Allosteric Effectors

DOI : 10.1002/cmdc.
201000366
Polyphosphates and Pyrophosphates of Hexopyranoses as
Allosteric Effectors of Human Hemoglobin: Synthesis,
Molecular Recognition, and Effect on Oxygen Release
Konstantina C. Fylaktakidou,
[a, b]
Carolina D. Duarte,
[a, c, d]
Alexandros E. Koumbis,
[a, e]
Claude Nicolau,*
[a, c, f]
and Jean-Marie Lehn*
[a]
Introduction
A most fundamental physiological process in the blood of
aerobic organisms resides in the delivery of oxygen bound to
hemoglobin (Hb) in red blood cells (RBCs) to all tissues.
Oxygen delivery is regulated by allosteric effectors that bind to
Hb and decrease its oxygen binding affinity. As numerous dis-
eases including cardiovascular disease and cancer involve hy-
poxia, achieving increased oxygen release is expected to re-
store normoxia, and to thereby possess significant therapeutic
potential. Therefore, finding allosteric effectors of Hb that in-
crease oxygen release and delivery by RBCs represents a goal
of very special interest.
In humans, allosteric regulation is effected by 2,3-bisphos-
phoglycerate (BPG, I, Figure 1),
[1]
the binding of which to the
allosteric pocket of the Hb tetramer is well characterized.
[2]
Among a variety of polyphosphates that are able to decrease
the affinity of human Hb for oxygen,
[1]
the natural substance
myo-inositol hexakisphosphate (IHP, II, Figure 1) is the most
powerful allosteric effector identified to date.
[1a]
It displaces
Hb-bound 2,3-BPG, occupying the allosteric pocket with higher
affinity.
[1b, 3]
Thus, it triggers a decrease in the affinity between
O
2
and Hb, and when loaded into circulating RBCs, it subse-
quently leads to increased and regulated release of oxygen
upon tissue demand.
[4]
We showed previously that myo-inositol trispyrophosphate
(ITPP, III, Figure 1), the derivative of IHP that contains three
seven-membered cyclic pyrophosphate groups, is a mem-
brane-permeant allosteric effector of Hb, which increases
oxygen release in vitro, both in free Hb and whole blood, in a
concentration-dependent manner.
[5]
As a result, ITPP was iden-
tified as a novel and highly effective anti-angiogenic and anti-
cancer agent, counteracting the effects of hypoxia and hinder-
ing cancer progression.
[6]
It suppresses HIF-1a and significantly
Polyphosphorylated and perphosphorylated hexopyranose
monosaccharides and disaccharides were synthesized from
parent or partially protected carbohydrates as potential alloste-
ric effectors of hemoglobin. A study toward the construction
of seven- and eight-membered cyclic pyrophosphates was also
performed on the sugars which had the proper orientation,
protection, and number of phosphates. All final compounds
were tested for their efficiency on oxygen release from human
hemoglobin. Several compounds presented higher potency
than myo-inositol hexakisphosphate, which is the most effi-
cient of the known allosteric effectors of hemoglobin. Struc-
tureactivity relationships were analyzed. The affinity and effi-
ciency depend on the number of phosphates attached to the
carbohydrate skeleton and are related primarily to the number
of negative charges present. Other effects operate, but play a
lesser role.
Figure 1. Structures of allosteric effectors of Hb (IIII). Structures of seven-
and eight-membered cyclic pyrophosphates (IV and V).
[a] Prof. K. C. Fylaktakidou, Dr. C. D. Duarte, Prof. A. E. Koumbis,
Prof. C. Nicolau, Prof. J.-M. Lehn
Institut de Science et dIngnierie Supramolculaires
Universit de Strasbourg
8 Alle Gaspard Monge, 67000 Strasbourg (France)
Fax: (+33) 368 855140
E-mail : lehn@isis.u-strasbg.fr
cnicolau@aol.com
[b] Prof. K. C. Fylaktakidou
Current address: Department of Molecular Biology and Genetics
Democritus University of Thrace, 68100 Alexandroupolis (Greece)
[c] Dr. C. D. Duarte, Prof. C. Nicolau
NormOxys Inc. , 200 Boston Avenue, Medford, MA 02155 (USA)
[d] Dr. C. D. Duarte
Current address: Quintiles Strasbourg, Parc dInnovation
Rue Jean Dominique Cassini, 67400, Illkirch Graffenstaden (France)
[e] Prof. A. E. Koumbis
Current address: Laboratory of Organic Chemistry
Aristotle University of Thessaloniki, 54124 Thessaloniki (Greece)
[f] Prof. C. Nicolau
Friedman School of Nutrition Science and Policy
Tufts University, Boston, MA 02115 (USA)
ChemMedChem 2011, 6, 153 168 2011 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim 153
decreases VEGF in cells, thus blocking the route that leads to
angiogenesis.
[6]
Furthermore, it was found that ITTP is capable
of increasing exercise capacity in both normal and transgenic
mice with severe heart failure.
[7]
Therefore, it appears that the
biological effects resulting from the enhanced release of
oxygen caused by ITPP may have general utility in the treat-
ment of a variety of disease states in which tissues suffer from
low oxygen tension, such as cardiovascular and oncological ail-
ments, ischemic insult, heart attacks, stroke, and tumor pro-
gression.
These results greatly warrant a more complete exploration
of other molecules that may present related properties. We
have therefore undertaken a wide-range research program to
this end and present herein the synthesis of an initial series of
such compounds, phosphates and cyclic pyrophosphates de-
rived from various carbohydrates, as well as data regarding
their effect on oxygen release from pure human Hb.
Rationale
Research into the action of organophosphates as allosteric ef-
fectors of Hb has not progressed for many years.
[8]
Indeed, or-
ganophosphates are rarely examined as drug candidates due
to their highly charged structure, which consequently prevents
high concentrations at the targeted site, thus showing poor
oral bioavailability and/or cell penetration. Nevertheless, the re-
markable properties of IHP and ITPP as allosteric effectors
[47]
prompted us to extend our investigations to compounds
closely related to inositols, such as carbohydrates. Polyphos-
phorylated carbohydrates (with three or more phosphate
groups) have been reported to possess numerous biological
activities. Specifically, various alkyl glycosides of phosphorylat-
ed d-galactose, di-galactose, or lactose derivatives exhibit insu-
linic
[9]
and anti-inflammatory activity,
[10]
or have been used as
scaffolds for mechanistic studies into the processes of cell sur-
face receptor recognition.
[11]
For the glucose series, tris phos-
phorylated analogues were found to exhibit anti-inflammatory
activity
[10a]
or to prevent restenosis.
[12]
Other derivatives such as
d-glucose 2,4,6-trisphosphate and its 1,3-deoxy analogues
showed moderate inhibition against acid sphingomyelinase rel-
ative to myo-inositol 1,3,5-triphosphate,
[13]
whereas 4-nitro-
phenyl 2,3,4,6-tetrakis phosphorylated a- and b-glucopyrano-
sides were used for mechanistic studies on hydrolysis.
[14]
Inter-
estingly, oligosaccharides composed of repeating glucose
units, such as maltotriose, cellobiose, or heparin-like pentasac-
charide analogues, were found to possess antithrombotic ac-
tivity.
[15]
Finally, sucrose phosphate ester derivatives were in
use some decades ago for the improvement of films for cine-
matographic and photographic materials owing to their gelling
properties.
[16]
The structural similarity of mannose tris phosphorylated de-
rivatives with a-trinositol (d-myo-inositol 1,2,6-trisphosphate
sodium salt), which has anti-inflammatory and analgesic activi-
ties, and with d-myo-inositol 1,4,5-trisphosphate, which is a
known secondary messenger, led to the synthesis of those car-
bohydrate derivatives as their mimics. It was found that
methyl-a-d-mannopyranoside 2,3,4-trisphosphate sodium salt
has less activity than a-trinositol, but is fivefold more resistant
to dephosphorylation.
[17]
The same 1- and/or 6-alkyl-protected
derivatives (as well as some rhamnose, fructopyranose, and
arabinitol analogues) were also found to prevent restenosis,
[12]
and to present growth factor modulating or other activities.
[18]
The linear diphosphate (DP, also termed linear pyrophos-
phate) moiety is a quite common unit in nature and is present
in ADP, for example. For the carbohydrate scaffolds, the glu-
cose pentakis linear DP derivative is a metabolite isolated from
bacteria.
[19]
However, seven-membered cyclic pyrophosphates
(PPs) are generally rather rare in nature, with the exception of
the cyclic 2,3-bisphosphoglycerate (cBPG, IV, Figure 1), which
was found in methanogenic bacteria.
[20]
Nevertheless, it would
seem that no seven-membered cyclic PP (like those present in
ITPP) has been ever constructed or identified on a carbohy-
drate scaffold.
In contrast, eight-membered cyclic PPs attached mainly to a
ribofuranose core (V, Figure 1), have received attention due to
cyclic ADP-ribose (or adenosine 3,5-PP),
[21]
a natural product
that was found to control calcium levels.
[22]
Other 3,5-PP nu-
cleotide analogues have shown effects on germinating
B. cereus 569 spores,
[23]
cytidilate cyclase activity,
[24]
or antitu-
mor activity.
[25]
3,5-PP nucleotide derivatives phosphorylated
at position 2 have been isolated from the red seaweed Por-
phyra umbilicalis, which is a constituent of herbal medicines.
[26]
Finally, several 3,5-deoxyribose phosphate and PP nucleotides
were proven to be human P2Y6 receptor ligands, P2Y1 recep-
tor antagonists and partial agonists, and recombinant rat P2X
receptor agonists and antagonists.
[27, 28]
In the work presented herein, we targeted polyphosphates
(with three or more phosphate groups) and seven- and/or
eight-membered cyclic PPs of hexopyranoses, which are all
novel compounds, except one. Furthermore, their binding to
human Hb and their effect on oxygen release were investigat-
ed in order to gain insight into molecular recognition features
and structureactivity relationships in the allosteric regulation
of Hb.
Results and Discussion
Synthesis of polyphosphate and cyclic pyrophosphate
derivatives of hexopyranoses
Our synthetic plan should provide valuable information on sev-
eral questions regarding molecular recognition in the allosteric
pocket of Hb, such as the effect of the number of phosphates,
the most appropriate conformation for binding (mannose and
galactose with one axial hydroxy group are more closely relat-
ed to IHP), and the role of the anomeric phosphate (a or b),
which is chemically the most labile. To serve these purposes,
double protection of positions 1 and 6 of monosaccharides
could lead to the corresponding tris phosphorylated deriva-
tives. Proper selective unmasking of positions 1 or 6 of hexoses
would allow the synthesis of tetrakis phosphorylated deriva-
tives, and subsequently the possible simultaneous formation
of two cyclic PPs in a row, which is the closest that can be ach-
ieved in mimicking the ITPP structure. In addition, protection
154 www.chemmedchem.org 2011 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim ChemMedChem 2011, 6, 153 168
MED J.-M. Lehn, C. Nicolau, et al.
of position 6 may allow the synthesis of both a- and b-anom-
ers of the tetrakis phosphorylated derivatives. Finally, perphos-
phorylation of naked monosaccharides and disaccharides is ex-
pected to give pentakis- and octakisphosphates, respectively.
For our purpose, we selected the three more common mono-
saccharidesglucose, mannose, and galactosewhich, upon
proper protection and deprotection reactions, would provide
tris- to pentakisphosphates and bispyrophosphates, and two
disaccharides (a reducing one: lactose, and a nonreducing
one: sucrose), expected to give octakisphosphates.
Synthesis of tris phosphorylated monosaccharides
The sodium salts of the tris phosphorylated glucose and man-
nose derivatives 8 and 10 were prepared from the known 6-O-
tert-butyldiphenylsilyl (TBDPS) glucose and mannose methyl
glycosides (1
[29]
and 2,
[30]
respectively), as depicted in
Scheme 1. Compounds 1 and 2 were individually subjected to
a phosphorylation reaction using dibenzyl N,N,-diisopropyl-
phosphoramidate and tetrazole in dry acetonitrile under argon
at room temperature for 24 h. The initially formed phosphites
were directly oxidized with meta-chloroperbenzoic acid
(mCPBA) to give compounds 3 and 4 in 76 and 73% yields for
glucose and mannose, respectively. Removal of the TBDPS pro-
tecting group was achieved with a buffered solution of tetra-
butylammonium fluoride (TBAF) at 08C, and yielded com-
pounds 5 and 6 (84% in both cases). This synthetic pathway
allows further substitution at position 6, for instance for the
preparation of more lipophilic derivatives.
The benzyl esters 5 and 6 were deprotected upon catalytic
hydrogenolysis (H
2
in the presence of Pd/C and triethylamine)
to give the triethylammonium salts 7 and 9. These were trans-
formed into sodium salts 8 and 10 by using a sequence of
cation exchange columns first in H
+
and subsequently in Na
+
forms (Scheme 1).
In general, the triethylammonium salts are required for the
preparation of the corresponding PPs, whereas the sodium
salts could also be directly obtained by performing the hydro-
genation reaction in the presence of sodium bicarbonate. The
direct formation of sodium salts was realized for several deriva-
tives; however, it is important to note that the gummy starting
material should be very carefully dried and weighted, because
an exact amount of sodium bicarbonate is required (one equiv-
alent per phosphate) in order to avoid contamination of the
final product. In contrast, the excess base (triethylamine) is
easily removed under vacuum when the corresponding triethyl-
ammonium salts are prepared. Transformation of the triethyl-
ammonium salt into the H
+
and then Na
+
forms using ion-ex-
change procedures provides an indirect and safe way to
obtain the sodium salts. In some cases this alternative ap-
proach is preferable. Finally, hydrogenations in the absence of
base should be avoided in order to prevent potential compli-
cations from the labile anomeric phosphates in an acidic envi-
ronment.
Compound 10 is the only derivative among those studied in
this work that has been previously reported;
[17]
however, it was
prepared by following a different pathway. Notably, under the
reaction conditions used, no migration of phosphate group(s)
was observed as indicated by NMR spectral data.
Synthesis of tetrakis phosphorylated monosaccharides
We first envisaged construction of the 1,2,3,4-tetrakis phos-
phorylated analogues in order to examine the role of the
anomeric orientation in molecular recognition of Hb. Reaction
of parent sugars with TBDPS chloride was used to selectively
block the primary hydroxy group at position 6. Whereas prepa-
ration of the silylated mannose derivative 15 has not been re-
ported, those of glucose and galactose, 14
[31]
and 16,
[32]
respec-
tively, have been. However, we followed a modified procedure
for the synthesis of all of them (Scheme 2).
Phosphorylation of these 6-O-silylated precursors proceeded
smoothly in acetonitrile and with a similar yield of ~80% in
each case. Both anomers were formed for all three sugars, al-
though in different proportions. Glucose and mannose gave a-
and b-anomers (17/18 and 19/20, respectively) in a ratio of
~5:3, whereas the opposite ratio (~3:5) was observed for the
galactose anomers 21/22. Glucose and mannose anomers
were easily separated by column chromatography. The galac-
tose derivatives were practically inseparable in large scale and
were taken forward as a mixture. Nevertheless, a small amount
of each anomer was isolated for full characterization. Removal
of the TBDPS protecting group was performed under carefully
controlled conditions (08C, near neutral pH) to prevent loss of
the sensitive and labile anomeric phosphate (yields 6181%,
compounds 2328). At this stage we were also able to sepa-
rate the galactose anomers.
This synthetic scheme again allows further substitution at
position 6, in case the synthesis of PPs or compounds with in-
creased lipophilicity are desired. Finally, hydrogenation in the
presence of sodium bicarbonate directly provided the sodium
salts of both anomers of all monosaccharides (2934) in excel-
Scheme 1. Synthesis of the tris phosphorylated derivatives 8 and 10 of glu-
cose and mannose respectively, from their silylated methyl glycoside precur-
sors 1 and 2: a) 1. (BnO)
2
PN(iPr)
2
, 1H-tetrazole, CH
3
CN, RT; 2. mCPBA, CH
2
Cl
2
,
408C!RT; b) TBAF, AcOH, THF, 08C; c) H
2
(1 atm), Pd/C, Et
3
N, EtOH/H
2
O
(1:1), RT; d) Dowex H
+
, H
2
O then Dowex Na
+
, H
2
O. [DBP=P(O)(OBn)
2
].
