 1H, H-3), 5.08 (m, 1H, H-5), 4.23 (m, 1H, H-4), 3.65 (m, 3H;

POOCH₃), 2.78 (m, 1H, H-6), 2.35 (m, 1H, H-6⁰), 2.05, 2.02 (2s, 6H, OAc),

1.92 (s, 3H, NHAc).

Galactose-Phosphonates

181

O-Methyl-O-[4,6-O-benzylidene-3-O-(1⁰-ethyl)-1,2-O-

0

´

isopropylidene-a-^D-galactopyranoside-2 -yl](3R,4R,5S)-

4-acetamido-3,5-diacetoxy-1-cyclohexene-

phosphonate (28) (h,l)

To a solution of acid 27 (15 mg, 0.043 mmol) in dry THF (1 mL) was added

galactose derivative 10 (30 mg, 0.086 mmol), Ph₃P (23 mg, 0.086 mmol), and

DIAD (17 mL, 0.086 mmol). The reaction was stirred at 608C for 48 h. The

solvent was removed in vacuo and the residue was puriﬁed by ﬂash chromato-

graphy (EtOAc:Tol 10:1 ! EtOAc:MeOH 10:1) to afford a mixture of diastereo-

mers 28 (h,l) (15 mg, 51%) as a colorless syrup. No attempt to separate them

was made. TLC (EtOAc:MeOH 10:1): R_f¼ 0.30–0.35; ¹H NMR (600 MHz,

CDCl₃) d 7.51–7.25 (m, 5H, C₆H₅), 6.44 (m, 1H, H-2a), 5.74 (s, 1H, CHPh),

5.61 (d, 1H, J_1–2¼ 5.4 Hz, H-1b), 5.50 (m, 1H, H-3a), 5.05 (m, 1H, H-5a),

4.63 (dd, 1H, J_2–3¼ 2.4 Hz, J_3–4¼ 8.4 Hz, H-3b), 4.41 (dd, 1H, H-2b), 4.36

(dd, 1H, J_3–4¼ 9.0 Hz, H-4a), 4.31 (dd, 1H, J_4–5¼ 1.8 Hz, H-4b), 4.20–4.06

(m, 4H, H-5b, 3H from ethyleneglycol), 3.74–3.60 (m, 6 H, POOMe, H-6⁰b,

H-6b, 1H from ethyleneglycol), 2.70 (m, 1H, H-6a), 2.43 (m, 1H, H-6a⁰), 2.04,

2.02 (2s, 6H, OAc), 1.90 (s, 3H, NHAc), 1.55, 1.33 (2s, 6H, C(CH₃)₂); ¹³C

NMR (151 MHz, CDCl₃) d 171.1 (NHCOCH₃), 171.0 (COCH₃), 170.1

(COCH₃), 139.1 (C-2a), 136.2 (C-Ph), 128.8–126.3 (C₆H₅), 110.0 (C(CH₃)₂),

103.7 (CHPh), 96.3 (C-1b), 71.8 (C-3b), 71.5 (C-4b, C-3a), 70.0 (C-6b), 69.9 (C-

2b), 68.6 (C-5a), 66.8 (C-5b), 65.3 (CH₂O), 53.0 (C-4a), 52.5 (POOCH₃), 30.4

(C-6a), 26.0, 24.9 (C(CH₃)₂), 23.3–20.8 (COCH₃); ³¹P NMR (151 MHz, CDCl₃)

d 18.3, 18.0 (2s, PO₃Me₂). MALDI-MS (DHB, THF, pos. mode) Calcd for

(C₃₁H₄₂NO₁₄P þ Na)^þ706.2. Found 706.4. Calcd for (C₃₁H₄₂NO₁₄P þ K)^þ

722.2. Found 722.5.

Triethylammonium [4,6-O-benzylidene-3-O-(1⁰-ethyl)-1,2-O-

isopropylidene-a-^D-galactopyranoside-2⁰-yl] (3R,4R,5S)-

4-acetamido-3,5-diacetoxy-1-cyclohexene-

phosphonate (29)

