Galactose-phosphonates as mimetics of the sialyltransfer by trypanosomal sialidases

Article abstract of DOI:10.1080/07328300701351532

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 ass

Full text of DOI:10.1080/07328300701351532

This article was downloaded by: [University of Rochester]
On: 06 June 2013, At: 09:47
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office:
Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Journal of Carbohydrate Chemistry
Publication details, including instructions for authors and subscription
information:
http://www.tandfonline.com/loi/lcar20
Galactose‐Phosphonates as Mimetics of the
Sialyltransfer by Trypanosomal Sialidases
Heike Busse a , Mitsuru Hakoda b , Matthew Stanley b & Hansjörg Streicher b
a Department of Chemistry, University of Konstanz, Konstanz, Germany
b Chemistry 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
To link to this article: http://dx.doi.org/10.1080/07328300701351532
PLEASE SCROLL DOWN FOR ARTICLE
Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-conditions
This article may be used for research, teaching, and private study purposes. Any substantial
or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or
distribution in any form to anyone is expressly forbidden.
The publisher does not give any warranty express or implied or make any representation that the
contents will be complete or accurate or up to date. The accuracy of any instructions, formulae,
and drug doses should be independently verified with primary sources. The publisher shall not
be liable for any loss, actions, claims, proceedings, demand, or costs or damages whatsoever or
howsoever caused arising directly or indirectly in connection with or arising out of the use of this
material.

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-R6 ¼ H), the most prominent member of the
family of sialic acids (R1–R6 ¼ 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 (IC50 values, 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 F254 plates. 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 H3PO4 was 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 NH4HCO3 as 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): Rf ¼ 0.07; [a]2D0 ¼ 240.2
(c ¼ 1, MeOH); 1H NMR (600 MHz, CD3OD) d 5.48 (d, 1H, J1–2 ¼ 4.8 Hz,
H-1), 4.63 (dd, 1H, J2–3 ¼ 2.4, J3–4 ¼ 7.8 Hz, H-3), 4.36 (dd, 1H, H-2),
4.30 (dd, 1H, J4–5 ¼ 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-CH3 and 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 CH3 of 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)þ 361.1. 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 NH4HCO23 buffer, and
1
lyophilized again to afford 7 (6 mg, 95%) as a white solid. H NMR (600 MHz,
CD3OD) d 5.15 (d, 1H, J1–2 ¼ 4.2 Hz, H-1a), 4.49 (d, 2H, J1–2 ¼ 8.4 Hz,
H-1b), 4.02–3.70 (m, 14H, H-4, H-5, H-6, H-60 both anomers and H-2a,
H-3a), 3.55 (dd, 2H, J2–3 ¼ 3.6, J3–4 ¼ 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)þ 281.1. 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 NaHCO23 solution, 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): Rf ¼ 0.4;
1
[a]2D0 ¼ 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, J1–2 ¼ 5.4 Hz, H-1), 4.65 (dd, 1H,
J2–3 ¼ 2.4, J3–4 ¼ 8.4 Hz, H-3), 4.45 (dd, 1H, H-2), 4.33 (dd, 1H,
0
J4–5 ¼ 1.8 Hz, H-4), 3.99 (ddd, 1H, J5–6 ¼ 4.8, J5–6 ¼ 6.6 Hz, H-5), 3.89 (dd,
1H, J6–6 ¼ 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
(C16H20O6 þ Na)þ 331.1. Found 331.1. Calcd for (C16H20O6 þ K)þ 347.1.
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 NaHCO3 was 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): Rf ¼ 0.27; [a]2D0 ¼ þ25.8 (c ¼ 1, MeOH); 1H NMR
(600 MHz, CD3OD) d 4.38 (pd, 1H, J3–4 ¼ 3.0 Hz, H-4), 4.18 (d, 1H,
0
J1–2 ¼ 7.2 Hz, H-1), 4.14 (dd, 1H, J5–6 ¼ 1.8, J6–6 ¼ 12.6 Hz, H-6),
3.83 (dd, 1H, H60), 3.72–3.65 (m, 4H, HOCH2CH2O), 3.62 (dd, 1H,
J2–3 ¼ 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 (C12H22O7 þ Na)þ 301.1. Found 301.3. Calcd for
(C12H22O7 þ K)þ 317.1. 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): Rf ¼ 0.57;
[a]2D0 ¼ 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, J1–2 ¼ 4.8 Hz, H-1), 4.66
(dd, 1H, J2–3 ¼ 2.4, J3–4 ¼ 8.4 Hz, H-3), 4.45 (dd, 1H, H-2), 4.35 (dd, 1H,
J4–5 ¼ 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
(C18H24O7 þ Na)þ 375.1. Found 375.0. Calcd for (C18H24O7 þ K)þ 391.1.
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 NaHCO32 solution, 1 M KH2PO24 solution, 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): Rf ¼ 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 NaHCO23 solution, 1M KH2PO42 solution, and NaCl2 solution
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): Rf ¼ 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. NaHCO23 solution 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
Rf ¼ 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, J1–2 ¼ 8.4 Hz, H-1),
0
4.45 (dd, 1H, J5–6 ¼ 1.3, J6–6 ¼ 12.6 Hz, H-6), 4.28 (bs, 1H, H-4), 4.15 (dd,
1H, J5–6 ¼ 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): Rf ¼ 0.5; [a]2D0 ¼ 255.7 (c ¼ 1, CHCl3);
1H NMR (600 MHz, CDCl3) d 7.38–7.25 (m, 5H, C6H5), 5.68 (d, 1H, J1–