ChemMedChem 2011, 6, 153 168 2011 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim www.chemmedchem.org 155
Allosteric Effectors of Human Hemoglobin
lent yields (>99%). Again, no phosphate migration was ob-
served for all of these derivatives.
The assignment of the a- and b-anomers for the glucose
and galactose derivatives was rather easy, in view of the differ-
ence in coupling constants between the two vicinal protons at
positions 1 and 2. A doublet of doublets was always present in
the spectra of all a-anomers (17, 23, 29 and 21, 27, 33) with a
small coupling constant indicating an equatorial/axial (eq/ax)
relative conformation of protons at positions 1 and 2 (
3
J
H-1,H-2
=
2.63.4 Hz), and a larger constant due to coupling of proton
H-1 with the neighboring phosphorous nucleus (
3
J
H-1,P
=5.4
7.1 Hz). In contrast, for the b-anomers (18, 24, 30 and 22, 28,
34), a triplet was observed, due to similar large values for the
coupling of vicinal protons and for the heteronucleus coupling
(
3
J
H-1,H-2
3
J
H-1,P
=6.57.9 Hz) thus indicating an ax/ax orienta-
tion of the protons. All the above results are in accordance
with published data for several a- and b-1-phosphorylated car-
bohydrates, in which substituents at position 2 are equatorially
oriented.
[33]
For the mannose derivatives, however, both eq/eq and ax/
eq couples of protons give small coupling constants. There-
fore, the assignment was based on comparison of the chemical
shifts with published values for a- and b-1-monophosphorylat-
ed mannose derivatives, which show considerable differences
in both
1
H and
13
C NMR spectra.
According to these data, for hexopyranonses such as man-
nose, which, based on NMR spectra, seems to present the
4
C
1
conformation, the anomeric proton signal of the a-anomer ap-
pears at lower field than that of the b-form.
[34]
This was the
case, as expected, for all glucose and galactose derivatives as
well. Shifts for H-5 of mannose a-anomers are found downfield
relative to the corresponding shifts of the b-isomers.
[33d, 35]
Moreover, the
13
C NMR data display downfield shifts for C-3
and C-5 for b-phosphates.
[33d, 35, 36]
These characteristic features
also apply for our derivatives, both protected phosphorylated
(19, 20, 25, 26) and sodium salts (31, 32), as shown in Table 1.
The data obtained for the perphosphorylated mannose deriva-
tives 44, 48, and 49 provide further evidence for the a-orienta-
tion of these carbohydrates (see Scheme 4 below). The anome-
ric configuration of the phosphates was, however, unambigu-
ously established on the basis of the values of the heteronu-
clear coupling constant J
C-1,H-1
, which was 174 and 175 Hz for
compounds 31 and 49 respectively, indicating an a-phosphate,
and 160 Hz for compound 32, indicating a b-anomer.
[35]
To gain access to 2,3,4,6-tetrakis phosphorylated glucose
and mannose derivatives, the commercially available glyco-
sides 35 and 36 were used (Scheme 3). The free hydroxy
groups in 35 and 36 were all simultaneously phosphorylated
(compounds 37 and 38) under the standard protocol (in 94
and 79% yields, respectively) to give, via the triethylammoni-
um salts 39 and 40, the final sodium salts 41 and 42 in excel-
lent overall yields. These glucose and mannose derivatives,
which have four phosphates in a row and the remaining
anomeric hydroxy group protected as a methyl ether, proved
to be suitable substrates to investigate the formation of PPs.
Synthesis of pentakis phosphorylated monosaccharides
Glucose (11), mannose (12), and galactose (13) were independ-
ently subjected to a phosphorylation reaction using dibenzyl
N,N,-diisopropylphosphoramidate and tetrazole in dry DMF/
acetonitrile, under argon at room temperature for 24 h. The in-
itially formed phosphites were directly oxidized with mCPBA to
Scheme 2. Synthesis of the tetrakis phosphorylated derivatives 2934 of glu-
cose, mannose and galactose from the silylated precursors 1416:
a) TBDPSCl, Et
3
N, DMAP, DMF, 08C!RT; b) 1. (BnO)
2
PN(iPr)
2
, 1H-tetrazole,
CH
3
2
Cl
2
, 408C!RT; c) TBAF, AcOH, THF, 08C; d) H
2
(1 atm), Pd/C, NaHCO
3
, EtOH/H
2
O (1:1), RT. [DBP=P(O)(OBn)
2
].
Table 1.
1
H and
13
C NMR chemical shifts for the comparative assignment
of a- and b-anomers for 1-O-phosphorylated mannose derivatives.
d [ppm]
Compd H-1 H-5 C-3 C-5
19 (a-anomer) 6.08 4.01 73.873.5 73.873.5
20 (b-anomer) 5.55 3.70 75.6 76.1
44 (a-anomer) 5.96 4.01 73.373.1 72.071.7
25 (a-anomer) 5.90 3.76 73.373.1 73.6
26 (b-anomer) 5.34 3.38 75.475.3 76.0
31 (a-anomer) 5.45 3.81 72.9 72.9
32 (b-anomer) 5.15 3.50 75.1 76.0
48 (a-anomer) 5.50 4.123.99 73.072.8 71.871.6
49 (a-anomer) 5.46 4.043.95 73.072.8 71.671.3
give compounds 43, 44, and 45 in 55, 62, and 65% yield, re-
spectively, (Scheme 4).
The benzyl esters were deprotected upon catalytic hydroge-
nolysis (H
2
in the presence of Pd/C) to give the triethylammo-
nium salts 46, 48, and 50 in very good yields (>94%). The
latter derivatives were then transformed into the sodium salts
47, 49, and 51 by applying a sequential ion exchange with
Dowex H
+
and subsequently Dowex Na
+
resins in quantitative
yields (Scheme 4).
Notably, in the cases of the perphosphorylated monosac-
charides there was a dramatic change regarding the selectivity
of the anomeric positions. In contrast to the 1,2,3,4-tetrakis
phosphorylated derivatives, only one anomer was formed for
the pentakis phosphorylated analogues. The bulkiness of the
TBDPS protecting group in conjunction with the effect of sol-
vent used (acetonitrile instead of DMF/acetonitrile), could be
considered the main factors that alter the anomeric effect and
which lead to the formation of both anomers.
The a-orientation for glucose and galactose derivatives was
indicated by the
1
H NMR spectra, in which the signals of the
anomeric protons appear as a doublet of doublets with a cou-
pling constant corresponding to an eq/ax relative conforma-
tion of protons at positions 1 and 2 (
3
J
H-1,H-2
) from 3.0 to 3.4 Hz.
The coupling constant of the anomeric proton with the neigh-
boring phosphorous nucleus (
3
J
H-1,P
) was in the range of 6.0 to
7.4 Hz. The coupling constants of compounds 43, 46, 47 and
45, 50, 51 were clearly quite similar to those of the a-anomers
of the tetrakis phosphorylated derivatives (17, 23, 29 and 21,
27, 33), shown in Scheme 2, and in accordance with published
data for a-1-phosphorylated carbohydrates, in which substitu-
ents at position 2 are equatorially oriented.
[33]
For the mannose derivatives 44, 48, and 49, comparison of
their
1
H and
13
C NMR spectra with those of the 1,2,3,4-tetrakis
phosphorylated derivatives and the value of the heteronuclear
coupling constant (J
C-1,H-1
=175 Hz) for compound 49 indicate
an a-orientation (Table 1).
Synthesis of octakis phosphorylated disaccharides
Lactose (52), a reducing disaccharide, was subjected to the
same sequence of reactions (phosphorylation, hydrogenation,
and ion exchange) as the monosaccharides above to give the
perphosphorylated lactose derivatives 53 (in a 1:4 ratio of a-
and b-anomers, Scheme 5). We managed to obtain, by column
chromatography, a small quantity of pure b-53, whereas the
rest remained as a mixture with the a-isomer.
The anomeric ratio of lactose derivatives was determined
based on their
1
H NMR spectra. Although it was relatively easy
to make the assignment of the anomeric proton of the minor
isomer of 53, it was practically impossible to observe the
anomeric proton for the major isomer (obscured by the meth-
ylene protons of benzyl groups). Therefore, it proved much
easier to assign the a- and b-anomer in the proton NMR spec-
tra of sodium salts 55. The minor anomer gives a signal at
5.57 ppm in the form of a doublet of doublets,
3
J
H-1,H-2
=3.5 Hz
and
3
J
H-1,P
=7.1 Hz. For the major lactose derivative 55 a triplet
appears at 5.02 ppm for the anomeric proton (
3
J
H-1,H-2
=
3
J
H1,P
=
8.0 Hz), thus indicating an ax/ax orientation of the protons.
The same distribution of anomers could be easily assigned for
Scheme 3. Synthesis of the tetrakis phosphorylated derivatives of glucose
41 and mannose 42 from the corresponding methyl glycosides 35 and 36:
a) 1. (BnO)
2
PN(iPr)
2
, 1H-tetrazole, DMF, CH
3
2
Cl
2
,
408C!RT; b) H
2
(1 atm), Pd/C, Et
3
N, EtOH/H
2
O (1:1), RT; c) Dowex H
+
, H
2
O
then Dowex Na
+
, H
2
O. [DBP=P(O)(OBn)
2
].
Scheme 4. Synthesis of the pentakisphosphorylated derivatives 4651 of
glucose (11), mannose (12), and galactose (13): a) 1. (BnO)
2
PN(iPr)
2
, 1H-tetra-
zole, DMF, CH
3
2
Cl
2
, 408C!RT; b) H
2
(1 atm), Pd/C,
Et
3
N, EtOH/H
2
O (1:1), RT; c) Dowex H
+
, H
2
O then Dowex Na
+
, H
2
O.
[DBP=P(O)(OBn)
2
].
Scheme 5. Synthesis of the perphosphorylated derivatives 54 and 55 of lac-
tose (52): a) 1. (BnO)
2
PN(iPr)
2
3
CN, RT; 2. mCPBA,
CH
2
Cl
2
, 408C!RT; b) H
2
(3 atm), Pd/C, Et
3
N, EtOH/H
2
O (1:1), RT; c) Dowex
H
+
, H
2
O then Dowex Na
+
, H
2
O.
the lactose derivatives 53 and 54. Because all perphosphoryla-
tion reactions were performed in the same solvent system, we
speculate that lactose gave a mixture of anomers, with the b-
anomer predominating, due solely to steric factors, whereas
the preference for the formation of a-anomers for glucose,
mannose, and galactose derivatives (43, 44, 45, respectively)
appears to result from the anomeric effect.
Sucrose (56), a nonreducing disaccharide, was subjected to
a phosphorylation reaction to give compound 57 in 77% yield
(Scheme 6). This benzyl ester was deprotected upon catalytic
hydrogenolysis to afford the triethylammonium salt 58 in very
good yield. The latter was then converted into the sodium salt
59 via ion exchange on resin columns Dowex H
+
and subse-
quently Dowex Na
+
in excellent yields. In this case, of course,
only one product was obtained, as this disaccharide lacks a
free anomeric hydroxy group.
Synthesis of cyclic pyrophosphates
For the synthesis of PPs, two vicinal phosphates, or an even
number of phosphates, all in pairs, are required. Condensation
reactions of phosphates, particularly in the conversion of IHP
into ITPP, were usually performed in pyridine by using the IHP
pyridinium salt.
[5, 37]
The rather large amount of an unpleasant
and toxic solvent, especially for synthesis on the multigram
scale, prompted us to develop a modified coupling reaction
for the synthesis of ITPP
[5, 7]
via its triethylammonium salt. The
method involves dissolution of the IHP triethylammonium salt
in a mixture of acetonitrile/water (ratio of 2:1) and heating the
solution at reflux in the presence of excess N,N-dicyclohexyl-
carbodiimide (DCC). The combination of these solvents was
used because the two solvents are miscible, and both polar
triethylammonium salt and lipophilic DCC are soluble in the
mixture. For other solvent ratios, either DCC or the salt may
remain partially undissolved. In line with these results, the for-
mation of the triethylammonium salts was also implemented
for the present synthesis of cyclic PP derivatives.
Tris phosphorylated derivatives (Scheme 1) are not good
candidates for the formation of cyclic PPs, as they lack an even
number of phosphates. However, we were interested in check-
ing the reactivity of the unprotected hydroxymethyl group
under the neutral reaction conditions described, and not in
pyridine, where formation of 4,6-cyclic phosphates of carbohy-
drates from their 6-monophosphorylated precursors has been
described.
[38]
Preliminary results showed mainly the formation
of two products, one that possibly contains the 3,4-pyrophos-
phate, and another that contains the 4,6-cyclic phosphate
along with the 2,3-pyrophosphate. We checked the formation
of cyclic phosphate by the heteronuclear coupling constant
observed for C-6 in
13
C NMR spectra. However, because the for-
mation of cyclic phosphates is outside the scope of this work,
we did not pursue further experiments with these derivatives,
nor with the 1,2,3,4-tetrakis phosphorylated derivatives
(Scheme 2), which both have the hydroxymethyl group unpro-
tected.
Complete and clean transformation into cyclic PPs could be
achieved from tetrakis phosphorylated hexopyranoses with the
remaining hydroxy group masked, that is, the anomeric group
in the case of the 2,3,4,6-tetrakis phosphorylated derivatives.
Although substrates 39 and 40 are structurally similar to IHP,
we failed to obtain products with two PPs in a row in a regio-
chemically controlled manner when the reactions were per-
formed under the same conditions as applied for ITPP.
We assumed then that water might be primarily responsible
for the failure of the preparation of these PPs. The reactions
were also conducted in neat acetonitrile, with the hope that
the triethylammonium salt, which is insoluble at room temper-
ature, would be slowly and totally solubilized in the solvent at
reflux. Indeed, when glucose salt derivative 39 was dissolved
in acetonitrile at reflux in the presence of excess DCC, the bis-
PP 60 was formed in 95% yield (Scheme 7). The same result
was obtained in the case of mannose salt 40. The reaction was
again successful, and after 24 h at reflux, the
31
P NMR spectrum
of the crude reaction mixture showed complete consumption
of the starting phosphate and the exclusive formation of 61.
Two pairs of doublets with coupling constants of 25.5 and
22.0 Hz appeared, indicating two AB systems that correspond
respectively to the eight- and seven-membered cyclic PPs. The
same pattern was observed in the spectra of glucose derivative
60, with coupling constants of 24.9 and 17.9 Hz, respectively.
The latter was easily purified by filtration to remove the
formed dicyclohexylurea (DCU) from the resulting aqueous so-
lution. It was then transformed into the corresponding sodium
salt 62 by ion exchange (Scheme 7). In contrast, mannose bis-
PP 61 was found to decompose during the aqueous workup,
possibly due to instability of the cis seven-membered pyro-
phosphate of this compound.
[39]
Scheme 6. Synthesis of the perphosphorylated derivatives 58 and 59 of su-
crose (56): a) 1. (BnO)
2
PN(iPr)
2
3
CN, RT; 2. mCPBA,
CH
2
Cl
2
, 408C!RT; b) H
2
(3 atm), Pd/C, Et
3
N, EtOH/H
2
O (1:1), RT; c) Dowex
H
+
, H
2
O then Dowex Na
+
, H
2
O.
Scheme 7. Synthesis of the pyrophosphates 60-62 of the tetrakis phosphory-
lated methyl glycosides of glucose 39 and mannose 40 derivatives: a) DCC,
CH
3
CN, 828C; b) Dowex H
+
, H
2
O then Dowex Na
+
, H
2
O.
Glucose, mannose, and galactose pentakis phosphorylated
analogues lack an even number of phosphate groups, and
they are not suitable substrates for the formation of PPs.
Indeed, triethylammonium salts (46, 48, and 50) failed to form
pyrophosphates in a regiochemically controlled manner. The
same was expected for the lactose derivative 54 (Scheme 6),
because the glucose subunit lacks two pairs of vicinal phos-
phates. Interestingly, the sucrose-derived salt 58, with all its
phosphates properly positioned for the formation of four PPs,
also failed to give a clean reaction. A total of 80% of phos-
phates were transformed into PPs (based on
31
P NMR investiga-
tions of the crude mixture) only after prolonged heating. This
reluctance toward PP formation might be due to improper
spatial orientation of the phosphate groups.