28 (h,l) (15 mg, 0.022 mmol) was dissolved in dry THF (500 mL). NEt₃

(42 mL, 0.31 mmol) and thiophenol (16 mL, 0.15 mmol) were added and the

reaction was stirred at rt for 2 days. The solvent was removed and the following

ﬂash chromatography (EtOAc:MeOH 10:1 ! 5:1, 1% NEt₃) yielded 29 (9 mg,

60%) as a colorless solid. Tlc (EtOAc:MeOH 5:1): R_f¼ 0.1; ¹H NMR

(600 MHz, CD₃OD) d 7.51–7.35 (m, 5H, C₆H₅), 6.22 (d, 1H, J_2-P¼ 18.6 Hz,

H-2a), 5.74 (s,1H, CHPh), 5.57 (d, 1H, J_1–2¼ 5.4 Hz, H-1b), 5.49 (pd, 1H,

J_3–4¼ 8.4 Hz, H-3a), 5.06 (dd, 1H, J_4–5¼ 9.6, J_5–6¼ 5.4 Hz, H-5a), 4.65 (dd,

1H, J_2–3¼ 2.4, J_3–4¼ 8.4 Hz, H-3b), 4.45 (dd, 1H, H-2b), 4.33 (dd, 1H,

J_4–5¼ 1.8 Hz, H-4b), 4.22 (dd, 1H, J_3–4¼ 8.4 Hz, H-4a), 4.08–4.06 (m, 1H,

H. Busse et al.

182

H-5b), 3.90–3.80 (m, 2H, from chain), 3.75–3.60 (m, 4H, 2H from chain, H-6b,

H-6b⁰), 3.15 (q, 6H, CH₂CH₃), 2.83 (m, 1H, H-6a), 2.42 (m, 1H, H-6a⁰), 2.08, 2.00

(2s, 6H, OAc), 1.89 (s, 3H, NHAc), 1.53, 1.34 (2s, 6H, C(CH₃)₂), 1.29 (t, 9H,

CH₂CH₃); ¹³C NMR (151 MHz, CD₃OD) d 170.6 (NHCOCH₃), 170.5 (COCH₃),

170.5 (COCH₃), 136.8 (C-2a), 133.1 (C-Ph), 129.3–127.5 (C₆H₅), 109.0

(C(CH₃)₂), 104.7 (CHPh), 97.6 (C-1b), 73.0 (C-3a, C-3b), 72.8 (C-4b), 72.1 (C-

6b), 71.5 (C-2b), 70.7 (C-5a), 68.0 (C-5b), 64.6 (CH₂O), 53.9 (C-4a), 47.1

(N(CH₂CH₃)₃), 32.0 (C-6a), 25.0, 23.7 (C(CH₃)₂), 21.3-19.5 (COCH₃), 19.4

(N(CH₂CH₃)₃); ³¹P NMR (151 MHz, CD₃OD) d 11.9 (s, PO₃R²). MALDI-MS

(CHCA, THF, pos. mode) Calcd for (C₃₀H₄₀NO₁₄P þ Na)^þ692.2. Found 692.5.

Calcd for (C₃₀H₄₀NO₁₄P þ K)^þ708.2. Found 708.5.

Ammonium [4,6-O-benzylidene-3-O-(1⁰-ethyl)-1,2-O-

isopropylidene-a-^D-galactopyranoside-2⁰-yl] (3R,4R,5S)-

4-acetamido-3,5-dihydroxy-1-

cyclohexenephosphonate (30)

