Synthesis and characterization of novel flavonoid derivatives via sequential phosphorylation of quercetin

Article abstract of DOI:10.1016/j.tetlet.2017.02.085

Flavonoids are naturally-occurring polyphenolics that have been implicated in a wide range of biological activities. The major obstacles to flavonoid applications are the poor solubility in common solvents. Phosphorylation of flavonoids yields a new class of flavonoid derivatives which are very soluble in aqueous solutions and hence have the potential to be used in biological studies. We hereby report the sequential phosphorylation of Quercetin leading to the synthesis of Quercetin pentaphosphate (QPP), Apigenin triphosphate (ATRP), 5,4′-Quercetin Diphosphate (5,4′-QDP) and 4′-Quercetin monophosphate (4′-QP) with solubility of 848?mg/mL, 367?mg/mL, 315?μg/mL and 106?μg/mL respectively. The synthesis of 4′-QP, 5,4′-QDP, QPP and ATRP was successful with 85%, 60.5%, 56% and 99% yield respectively. These compounds have been characterized using ¹H NMR, ¹³C NMR, ³¹P NMR.

Full text of DOI:10.1016/j.tetlet.2017.02.085

Accepted Manuscript
Synthesis and characterization of novel flavonoid derivatives via sequential
phosphorylation of Quercetin
Francis J. Osonga, Joab O. Onyango, Samuel K. Mwilu, Naomih M. Noah,
Jürgen Schulte, Ming An, Omowunmi A. Sadik
PII:
S0040-4039(17)30271-X
DOI:
http://dx.doi.org/10.1016/j.tetlet.2017.02.085
TETL 48696
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Accepted Date:
17 January 2017
27 February 2017
Please cite this article as: Osonga, F.J., Onyango, J.O., Mwilu, S.K., Noah, N.M., Schulte, J., An, M., Sadik, O.A.,
Synthesis and characterization of novel flavonoid derivatives via sequential phosphorylation of Quercetin,
Tetrahedron Letters (2017), doi: http://dx.doi.org/10.1016/j.tetlet.2017.02.085
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Graphical Abstract
Synthesis and characterization of novel
flavonoid derivatives via sequential
phosphorylation of Quercertin
Leave this area blank for abstract info.
Francis J. Osongaa, Joab O. Onyangoa, Samuel K. Mwilua, Naomih M.Noaha, Jürgen Schulte, Ming Ana,
Omowunmi A. Sadika, ∗

1
Tetrahedron Letters
jour nal homepage: w ww.elsevier. com
Synthesis and characterization of novel flavonoid
derivatives via sequential phosphorylation of
Quercetin
Francis J. Osongaa , Joab O. Onyangob, Samuel K. Mwilua, Naomih M.Noaha , Jürgen Schulteb, Ming Anb,
Omowunmi A. Sadika,∗
a Department of Chemistry, Center for Research in Advanced Sensing Technologies & Environmental Sustainability (CREATES), State University of New York at
Binghamton, P.O Box 6000 Binghamton, NY, 13902 USA.
b Department of Chemistry, State University of New York at Binghamton, P.O Box 6000 Binghamton, NY, 13902 USA
ARTICLE INFO
ABSTRACT
Article history:
Received
Flavonoids are naturally-occurring polyphenolics that have been implicated in a wide range of
biological activities. The major obstacles to flavonoid applications are the poor solubility in
common solvents. Phosphorylation of flavonoids yields a new class of flavonoid derivatives
which are very soluble in aqueous solutions and hence have the potential to be used in biological
studies. We hereby report the sequential phosphorylation of Quercetin leading to the synthesis of
Quercetin pentaphosphate (QPP), Apigenin triphosphate (ATRP), 5, 4’-Quercetin Diphosphate
(5, 4’-QDP) and 4’- Quercetin monophosphate (4’-QP) with solubility of 848 mg/mL, 367
mg/mL, 315 µg/mL and 106 µg/mL respectively. The synthesis of 4’- QP, 5, 4’- QDP, QPP and
ATRP) was successful with 85%, 60.5 %, 56% and 99% yield respectively These compounds
have been characterized using 1H-NMR, 13C-NMR, 31 P-NMR.
Received in revised form
Accepted
Available online
Keywords:
Keyword_1
Keyword_2
Keyword_3
Keyword_4
Keyword_5
2009 Elsevier Ltd. All rights reserved.
Flavonoids constitute a large family of plant–derived polyphenolic compounds that are found in fruits,
vegetables, spices, wines and juices1. Examples of flavonoids include quercetin, fisetin, luteolin, apeginin,
myricetin and many others. Previous studies have shown that these compounds exhibit an array of
biological effects that are beneficial to humans, including antiviral, antioxidative, anti-inflammatory and
anticarcinogenic.
The solubility of these polyphenolic compounds can be increased through the modification of the chemical
structure, which may improve oral bioavailability2. As a result, considerable efforts have been directed
towards the development of novel, highly soluble conjugates having clinical profiles that are similar or
even superior to those exhibited by the parent molecules in vitro. These include synthetic esters, acyl
———
∗ Corresponding author. Tel.: +16077774132; fax: +1-607-777-4478
; e-mail: osadik@binghamton.edu