Binding affinity of the compounds for and enhancement of
oxygen release from hemoglobin
As indicated above, the phosphorylated compounds BPG and
IHP as well as the trispyrophosphate ITPP (Figure 1) bind to Hb
and significantly enhance its oxygen release capacity. After
having successfully synthesized the tris, tetrakis, pentakis and
octakis phosphorylated carbohydrate derivatives and a bispyr-
ophosphate described herein, all final compounds were evalu-
ated for their effect on stripped human Hb. To determine their
ability to shift the oxygen saturation curve to higher pO
2
values, we assessed the partial pressure of oxygen for half-sat-
uration (P
50
) of Hb in the presence of the compounds and their
corresponding dissociation constants (K
d
) to Hb. The results are
depicted below in Table 2 and in Figures 27.
All compounds were able to shift the Hb oxygenation
curves up from 58 to 550%, and the relationship between
binding to Hb and oxygen release is illustrated in Figure 2. In
general, the ability of the compounds to lower the Hb affinity
for oxygen is directly related to the number of negative charg-
es present; that is, a greater number of phosphates gives a
higher P
50
value. For instance, octakisphosphate carbohydrates
55 (550%) and 59 (550%) were more effective than trisphos-
phate compounds 8 (113%) and 10 (144%). The same trend
was observed for the lead compounds BPG (I) < ITPP (III) <
IHP (II) (see Table 2 and Figures 27).
Benesch, Edalji, and Benesch
[40]
previously observed the
same phenomenon regarding the relationship between the
number of negative charges of allosteric effectors and oxygen
displacement from Hb. These observations are in agreement
with the fact that the allosteric pocket of Hb is particularly rich
in positively charged amino acid residues, located on the b
subunits at the entrance to the Hb central cavity,
[2]
which
would favor the tight docking of polyanions by electrostatic in-
teraction. As a result, with the number of negative charges
being directly proportional to the strength of electrostatic in-
teraction, a greater number would be expected to induce the
formation of a tighter Hbeffector complex.
Indeed, the direct correlation between the affinity of the
present compounds toward Hb and their ability to induce
oxygen release is consistent with the above. Figure 2 clearly
shows that the larger the P
50
shift, the higher the affinity of the
Table 2. P
50
values, Hill coefficients, and dissociation constants in stripped
human Hb for carbohydrate-derived polyphosphate allosteric effectors of
hemoglobin.
Compd P
50
[Torr]
[a]
P
50
shift [%]
[b]
Hill coeff.
[c]
K
d
[m]
[d]
Control 10.20.7 1.20.1
BPG (I) 17.51.0 71 1.20.1 8.410
5
ITPP (III) 22.20.4 118 1.60.1 1.710
7
IHP (II) 57.70.6 466 2.30.1 3.210
8
8 21.70.2 113 2.30.1 4.910
4
10 24.91.4 144 2.10.1 5.210
5
29 37.31.0 266 2.20.1 1.310
6
30 30.70.2 201 2.30.1 1.510
5
31 48.33.7 374 2.20.1 9.010
8
32 45.60.3 347 2.20.1 2.010
7
33 33.21.1 226 1.90.1 2.210
7
34 36.60.6 259 2.40.1 4.910
6
41 37.10.7 264 2.30.1 2.210
6
42 34.40.5 238 2.20.1 3.110
6
47 57.63.4 466 2.20.2 1.610
8
49 63.23.7 520 2.20.1 8.110
9
51 56.01.0 449 2.50.1 1.810
7
55 66.24.0 550 2.10.1 1.310
9
59 62.21.3 510 2.10.1 1.410
9
62 16.10.9 58 1.90.1 2.610
3
[a] P
50
values were assessed by linear regression analysis from data points
obtained between 40 and 60% blood oxygen saturation. For this screen-
ing evaluation, we elected a compound/Hb molar ratio of 20. [b] P
50
shift
was calculated from the ratio between P
50
values in the presence and ab-
sence of the compounds (control). [c] Hill coefficients were determined
from the corresponding oxygen saturation curves to evaluate the effect
of the compounds on Hboxygen binding cooperativity. [d] The dissocia-
tion constant (K
d
) was determined from the equation:
[2b, 40]
logP
50
=con-
stant +(1/n) log(1+C/K
d
), in which n is the Hill coefficient and C is the
concentration of the effector; standard error is between 5 and 10%.
Figure 2. Relationship between Hb-O
2
binding (P
50
) and dissociation con-
stants from Hb (K
d
) for compounds BPG (I), IHP (II), ITPP (III), 1-O-methyl-a-
glucose 2,3,4-trisphosphate (8), 1-O-methyl-a-mannose 2,3,4-trisphosphate
(10), a-glucose 1,2,3,4-tetrakisphosphate (29), b-glucose 1,2,3,4-tetrakisphos-
phate (30), a-mannose 1,2,3,4-tetrakisphosphate (31), b-mannose 1,2,3,4-tet-
rakisphosphate (32), a-galactose 1,2,3,4-tetrakisphosphate (33), b-galactose
1,2,3,4-tetrakisphosphate (34), 1-O-methyl-a-glucose tetrakisphosphate (41),
1-O-methyl-a-mannose tetrakisphosphate (42), a-glucose pentakisphosphate
(47), a-mannose pentakisphosphate (49), a-galactose pentakisphosphate
(51), lactose octakisphosphate (55), sucrose octakisphosphate (59), and 1-O-
methyl-a-glucose bispyrophosphate (62). The line corresponds to the linear
regression function (R
2
=0.795) for all compounds studied. All values were
taken from Table 2. The rectangles group the compounds according to the
number of negative charges (indicated in bold) in the fully ionized state.
compounds for Hb, which is directly linked to the number of
charges, that is, phosphate groups. This trend is represented
by the rectangles in Figure 2, which collect the compounds
that bear the same number of negative charges in their fully
ionized state. Higher charge may be expected to result in
tighter electrostatic docking of the compounds into the allo-
steric pocket of Hb.
With respect to the specificities observed within the distinct
series of hexopyranose polyphosphate compounds, mannose
derivatives (10, 31, 32, and 49) were generally more effective
than their corresponding glucose (8, 29, 30, and 47) and galac-
tose (33, 34, and 51) analogues (Figures 36). More specifically,
comparing the activity observed between analogous deriva-
tives of glucose and mannose, 1-O-methyl-a-glucose 2,3,4-tri-
sphosphate (8) and 1-O-methyl-a-mannose 2,3,4-trisphosphate
(10) exhibited quite similar P
50
values. The same was observed
for derivatives 41 and 42, meaning that C-2 equatorial versus
C-2 axial orientation of glucose and mannose, respectively, is
not important. However, the large enhancement of the activity
of the mannose derivatives 31, 32, and 49 versus glucose de-
rivatives 29, 30, and 47 may suggest that the C-1 phosphate in
conjunction with the axial C-2 phosphate of the mannose de-
rivative is most probably responsible for the effect, whereas
the presence of a C-6 phosphate is not so important (Figure 4).
Interestingly, compound 42, 1-O-methyl-a-mannose tetraki-
sphosphate, was less effective than the other mannose tetraki-
sphosphate derivatives (31 and 32). Such a decrease in effec-
tiveness could be due to the absence in 42 of the C-1 or the
presence of a C-6 phosphate group, which would be important
for the molecular recognition of the mannose polyphosphates.
However, as the C-6 phosphate does not affect the similar ac-
tivity between glucose tetrakis phosphorylated derivatives 41
and 29, 30, we may speculate that the C-1 phosphate group is
responsible for the enhancement of affinity with Hb in the
mannose series. Finally, the anomeric configuration of the
mannose derivatives seems to play a less important role
(Figure 5).
For the glucose derivatives, the dependence of the effective-
ness on the number of phosphates on the carbohydrate scaf-
fold seems to be more linear. The absence of either C-1 or C-6
phosphate groups leads to the same effect within the a-anom-
ers, that is, compounds 29 and 41 (266 and 264%, respective-
ly) ; however, the corresponding a-anomer of glucose 1,2,3,4-
tetrakisphosphate (30) was less effective (201%). Conversely,
within the galactose series, the b-anomer of galactose 1,2,3,4-
tetrakisphosphate (34) is more effective than its a-anomer 33
(259 versus 226%, respectively). Furthermore, compounds a-
glucose 1,2,3,4-tetrakisphosphate (29) and b-galactose 1,2,3,4-
tetrakisphosphate (34) showed similar effects on oxygen re-
lease.
The quite similar P
50
values between analogous derivatives
of glucose and galactose carbohydrates show that the C4-eq
versus C4-ax phosphate group does not primarily affect the
binding with Hb. Finally, the anomeric configuration seems to
play a role for glucose and galactose derivatives, yet less im-
portantly than the number of phosphate charges.
Figure 3. P
50
values for stripped human Hb and corresponding Hill coeffi-
cients for compounds BPG (I), ITPP (III), IHP (II), 1-O-methyl-a-glucose 2,3,4-
trisphosphate (8), and 1-O-methyl-a-mannose 2,3,4-trisphosphate (10). All
data were extracted from oxygen saturation curves, which were measured in
triplicate. Error bars represent the standard deviation.
Figure 4. P
50
cients for compounds IHP (II), a-glucose 1,2,3,4-tetrakisphosphate (29), b-
glucose 1,2,3,4-tetrakisphosphate (30), a-mannose 1,2,3,4-tetrakisphosphate
(31), b-mannose 1,2,3,4-tetrakisphosphate (32), a-galactose 1,2,3,4-tetraki-
sphosphate (33), and b-galactose 1,2,3,4-tetrakisphosphate (34). All data
were extracted from oxygen saturation curves, which were measured in trip-
licate. Error bars represent the standard deviation.
Figure 5. P
50
cients for compounds IHP (II), 1-O-methyl-a-glucose tetrakisphosphate (41),
1-O-methyl-a-mannose tetrakisphosphate (42), and 1-O-methyl-a-glucose
bispyrophosphate (62). All data were extracted from oxygen saturation
curves, which were measured in triplicate. Error bars represent the standard
deviation.
Octakisphosphate disaccharides 55 (550%) and 59 (510%)
were able to lower Hb affinity for oxygen in a moderately
higher fashion than IHP itself (466%, Figure 7), relative to mo-
nosaccharides 47 (466%), 49 (520%), and 51 (449%). The fact
that the latter compounds, which contain only five phosphate
groups, nevertheless induce allosteric effects similar to that of
IHP (Figure 6) might be related to access to the allosteric bind-
ing site. This suggests that for the disaccharides 55 and 59
(Figure 7), which are bulkier than the monosaccharides 47, 49,
and 51, steric effects also play a significant role (although only
secondary to electrostatic factors) regarding access to the
binding site.
As a result, the observed increment on activity for disacchar-
ides 55 and 59 is most probably related to the docking mode
of these compounds, as they are able to bind Hb through in-
teraction with either the hexopyranose or pentofuranose subu-
nits. As a result, the docking of these compounds to Hb is stat-
istically increased by their dual binding mode. Furthermore,
the latter may explain why there is only a slight difference in
activity between lactose 55 and sucrose 59, with the sugar
scaffold apparently playing a minor role in selectivity for phos-
phorylated disaccharides. The present data suggest that the
larger polyphosphate derivatives may interact with Hb both
inside and at the entry of the allosteric pocket.
The above analysis of the structureactivity relationships
provides an initial insight into the molecular recognition of
phosphorylated hexoses by Hb. Based on our results we can
list the roles of specific features for binding to Hb in the fol-
lowing order of decreasing importance: 1) electrostatic effects
related to the number of phosphate groups, 2) spatial disposi-
tion of the phosphates (stereochemistry at the corresponding
carbon atoms), and 3) secondary interactions involving hydro-
gen bonding and hydrophobic effects.
In addition, for the more extended structures, the spatial dis-
position of exo-cavity phosphates may also contribute through
a combination of the same three factors. Further exploration
using other types of phosphorylated compounds should allow
refinement of the picture provided by the present data. Such
studies will be presented in due course.
Conclusions
The synthesis of polyphosphate derivatives of hexopyranoses
provides a range of compounds that may have significant bio-
logical activities in various areas of medicinal research. In par-
ticular, they were found to act as allosteric effectors of human
hemoglobin, inducing an increase in oxygen release capacity.
The effects increase with the strength of binding to Hb, which
may be traced primarily to electrostatic interactions as they
follow the number of negative charges present. Stereochemi-
cal and steric factors also play a role, although a less important
one. Some compounds were found to present an even stron-
ger effect on oxygen release from Hb than the most efficient
compounds known. In view of the central role played by hypo-
xia in numerous types of disease, the exploration of phosphate
derivatives of saccharides and related compounds represents
an important avenue in the search for substances that act on
the oxygenation status of tissues. Such exploration may there-
fore have significant potential in the discovery and develop-
ment of a range of novel drug candidates.
Experimental Section
All chemicals were purchased from Sigma, Aldrich, or Fluka and
were used without further purification. The resins Dowex 50WX8
200 and Marathon C Na
+
were purchased from SigmaAldrich and
washed with distilled H
2
O before the first use.
1
H,
13
C and
31
P NMR
spectra were recorded on a Bruker AC-400 spectrometer. Mass
spectra were determined by the Service Commun de Spectrom-
trie de Masse at the Institut dIngnierie Supramolculaire. ITPP
(myo-inositol trispyrophosphate hexasodium salt) was manufac-
tured by Carbogen AMCIS (Switzerland) by following improved
synthetic procedures, derived from those previously described.
[5, 7]
BPG (2,3-bisphospho-d-glyceric acid pentasodium salt) was pur-
chased from Sigma (USA), and IHP (myo-inositol hexakisphosphate)
was purchased from SigmaAldrich (Italy).
General procedure for the phosphorylation reactions: In a solu-
tion of the carbohydrate (1 mmol) in DMF (20 mL) a 0.45m solution
of tetrazole in CH
3
CN (2.25 equiv for each OH group) and dibenzyl
Figure 6. P
50
cients for compounds IHP (II), a-glucose pentakisphosphate (47), a-mannose
pentakisphosphate (49), and a-galactose pentakisphosphate (51). All data
were extracted from oxygen saturation curves, which were measured in trip-
licate. Error bars represent the standard deviation.
Figure 7. P
50
cients for compounds IHP (III), lactose octakisphosphate (55), and sucrose
octakisphosphate (59). All data were extracted from oxygen saturation
curves, which were measured in triplicate. Error bars represent the standard
deviation.
N,N-diisopropylphosphoramidite (1.5 equiv for each OH group)
were added under Ar atmosphere at room temperature. The result-
ing slurry was vigorously stirred at room temperature for 24 h. The
mixture was then cooled to 408C, and a solution of 70% mCPBA
(1.75 equiv for each OH group) in CH
2
Cl
2
(1.5 mL per mmol
mCPBA) was added dropwise, and the mixture was left to stir for a
total of 12 h while it was allowed to warm to room temperature.
The mixture was then diluted with CH
2
Cl
2
(150 mL per mmol start-
ing material) and washed with a 10% aqueous solution of Na
2
SO
3
(210 mL per mmol mCPBA), a saturated aqueous solution of
NaHCO
3
(210 mL per mmol mCPBA), H
2
O (5 mL per mmol
mCPBA) and saturated brine (5 mL per mmol mCPBA). The organic
phase was dried (MgSO
4
) and the solvents were removed under re-
duced pressure. The obtained residue was purified by flash column
chromatography. Note that DMF was used for the naked sugars. In
case of the 6-O-TBDPS protected sugars, the tetrazole solution in
CH
3
CN was directly poured into the flask containing the carbohy-
drate derivative and thus CH
3
CN was used as a solvent for the
sugar as well.