29 (8 mg, 0.01 mmol) was dissolved in water/dioxane (1:1, 1 mL) and 8

drops of 25% NH₃aq solution were added. The reaction was stirred at rt for

4 days. The solvents were evaporated and the crude product was lyophilized

1

from water to afford 30 (6 mg, qu) as a colorless solid. H NMR (600 MHz,

CD₃OD) d 7.51–7.35 (m, 5H, C₆H₅), 6.28 (d, 1H, J_2-P¼ 13.2 Hz, H-2a), 5.73

(s, 1H, CHPh), 5.59 (d, 1H, J_1–2¼ 4.8 Hz, H-1b), 4.65 (dd, 1H, J_2–3¼ 2.4,

J_3–4¼ 8.4 Hz, H-3b), 4.45 (dd, 1H, H-2b), 4.34 (dd, 1H, J_4–5¼ 1.8 Hz, H-4b),

4.10–4.00 (m, 2H, H-5b, H-3a), 3.90–3.85 (m, 2H from chain), 3.78–3.60

(m, 6H, 2H from chain, H-4a, H-5a, H-6b, H-6b⁰), 2.72 (m, 1H, H-6a), 2.25

(m, 1H, H-6a⁰), 2.08 (1s, 3H, NHAc), 1.53, 1.34 (2s, 6H, C(CH₃)₂); ¹³C NMR

(151 MHz, CD₃OD) d 174.5 (NHCOCH₃), 138.2 (C-2a), 130.7–127.5 (C₆H₅),

110.0 (C(CH₃)₂), 104.5 (CHPh), 97.4 (C-1b), 72.9 (C-3b), 72.5 (C-4b), 72.4 (C-

6b), 71.9 (CH₂O), 71.2 (C-2b), 70.8 (C-5b), 69.4 (C-5a), 67.8 (C-3a), 64.3

(CH₂O), 59.9 (C-4a), 35.1 (C-6a), 26.3-25.1 (C(CH₃)₂), 23.2 (COCH₃); ³¹P

NMR (151 MHz, CD₃OD) d 13.9 (s, PO₃R²). MALDI-MS (CHCA, THF, pos.

mode) Calcd for (C₂₆H₃₆NO₁₂P þ Na)^þ608.2. Found 608.3. Calcd for

(C₂₆H₃₆NO₁₂P þ K)^þ624.2. Found 624.4.

Ammonium [3-O-(1⁰-ethyl)-a,b -D-galactopyranoside-2⁰-

yl](3R,4R,5S)-4-acetamido-3,5-dihydroxy-1-

cyclohexenephosphonate (31)

30 (5 mg, 9 mmol) was dissolved in TFA (50%, 1 mL) and stirred overnight.

The solvent was evaporated and the residue was lyophilized from water to

afford 31 (3 mg, qu) as a colorless solid. ¹H NMR (600 MHz, D₂O) d 6.16

Galactose-Phosphonates

183

(d, 2H, J_2-P¼ 19.8 Hz, H-2a), 5.14 (d, 0.8H, J_1–2¼ 4.2 Hz, H-1ba), 4.47 (dd, 1.3

H, J_1–2¼ 7.8 Hz, H-1bb), 4.20–4.10 (m, 3H, H-3a both anomers), 3.86–3.59

(m, 25 H, H-4a, H-5a, H-6b, H-6b⁰both anomers, CH₂CH₂O, H-3ba, H-2ba,

H-5b, H-4b both anomers), 3.53 (dd, 1.3 H, J_2–3¼ 9.6 Hz, J_3–4¼ 3.6 Hz,

H-3bb), 3.37 (dd, 1.3 H, H-2bb), 2.57 (m, 2H, H-6a), 2.14 (m, 2H, H-6a⁰), 1.95

(1s, 6H, NHAc); ¹³C NMR (151 MHz, D₂O, selected data) d 174.5 (NHCOCH₃),

138.1 (C-2a), 96.2 (C-1bb), 92.1 (C-1ba), 72.5 (C-3bb), 71.6 (C-2bb), 33.2 (C-6a),

22.2 (COCH₃); ³¹P NMR (162 MHz, D₂O) d 15.6 (s, PO₃R²). MALDI-MS

(CHCA, THF, pos. mode) Calcd for (C₁₆H₂₈NO₁₂P þ Na)^þ480.1. Found 480.2.