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, J4–5 ¼ 3.0,
J5–5 ¼ 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)þ 417.2. Found 417.8. Calcd for (C21H34O5Si þ K)þ 433.2.
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): Rf ¼ 0.25;
[a]2D0 ¼ 282.7 (c ¼ 1, CHCl3); H NMR (600 MHz, CDCl3) d 7.39–7.26 (m, 5H,
1
C6H5), 5.73 (d, 1H, J1–2 ¼ 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, J2–3 ¼ 8.4 Hz, H-2), 4.12
0
(ddd, 1H, J3–4 ¼ 4.2, J4–5 ¼ 2.4 Hz, J4–5 ¼ 9.0 Hz, H-4), 3.92 (dd, 1H,
J4–5 ¼ 2.4 Hz, J5–5 ¼ 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 (C15H20O5 þ Na)þ 303.1. Found 303.2. Calcd for
(C15H20O5 þ K)þ 319.1. 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 NaHCO23 solution and KH2PO42 solution 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 MgSO4 and 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): Rf ¼ 0.3; [a]2D0 ¼ 224.3 (c ¼ 1, CHCl3); H NMR (600 MHz,
1
CDCl3) d 7.36–7.25 (m, 5H, C6H5), 5.68 (d, 1H, J1–2 ¼ 3.7 Hz, H-1), 4.75
(d, 2H, J ¼ 11.94 Hz, CH2Ph), 4.53 (dd, 1H, J2–3 ¼ 8.8 Hz, H-2), 4.29 (ddd,
0
1H, J3–4 ¼ 4.2, J4–5 ¼ 9.2, J4–5 ¼ 12.9 Hz, H-4), 3.79 (12H, 4 OCH3), 3.40
0
(dd, 1H, H-3), 2.83 (dd, 1H, J5 -6 ¼ 3.8, J5–6 ¼ 8.8 Hz, H-6), 2.29 (ddd, 1H, J5–
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 (C20H32O10P2 þ Na)þ 517.2.
Found 516.8. Calcd for (C20H32O10P2 þ K)þ 533.2. 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 NaHCO23 solution, 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): Rf ¼ 0.4; 1H NMR (600 MHz, CDCl3, major anomer) d
7.37–7.33 (m, 5H, C6H5), 5.23 (d, 1H, J1–2 ¼ 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 (C17H28O10P2 þ Na)þ 477.1. Found 477.0. Calcd for
(C17H28O10P2 þ K)þ 493.1. 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- Hþ, 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): Rf ¼ 0.6; [a]2D0 ¼ 277.0 (c ¼ 1, CHCl3); 1H NMR (600 MHz,
CDCl3) d 7.36–7.32 (m, 5H, C6H5), 6.67 (d, 1H, J2-P ¼ 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)þ 351.1. 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): Rf ¼ 0.5; [a]D
(600 MHz, CDCl3) d 7.33–7.27 (m, 5H, C6H5), 6.46 (d,1H, J2-P ¼ 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)þ 435.1. 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 NaHCO23 solution, and the organic phase was dried over MgSO4 and
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): Rf ¼ 0.48;
[a]2D0 ¼ 232.5 (c ¼ 1, CHCl3); 1H NMR (600 MHz, CDCl3) d 7.36–7.21
(m, 5H, C6H5), 6.53 (d, 1H, J2-P ¼ 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, JP-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)þ 439.2. Found 439.7. Calcd for (C19H29O8P þ K)þ 455.2.
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): Rf ¼ 0.26;
[a]2D0 ¼ 210.6 (c ¼ 1, CHCl3); 1H NMR (600 MHz, CDCl3) d 6.50 (d, 1H,
J2-P ¼ 21.6 Hz, H-2), 4.78, 4.73 (dd, 4H, Ja-b ¼ 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, JP-OMe ¼ 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)þ 349.1. 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
NaHCO23 solution and KH2PO42 solution 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): Rf ¼ 0.3; [a]2D0 ¼ 238.6 (c ¼ 1, CHCl3); 1H
NMR (600 MHz, CDCl3) d 6.52 (d, 1H, J2-P ¼ 21.6 Hz, H-2), 4.78, 4.76 (dd,
4H, CH2O), 4.08 (bd, 1H, H-3), 3.71 (2d, 6H, JP-OMe ¼ 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)þ 374.1. 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): Rf ¼ 0.45; [a]D20 ¼ 214.6 (c ¼ 1, CHCl3); 1H NMR (600 MHz,
CDCl3) d 6.36 (d, 1H, J2-P ¼ 22.2 Hz, H-2), 4.16 (m, 1H, H-3), 3.76 (ddd, 1H,
0
J5–6 ¼ 5.4, J4–5 ¼ 10.2 Hz, J5–6 ¼ 15.2 Hz, H-5), 3.64–3.60 (2d, 6H,
JP-OMe ¼ 11.4 Hz, POOCH3), 3.35 (dd, 1H, J3–4 ¼ 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)þ 286.1. 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 PMe3 in 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): Rf ¼ 0.26; [a]2D0 ¼ 244.8 (c ¼ 1, CHCl3); 1H NMR
(600 MHz, CDCl3) d 6.47 (d, 1H, J2-P ¼ 19.2 Hz, H-2), 5.55 (m, 1H, H-3),
0
5.10 (ddd, 1H, J4–5 ¼ 9.0, J5–6 ¼ 5.4, J5–6 ¼ 10.8 Hz, H-5), 4.25 (dd, 1H,