General procedure for the hydrogenation reactions; preparation
of triethylammonium salts: Benzyl phosphate (1 mmol) was dis-
solved in a 1:1 mixture of EtOH and H
2
O (60 mL). Et
3
N (5 equiv for
each phosphate) was added to the resulting emulsion followed by
10% Pd/C (0.3 g for each phosphate). This mixture was left to vigo-
rously stir under H
2
atmosphere (1 atm) or shaken in a high pres-
sure hydrogenator (3 atm) at room temperature for 24 h. The cata-
lyst was removed by filtration through an LCR/PTFE hydrophilic
membrane (0.5 mm), and the filtrate was washed with a 1:1 mixture
of EtOH and H
2
O (250 mL per mmol starting material). The com-
bined filtrates were evaporated under reduced pressure (608C),
and the obtained residue was dried under high vacuum to give
the corresponding triethylammonium salt.
General procedure for the hydrogenation reactions; direct prep-
aration of sodium salts: The above described procedure was used
for the preparation of sodium salts, but NaHCO
3
(1 equiv for each
phosphate) was used instead of Et
3
N.
General procedure for the synthesis of sodium phosphates from
the triethylammonium phosphates: A solution of ammonium salt
of phosphate (1 mmol) in H
2
O (10 mL) was passed through a
column containing Dowex H
+
and eluted with distilled H
2
O until
all the acidic fractions were collected. They were then poured into
a flask containing Dowex Na
+
(50 g for both resins of the same ca-
pacity). The mixture was stirred for 30 min, filtered off through a
sintered funnel, the resin was washed with distilled H
2
O (2
30 mL), and the clear solution was evaporated to dryness. Alterna-
tively, the acidic fractions could be adjusted to neutral pH upon ti-
tration with a solution of NaOH. For some ammonium salts a
single direct passage through Dowex Na
+
was sufficient for the ex-
change of the countercations.
General procedure for the synthesis of sodium pyrophosphates
from the triethylammonium pyrophosphates: The above de-
scribed procedure was used for the preparation of these sodium
salts as well.
General procedure for the silylation reactions: The desired carbo-
hydrate (1 mmol) was dissolved in dry DMF (20 mL) and cooled to
08C under Ar. Et
3
N (1.4 equiv) and DMAP (10 mg) were added, fol-
lowed by the slow addition (2 h) of TBDPSCl (1 mmol). The reaction
mixture was allowed to warm to room temperature and was left
stirring for 24 h. EtOAc (50 mL) was added, and the mixture was
washed with H
2
O (250 mL) and saturated brine (250 mL). The
organic layer was dried (Na
2
SO
4
), the solvents were removed under
reduced pressure, and the obtained residue was purified with
column chromatography.
General procedure for the desilylation reactions: The silylated
compound (1 mmol) was dissolved in THF (40 mL), and the mixture
was cooled to 08C. A mixture of TBAF (5 mmol, 1m in THF) and
AcOH (5 mmol) dissolved in ice-cold THF (20 mL) was then added
dropwise under Ar over a period of 1 h. The reaction mixture was
allowed to warm to room temperature and was stirred for 512 h
(checked by TLC). EtOAc (50 mL) was added, and the organic
phases were washed with H
2
O (50 mL) and saturated brine
(50 mL). The organic phase was dried (MgSO
4
), and the solvents
were removed under reduced pressure. The obtained residue was
purified by flash column chromatography.
General procedure for the formation of pyrophosphates: The
triethylammonium salt of the carbohydrate phosphate (1 mmol)
was dissolved in a mixture of CH
3
CN/H
2
O in a ratio 2:1 (45 mL), or
in neat CH
3
CN (30 mL), and N,N-dicyclohexylcarbodiimide (DCC;
1 equiv for each phosphate) was added in one portion. The mix-
ture was stirred at reflux for 1824 h, and then cooled and concen-
trated under vacuum. H
2
O (230 mL) was added, and the N,N-dicy-
clohexylurea was filtered off through a sintered funnel. The filtrate
was concentrated under vacuum to give the pure triethylammoni-
um salt of the pyrophosphate.
Hexabenzyl-2,3,4-(6-O-tert-butyldiphenylsilyl-1-O-methyl-a-d-
glucopyranosyl) trisphosphate (3): Flash column chromatography
(heptanes ! 40% EtOAc in heptanes), thick colorless oil (76%):
R
f
=0.24 (EtOAc/heptanes, 2:3) ; [a]
20
D
=+29.9 (c=1.0 in CHCl
3
) ;
1
H NMR (CDCl
3
, 400 MHz): d=7.79 (d, J =8.0 Hz, 2H), 7.77 (d, J =
8.5 Hz, 2H), 7.477.15 (m, 36H), 5.25 (d, J =3.4 Hz, 1H, H-1), 5.19
5.04 (m, 10H, 9CHHPh, H-3), 5.024.90 (m, 2H, CH
2
Ph), 4.85 (dd,
J =11.7, 9.6 Hz, 1H, CHHPh), 4.55 (q, J =9.5 Hz, 1H, H-4), 4.40 (ddd,
J =9.5, 5.6, 3.8 Hz, 1H, H-2), 4.17 (d, J =9.6 Hz, 1H, H-6), 4.073.96
(m, 2H, H-5, H-6), 3.43 (s, 3H, OCH
3
), 1.16 (s, 9H, C(CH
3
)
3
);
13
C NMR
(CDCl
3
, 100 MHz): d=135.9 (d,
3
J =7.3 Hz), 135.6135.3 (m), 133.3,
133.2, 129.4, 128.4127.2 (m), 96.7 (C-1), 76.7 (bs, C-3), 75.0 (d,
2
J
CP
=3.3 Hz, C-2), 74.1 (br s, C-4), 70.5 (br d,
3
J
CP
=2.6 Hz, C-5), 69.5
69.1 (m, CH
2
Ph), 62.5 (s, C-6), 55.0 (OCH
3
), 26.6 (C(CH
3
)
3
), 19.0
(C(CH
3
)
3
) ;
31
P NMR (162 MHz): d=1.02, 1.81, 2.06; HRMS (ESI):
m/z [M+Li]
+
calcd for C
65
H
71
LiO
15
P
3
Si : 1219.3930, found: 1219.3939.
Hexabenzyl-2,3,4-(1-O-methyl-a-d-glucopyranosyl) trisphos-
phate (5): Flash column chromatography (90% EtOAc in heptanes
! EtOAc), thick colorless oil (84%): R
f
=0.17 (EtOAc/heptanes, 2:1);
[a]
20
D
=+34.9 (c=1.0 in CH
2
Cl
2
);
1
H NMR (CDCl
3
, 400 MHz): d=
7.357.15 (m, 30H), 5.16 (d, J =3.5 Hz, 1H, H-1), 5.124.91 (m, 13H,
CH
2
Ph, H-3), 4.48 (q, J =9.5 Hz, 1H, H-4), 4.36 (ddd, J =9.8, 6.4,
3.6 Hz, 1H, H-2), 4.02 (d, J =13.2 Hz, 1H, H-6), 3.76 (d, J =13.0 Hz,
1H, H-6), 3.69 (d, J =9.8 Hz, 1H, H-5), 3.36 (s, 3H, OCH
3
) ;
13
C NMR
(CDCl
3
, 100 MHz): d=135.75 (d,
3
J =7.1 Hz), 135.65 (d,
3
J
CP
=7.5 Hz),
135.4 (d,
3
J
CP
=7.5 Hz), 135.3 (d,
3
J
CP
=6.1 Hz), 135.1 (d,
3
J
CP
=
7.0 Hz), 128.4127.4 (m), 96.3 (C-1), 76.175.8 (m, C-3), 74.8 (d,
2
J
CP
=3.9 Hz, C-2), 72.7 (br s, C-4), 70.2 (br s, C-5), 70.1 (d,
2
J
CP
=
5.8 Hz, 1CH
2
Ph), 69.8 (d,
2
J
CP
=5.9 Hz, 1CH
2
Ph), 69.5 (d,
2
J
CP
=
5.6 Hz, 1CH
2
Ph), 69.4 (d,
2
J
CP
=5.6 Hz, 1CH
2
Ph), 69.3 (d,
2
J
CP
=
5.5 Hz, 1CH
2
Ph), 69.1 (d,
2
J
CP
=5.4 Hz, 1CH
2
Ph), 59.9 (C-6), 55.3
(OCH
3
);
31
P NMR (162 MHz): d=0.14, 0.92, 1.99; HRMS (ESI): m/z
[M+Li]
+
calcd for C
49
H
53
LiO
15
P
3
: 981.2753, found: 981.2873.
Tris(triethylammonium)-2,3,4-(1-O-methyl-a-d-glucopyranosyl)
trisphosphate (7): Glassy pale-brown solid (99%):
1
H NMR (D
2
O,
400 MHz): d=4.85 (d, J =3.5 Hz, 1H, H-1), 4.32 (q, J =9.2 Hz, 1H, H-
3), 3.98 (dt, J =9.4, 3.6 Hz, 1H, H-2), 3.92 (q, J =9.6 Hz, 1H, H-4),
3.68 (d, J =2.9 Hz, 2H, H-6, H-6), 3.61 (dt, J =9.9, 3.0 Hz, 1H, H-5),
3.28 (s, 3H, OCH
3
), 3.04 (q, J =7.3 Hz, 18H, CH
2
CH
3
), 1.13 (t, J =
7.3 Hz, 27H, CH
2
CH
3
) ;
13
C NMR (D
2
O, 100 MHz): d=97.9 (C-1), 76.5
(br s, C-3), 73.8 (d,
2
J
CP
=5.4 Hz, C-2), 72.7 (br s, C-4), 70.5 (br s, C-5),
60.2 (C-6), 55.0 (OCH
3
), 46.6 (CH
2
CH
3
), 8.2 (CH
2
CH
3
);
31
P NMR
(162 MHz): d=0.34, 0.07, 0.39.
Trisodium-2,3,4-(1-O-methyl-a-d-glucopyranosyl) trisphosphate
(8): Pale-white solid (96%): [a]
20
D
=+56.4 (c=0.5 in H
2
O);
1
H NMR
(D
2
O, 400 MHz): d=4.91 (d, J =3.7 Hz, 1H, H-1), 4.35 (q, J =9.3 Hz,
1H, H-3), 4.02 (dt, J =9.5, 3.6 Hz, 1H, H-2), 3.98 (q, J =9.5 Hz, 1H,
H-4), 3.74 (d, J =3.2 Hz, 2H, H-6, H-6), 3.67 (br dd, J =9.8, 3.2 Hz,
1H, H-5), 3.34 (s, 3H, OCH
3
) ;
13
C NMR (D
2
O, 100 MHz): d=97.9 (C-
1), 76.5 (br s, C-3), 73.8 (br s, C-2), 72.7 (br d,
2
J
CP
=4.9 Hz, C-4), 70.5
(br d,
3
J
CP
=3.6 Hz, C-5), 60.2 (C-6), 55.1 (OCH
3
) ;
31
P NMR (162 MHz):
d=0.59, 0.29, 0.16; HRMS (ESI): m/z [MNa]
calcd for
C
7
H
14
Na
2
O
15
P
3
: 476.9335, found: 476.9342.
Hexabenzyl-2,3,4-(6-O-tert-butyldiphenylsilyl-1-O-methyl-a-d-
mannopyranosyl) trisphosphate (4): Flash column chromatogra-
phy (heptanes ! 40% EtOAc in heptanes), thick colorless oil
(73%): R
f
20
D
=+1.3 (c=1.0 in
CHCl
3
) ;
1
H NMR (CDCl
3
, 400 MHz): d=7.797.74 (m, 4H), 7.407.14
(m, 36H), 5.174.79 (m, 16H, CH
2
Ph, H-1, H-2, H-3, H-4), 4.17 (d, J =
10.9 Hz, 1H, H-6), 4.02 (dd, J =11.3, 6.3 Hz, 1H, H-6), 3.91 (br t, J =
7.2, Hz, 1H, H-5), 3.43 (s, 3H, OCH
3
), 1.12 (s, 9H, C(CH
3
)
3
) ;
13
C NMR
(CDCl
3
, 100 MHz): d=136.0135.5 (m), 133.6, 133.5, 129.6, 128.6
127.5 (m), 98.4 (C-1), 75.0 (d,
2
J
CP
=4.6 Hz, C-2), 74.6 (br s, C-3), 72.3
(d,
3
J
CP
=4.3 Hz, C-5), 72.0 (t, J
CP
=6.5 Hz, C-4), 69.869.2 (m, CH
2
Ph),
62.9 (C-6), 55.0 (OCH
3
), 26.8 (C(CH
3
)
3
), 19.3 (C(CH
3
)
3
);
31
P NMR
(162 MHz): d=1.23, 1.44, 1.91; HRMS (ESI): m/z [M+Li]
+
calcd
for C
65
H
71
LiO
15
P
3
Si : 1219.3930, found: 1219.3778.
Hexabenzyl-2,3,4-(1-O-methyl-a-d-mannopyranosyl) trisphos-
phate (6): Flash column chromatography (EtOAc), thick colorless
oil (84%): R
f
20
D
=12.4 (c=1.0 in
CH
2
Cl
2
) ;
1
H NMR (CDCl
3
, 400 MHz): d=7.417.19 (m, 30H), 5.15
4.89 (m, 16H, CH
2
Ph, H-1, H-2, H-3, H-4), 4.04 (dd, J =13.2, 2.3 Hz,
1H, H-6), 3.86 (d, J =12.9 Hz, 1H, H-6), 3.69 (br d, J =8.7 Hz, 1H, H-
5), 3.35 (s, 3H, OCH
3
) ;
13
C NMR (CDCl
3
, 100 MHz): d=135.3134.8
(m), 128.3127.5 (m), 98.4 (C-1), 74.6 (d,
2
J
CP
=4.3 Hz, C-2), 73.9
73.7 (m, C-3), 71.3 (d,
3
J
CP
=2.1 Hz, C-5), 70.8 (dd, J
CP
=6.8, 5.0 Hz,
C-4), 69.869.2 (m, CH
2
Ph), 60.3 (C-6), 54.9 (OCH
3
) ;
31
P NMR
(162 MHz): d=0.04, 1.19, 1.92; HRMS (ESI): m/z [M+Li]
+
calcd
for C
49
H
53
LiO
15
P
3
: 981.2752, found: 981.2668.
Tris(triethylammonium)-2,3,4-(1-O-methyl-a-d-mannopyranosyl)
trisphosphate (9): Glassy pale-brown solid, (0.49 g, 99%):
1
H NMR
(D
2
O, 400 MHz): d=4.79 (s, 1H, H-1), 4.384.29 (m, 2H, H-2, H-3),
4.17 (q, J =9.6 Hz, 1H, H-4), 3.72 (d, J =3.3 Hz, 1H, H-6, H-6), 3.61
(dt, J =9.8, 3.2 Hz, 1H, H-5), 3.28 (s, 3H, OCH
3
), 3.05 (q, J =7.3 Hz,
18H, CH
2
CH
3
), 1.14 (t, J =7.3 Hz, 27H, CH
2
CH
3
) ;
13
C NMR (D
2
O,
100 MHz): d=99.3 (C-1), 73.4 (br s, C-3), 73.2 (d,
2
J
CP
=3.6 Hz, C-2),
71.9 (d,
3
J
CP
=4.22 Hz, C-5), 70.2 (t, J
CP
=5.4 Hz, C-4), 60.5 (C-6), 54.9
(OCH
3
), 46.6 (CH
2
CH
3
), 8.2 (CH
2
CH
3
) ;
31
P NMR (162 MHz): d=0.08
(2P), 0.30 (1P).
Trisodium-2,3,4-(1-O-methyl-a-d-mannopyranosyl trisphosphate
(10): Pale-white solid, (99%): all data were in accordance with pub-
lished data.
[17]
6-O-tert-Butyldiphenylsilyl-d-glucopyranose (14):
[31]
Flash column
chromatography (EtOAc ! 2% CH
3
OH in EtOAc) as a white solid
(75%), ratio of a/b anomers 1/0.8: R
f
=0.29 (2% CH
3
OH in EtOAc).