Calcd for (C₁₆H₂₈NO₁₂P þ K)^þ496.1. Found 496.2. [HR-ESI-MS] Calcd for

[M þ 2Na-H]^þ502.1072. Found 502.1060.

Ammonium [methyl (3R,4R,5S)-4-acetamido-3,5-dihydroxy-

1-cyclohexenephosphonate] (32)

27 (7 mg, 0.02 mmol) was dissolved in dioxane/water (1:1, 1 mL), 12 drops

of 25% NH₃aq solution were added, and the reaction was stirred at rt for 4

days. The solvents were removed in vacuo and the product was lyophilized

from water to afford 32 (4 mg, qu) as a white lyophylisate. [a]²_D⁰¼ 25.4

(c ¼ 0.5, H₂O); ¹H NMR (600 MHz, D₂O) d 6.13 (d, 1H, J_2-P¼ 19.8 Hz,

H-2), 4.15 (bs, 1H, H-3), 3.71 (m, 2H, H-5, H-4), 3.49 (1d, 3H, POOCH₃),

2.56 (m, 1H, H-6), 2.16 (m, 1H, H-6⁰), 1.95 (s, 3H, NHAc); ¹³C NMR

(151 MHz, D₂O) d 175.0 (NHCOCH₃), 137.5 (C-2), 70.8 (C-3), 67.6 (C-5),

58.0 (C-4), 51.4 (POOCH₃), 33.3 (C-6), 22.0 (COCH₃); ³¹P NMR (162 MHz,

D₂O) d 16.2 (s, PO₃Me²). MALDI-MS (DHB, THF, pos. mode) Calcd for

(C₉H₁₆NO₆P þ Na)^þ288.1. Found 288.2. Calcd for (C₉H₁₆NO₆P þ K)^þ304.1.

Found 304.3.

Triethylammonium O-[4,6-O-benzylidene-3-O-(1⁰-ethyl)-1,2-

O-isopropylidene-a-^D-galactopyranoside-2⁰-yl]

methylphosphonate (33)

Methanephosphonic acid (15 mg, 0.16 mmol) was dissolved in dry THF

(2 mL), and galactose 10 (82 mg, 0.23 mmol), Ph₃P (61 mg, 0.23 mmol), and

ﬁnally DIAD (46 mL, 0.23 mmol) were added under an argon atmosphere.