H. Busse et al.
180
J3–4 ¼ 10.8 Hz, H-4), 3.76 (2d, 6H, JP-OMe ¼ 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)þ 386.1. 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): Rf ¼ 0.1; [a]2D0 ¼ 23.2 (c ¼ 0.5, MeOH); 1H NMR
(600 MHz, CD3OD) d 6.21 (d, 1H, J2-P ¼ 18.6 Hz, H-2), 5.53 (pd, 1H, H-3),
5.08 (ddd, 1H, J4–5 ¼ 9.0, J5–6 ¼ 5.4 Hz, H-5), 4.21 (dd, 1H J3–4 ¼10.8 Hz,
H-4), 3.48 (1d, 3H, JP-OMe ¼ 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)þ 372.1. 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 (Hþ) 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]2D0 ¼ 28.34
1
(c ¼ 0.5, MeOH); H NMR (600 MHz, CD3OD) d 6.38 (d, 1H, J2-P ¼ 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): Rf ¼ 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, J1–2 ¼ 5.4 Hz, H-1b), 5.50 (m, 1H, H-3a), 5.05 (m, 1H, H-5a),
4.63 (dd, 1H, J2–3 ¼ 2.4 Hz, J3–4 ¼ 8.4 Hz, H-3b), 4.41 (dd, 1H, H-2b), 4.36
(dd, 1H, J3–4 ¼ 9.0 Hz, H-4a), 4.31 (dd, 1H, J4–5 ¼ 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)þ 706.2. 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): Rf ¼ 0.1; 1H NMR
(600 MHz, CD3OD) d 7.51–7.35 (m, 5H, C6H5), 6.22 (d, 1H, J2-P ¼ 18.6 Hz,
H-2a), 5.74 (s,1H, CHPh), 5.57 (d, 1H, J1–2 ¼ 5.4 Hz, H-1b), 5.49 (pd, 1H,
J3–4 ¼ 8.4 Hz, H-3a), 5.06 (dd, 1H, J4–5 ¼ 9.6, J5–6 ¼ 5.4 Hz, H-5a), 4.65 (dd,
1H, J2–3 ¼ 2.4, J3–4 ¼ 8.4 Hz, H-3b), 4.45 (dd, 1H, H-2b), 4.33 (dd, 1H,
J4–5 ¼ 1.8 Hz, H-4b), 4.22 (dd, 1H, J3–4 ¼ 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)þ 692.2. Found 692.5.
Calcd for (C30H40NO14P þ K)þ 708.2. 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% NH3 aq 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, J2-P ¼ 13.2 Hz, H-2a), 5.73
(s, 1H, CHPh), 5.59 (d, 1H, J1–2 ¼ 4.8 Hz, H-1b), 4.65 (dd, 1H, J2–3 ¼ 2.4,
J3–4 ¼ 8.4 Hz, H-3b), 4.45 (dd, 1H, H-2b), 4.34 (dd, 1H, J4–5 ¼ 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)þ 608.2. Found 608.3. Calcd for
(C26H36NO12P þ K)þ 624.2. 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, J2-P ¼ 19.8 Hz, H-2a), 5.14 (d, 0.8H, J1–2 ¼ 4.2 Hz, H-1ba), 4.47 (dd, 1.3
H, J1–2 ¼ 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-6b0 both anomers, CH2CH2O, H-3ba, H-2ba,
H-5b, H-4b both anomers), 3.53 (dd, 1.3 H, J2–3 ¼ 9.6 Hz, J3–4 ¼ 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)þ 480.1. Found 480.2.
Calcd for (C16H28NO12P þ K)þ 496.1. Found 496.2. [HR-ESI-MS] Calcd for
[M þ 2Na-H]þ 502.1072. 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% NH3 aq 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]2D0 ¼ 25.4
(c ¼ 0.5, H2O); 1H NMR (600 MHz, D2O) d 6.13 (d, 1H, J2-P ¼ 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)þ 288.1. Found 288.2. Calcd for (C9H16NO6P þ K)þ 304.1.
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): Rf ¼ 0.1; [a]2D0 ¼ 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, J1–2 ¼ 4.8 Hz, H-1), 4.66 (dd, 1H, J2–3 ¼ 2.4,
J3–4 ¼ 8.4 Hz, H-3), 4.46 (dd, 1H, H-2), 4.31 (dd, 1H, J4–5 ¼ 1.8 Hz, H-4),
4.08 (m, 1H, H-5), 4.02–3.98 (m, 2H, CH2CH2O), 3.73 (dd, 1H, J5–6 ¼ 5.4,