Octabenzyl-1,2,3,4-(6-O-tert-butyldiphenylsilyl-a-d-glucopyrano-
syl) tetrakisphosphate (17) and octabenzyl-1,2,3,4-(6-O-tert-bu-
tyldiphenylsilyl-b-d-glucopyranosyl) tetrakisphosphate (18):
Flash column chromatography (heptanes ! 40% EtOAc in hep-
tanes) to afford first the a-anomer 17 (52%) and subsequently the
b-anomer 18 (30%) as thick colorless oils. 17: R
f
=0.20 (EtOAc/hep-
tanes, 2:1) ; [a]
20
D
=+42.8 (c=0.5 in CHCl
3
) ;
1
H NMR (CDCl
3
,
400 MHz): d=7.78 (d, J =7.6 Hz, 2H), 7.75 (d, J =7.5 Hz, 2H), 7.48
7.20 (m, 46H), 6.30 (dd, J =5.4, 3.3 Hz, 1H, H-1), 5.264.98 (m, 17H,
CH
2
Ph, H-3), 4.934.82 (m, 1H, H-4), 4.534.46 (m, 1H, H-2), 4.18
4.04 (m, 2H, H-5, H-6), 3.86 (br d, J =11.4 Hz, 1H, H-6), 1.17 (s, 9H,
C(CH
3
)
3
);
13
C NMR (CDCl
3
, 100 MHz): d=135.7135.0 (m), 133.2,
132.9, 129.3, 129.3, 128.3127.2 (m), 94.4 (d,
2
J
CP
=5.3 Hz, C-1), 75.8
(br s, C-3), 74.0 (br t, J
CP
=6.5 Hz, C-2), 72.8 (br s, C-4), 72.3 (br s, C-5),
69.5 (d,
2
J
CP
=5.7 Hz, 1CH
2
Ph), 69.4 (d,
2
J
CP
=5.4 Hz, 1CH
2
Ph),
69.368.1 (m, 4CH
2
Ph), 69.1 (d,
2
J
CP
=5.4 Hz, 1CH
2
Ph), 69.0 (d,
2
J
CP
=5.4 Hz, 1CH
2
Ph), 61.2 (C-6), 26.6 (C(CH
3
)
3
), 19.0 (C(CH
3
)
3
) ;
31
P NMR (162 MHz): d=1.11, 1.63, 2.28, 2.58; HRMS (ESI): m/
z [M+Na]
+
calcd for C
78
H
82
NaO
18
P
4
Si : 1481.4113, found: 1481.3956.
18: R
f
=0.075 (EtOAc/heptanes, 2:1); [a]
20
D
=+7.5 (c=1.0 in CHCl
3
) ;
1
H NMR (CDCl
3
, 400 MHz): d=7.727.66 (m, 4H), 7.407.14 (m,
46H), 5.52 (t, J =6.9 Hz, 1H, H-1), 5.134.67 (m, 19H, CH
2
Ph, H-2, H-
3, H-4), 4.09 (dd, J =11.7, 2.8 Hz, 1H, H-6), 3.99 (dd, J =11.7, 5.4 Hz,
1H, H-6), 3.903.83 (m, 1H, H-5), 1.08 (s, 9H, C(CH
3
)
3
) ;
13
C NMR
(CDCl
3
, 100 MHz): d=135.8135.2 (m), 133.1, 132.9, 129.6, 129.6,
128.4127.5 (m), 96.2 (t, J
CP
=4.3 Hz, C-1), 77.7 (br d,
2
J
CP
=4.4 Hz, C-
3), 76.376.0 (m, C-2, C-5), 73.2 (br s, C-4), 69.769.3 (m, CH
2
Ph),
62.7 (C-6), 26.7 (C(CH
3
)
3
), 19.2 (C(CH
3
)
3
) ;
31
P NMR (162 MHz): d=
1.34, 1.87, 2.01, 2.78; HRMS (ESI): m/z [M+K]
+
calcd for
C
78
H
82
KO
18
P
4
Si : 1497.3852, found: 1497.3958.
Octabenzyl-1,2,3,4-a-d-glucopyranosyl tetrakisphosphate (23):
Flash column chromatography (EtOAc ! 5% CH
3
OH in EtOAc), as
a thick colorless oil (61%): R
f
20
D
=
+30.2 (c=2.5 in CHCl
3
) ;
1
H NMR (CDCl
3
, 400 MHz): d=7.397.15
(m, 40H), 6.18 (dd, J =5.7, 3.4 Hz, 1H, H-1), 5.154.95 (m, 17H,
CH
2
Ph, H-3), 4.51 (q, J =9.7 Hz, 1H, H-4), 4.484.40 (m, 1H, H-2),
3.89 (dd, J =13.6, 2.0, 1H, H-6), 3.83 (br d, J =10.0 Hz, 1H, H-5), 3.60
(br d, J =13.0 Hz, 1H, H-6);
13
C NMR (CDCl
3
, 100 MHz): d=135.8
135.0 (m), 128.6127.5 (m), 94.8 (d,
2
J
CP
=5.8 Hz, C-1), 75.2 (br d,
2
J
CP
=6.7 Hz, C-3), 74.274.0 (m, C-2), 72.5 (br s, C-5), 71.9 (br s, C-4),
70.2 (d,
2
J
CP
=5.7 Hz, 1CH
2
Ph), 70.0 (d,
2
J
CP
=5.7 Hz, 1CH
2
Ph),
69.8 (d,
2
J
CP
=5.5 Hz, 1CH
2
Ph), 69.769.4 (m, 4CH
2
Ph), 69.3 (d,
2
J
CP
=5.5 Hz, 1CH
2
Ph), 59.4 (C-6);
31
P NMR (162 MHz): d=0.16,
0.96, 1.49, 2.70; HRMS (ESI): m/z [M+Na]
+
calcd for
C
62
H
64
NaO
18
P
4
: 1243.2935, found: 1243.3035.
Tetrasodium-1,2,3,4-a-d-glucopyranosyl tetrakisphosphate (29):
White solid (99%): [a]
20
D
=+69.0 (c=0.5 in H
2
O);
1
H NMR (D
2
O,
400 MHz): d=5.57 (dd, J =7.2, 3.1 Hz, 1H, H-1), 4.43 (q, J =9.2 Hz,
1H, H-3), 4.103.99 (m, 2H, H-2, H-4), 3.90 (br d, J =9.9, 1H, H-5),
3.823.70 (m, 2H, H-6, H-6) ;
13
C NMR (D
2
O, 100 MHz): d=93.3 (d,
2
J
CP
=3.4 Hz, C-1), 76.1 (br s, C-3), 73.8 (br s, C-2), 72.4 (d,
2
J
CP
=
5.6 Hz, C-4), 71.7 (C-5), 60.1 (C-6);
31
P NMR (D
2
O, 162 MHz): d=0.85,
0.51, 0.095, 1.21; HRMS (ESI): m/z [M+H]
+
calcd for
C
6
H
13
Na
4
O
18
P
4
: 588.8638, found: 588.8600.
Octabenzyl-1,2,3,4-b-d-glucopyranosyl tetrakisphosphate (24):
Flash column chromatography (95% EtOAc in heptanes), thick col-
orless oil (64%): R
f
20
D
=6.4 (c=
2.0 in CHCl
3
) ;
1
H NMR (CDCl
3
, 400 MHz): d=7.397.19 (m, 40H),
5.48 (t, J =6.5 Hz, 1H, H-1), 5.184.78 (m, 16H, CH
2
Ph), 4.784.62
(m, 3H, H-2, H-3, H-4), 3.95 (dd, J =13.6, 2.5 Hz, 1H, H-6), 3.83 (d,
J =12.3 Hz, 1H, H-6), 3.56 (br d, J =9.1 Hz, 1H, H-5) ;
13
C NMR
(CDCl
3
, 100 MHz): d=135.7135.0 (m), 128.4127.6 (m), 96.1 (br t,
J
CP
=3.1 Hz, C-1), 77.5 (d,
2
J
CP
=5.7 Hz, C-3), 76.375.9 (m, C-2), 75.3
(br s, C-5), 72.0 (br s, C-4), 70.05 (d,
2
J
CP
=5.6 Hz, 1CH
2
Ph), 70.0 (d,
2
J
CP
=5.5 Hz, 1CH
2
Ph), 69.669.2 (m, 6CH
2
Ph), 59.8 (C-6);
31
P NMR (162 MHz): d=0.05, 1.02, 1.76, 2.95; HRMS (ESI): m/z
[M+Na]
+
calcd for C
62
H
64
NaO
18
P
4
: 1243.2935, found: 1243.2935.
Tetrasodium-1,2,3,4-b-d-glucopyranosyl tetrakisphosphate (30):
White solid (99%): [a]
20
D
=+7.0 (c=0.5 in H
2
O);
1
H NMR (D
2
O,
400 MHz): d=4.96 (t, J =7.9 Hz, 1H, H-1), 4.24 (q, J =9.2 Hz, 1H, H-
3), 3.983.87 (m, 2H, H-2, H-4), 3.80 (br d, J =12.6, 1H, H-6), 3.67
(dd, J =12.8, 5.3, 1H, H-6), 3.54 (dd, J =9.1, 4.8, 1H, H-5);
13
C NMR
(D
2
O, 100 MHz): d=96.496.2 (m, C-1), 78.878.6 (m, C-3), 77.3
77.1 (m, C-2), 75.5 (d,
3
J
CP
=4.0 Hz, C-5), 72.572.35 (m, C-4), 60.5
(C-6);
31
P NMR (D
2
O, 162 MHz): d=1.28, 0.36, 0.01, 0.84; HRMS
(ESI): m/z [MNa]
calcd for C
6
H
12
Na
3
O
18
P
4
: 564.8662, found:
564.8681.
6-O-tert-Butyldiphenylsilyl-d-mannopyranose (15): Flash column
3
OH in EtOAc). White solid
(78%), ratio of a/b anomers 1:0.6: R
f
=0.29 (2% CH
3
OH in EtOAc) ;
1
H NMR (CDCl
3
, 400 MHz): d=7.727.60 (m, 6.4H), 7.407.30 (m,
9.6H), 5.09 (s, 1H), 4.50 (s, 0.6H), 3.973.73 (m, 13.8H), 3.64 (t, J =
9.3 Hz, 1H), 3.39 (dd, J =9.2, 2.6 Hz, 0.6H), 3.253.16 (m, 0.6H), 1.03
(s, 14.4H);
13
C NMR (CDCl
3
, 100 MHz): d=135.5, 133.1, 132.95,
129.8, 129.8, 127.7, 127.7, 94.1, 93.9, 74.8, 74.0, 71.5, 71.0, 70.6,
69.4, 65.15, 65.0, 26.8, 19.15, 19.1; HRMS (ESI): m/z: [M+Li]
+
calcd
for C
22
H
30
LiO
6
Si : 425.1966, found: 425.1949.
Octabenzyl-1,2,3,4-(6-O-tert-butyldiphenylsilyl-a-d-mannopyra-
nosyl) tetrakisphosphate (19) and octabenzyl 1,2,3,4-(6-O-tert-
butyldiphenylsilyl)-b-d-mannopyranosyl) tetrakisphosphate (20):
tanes) to yield a-anomer 19 (50%) and (60% EtOAc in heptanes)
to yield b-anomer 20 (36%) as thick colorless oils: 19: R
f
=0.24
(EtOAc/heptanes, 1:1) ; [a]
20
D
=+8.3 (c=2.4 in CHCl
3
) ;
1
H NMR
(CDCl
3
, 400 MHz): d=7.78 (d, J =6.7 Hz, 4H), 7.487.20 (m, 46H),
6.08 (dd, J =5.9, 1.9 Hz, 1H, H-1), 5.31 (q, J =9.5 Hz, 1H, H-4), 5.28
(dd, J =8.2, 2.5 Hz, 1H, H-2), 5.205.00 (m, 16H, 15CHHPh, H-3),
4.90 (dd, J =11.8, 9.0 Hz, 1H, CHHPh), 4.08 (dd, J =11.9, 3.3 Hz, 1H,
H-6), 4.01 (bd, J =9.7 Hz, 1H, H-5), 3.86 (br d, J =10.6 Hz, 1H, H-6),
1.13 (s, 9H, C(CH
3
)
3
) ;
13
C NMR (CDCl
3
, 100 MHz): d=135.7135.0
(m), 133.4, 133.0, 129.4, 128.5127.3 (m), 95.4 (br d,
2
J
CP
=5.7 Hz, C-
1), 74.374.1 (m, C-2), 73.873.5 (m, C-3, C-5), 70.670.4 (m, C-4),
69.869.1 (m, 7CH
2
Ph), 67.0 (d,
2
J
CP
=5.4 Hz, 1CH
2
Ph), 61.5 (s, C-
6), 26.6 (C(CH
3
)
3
), 19.1 (C(CH
3
)
3
);
31
P NMR (162 MHz): d=1.28,
1.67, 1.87, 3.05; HRMS (ESI): m/z [M+Li]
+
calcd for
C
78
H
82
LiO
18
P
4
Si : 1465.4375, found: 1465.4205. 20: R
f
=0.14 (EtOAc/
heptanes, 1:1) ; [a]
20
D
=10.4 (c=1.0 in CHCl
3
) ;
1
H NMR (CDCl
3
,
400 MHz): d=7.807.73 (m, 4H), 7.407.15 (m, 46H), 5.55 (d, J =
8.0 Hz, 1H, H-1), 5.39 (dd, J =9.2, 2.4 Hz, 1H, H-2), 5.314.91 (m,
16H, 15CHHPh, H-4), 4.86 (dd, J =11.8, 9.2 Hz, 1H, CHHPh), 4.69
(br t, J =9.0 Hz, 1H, H-3), 4.184.12 (m, 2H, H-6, H-6), 3.70 (br d, J =
5.3 Hz, 1H, H-5), 1.14 (s, 9H, C(CH
3
)
3
) ;
13
C NMR (CDCl
3
, 100 MHz): d
135.8135.2 (m), 133.2, 132.9, 129.5, 128.4127.4 (m), 94.0 (br s, C-
1), 76.1 (br d,
3
J
CP
=4.4 Hz, C-5), 75.7 (br s, C-3), 75.575.2 (m, C-2),
70.9 (t, J
CP
=5.8 Hz, C-4), 69.969.2 (m, CH
2
Ph), 62.3 (C-6), 26.6
(C(CH
3
)
3
), 19.1 (C(CH
3
)
3
) ;
31
P NMR (162 MHz): d=1.28, 1.46,
2.06, 2.49; HRMS (ESI): m/z [M+Li]
+
calcd for C
78
H
82
LiO
18
P
4
Si :
1465.4375, found: 1465.4283.
Octabenzyl-1,2,3,4-a-d-mannopyranosyl tetrakisphosphate (25):
tanes). Thick colorless oil (60%): R
f
[a]
20
D
=0.2 (c=1.0 in CHCl
3
) ;
1
H NMR (CDCl
3
, 400 MHz): d=7.32
7.13 (m, 40H), 5.90 (d, J =4.5 Hz, 1H, H-1), 5.104.90 (m, 17H,
CH
2
Ph, H-2), 4.88 (q, J =9.5 Hz, 1H, H-4), 4.844.78 (m, 1H, H-3),
3.82 (br d, J =13.0 Hz, 1H, H-6), 3.76 (br d, J =9.5 Hz, 1H, H-5), 3.61
(d, J =13.2 Hz, 1H, H-6) ;
13
C NMR (CDCl
3
, 100 MHz): d=135.5
135.0 (m), 128.8128.3 (m), 128.2127.6 (m), 95.6 (d,
2
J
CP
=5.1 Hz,
C-1), 74.574.2 (m, C-2), 73.6 (br s, C-5), 73.373.1 (m, C-3), 70.2
70.1 (m, C-4), 70.169.5 (m, CH
2
Ph), 60.0 (C-6);
31
P NMR (162 MHz):
d=0.12, 1.34, 1.90, 3.34; HRMS (ESI): m/z: [M+Na]
+
calcd for
C
62
H
64
NaO
18
P
4
: 1243.2935, found: 1243.2767.