The mixture was stirred at 608C for 2 days. The solvent was removed in

vacuo and the crude product was puriﬁed by ﬂash chromatography

(EtOAc:Tol 10:1 ! EtOAc:MeOH 10:1 ! 4:1, 1% NEt₃) to afford 33 (37 mg,

42%) as a colorless syrup. TLC (EtOAc:Tol 10:1): R_f¼ 0.1; [a]²_D⁰¼ 240.3

(c ¼ 1, CH₃OH); ¹H NMR (600 MHz, CD₃OD) d 7.51–7.35 (m, 5H, C₆H₅),

5.72 (s, 1H, HPh;), 5.57 (d, 1H, J_1–2¼ 4.8 Hz, H-1), 4.66 (dd, 1H, J_2–3¼ 2.4,

J_3–4¼ 8.4 Hz, H-3), 4.46 (dd, 1H, H-2), 4.31 (dd, 1H, J_4–5¼ 1.8 Hz, H-4),

4.08 (m, 1H, H-5), 4.02–3.98 (m, 2H, CH₂CH₂O), 3.73 (dd, 1H, J_5–6¼ 5.4,

H. Busse et al.

184

J_6–6¼ 10.2 Hz, H-6), 3.65–3.60 (m, 3H, H-6⁰, CH₂CH₂O), 3.15 (q, 6H,

0

CH₂CH₃), 1.51 (s, 3H, C(CH₃)₂), 1.34 (s, 3H, C(CH₃)₂), 1.32⁰1.25 (m, 12H,

P-CH₃and CH₂CH₃); ¹³C NMR (151 MHz, CD₃OD) d 138.2 (C-Ph), 130.7–

127.5 (C₆H₅), 110.1 (C(CH₃)₂), 104.5 (CHPh), 97.5 (C-1), 72.9 (C-2), 72.5 (C-

4), 71.7 (C-a), 71.1 (C-3), 70.8 (C-6), 67.8 (C-5), 64.4 (C-b), 47.2 (N(CH₂CH₃),

26.3, 25.1 (C(CH₃)₂), 12.6 (N(CH₂CH₃)₃), 11.7 (PCH₃); ³¹P NMR (151 MHz,

CD₃OD) d 27.3 (s, PCH₃). MALDI-MS (CHCA, THF, pos. mode) Calcd for (C_20-

H₂₉O₉P þ Na þ H)^þ468.2. Found 468.8.

Ammonium O-[3-O-(1⁰-ethyl)-a,b-D-galactopyranoside-2⁰-yl]

methylphosphonate (34)

33 (10 mg, 0.018 mmol) was dissolved in TFA (50%, 2 mL) and stirred at rt

overnight. The reaction product was lyophilized, dissolved in 1M NH₄HCO₃²

1

buffer, and lyophilized again to afford 34 (7 mg, 93%) as a colorless solid. H

NMR (600 MHz, D₂O) d 5.14 (d, 1H, J_1–2¼ 3.6 Hz, H-1a), 4.45 (d, 2H,

J_1–2¼ 7.8 Hz, H-1b), 3.97–3.60 (m, 25 H, H-4, H-5,H-6, H-6⁰, CH₂CH₂O both

anomers and H-2 a, H-3 a), 3.51 (dd, 2H, J_2–3¼ 3.6 Hz, J_3–4¼ 9.0 Hz,

H-3b), 3.35 (dd, 2H, H-2b), 3.05 (q, 6H, N(CH₂CH₃)₃), 1.32–1.28 (2s, 6H,

PCH₃), 1.12 (t, 9H, N(CH₂CH₃)₃); ¹³C NMR (151 MHz, D₂O) d 96.5 (C-1 b),

92.2 (C-1 a), 72.7 (C-3), 71.8 (C-2), 46.8 (N(CH₂CH₃)₃), 11.9 (PMe), 11.5

(N(CH₂CH₃)₃) ppm, 4C unresolved; ³¹P NMR (162 MHz, D₂O) d 27.9

(s, PCH₃). [HR-ESI-MS] Calcd for [M þ 2Na-H]^þ347.0459. Found 347.0478.

Methyl 2,4,6-tri-O-acetyl-3-O-benzyl-b-D-galactopyranoside

(35)

Dibutyltin oxide (1.28 g, 5.1 mmol) was added to a solution of methyl b-D-

galactopyranoside (995 mg, 5.1 mmol) in dry methanol (20 mL). The solution

was stirred under reﬂux for 2 h and the methanol was removed by evaporation

in vacuo by repeated coevaporation with toluene. The residue was taken up in

dry toluene (40 mL), TBAI (2.28 g, 6.2 mmol) and benzyl bromide (1.2 mL,

10.3 mmol) were added, and the solution was stirred under reﬂux for 2 h.

Excess benzyl bromide was destroyed by addition of sodium methoxide. The

solution was evaporated and acetic anhydride (5 mL) and pyridine (1:2,

10 mL) was added to the residue and the mixture was stirred overnight at rt.