H. Busse et al.
184
J6–6 ¼ 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-CH3 and 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 (C20-
H29O9P þ Na þ H)þ 468.2. 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, J1–2 ¼ 3.6 Hz, H-1a), 4.45 (d, 2H,
J1–2 ¼ 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, J2–3 ¼ 3.6 Hz, J3–4 ¼ 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]þ 347.0459. 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): Rf ¼ 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): Rf ¼ 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): Rf ¼ 0.35; H NMR (300 MHz, CDCl3) d
7.38–7.36 (m, 5H, -C6H5), 6.88 (d, 0.5H, JP-H ¼ 724 Hz, PH (1st diastereomer)),
6.80 (d, 0.5H, JP-H ¼ 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]þ 497.1183. 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 NH4HCO3 buffer). 1H
NMR (300 MHz, D2O)
d
6.66 (d, 0.7H, JP-H ¼ 647 Hz, PH (1st

H. Busse et al.
186
diastereoisomer)), 6.46 (d, 0.3H, JP-H ¼ 634 Hz, PH (2nd diastereoisomer)),
4.22 (d, 1H, J1–2 ¼ 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]þ 281.0395. 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
NH4Cl2 solution (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 NH4Cl2 solution,
dried, and evaporated. Flash chromatography (Tol:EtOAc 1:1) gave 40 as a color-
less oil (70 mg, 75%). TLC (EtOAc: Tol 1:1): Rf ¼ 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, J2–3 ¼ 9.7, J1–2 ¼ 8.0 Hz, H-2), 4.99–4.85 (m, 4H, 2 OCH2Ph), 4.53 (ddd,
1H, J3-P ¼ ꢀ10Hz, H-3), 4.27 (d, 1H, H-1), 4.10–4.02 (m, 2H, H-6, H-60), 3.74
0
(dd, J5–6 ¼ J5–6 ¼ 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]þ 603.1607760. 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 NH4HCO3 buffer) and lyophilized. 1H NMR (300 MHz, D2O) d 4.21
(d, 1H, J1–2 ¼ 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]þ 297.0355. 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): Rf ¼ 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]þ 660.1816. 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 NH4HCO3 buffer), 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]þ 444.1041. 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): Rf ¼ 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]þ 587.51. 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,