2
Tetrahedron
(methyl, ethyl, propyl etc), phenylisocyanated quercetin derivatives, as well as the quercetin conjugates
(methylated, sulfated, glucuronidated, glutathionated, and the isomers of mono, di- and mixed conjugates) 3,
4. Recently, some phosphorylated quercetin glycosides were synthesized and reported to exhibit improved
solubility with comparable activity towards cultured human malignant cells5. Several attempts have also
been made to phosphorylate quercetin and some of its derivatives 6-10 so as to improve its pharmaceutical or
physiological activities but none of these studies have been successful in completely phosphorylating the
parent molecule. Studies have shifted to the protection of the hydroxyl groups of polyphenols.3, 6, 7, 11.
Currently there is significant interest in the modification of flavonoids to improve the solubility and
3, 11
stability under intracellular conditions by carrying out selective protection of hydroxyl groups.
Recently, Quercetin pivaloxymethyl (POM) analogues were synthesized and it was revealed that the 7-
POM was more soluble in PBS (pH 7.4) buffer than 3-POM. Although 3-POM was found to be stable, the
study revealed that it could not penetrate the cells.11 Recently Yingling et al (2013) investigated in vitro
studies of the synthesized ester phosphorylated flavonoids as inhibitors of pancreatic cholesterol esterase
and acetylcholinesterase.3 The study revealed that the modified flavonoids showed remarkable potency
against those targets than the corresponding parent flavonoids and the position of the phosphate group has
significant effect on the inhibitory activities with the 7-phosphate most potent.
In the last decade, our group has been actively involved in the study of polyphenols, ranging from their
ability to affect the viability and proliferation of cancerous cells and to understand their mode of action12-13
.
Until recently, the degradation products of quercetin in biological-like conditions has not been
systematically isolated, characterized nor the degradation mechanisms successfully elucidated 12. We
reported the first complete isolation and structural elucidation of 18 intermediates following the
14
electrochemical oxidation of quercetin . Extensive studies by Zhou et al. have been critical in providing
deeper insights into underlying mechanisms of quercetin oxidation in different systems14.The nature of
interaction of quercetin with nucleotides has also been reported 13.
It is desirable to selectively add only one phosphate group via sequential phosphorylation and selective
protection in order to investigate the biological implication of each phosphate on the flavonoid. Hence the