The solvents were evaporated in vacuo and the residue was puriﬁed by

medium performance liquid chromatography (MPLC) (Tol:EtOAc 3:1) to give

35 (1.35 g, 64%). TLC (EtOAc:Tol 1:3): R_f¼ 0.3. Physical data correspond to

those reported.^[32]

Galactose-Phosphonates

185

Methyl 2,4,6-tri-O-acetyl-b-D-galactopyranoside (36)

35 (486 mg, 1.18 mmol) was dissolved in dry methanol (20 mL), Pd/C

(20 mg) was added, and the mixture was stirred under an atmosphere of

hydrogen for 2 h. Following ﬁltration through celite the solvent was evaporated

and the residue was puriﬁed by ﬂash chromatography (Tol:EtOAc 1:3) to give

compound 36 (368 mg, 97%). TLC (EtOAc:Tol 3:1): R_f¼ 0.5. Physical data cor-

respond to those reported.^[32]

O,O-Dibenzyl O-(methyl-2,4,6-tri-O-acetyl-b-D-

galactopyranoside-3-yl) phosphonate (37)

36 (198 mg, 0.64 mmol) and tetrazole (118 mg, 1.66 mmol, 2.6 Eq) were

mixed and dried thoroughly under vacuum. Dry DCM (10 mL) was added to

the mixture under a nitrogen atmosphere and the mixture was stirred at rt

until a clear solution was obtained. Dibenzyl diisopropylphosphoramidite

(424 mL, 1.28 mmol) was added and the solution was stirred until no starting

material was detectable. The triester 37 was not isolated but directly converted

into the H-phosphonate 38 or the phosphate 40.

O-Benzyl O-(methyl-2,4,6-tri-O-acetyl-b-D-

galactopyranoside-3-yl) phosphonic acid (38)

HCl solution (0.2 M, 8 mL) and 1,4-dioxane (4 mL) were added to the

triester solution obtained from 0.64 mmol of 36 followed by stirring for

further 4 h. The organic phase was washed with water, dried over MgSO₄,

and evaporated. The crude product was puriﬁed by ﬂash chromatography

(Tol:EtOAc 1:2) to give 38 as a mixture of both diastereomeric H-phosphonates

1

(240 mg, 84%). TLC (EtOAc:Tol 2:1): R_f¼ 0.35; H NMR (300 MHz, CDCl₃) d

7.38–7.36 (m, 5H, -C₆H₅), 6.88 (d, 0.5H, J_P-H¼ 724 Hz, PH (1st diastereomer)),

6.80 (d, 0.5H, J_P-H¼ 727 Hz, PH (2nd diastereomer)), 5.46 (br s, 1H, H-4), 5.24–

4.98 (m, 3H, H-2, CH₂Ph), 4.75–4.64 (m, 1H, H-3), 4.37, 4.33 (2d, 1H, H-1),

4.23–4.08 (m, 2H, H-6,H-6⁰), 3.87–3.79 (m, 1H, H-5), 3.51 (s, 3H, OCH₃), 2.18–

2.06 (m, 9H, 3 OAc). ³¹P NMR (121.4 MHz, CDCl₃) d 8.45, 7.95 (2s, 1H, PH).

[HR-ESI-MS] Calcd for [C₂₀H₂₇O₁₁PNa]^þ497.1183. Found 497.1196.

Ammonium O-(methyl b-D-galactopyranoside-3-yl)

phosphonate (39)

38 was debenzylated as described for 36. The debenzylated compound was

then treated with a mixture of 1,4-dioxane/25% NH₄OH solution (1:1, 4 mL)

overnight at rt. The reaction mixture was lyophilized and 39 was puriﬁed by

gel permeation chromatography (Biogel P4, 0.1 M NH₄HCO₃buffer). ¹H

NMR (300 MHz, D₂O)

d

6.66 (d, 0.7H, J_P-H¼ 647 Hz, PH (1st

H. Busse et al.

186

diastereoisomer)), 6.46 (d, 0.3H, J_P-H¼ 634 Hz, PH (2nd diastereoisomer)),

4.22 (d, 1H, J_1–2¼ 8.1 Hz, H-1), 3.94–3.87 (m, 2H, H-3, H-4), 3.63–3.54

(m, 3H, H-5, H-6, H-6⁰), 3.46 (dd, H-2), 3.41 (s, 3H, OCH₃). ³¹P NMR

(121.4 MHz, D₂O) d 9.14, 6.72 (2s, 1H, PH). [HR-ESI-MS] Calcd for

[C₇H₁₅O₈PNa]^þ281.0395. Found 281.0396.