Tetrasodium-1,2,3,4-a-d-mannopyranosyl tetrakisphosphate
(31): White solid (99%): [a]
20
D
=+19.8 (c=0.5 in H
2
O);
1
H NMR (D
2
O,
400 MHz): d=5.45 (d, J =7.7 Hz, 1H, H-1), 4.444.37 (m, 2H, H-2, H-
3), 4.22 (q, J =9.6 Hz, 1H, H-4), 3.81 (br d, J =10.1, 1H, H-5), 3.79
3.65 (m, 2H, H-6, H-6) ;
13
C NMR (D
2
O, 100 MHz): d=94.3 (d,
2
J
CP
=
3.2 Hz, C-1), 73.873.6 (m, C-2), 72.9 (br s, C-3, C-5), 69.8 (br s, C-4),
60.3 (C-6);
31
P NMR (D
2
O, 162 MHz): d=0.61 (2P), 0.01, 1.87;
HRMS (ESI): m/z [MNa]
calcd for C
6
H
12
Na
3
O
18
P
4
: 564.8662, found:
564.8746.
Octabenzyl-1,2,3,4-b-d-mannopyranosyl tetrakisphosphate (26):
Flash column chromatography (EtOAc ! 5% CH
3
OH in EtOAc),
thick colorless oil (70%): R
f
20
D
=
25.0 (c=1.0 in CHCl
3
) ;
1
H NMR (CDCl
3
, 400 MHz): d=7.347.15 (m,
40H), 5.34 (d, J =7.3 Hz, 1H, H-1), 5.19 (dd, J =9.0, 2.2 Hz, 1H, H-2),
5.134.90 (m, 16H, CH
2
Ph), 4.88 (q, J =9.6 Hz, 1H, H-4), 4.51 (br t,
J =9.1 Hz, 1H, H-3), 3.90 (dd, J =13.2, 2.1, 1H, H-6), 3.79 (br d, J =
13.2 Hz, 1H, H-6), 3.38 (br d, J =9.2 Hz, 1H, H-5) ;
13
C NMR (CDCl
3
,
100 MHz): d=136.0135.0 (m), 128.7127.5 (m), 94.394.1 (m, C-1),
76.0 (br s, C-5), 75.475.3 (m, C-2, C-3), 70.369.2 (m, CH
2
Ph, C-4),
60.3 (C-6);
31
P NMR (162 MHz): d=0.19, 1.08, 2.02, 3.03; HRMS
(ESI): m/z [M+Li]
+
calcd for C
62
H
64
LiO
18
P
4
: 1227.3198, found:
1227.3066.
Tetrasodium-1,2,3,4-b-d-mannopyranosyl tetrakisphosphate
(32): White solid (99%): [a]
20
D
=12.6 (c=0.5 in H
2
O);
1
H NMR (D
2
O,
400 MHz): d=5.15 (d, J =8.4 Hz, 1H, H-1), 4.57 (dd, J =9.9, 1.5 Hz,
1H, H-2), 4.25 (br t, J =8.7 Hz, 1H, H-3), 4.13 (q, J =9.6 Hz, 1H, H-4),
3.82 (dd, J =12.6, 1.4 Hz, 1H, H-6), 3.71 (dd, J =12.7, 5.8 Hz, 1H, H-
6), 3.50 (dd, J =8.2, 5.0 Hz, 1H, H-5);
13
C NMR (D
2
O, 100 MHz): d=
94.294.0 (m, C-1), 76.0 (d,
3
J
CP
=3.8 Hz, C-5), 75.1 (br s, C-3), 74.9
(br t, J
CP
=6.0 Hz, C-2), 69.95 (br t, J
CP
=5.2 Hz, C-4), 60.7 (C-6);
31
P NMR (D
2
O, 162 MHz): d=0.61, 0.45, 0.025, 0.97; HRMS (ESI):
m/z [MNa]
calcd for C
6
H
12
Na
3
O
18
P
4
: 564.8662, found: 564.8692.
6-O-tert-Butyldiphenylsilyl-d-glucopyranose (16):
[32]
Flash column
3
OH in EtOAc) as a white solid
(77%), ratio of a/b anomers 1/0.7: R
f
=0.1 (EtOAc).
Octabenzyl-1,2,3,4-(6-O-tert-butyldiphenylsilyl-a-d-galactopyra-
nosyl) tetrakisphosphate (21) and octabenzyl 1,2,3,4-(6-O-tert-
butyldiphenylsilyl-b-d-galactopyranosyl) tetrakisphosphate (22):
tanes) to yield inseparable a- and b-anomers (80%, a/b 1:1.7) as a
thick colorless oil. Small amounts of each anomer were purified for
data collection. 21: R
f
20
D
=+39.4
(c=1.0 in CHCl
3
);
1
H NMR (CDCl
3
, 400 MHz): d=7.707.60 (m, 4H),
7.487.10 (m, 46H), 6.15 (dd, J =6.4, 2.6 Hz, 1H, H-1), 5.34 (br d, J =
9.1 Hz, 1H, H-4), 5.264.82 (m, 17H, 15CHHPh, H-2, H-3), 4.82 (dd,
J =11.8, 8.2 Hz, 1H, CHHPh), 4.18 (br t, J =5.6 Hz, 1H, H-5), 3.91 (dd,
J =10.6, 6.5 Hz, 1H, H-6), 3.78 (dd, J =10.5, 6.6 Hz, 1H, H-6), 1.06
(s, 9H, C(CH
3
)
3
) ;
13
C NMR (CDCl
3
, 100 MHz): d=135.8135.3 (m),
132.8, 129.8, 129.7, 128.4127.4 (m), 95.4 (d,
2
J
CP
=5.5 Hz, C-1), 75.0
(d,
2
J
CP
=4.2 Hz, C-4), 72.872.6 (m, C-2 or C-3), 71.8 (d,
2
J
CP
=4.3 Hz,
C-2 or C-3), 71.7 (br d, J
CP
=3.6 Hz, C-5), 69.8 (d,
2
J
CP
=5.5 Hz, 1
CH
2
Ph), 69.7 (d,
2
J
CP
=5.6 Hz, 1CH
2
Ph), 69.769.5 (m, 4CH
2
Ph),
69.4 (d,
2
J
CP
=5.4 Hz, 1CH
2
Ph), 69.1 (d,
2
J
CP
=5.5 Hz, 1CH
2
Ph),
61.9 (C-6), 26.6 (C(CH
3
)
3
), 19.0 (C(CH
3
)
3
) ;
31
P NMR (162 MHz): d=
0.92, 1.08, 1.41, 2.30; HRMS (ESI): m/z [M+Li]
+
calcd for
C
78
H
82
LiO
18
P
4
Si : 1465.4375, found: 1465.4289. 22: R
f
=0.29 (EtOAc/
heptanes, 1:1) ; [a]
20
D
=+6.6 (c=2.75 in CHCl
3
) ;
1
H NMR (CDCl
3
,
400 MHz): d=7.747.68 (m, 4H), 7.507.23 (m, 46H), 5.35 (t, J =
7.2 Hz, 1H, H-1), 5.30 (dd, J =9.3, 2.6 Hz, 1H, H-4), 5.274.80 (m,
17H, CH
2
Ph, H-2), 4.51 (t, J =9.7 Hz, 1H, H-3), 3.93 (dd, J =10.6,
6.6 Hz, 1H, H-6), 3.84 (dd, J =10.6, 6.6 Hz, 1H, H-6), 3.70 (br t, J =
6.5 Hz, 1H, H-5), 1.09 (s, 9H, C(CH
3
)
3
) ;
13
C NMR (CDCl
3
, 100 MHz):
d=135.8135.2 (m), 132.7, 132.6, 129.8, 129.7, 128.4127.3 (m),
96.8 (br s, C-1), 75.7 (br s, C-3), 74.774.5 (m, C-2, C-5), 73.9 (d,
2
J
CP
=
5.0 Hz, C-4), 69.9 (d,
2
J
CP
=5.5 Hz, 1CH
2
Ph), 69.769.2 (m, 6
CH
2
Ph), 69.0 (d,
2
J
CP
=5.4 Hz, 1CH
2
Ph), 61.5 (C-6), 26.6 (C(CH
3
)
3
),
18.9 (C(CH
3
)
3
) ;
31
P NMR (162 MHz): d=1.16, 1.21, 1.60, 2.81;
HRMS (ESI): m/z [M+Na]
+
calcd for C
78
H
82
NaO
18
P
4
Si : 1481.4113,
found: 1481.4313.
Octabenzyl-1,2,3,4-a-d-galactopyranosyl tetrakisphosphate (27)
and octabenzyl 1,2,3,4-b-d-galactopyranosyl tetrakisphosphate
(28): Flash column chromatography (EtOAc ! 2% CH
3
OH in
EtOAc) to afford first the a-anomer 27 (29%) and subsequently (4
5% CH
3
OH in EtOAc) the b-anomer 28 (51%), as thick colorless
oils: 27: R
f
20
D
=+60.5 (c=2.0 in
CHCl
3
) ;
1
H NMR (CDCl
3
, 400 MHz): d=7.407.20 (m, 40H), 6.09 (br d,
J =5.7 Hz, 1H, H-1), 5.155.00 (m, 17H, CH
2
Ph, H-4), 4.854.80 (m,
2H, H-2, H-3), 4.00 (br t, J =7.3 Hz, 1H, H-5), 3.66 (dd, J =11.8,
8.9 Hz, 1H, H-6), 3.56 (dd, J =11.8, 6.0 Hz, 1H, H-6) ;
13
C NMR
(CDCl
3
, 100 MHz): d=135.3 (d,
3
J
CP
=7.26 Hz), 128.7128.3 (m),
128.1127.7 (m), 95.2 (d,
2
J
CP
=5.7 Hz, C-1), 77.8 (d,
2
J
CP
=4.8 Hz, C-
4), 72.372.0 (m), 71.671.3 (m), 70.7 (br s), 70.3 (d,
2
J
CP
=5.9 Hz, 1
CH
2
Ph), 69.9 (d,
2
J
CP
=5.9 Hz, 1CH
2
Ph), 69.969.4 (m, 6CH
2
Ph),
58.8 (C-6);
31
P NMR (162 MHz): d=0.40, 1.19, 1.29, 2.25; HRMS
(ESI): m/z [M+Li]
+
calcd for C
62
H
64
LiO
18
P
4
: 1227.3198, found:
1227.3269. 28: R
f
20
D
=+31.4 (c=
0.5 in CHCl
3
) ;
1
H NMR (CDCl
3
, 400 MHz): d=7.357.23 (m, 40H),
5.33 (t, J =7.0 Hz, 1H, H-1), 5.225.00 (m, 17H, CH
2
Ph, H-4), 4.89 (q,
J =9.0 Hz, 1H, H-2), 4.51 (br t, J =9.3 Hz, 1H, H-3), 3.783.70 (m,
3H, H-5, H-6, H-6);
13
C NMR (CDCl
3
, 100 MHz): d=135.6134.7 (m),
128.6127.5 (m), 96.8 (br s, C-1), 75.1 (br s, C-3), 74.874.5 (m, C-2),
73.8 (br s, C-5), 73.072.8 (m, C-4), 70.3 (d,
2
J
CP
=6.0 Hz, 1CH
2
Ph),
70.069.7 (m, 3CH
2
Ph), 69.6 (d,
2
J
CP
=5.5 Hz, 1CH
2
Ph), 69.569.3
(m, 3CH
2
Ph), 58.6 (C-6);
31
P NMR (162 MHz): d=0.49, 1.23,
1.60, 2.89; HRMS (ESI): m/z [M+Na]
+
calcd for C
62
H
64
NaO
18
P
4
:
1243.2935, found: 1243.3018.
Tetrasodium-1,2,3,4-a-d-galactopyranosyl tetrakisphosphate
(33): White solid (99%): [a]
20
D
=+68.0 (c=0.5 in H
2
O);
1
H NMR (D
2
O,
400 MHz): d=5.59 (dd, J =7.1, 3.1 Hz, 1H, H-1), 4.62 (d, J =9.8 Hz,
1H, H-4), 4.41 (br t, J =9.3 Hz, 1H, H-3), 4.32 (br t, J =9.4 Hz, 1H, H-
2), 4.12 (t, J =6.3 Hz, 1H, H-5), 3.64 (d, J =6.2 Hz, 2H, H-6, H-6);
13
C NMR (D
2
O, 100 MHz): d=93.8 (d,
2
J
CP
=4.7 Hz, C-1), 73.0 (d,
2
J
CP
=5.4 Hz, C-4), 71.6 (br t, J
CP
=4.5 Hz, C-3), 70.9 (br s, C-2), 70.7
(br d,
3
J
CP
=5.2 Hz, C-5), 60.5 (C-6);
31
P NMR (D
2
O, 162 MHz): d=0.28
(3P), 1.16; HRMS (ESI): m/z [MNa]
calcd for C
6
H
12
Na
3
O
18
P
4
:
564.8662, found: 564.8698.
Tetrasodium-1,2,3,4-b-d-galactopyranosyl tetrakisphosphate
(34): White solid (99%): [a]
20
D
=+28.4 (c=0.5 in H
2
O);
1
H NMR (D
2
O,
400 MHz): d=4.95 (t, J =7.8 Hz, 1H, H-1), 4.52 (br d, J =10.1 Hz, 1H,
H-4), 4.20 (br t, J =9.5 Hz, 1H, H-3), 4.13 (q, J =8.7 Hz, 1H, H-2),
3.77 (br t, J =5.7 Hz, 1H, H-5), 3.743.62 (m, 2H, H-6, H-6) ;
13
C NMR
(D
2
O, 100 MHz): d=96.896.6 (m, C-1), 75.274.9 (m, C-2, C-3), 74.8
(br d,
3
J
CP
=2.6 Hz, C-5), 72.2 (br d,
2
J
CP
=4.6 Hz, C-4), 60.7 (C-6);
31
P NMR (D
2
O, 162 MHz): d=0.45, 0.22 (2P), 0.68; HRMS (ESI): m/z
[MNa]
calcd for C
6
H
12
Na
3
O
18
P
4
: 564.8662, found: 564.8627.
Octabenzyl-2,3,4,6-(1-O-methyl-a-d-glucopyranosyl) tetraki-
sphosphate (37): Flash column chromatography (80% EtOAc in
heptanes ! EtOAc), thick colorless oil (94%): R
f
=0.18 (EtOAc/hep-
tanes, 8:1) ; [a]
20
D
=+45.5 (c=1.0 in CH
2
Cl
2
);
1
H NMR (CDCl
3
,
400 MHz): d=7.417.20 (m, 40H), 5.185.02 (m, 18H, CH
2
Ph, H-1,
H-3), 4.594.38 (m, 3H, H-4, H-6, H-6), 4.31 (ddd, J =9.6, 5.9, 3.5 Hz,
1H, H-2), 3.96 (br dd, J =9.7, 4.4 Hz, 1H, H-5), 3.33 (s, 3H, OCH
3
) ;
13
C NMR (CDCl
3
, 100 MHz): d=135.9135.3 (m), 128.4127.5 (m),
96.9 (C-1), 76.376.0 (m, C-3), 74.6 (br s, C-2), 73.4 (br s, C-4), 69.7 (d,
2
J
CP
=5.8 Hz, 1CH
2
Ph), 69.6 (d,
2
J
CP
=5.3 Hz, 1CH
2
Ph), 69.5 (d,
2
J
CP
=5.5 Hz, 1CH
2
Ph), 69.469.2 (m, 3CH
2
Ph), 69.0 (d,
2
J
CP
=
4.2 Hz, 2CH
2
Ph), 68.5 (br d,
3
J
CP
=6.9 Hz, C-5), 65.6 (d,
2
J
CP
=5.5 Hz,
C-6), 55.4 (OCH
3
) ;
31
P NMR (162 MHz): d=0.81, 1.10, 1.72,
2.04; HRMS (ESI): m/z [M+Na]
+
calcd for C
63
H
66
NaO
18
P
4
:
1257.3092, found: 1257.2927.
