allosteric effectors
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DOI: 10.1002/cmdc.201000366
Polyphosphates and Pyrophosphates of Hexopyranoses asAllosteric 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 therapeuticpotential. 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
O2 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 increasesoxygen 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 (IVandV).
[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
Universitde 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)
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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 allnovel 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
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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 (H2 in the presence of Pd/C and triethylamine)
to give the triethylammonium salts7 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.
Compound10 is the only derivative among those studied inthis 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] and16 ,[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 (0 8C, 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 derivatives8 and 10 of glu-
cose and mannose respectively, from their silylated methyl glycoside precur-
sors1 and 2 : a) 1. (BnO)2PN(iPr)2, 1H-tetrazole, CH3CN, RT; 2.mCPBA, CH2Cl2,
40 8C!RT; b) TBAF, AcOH, THF, 0 8C; c) H2 (1 atm), Pd/C, Et3N, EtOH/H2O
(1:1), RT; d) Dowex H+ , H 2O then Dowex Na+, H2O. [DBP=P(O)(OBn)2].
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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 (3JH-1,H-2=
2.63.4 Hz), and a larger constant due to coupling of proton
H-1 with the neighboring phosphorous nucleus (3JH-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
(3JH-1,H-23JH-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 severala- 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 1H and 13C NMR spectra.
According to these data, for hexopyranonses such as man-
nose, which, based on NMR spectra, seems to present the 4C1conformation, the anomeric proton signal of thea-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 13C 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-
tives44 ,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 JC-1,H-1, which was 174 and 175 Hz for
compounds31 and 49 respectively, indicating ana-phosphate,
and 160 Hz for compound 32 , indicating ab-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 2934of glu-
cose, mannose and galactose from the silylated precursors 1416 :
a) TBDPSCl, Et3N, DMAP, DMF, 0 8C!RT; b) 1. (BnO)
2PN(iPr)
2, 1H-tetrazole,
CH3CN, RT; 2.mCPBA, CH2Cl2, 40 8C!RT; c) TBAF, AcOH, THF, 0 8C; d) H2(1 atm), Pd/C, NaHCO3, EtOH/H2O (1:1), RT. [DBP=P(O)(OBn)2].
Table 1. 1H and 13C NMR chemical shifts for the comparative assignment
ofa- andb-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
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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 (H2 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-tetrakisphosphorylated 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 1H 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 (3JH-1,H-2) from 3.0 to 3.4 Hz.
The coupling constant of the anomeric proton with the neigh-
boring phosphorous nucleus (3JH-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 1H and 13C NMR spectra with those of the 1,2,3,4-tetrakis
phosphorylated derivatives and the value of the heteronuclear
coupling constant (JC-1,H-1=175 Hz) for compound 49 indicate
ana-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 ofa-
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 1H 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, 3JH-1,H-2=3.5 Hz
and 3JH-1,P=7.1 Hz. For the major lactose derivative 55 a triplet
appears at 5.02 ppm for the anomeric proton (3JH-1,H-2=3JH1,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
41and mannose 42 from the corresponding methyl glycosides 35 and 36 :
a) 1. (BnO)2PN(iPr)2, 1H-tetrazole, DMF, CH3CN, RT; 2.mCPBA, CH2Cl2,
40 8C!RT; b) H2(1 atm), Pd/C, Et3N, EtOH/H2O (1:1), RT; c) Dowex H+, H2O
then Dowex Na+ , H2O. [DBP=P(O)(OBn)2].
Scheme 4.Synthesis of the pentakisphosphorylated derivatives 4651of
glucose (11), mannose (12), and galactose (13): a) 1. (BnO)2PN(iPr)2, 1H-tetra-
zole, DMF, CH3CN, RT; 2.mCPBA, CH2Cl2, 40 8C!RT; b) H2(1 atm), Pd/C,
Et3N, EtOH/H2O (1:1), RT; c) Dowex H+ , H2O then Dowex Na
+, H2O.
[DBP=P(O)(OBn)2].
Scheme 5.Synthesis of the perphosphorylated derivatives 54 and 55 of lac-
tose (52): a) 1. (BnO)2PN(iPr)2, 1H-tetrazole, DMF, CH3CN, RT; 2.mCPBA,
CH2Cl2, 40 8C!RT; b) H2(3 atm), Pd/C, Et3N, EtOH/H2O (1:1), RT; c) Dowex
H+, H2O then Dowex Na+ , H 2O.
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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 compound57 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 13C 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 31P NMR spectrum
of the crude reaction mixture showed complete consumptionof 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)2PN(iPr)2, 1 H-tetrazole, DMF, CH3CN, RT; 2.mCPBA,
CH2Cl2, 40 8C!RT; b) H2 (3 atm), Pd/C, Et3N, EtOH/H2O (1:1), RT; c) Dowex
H+, H2O then Dowex Na+, H2O.
Scheme 7.Synthesis of the pyrophosphates 60-62of the tetrakis phosphory-
lated methyl glycosides of glucose 39 and mannose 40 derivatives: a) DCC,
CH3CN, 828
C; b) Dowex H+
, H2O then Dowex Na+
, H2O.
158 www.chemmedchem.org 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemMedChem 2011, 6, 153 168
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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 31P 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 Hband 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 pO2values, we assessed the partial pressure of oxygen for half-sat-
uration (P50) of Hb in the presence of the compounds and their
corresponding dissociation constants (Kd) 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 betweenbinding 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
higherP50 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)
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