Dibenzyl O-(methyl-2,4,6-tri-O-acetyl-b-D-

galactopyranoside-3-yl) phosphate (40)

A solution of phosphite 37 (from 50 mg, 0.32 mmol of 36), prepared from 36

as described above, was diluted with DCM (5 mL) and extracted with saturated

NH₄Cl²solution (5 mL). The organic phase was dried over MgSO₄, ﬁltered, and

evaporated to dryness. The crude phosphite was taken up in dry DCM (5 mL)

and treated with tertbutyl hydroperoxyde (0.1 mL, 6M in decane) for 30 min

at rt. Sodium bisulﬁte solution was added (0.5 M, 5 mL), the mixture was

stirred for 15 min, and the organic phases were washed with NH₄Cl²solution,

dried, and evaporated. Flash chromatography (Tol:EtOAc 1:1) gave 40 as a color-

less oil (70 mg, 75%). TLC (EtOAc: Tol 1:1): R_f¼ 0.33; ¹H NMR (300 MHz,

CDCl₃) d 7.30–7.10 (m, 10H, 2 C₆H₅), 5.45 (d, 1H, J ¼ 3.3 Hz, H-4), 5.14 (dd,

1H, J_2–3¼ 9.7, J_1–2¼ 8.0 Hz, H-2), 4.99–4.85 (m, 4H, 2 OCH₂Ph), 4.53 (ddd,

1H, J_3-P¼ ꢀ10Hz, H-3), 4.27 (d, 1H, H-1), 4.10–4.02 (m, 2H, H-6, H-6⁰), 3.74

0

(dd, J_5–6¼ J_5–6¼ 6.6 Hz, H-5), 3.43 (s, 3H, OCH₃), 2.02, 1.99, 1.78 (3s, 9H, 3

OAc); ³¹P NMR (121.4 MHz, CDCl₃) d 20.75 (1P, PO(OR)₃). [HR-ESI-MS]

Calcd for [C₂₇H₃₃O₁₂PNa]^þ603.1607760. Found 603.1601841.

Ammonium O-(methyl 2,4,6-tri-O-acetyl-b-D-

galactopyranoside-3-yl) phosphate (41)

40 was debenzylated and deacetylated as described for 36 and 38. The

crude product was puriﬁed by gel permeation chromatography (Biogel P4,

0.1 M NH₄HCO₃buffer) and lyophilized. ¹H NMR (300 MHz, D₂O) d 4.21

(d, 1H, J_1–2¼ 8.1 Hz, H-1), 3.89–3.82 (m, 2H, H-3, H-4), 3.64–3.55 (m, 3H,

H-5, H-6, H-6⁰), 3.48 (dd, 1H, J ¼ 7.9, 9.0 Hz, H-2), 3.41 (s, 3H, OCH₃). ³¹P

NMR (121.4 MHz, D₂O) d 21.42 (s, 1P, OPO²₃²). [HR-ESI-MS] Calcd for

[C₇H₁₅O₉PNa]^þ297.0355. Found 297.0345.

O-Benzyl O-(methyl-2,4,6-tri-O-acetyl-b-D-

galactopyranoside-3-yl) [1-(4-acetamido)phenyl-

hydroxymethyl)] phosphonate (42)

Compound 38 (50 mg, 0.10 mmol) was dissolved in dry DCM (2 mL). Para-

acetamido benzaldehyde (21 mg, 0.12 mmol) and Et₃N (30 mL, 0.22 mmol) were

added to the solution and the mixture was stirred for 3 h at rt. After removing

Galactose-Phosphonates

187

the solvent in vacuo, the compound was puriﬁed by ﬂash chromatography

(EtOAc: MeOH ¼ 10:0.4) to afford compound 42 (54.6 mg, 81%) as a mixture

1

of the four possible diastereomers. TLC (EtOAc: MeOH 10:0.4): R_f¼ 0.20; H

NMR (300 MHz, CDCl₃, complex, only selected signals given) d 3.49, 3.50 (2s,

3H, OCH₃), 2.24–2.05 (m, 12 H, OAc, NHAc). ³¹P NMR (121.4 MHz, CDCl₃)

d 23.76, 23.08, 22.28, 22.19 (4s, 4 RPO(OR)₂. [HR-ESI-MS] Calcd for

[C₂₉H₃₆NO₁₃PNa]^þ660.1816. Found 660.1777.