Tetrakis(triethylammonium)-2,3,4,6-(1-O-methyl-a-d-glucopyra-
nosyl) tetrakisphosphate (39): Glassy pale-brown solid (99%):
1
H NMR (D
2
O, 400 MHz): d=4.89 (br d, J =3.4 Hz, 1H, H-1), 4.35 (q,
J =9.1 Hz, 1H, H-3), 4.103.90 (m, 4H, H-2, H-4, H-6, H-6), 3.82
(br dd, J =8.8, 3.5 Hz, 1H, H-5), 3.35 (s, 3H, OCH
3
), 3.10 (q, J =
7.3 Hz, 24H, CH
2
CH
3
), 1.18 (t, J =7.3 Hz, 36H, CH
2
CH
3
) ;
13
C NMR
(D
2
O, 100 MHz): d=97.9 (C-1), 76.4 (br s, C-3), 73.873.7 (m, C-2),
73.072.8 (m, C-4), 69.7569.6 (m, C-5), 63.763.6 (m, C-6), 55.1
(OCH
3
), 46.6 (CH
2
CH
3
), 8.2 (CH
2
CH
3
) ;
31
P NMR (162 MHz): d=0.73,
0.64, 0.15, 0.76.
Tetrasodium-2,3,4,6-(1-O-methyl-a-d-glucopyranosyl) tetraki-
sphosphate (41): Pale-white solid (99%): [a]
20
D
=+52.6 (c=0.5 in
H
2
O);
1
H NMR (D
2
O, 400 MHz): d=4.91 (d, J =3.6 Hz, 1H, H-1), 4.37
(q, J =9.3 Hz, 1H, H-3), 4.154.00 (m, 3H, H-2, H-4, H-6), 4.003.92
(m, 1H, H-6), 3.85 (br dd, J =9.7, 3.9 Hz, 1H, H-5), 3.36 (s, 3H,
OCH
3
) ;
13
C NMR (D
2
O, 100 MHz): d=97.8 (C-1), 76.576.4 (m, C-3),
73.773.6 (m, C-2), 73.0 (br d,
2
J
CP
=5.9 Hz, C-4), 69.669.5 (m, C-5),
63.7 (d,
2
J
CP
=4.6 Hz, C-6), 55.1 (OCH
3
) ;
31
P NMR (162 MHz): d=0.66
(2P), 0.15, 0.70; HRMS (ESI): m/z [MNa]
calcd for
C
7
H
14
Na
3
O
18
P
4
: 578.8818, found: 578.8870.
Octabenzyl-2,3,4,6-(1-O-methyl-a-d-mannopyranosyl) tetraki-
sphosphate (38): Flash column chromatography (90% EtOAc in
heptanes ! EtOAc), thick colorless oil (79%): R
f
=0.25 (EtOAc/hep-
tanes, 4:1) ; [a]
20
D
=+4.4 (c=1.0 in CH
2
Cl
2
);
1
H NMR (CDCl
3
,
400 MHz): d=7.357.24 (m, 40H), 5.134.95 (m, 17H, CH
2
Ph, H-2),
4.90 (d, J =1.5 Hz, 1H, H-1), 4.884.80 (m, 1H, H-3), 4.81 (q, J =
9.2 Hz, 1H, H-4), 4.57 (ddd, J =11.3, 4.3, 1.8 Hz, 1H, H-6), 4.384.30
(m, 1H, H-6), 3.91 (br t, J =7.2 Hz, 1H, H-5), 3.32 (s, 3H, OCH
3
);
13
C NMR (CDCl
3
, 100 MHz): d=135.8135.3 (m), 128.4128.2 (m),
128.0127.7 (m), 98.3 (C-1), 74.5 (d,
2
J
CP
=4.2 Hz, C-2), 74.073.9 (m,
C-3), 71.25 (t, J
CP
=6.5 Hz, C-4), 70.0 (dd, J
CP
=7.2, 3.5 Hz, C-5), 69.7
69.4 (m, 6CH
2
Ph), 69.1 (d,
2
J
CP
=4.8 Hz, 1CH
2
Ph), 69.0 (d,
2
J
CP
=
4.8 Hz, 1CH
2
Ph), 66.0 (d,
2
J
CP
=5.5 Hz, C-6), 55.3 (OCH
3
) ;
31
P NMR
(162 MHz): d=0.91, 1.30, 1.33, 1.88; HRMS (ESI): m/z
[M+Na]
+
calcd for C
63
H
66
NaO
18
P
4
: 1257.3092, found: 1257.3069.
Tetrakis(triethylammonium)-2,3,4,6-(1-O-methyl-a-d-mannopyra-
nosyl) tetrakisphosphate (40): Glassy pale-brown solid (75%):
1
H NMR (D
2
O, 400 MHz): d=4.86 (s, 1H, H-1), 4.41 (d, J =9.2 Hz,
1H, H-2), 4.394.35 (m, 1H, H-3), 4.28 (q, J =9.3 Hz, 1H, H-4), 4.13
(dd, J =11.2, 5.1 Hz, 1H, H-6), 4.033.95 (m, 1H, H-6), 3.85 (br t, J =
6.8 Hz, 1H, H-5), 3.37 (s, 3H, OCH
3
), 3.11 (q, J =7.3 Hz, 24H,
CH
2
CH
3
), 1.20 (t, J =7.3 Hz, 36H, CH
2
CH
3
);
13
C NMR (D
2
O, 100 MHz):
d=99.4 (C-1), 73.473.2 (m, C-3), 73.0 (d,
2
J
CP
=4.5 Hz, C-2), 71.0
70.8 (m, C-5), 70.5 (br t, J
CP
=6.6 Hz, C-4), 63.9 (d,
2
J
CP
=3.0 Hz, C-6),
55.0 (OCH
3
), 46.5 (CH
2
CH
3
), 8.2 (CH
2
CH
3
) ;
31
P NMR (162 MHz): d=
0.65, 0.44, 0.16, 0.61.
Tetrasodium-2,3,4,6-(1-O-methyl-a-d-mannopyranosyl) tetraki-
sphosphate (42): Pale-white solid (99%): [a]
20
D
=+24.2 (c=0.5 in
H
2
O);
1
H NMR (D
2
O, 400 MHz): d=4.87 (s, 1H, H-1), 4.44 (br d, J =
9.5 Hz, 1H, H-2), 4.38 (br d, J =10.5 Hz, 1H, H-3), 4.33 (q, J =9.3 Hz,
1H, H-4), 4.14 (dd, J =11.2, 4.6 Hz, 1H, H-6), 4.084.00 (m, 1H, H-6),
3.87 (br t, J =6.7 Hz, 1H, H-5), 3.38 (s, 3H, OCH
3
) ;
13
C NMR (D
2
O,
100 MHz): d=99.3 (C-1), 73.4 (br s, C-3), 73.1 (d,
2
J
CP
=4.7 Hz, C-2),
70.970.6 (m, C-5), 70.670.4 (m, C-4), 63.9 (bd,
2
J
CP
=3.5 Hz, C-6),
55.1 (OCH
3
) ;
31
P NMR (162 MHz): d=0.70, 0.42, 0.13, 0.51; HRMS
(ESI): m/z [MNa]
calcd for C
7
H
14
Na
3
O
18
P
4
: 578.8818, found:
578.8845.
Decabenzyl-1,2,3,4,6-(a-d-glucopyranosyl) pentakisphosphate
(43): Flash column chromatography (80% EtOAc in heptanes !
EtOAc, thick colorless oil (55%): R
f
[a]
20
D
=+43.9 (c=1.0 in CHCl
3
) ;
1
H NMR (CDCl
3
, 400 MHz): d=7.38
7.22 (m, 50H), 6.18 (dd, J =6.0, 3.3 Hz, 1H, H-1), 5.224.90 (m, 21H,
CH
2
Ph, H-3), 4.58 (q, J =9.7 Hz, 1H, H-4), 4.404.32 (m, 2H, H-2, H-
6), 4.26 (ddd, J =11.4, 5.9, 1.4 Hz, 1H, H-6), 4.01 (br d, J =9.0 Hz,
1H, H-5);
13
C NMR (CDCl
3
, 100 MHz): d=135.9135.1 (m), 128.6
127.7 (m), 94.4 (d,
2
J
CP
=5.2 Hz, C-1), 75.675.4 (m, C-3), 73.973.7
(m, C-2), 72.572.4 (m, C-4), 70.55 (dd, J
CP
=8.2, 2.6 Hz, C-5), 70.1
69.0 (m, CH
2
Ph), 64.7 (d,
2
J
CP
=4.7 Hz, C-6) ;
31
P NMR (162 MHz): d=
1.03 (2P), 1.49, 1.83, 2.56; HRMS (ESI): m/z [M+Li]
+
calcd for
C
76
H
77
LiO
21
P
5
: 1487.3800, found: 1487.3813.
Pentakis(triethylammonium)-1,2,3,4,6-(a-d-glucopyranosyl) pen-
takisphosphate (46): Glassy hydroscopic pale-brown solid (97%):
1
H NMR (D
2
O, 400 MHz): d=5.30 (dd, J =7.4, 3.2 Hz, 1H, H-1), 4.17
(q, J =9.3 Hz, 1H, H-3), 3.89 (q, J =9.5 Hz, 1H, H-4), 3.803.70 (m,
4H, H-2, H-5, H-6, H-6), 2.86 (q, J =7.3 Hz, 30H, CH
2
CH
3
), 0.96 (t,
J =7.3 Hz, 45H, CH
2
CH
3
) ;
13
C NMR (D
2
O, 100 MHz): d=93.2 (d,
2
J
CP
=5.1 Hz, C-1), 76.0 (br s, C-3), 73.873.6 (m, C-2), 72.5 (br s, C-4),
70.4 (br s, C-5), 63.3 (br s, C-6), 46.3 (CH
2
CH
3
), 8.2 (CH
2
CH
3
) ;
31
P NMR
(162 MHz): d=0.69, 0.38, 0.23, 1.05, 1.77.
Pentasodium-1,2,3,4,6-(a-d-glucopyranosyl) pentakisphosphate
(47): Glassy white solid (99%): [a]
20
D
=+50.2 (c=1.0 in H
2
O) ;
1
H NMR (D
2
O, 400 MHz): d=5.59 (dd, J =7.3, 3.2 Hz, 1H, H-1), 4.47
(q, J =9.3 Hz, 1H, H-3), 4.20 (q, J =9.4 Hz, 1H, H-4), 4.144.00 (m,
4H, H-2, H-5, H-6, H-6) ;
13
C NMR (D
2
O, 100 MHz): d=93.2 (br s, C-
1), 76.2 (br s, C-3), 73.6 (br s, C-2), 72.772.6 (m, C-4), 70.670.3 (br s,
C-5), 63.6 (C-6);
31
P NMR (162 MHz): d=0.81, 0.67, 0.01, 0.76,
1.46; HRMS (ESI): m/z [MNa]
calcd for C
6
H
12
Na
4
O
21
P
5
: 666.8144,
found 666.8215.
Decabenzyl-1,2,3,4,6-(a-d-mannopyranosyl) pentakisphosphate
(44): Flash column chromatography (80% EtOAc in heptanes !
EtOAc), thick colorless oil (62%): R
f
=0.38 (EtOAc/heptanes, 3:1) ;
[a]
20
D
=+6.0 (c=1.5 in CHCl
3
) ;
1
H NMR (CDCl
3
, 400 MHz): d=7.38
7.22 (m, 50H), 5.96 (dd, J =6.2, 2.0 Hz, 1H, H-1), 5.204.83 (m, 23H,
CH
2
Ph, H-2, H-3, H-4), 4.36 (ddd, J =11.7, 6.3, 1.8 Hz, 1H, H-6), 4.29
(dt, J =11.6, 4.8 Hz, 1H, H-6), 4.01 (br dt, J =9.4, 2.0 Hz, 1H, H-5) ;
13
C NMR (CDCl
3
, 100 MHz): d=135.8135.1 (m), 128.9127.8 (m),
95.2 (br d,
2
J
CP
=6.9 Hz, C-1), 74.273.9 (m, C-2), 73.373.1 (m, C-3),
72.071.7 (m, C-5), 70.4 (t, J
CP
=9.0 Hz, C-4), 70.169.2 (m, CH
2
Ph),
65.265.0 (m, C-6) ;
31
P NMR (162 MHz): d=1.11, 1.43 (2P),
1.92, 3.18; HRMS (ESI): m/z [M+K]
+
calcd for C
76
H
77
KO
21
P
5
:
1519.3277, found: 1519.3447.
Pentakis(triethylammonium)-1,2,3,4,6-(a-d-mannopyranosyl)
pentakisphosphate (48): Glassy hydroscopic pale-brown solid
(95%):
1
H NMR (D
2
O, 400 MHz): d=5.50 (d, J =7.6 Hz, 1H, H-1),
4.494.42 (m, 2H, H-2, H-3), 4.36 (q, J =9.2 Hz, 1H, H-4), 4.123.99
(m, 3H, H-5, H-6, H-6), 3.12 (q, J =7.3 Hz, 30H, CH
2
CH
3
), 1.19 (t, J =
7.3 Hz, 45H, CH
2
CH
3
) ;
13
C NMR (D
2
O, 100 MHz): d=94.394.1 (m, C-
1), 73.773.5 (m, C-2), 73.072.8 (m, C-3), 71.871.6 (m, C-5), 70.4
70.2 (m, C-4), 64.063.8 (m, C-6), 46.6 (CH
2
CH
3
), 8.2 (CH
2
CH
3
) ;
31
P NMR (D
2
O, 162 MHz): d=0.62, 0.30, 0.35, 0.68, 2.35.
Pentasodium-1,2,3,4,6-(a-d-mannopyranosyl) pentakisphos-
phate (49): Glassy pale-brown solid (95%): [a]
20
D
=+10.5 (c=1.0 in
H
2
O);
1
H NMR (D
2
O, 400 MHz): d=5.46 (d, J =7.6 Hz, 1H, H-1),
4.464.32 (m, 3H, H-2, H-3, H-4), 4.043.95 (m, 3H, H-5, H-6, H-6);
13
C NMR (D
2
O, 100 MHz): d=94.1 (br d,
2
J
CP
=4.6 Hz, C-1), 73.5 (dd,
J
CP
=6.7, 4.3 Hz, C-2), 73.072.8 (m, C-3), 71.671.3 (m, C-5), 70.2
(br t, J
CP
=5.0 Hz, C-4), 63.8 (br s, C-6) ;
31
P NMR (D
2
O, 162 MHz): d=
0.57, 0.28, 0.35, 0.60, 2.26; HRMS (ESI) calcd for C
6
H
12
Na
4
O
21
P
5
m/z: [MNa]
666.8144, found: 666.8081.

Decabenzyl-1,2,3,4,6-(a-d-galactopyranosyl) pentakisphosphate
(45): Flash column chromatography (65% EtOAc in heptanes),
thick colorless oil (63%): R
f
20
D
=+
46.0 (c=2.0 in CHCl
3
) ;
1
H NMR (CDCl
3
, 400 MHz): d=7.387.20 (m,
50H), 6.18 (dd, J =6.5, 3.0 Hz, 1H, H-1), 5.265.00 (m, 21H, CH
2
Ph,
H-4), 4.964.84 (m, 2H, H-2, H-3), 4.264.15 (m, 2H, H-6, H-6), 4.02
(br t, J =5.8 Hz, 1H, H-5) ;
13
C NMR (CDCl
3
, 100 MHz): d=135.3
134.8 (m), 128.2128.0 (m), 127.8127.5 (m), 94.8 (d,
2
J
CP
=5.4 Hz,
C-1), 74.2 (d,
2
J
CP
=3.3 Hz, C-4), 71.871.7 (m, C-3), 71.170.9 (m, C-
2), 69.769.0 (m, CH
2
Ph, C-5), 64.5 (d,
2
J
CP
=4.6 Hz, C-6) ;
31
P NMR
(162 MHz): d=0.98, 1.00, 1.06, 1.38, 2.26; HRMS (ESI): m/z:
[M+Li+Na]
2+
calcd for C
76
H
77
LiNaO
21
P
5
: 755.1846, found: 755.1753.