Ammonium O-(methyl b-D-galactopyranoside-3-yl) [1-(4-

acetamido)phenyl-hydroxymethyl)] phosphonate (43)

Compound 42 was debenzylated and deacetylated as described for 36, 38,

and 40 (yield 81%), puriﬁed by gel permeation chromatography (Biogel P4,

0.1 M NH₄HCO₃buffer), and lyophilized.¹H NMR (300 MHz, D₂O) d 7.24–

7.44 (m, 4H, C₆H₄), 4.88–4.40 (1H, CHOH, below water signal), 4.28, 4.26

(2d, 1H, J ¼ 7.9 Hz, H-1), 4.15–4.10 (m, 1H, H-3), 3.90, 3.82 (2d, 1H, H-4),

3.72–3.51 (m, 4H, H-2, H-5, H-6, H-6⁰), 3.47 (s, 3H, OCH₃), 2.05 (s, 3H,

NHAc). ³¹P NMR (121.4 MHz, D₂O) d 19.0–18.5 (br s, RPO²₂(OR)). [HR-ESI-

MS] Calcd for [C₁₆H₂₄NO₁₀PNa]^þ444.1041. Found 444.1030.

O-Benzyl O-(methyl-2,4,6-tri-O-acetyl-b-D-

galactopyranoside-3-yl) (phenylmethyl)

phosphonate (44)

38 (30 mg, 0.06 mmol) was dissolved in dry DCM (1 mL). DBU (9.6 mL,

0.06 mmol) and BnBr (7.5 mL, 0.06 mmol) were added and the mixture was

stirred for 1 day under nitrogen atmosphere at rt. The reaction mixture was

diluted with DCM (10 mL), washed with presaturated ammonium chloride

solution (2 ꢀ 5 mL), dried with MgSO₄, ﬁltered, and evaporated. Puriﬁcation

by ﬂash chromatography (Tol:EtOAc ¼ 1:1) yielded diastereomeric phospho-

1

nates 44 (13.3 mg, 37%, mixture). TLC (EtOAc:Tol 2:1): R_f¼ 0.42; H NMR

(300 MHz, CDCl₃) d 7.32–7.23 (m, 10H, C₆H₅), 5.43–5.39 (m, 1H, H-4),

5.20–5.14 (m, 1H, H-2), 4.96–4.79 (m, 4H, 2OCH₂Ph), 4.60–4.45 (m, 1H,

H-3), 4.33–4.28 (m, 1H, H-1), 4.18–4.07 (m, 2H, H-6, H-6⁰), 3.86–3.74

(m, 1H, H-5), 3.50, 3.49 (2s, 3H, 2 OCH₃), 3.21–3.00 (2dd, 2H, 2 CH₂P),

2.09–1.80 (m, 9H, 3 OAc). ³¹P NMR (121.4 MHz, CDCl₃) d 28.86, 28.72 (2s,

1P, RPO(OR)₂). [EI-MS] Calcd for [C₂₇H₃₃O₁₁PNa]^þ587.51. Found 587.34.

Ammonium O-(methyl b-D-galactopyranoside-3-yl)

(phenylmethyl) phosphonate (45)

44 was debenzylated and deacetylated under the standard conditions

described above. Puriﬁcation by gel permeation chromatography (Biogel P4,

Article Doi

Galactose-phosphonates as mimetics of the sialyltransfer by trypanosomal sialidases

DOI: 10.1080/07328300701351532

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Full text of DOI:10.1080/07328300701351532

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