Pentakis(triethylammonium)-1,2,3,4,6-(a-d-galactopyranosyl)
pentakisphosphate (50): Glassy hydroscopic pale-brown solid
(94%):
1
H NMR (D
2
O, 400 MHz): d=5.59 (dd, J =7.3, 3.4 Hz, 1H, H-
1), 4.704.65 (obscured, 1H, H-4), 4.41 (dt, J =9.9, 2.1 Hz, 1H, H-3),
4.31 (br d, J =9.4 Hz, 1H, H-2), 4.27 (br t, J =6.5 Hz, 1H, H-5), 3.98
3.82 (m, 2H, H-6, H-6), 3.08 (q, J =7.3 Hz, 30H, CH
2
CH
3
), 1.17 (t, J =
7.3 Hz, 45H, CH
2
CH
3
) ;
13
C NMR (D
2
O, 100 MHz): d=93.7 (d,
2
J
CP
=
4.9 Hz, C-1), 73.1 (br d,
2
J
CP
=6.0 Hz, C-4), 71.871.6 (m, C-3), 70.7
(br s, C-2), 69.7 (br dd, J
CP
=7.5, 3.7 Hz, C-5), 63.863.7 (m, C-6), 46.6
(CH
2
CH
3
), 8.2 (CH
2
CH
3
) ;
31
P NMR (162 MHz): d=0.55, 0.45, 0.16,
0.30, 1.21.
Pentasodium-1,2,3,4,6-(a-d-galactopyranosyl) pentakisphos-
phate (51): Glassy pale-yellow solid (99%): [a]
20
D
=+59.0 (c=1.0 in
H
2
O);
1
H NMR (D
2
O, 400 MHz): d=5.62 (dd, J =7.3, 3.4 Hz, 1H, H-1),
4.70 (obscured, 1H, H-4), 4.45 (dt, J =9.8, 2.4 Hz, 1H, H-3), 4.36 (td,
J =9.6, 2.9 Hz, 1H, H-2), 4.30 (br t, J =6.6 Hz, 1H, H-5), 4.103.77 (m,
2H, H-6, H-6) ;
13
C NMR (D
2
O, 100 MHz): d=93.7 (d,
2
J
CP
=5.5 Hz, C-
1), 73.3 (d,
2
J
CP
=5.9 Hz, C-4), 71.871.7 (m, C-3), 70.770.6 (m, C-2),
69.7 (br dd, J
CP
=7.8, 3.7 Hz, C-5), 63.9 (d,
2
J
CP
=4.5 Hz, C-6) ;
31
P NMR
(D
2
O, 162 MHz): d=0.49, 0.40, 0.20, 0.42, 1.34; HRMS (ESI): m/z
[MNa]
calcd for C
6
H
12
Na
4
O
21
P
5
: 666.8144, found: 666.8152.
Hexadecabenzyllactose octakisphosphate (53): Flash column
chromatography (70% EtOAc in heptanes ! EtOAc), thick color-
less oil, ratio a/b 1:4 (65%). A small amount of pure b-anomer was
obtained and characterized: R
f
20
D
=
0.3 (c=1.0 in CHCl
3
);
1
H NMR (CDCl
3
, 400 MHz): d=7.407.10 (m,
80H), 5.204.79 (m, 36H), 4.754.69 (m, 1H), 4.464.27 (m, 5H),
4.04 (br d, J =12.5 Hz, 1H), 3.88 (t, J =9.3 Hz, 1H), 3.70 (br t, J =
5.9 Hz, 1H), 2.85 (br d, J =9.6 Hz, 1H) ;
13
C NMR (CDCl
3
, 100 MHz):
d=136.5135.4 (m), 129.0127.6 (m), 99.9 (br s), 96.0 (d, J
CP
=
5.8 Hz), 77.2 (obscured), 75.675.4 (m), 75.2 (br t, J
CP
=5.7 Hz), 74.2
(br d, J
CP
=4.3 Hz), 73.7 (br s), 72.7 (br d, J
CP
=8.9 Hz), 71.7 (dd, J
CP
=
9.1, 4.4 Hz), 70.0 (d, J
CP
=5.3 Hz), 69.969.2 (m), 64.1 (br s), 63.9
(br d, J
CP
=5.0 Hz) ;
31
P NMR (162 MHz): d=0.20, 0.50, 1.01,
1.18, 1.29, 1.60, 1.69, 3.09; HRMS (ESI): m/z [M+Li]
+
calcd
for C
124
H
126
LiO
35
P
8
: 2429.6135, found: 2430.6325. Representative
shifts for the a-anomer: 6.21 (dd, J =5.6, 3.2 Hz, 1H), 4.61 (dd, J =
10.6, 5.5 Hz, 1H).
Octakis(triethylammonium)lactose octakisphosphate (54): Glassy
hydroscopic pale-brown solid (99%):
1
H NMR (D
2
O, 400 MHz): d=
4.95 (t, J =8.0 Hz, 1H), 4.70 (obscured 1H), 4.60 (d, J =9.7 Hz, 1H),
4.28 (q, J =9.0 Hz, 1H), 4.234.08 (m, 4H), 3.953.87 (m, 4H), 3.84
(t, J =9.9 Hz, 1H), 3.72 (br d, J =10.7 Hz, 1H), 3.08 (q, J =7.3 Hz,
48H), 1.16 (t, J =7.3 Hz, 72H);
13
C NMR (D
2
O, 100 MHz): d=101.3
101.2 (m), 96.296.1 (m), 77.877.7 (m), 77.377.2 (m), 75.6 (br s),
75.575.3 (m), 74.473.9 (m), 73.272.9 (m), 72.5 (br d, J
CP
=5.0 Hz),
63.162.9 (m), 62.962.8 (m), 46.6, 8.2;
31
P NMR (162 MHz): d=0.30,
0.08, 0.17, 0.28, 0.56 (total integration 7P), 1.31.
Octasodium lactose octakisphosphate (55): Glassy pale-white
solid (99%): [a]
20
D
=+3.6 (c=0.5 in H
2
O) ;
1
H NMR (D
2
O, 400 MHz):
d=5.00 (t, J =8.0 Hz, 1H), 4.70 (obscured 1H), 4.63 (dd, J =10.2,
2.8 Hz, 1H), 4.30 (q, J =9.3 Hz, 1H), 4.264.08 (m, 4H), 4.003.93
(m, 4H), 3.92 (t, J =9.4 Hz, 1H), 3.76 (br d, J =9.5 Hz, 1H);
13
C NMR
(D
2
O, 100 MHz): d=101.4 (d, J
CP
=3.5 Hz), 96.396.1 (m), 77.977.7
(m), 77.277.0 (m), 75.6 (br s), 75.575.4 (m), 74.373.9 (m), 72.9
(dd, J
CP
=9.0, 3.5 Hz), 72.7 72.6 (m), 63.262.7 (m);
31
P NMR
(162 MHz): d=0.50, 0.38, 0.31, 0.03, 0.12, 0.27, 0.37, 1.00;
HRMS (ESI): m/z [M+Na]
+
calcd for C
12
H
22
Na
9
O
35
P
8
: 1180.6916,
found: 1180.6955. Representative shifts for the a-anomer: 5.57 (dd,
J =7.1, 3.5 Hz, 1H), 4.49 (q, J =9.3 Hz, 1H).
Hexadecabenzylsucrose octakisphosphate (57): Flash column
chromatography, (70% EtOAc in heptanes ! EtOAc), thick color-
less oil (77%): R
f
20
D
=+13.8 (c=1.0
in CHCl
3
) ;
1
H NMR (CDCl
3
, 400 MHz): d=7.407.10 (m, 80H), 6.03
(d, J =3.5 Hz, 1H), 5.43 (t, J =8.6 Hz, 1H), 5.254.90 (m, 34H), 4.76
4.67 (m, 2H), 4.664.60 (m, 2H), 4.584.43 (m, 4H), 4.424.30 (m,
2H);
13
C NMR (CDCl
3
, 100 MHz): d=137.0135.3 (m), 128.4127.7
(m), 102.3, 89.5, 78.8 (br s), 77.777.6 (m), 76.276.0 (m), 73.6 (br s),
72.9 (br s), 70.169.6 (m), 69.369.0 (m), 66.9 (br d, J
CP
=4.9 Hz), 66.0
(br d, J
CP
=5.0 Hz), 64.9 (br s);
31
P NMR (162 MHz): d=0.41, 0.63,
0.93, 1.04, 1.10, 1.21, 1.26, 1.57; HRMS (ESI): m/z [M+Li]
+
calcd for C
124
H
126
LiO
35
P
8
: 2429.6135, found: 2429.6423.
Octakis(triethylammonium)sucrose octakisphosphate (58):
Glassy hydroscopic pale-brown solid (92%):
1
H NMR (D
2
O,
400 MHz): d=5.41 (d, J =3.5 Hz, 1H), 4.77 (t, J =9.8 Hz, 1H), 4.47
(q, J =8.0 Hz, 1H), 4.34 (q, J =8.3 Hz, 1H), 4.163.86 (m, 9H), 3.72
(dd, J =11.3, 3.2 Hz, 1H), 3.03 (q, J =7.3 Hz, 48H), 1.12 (t, J =7.3 Hz,
72H) ;
13
C NMR (D
2
O, 100 MHz): d=103.3 (dd, J
CP
=10.6, 4.7 Hz),
90.7 (br s), 80.1 (br s), 76.7 (br s), 76.476.1 (m), 73.5 (br s), 72.9 (br s),
70.4 (br s), 65.9 (br s), 63.763.4 (m), 46.4, 8.2;
31
P NMR (162 MHz):
d=0.78 (2P), 0.05, 0.06, 0.26, 0.37, 0.49, 0.85.
Octasodiumsucrose octakisphosphate (59): Glassy white solid
(99%): [a]
20
D
+26.4 (c=0.5 in H
2
O);
1
H NMR (D
2
O, 400 MHz): d=
5.44 (bs, 1H), 4.70 (obscured, 1H), 4.47 (br q, J =7.8 Hz, 1H), 4.33
(br q, J =8.2 Hz, 1H), 4.083.80 (m, 9H), 3.72 (br d, J =10.4 Hz, 1H) ;
13
C NMR (D
2
O, 100 MHz): d=102.9 (dd, J
CP
=11.3, 2.7 Hz), 90.4 (br s),
79.5 (dd, J
CP
=8.9, 4.5 Hz), 77.0 (br s), 76.4 (br s), 76.176.0 (m), 73.2
(br s), 72.8 (br s), 70.12 (dd, J
CP
=7.8, 4.6 Hz), 65.5 (br s), 64.0 (br s),
63.7 (br s) ;
31
P NMR (162 MHz): d=0.65, 0.57, 0.10, 0.10, 0.39,
0.63, 0.75, 0.81; HRMS (ESI): m/z [M2Na]
2
calcd for
C
12
H
22
Na
6
O
35
P
8
: 555.8609, found: 555.8655.
Tetrakis(triethylammonium)-1-O-methyl-a-d-glucopyranose-
2,3:4,6-bispyrophosphate (60): Glassy pale-white solid (93%)
1
H NMR (D
2
O, 400 MHz): d=4.86 (d, J =3.6 Hz, 1H, H-1), 4.43 (q, J =
8.9 Hz, 1H, H-3), 4.304.20 (m, 3H, H-2, H-4, H-6), 4.03 (br t, J =
11.2 Hz, 1H, H-6), 3.95 (br d, J =9.2 Hz, 1H, H-5), 3.34 (s, 3H, OCH
3
),
3.09 (q, J =7.2 Hz, 24H, CH
2
CH
3
), 1.17 (t, J =7.2 Hz, 36H, CH
2
CH
3
);
13
C NMR (D
2
O, 100 MHz): d=97.9 (d,
3
J
CP
=9.8 Hz, C-1), 76.976.7
(m, C-3), 75.4 (d,
2
J
CP
=6.8 Hz, C-2), 72.3 (t, J
CP
=7.3 Hz, C-4), 68.2
(br s, C-5), 65.1 (d,
2
J
CP
=4.6 Hz, C-6), 55.0 (OCH
3
), 46.6 (CH
2
CH
3
), 8.2
(CH
2
CH
3
) ;
31
P NMR (162 MHz): d=10.52 (d, J =24.9 Hz), 11.31 (d,
J =17.9 Hz), 11.65 (d, J =17.9 Hz), 12.45 (d, J =24.9 Hz).
Tetrasodium-1-O-methyl-a-d-glucopyranose-2,3:4,6-bispyro-
phosphate (62): Glassy pale-white solid (99%): [a]
20
D
=+60.8 (c=
0.5 in H
2
O) ;
1
H NMR (D
2
O, 400 MHz): d=4.92 (br d, J =3.7 Hz, 1H,
H-1), 4.514.43 (m, 1H, H-3), 4.364.27 (m, 3H, H-2, H-4, H-6), 4.07
(br t, J =11.3 Hz, 1H, H-6), 4.01 (br d, J =9.8 Hz, 1H, H-5), 3.38 (s,
3H, OCH
3
) ;
13
C NMR (D
2
O, 100 MHz): d=97.9 (d,
3
J
CP
=10.9 Hz, C-1),
76.976.7 (m, C-3), 75.4 (d,
2
J
CP
=6.8 Hz, C-2), 72.372.1 (m, C-4),
68.2 (br s, C-5), 65.1 (d,
2
J
CP
=4.6 Hz, C-6), 55.1 (OCH
3
) ;
31
P NMR
(162 MHz): d=10.13 (d, J =24.5 Hz), 11.02 (d, J =18.0 Hz),
11.45 (d, J =18.0 Hz), 11.96 (d, J =24.5 Hz) ; HRMS (ESI): m/z
[MNa]
calcd for C
7
H
10
Na
3
O
16
P
4
: 542.8607, found: 542.8555.
Tetrakis(triethylammonium)-1-O-methyl-a-d-mannopyranose
2,3:4,6-bispyrophosphate (61): Data for crude material.
1
H NMR
(D
2
O, 400 MHz): d=4.85 (s, 1H, H-1), 4.78 (dd, J =11.8, 4.1 Hz, 1H,
H-2), 4.52 (q, J =10.4 Hz, 1H, H-4), 4.404.26 (m, 2H, H-3, H-6), 4.07
(br ddd, J =12.6, 9.7, 2.8 Hz, 1H, H-6), 3.97 (br d, J =9.8 Hz, 1H, H-
5), 3.33 (s, 3H, OCH
3
), 3.12 (q, J =7.3 Hz, 24H, CH
2
CH
3
), 1.20 (t, J =
7.3 Hz, 36H, CH
2
CH
3
);
31
P NMR (162 MHz): d=9.85 (d, J =22.0 Hz),
10.34 (d, J =25.5 Hz), 12.25 (bd, J =25.2 Hz), 14.17 (d, J =
22.0 Hz).
Preparation of stripped hemoglobin: Human blood was with-
drawn from a nonsmoking healthy volunteer (C.D.D.) in hepari-
nized microtubes and treated according to the procedure de-
scribed by Riggs.
[41]
Red blood cells were washed three times with
0.85% saline and lysed by the addition of 1 volume of purified H
2
O
per volume of packed red blood cells. Hemolysate was kept cold
and passed through a column of Sephadex G-25 (1.020 cm) equi-
librated with 0.1m NaCl + 10
5
m EDTA pH 7.5, in order to remove
BPG. The concentration of oxy-Hb was assessed by UV/Vis spectro-
photometry (e=58400m
1
cm
1
at l=577 nm per oxy-Hb tetra-
mer).
General procedure for oxygen equilibration curve measure-
ments: Solutions of the test compounds (100 mm) were prepared
in purified H
2
O and the pH was adjusted to 7.07.4 prior to incuba-
tions with stripped Hb in a molar ratio of 20:1. The mixtures were
diluted in 3 mL TES-saline buffer (30 mm, 140 mm saline, pH 7.4),
and oxygen equilibrium curves were measured with a Hemox Ana-
lyzer apparatus (TCS Scientific Corp. , USA). The P
50
values and Hill
coefficients were calculated by linear regression analysis from data
points obtained between 40 and 60% oxygen saturation.
Acknowledgements
K.C.F. and A.E.K. thank Democritus University of Thrace and Aris-
totle University of Thessaloniki, respectively, for financial support
during their sabbatical leave. We thank Dr. J.-L. Schmitt for his
assistance regarding NMR experiments. C.D.D. thanks CAPES-BR
and Collge de France for a postdoctoral fellowship. The authors
thank NormOxys Inc. for financial support of the work and Firme-
nich SA for a research grant.
Keywords: allosteric effectors hemoglobin oxygen release
phosphorylated carbohydrates structureactivity relationships
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Received: August 30, 2010
Revised: October 10, 2010
Published online on November 24, 2010

Allosteric Effectors

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