3
goal of this work is to (i) synthesize a new class of phosphorylated Quercetin derivatives via sequential
phosphorylation (ii) account for the position and nature of the global phosphorylated Quercetin (QCR) and
demonstrate the sequential phosphorylated synthesis of Quercetin diphosphate (QDP), Quercetin
monophosphate (QP) and Apigenin triphosphate (ATRP).
15
In this phosphorylation work, we adopted Silverberg’s method
which employs in situ generation of
chlorodibenzylphosphate as the electrophiles starting from a stable dibenzyl phosphite.11, 16-20 It is simple,
rapid and clean compared to other methods Scheme 1. The elegance of the reaction rests in the instability of
the in situ generated chlorodibenzylphosphite as well as the nucleophilic phenoxide generated in situ.
Scheme 1: General Phosphorylation using Dibenzyl Phosphite, N, N-Diisopropylethylamine (DIPEA),
Dimethylaminopyridine (DMAP) and carbon tetrachloride (CCl4)
This reaction was used to install dibenzyl phosphate moieties on free hydroxyls groups on Quercetin.
Organophosphates form a principal means in the multi-billion dollar agricultural, household and pest
control industry among other commercial uses.21 Even though phosphate exists in many biological systems
from small molecules as phosphoenol pyruvate (PEP) to fairly large conjugates such as adenosine
triphosphate (ATP), not many drugs of organophosphorus nature have advanced to clinical trials. Our
encouraging bioactivity result on polyphosphorylated flavonoids is supported by the existence of
fosfomycin - an organophosphonate broad spectrum antibiotic marketed as monurol.22 With advances in
nucleic acid chemistry, newer phosphorylation methods have been developed including tetrazole-catalyzed
23, 24
method involving phosphoramidite compounds.
Furthermore, Lewis acid catalysis have been used in
the cases where the donor is not sufficiently nucleophilic.25 Recently, development of electrochemical
26
synthesis of organophosphorus was reported.
Phosphorylation of phenols with these methods gives
moderate yields and may not be great if applied to late stage synthetic products. The matter is complicated
if other functionalities exist in the molecule that need to be handled carefully with appropriate protection-
deprotection manipulations.

4
Tetrahedron
Scheme 2: Synthesis of Quercetin Pentaphosphate Intermediate (QPPI) (2) and Quercetin Pentaphosphate
(QPP) (3)
Compounds 2, 3 5, 6, 7, 8, 9 and 10 were synthesized following the procedure highlighted in the
Supplementary section. Scheme 2 shows the synthesis of compounds 2, 3 and in this case we used DMSO-
d6 as NMR solvent as opposed to CDCl3. The latter has δ ppm resonating at 7.26ppm in the 1H NMR which
coincides with the chemical shifts of the aromatic protons in the molecule reducing the ability to integrate
the area under the peaks corresponding to the proton number. Hence, use of DMSO-d6 with chemical shift
1
at 2.50 ppm in the H NMR allows us to account for all the protons in the molecule. We were therefore
able to account for 20 methylene protons and 55 aromatic protons for QPPI (2) by integration
corresponding to the claimed structure (Figure S1C).
The synthesis of 4’- Quercetin monophosphate (QP), 5, 4’-Quercetin Diphosphate (QDP), Quercetin
pentaphosphate (QPP) 3 and Apigenin triphosphate (ATRP) was successful with 85%, 60.5 %, 56% and
99% yield respectively. However, the synthesis of 5- monophosphate intermediate (QPI) failed. The NMR
of compounds 8 and 9 were in agreement with the literature.1 The NMR data (Figures 2-3 and Figure S1 -
S3) of the compounds 2, 3, 7, 8, 9 and 10 were in agreement with the structures. From the synthesis of

5
compounds 5 and 6, (Scheme 3) it can be deduced that the benzylation of Quercetin with 3.5 equivalent of
benzyl bromide and K2CO3 results mainly in the production of compound 5 (59.84 %) and 6 (19.65 %)
which is in agreement with ratio obtained in literature.11
1
13
The H and C NMR results in the experimental section depicts similar trend with slight variation caused
by CDCl3 used by the literature11 source as opposed to DMSO-d6 which we used. The choice of DMSO-d6
is justified because CDCl3 resonates around 7ppm which is the region for benzylic protons. From literature
studies it is a fact that the 5 position of Quercetin or any other flavonoids cannot be benzylated easily in the
conditions stated in the synthesis since the 5-OH is less acidic than the rest of the phenolic moieties, thus
providing justification about why it is not possible to install phosphate at position 5 of the parent Quercetin.
This fact is attributed to an acid weakening effect of an intramolecular H-bond between the 5-hydroxyl
group and the 4-keto group.27, 28 Selective protection of the Quercetin catechol ring is a prerequisite for
effective sequential phosphorylation of Quercetin. In recent years synthesis of 4’, 5, 7 monophosphate
analogues have been attempted by employing the protection strategy of the O-dihydroxyphenyl group using
dichlorophenylmethane.9, 11 The strategy being adopted in this study depends heavily on the difference in
reactivity sites of the phenolic groups and the selective protection of the catechol group. 3, 11
Scheme 3: Sequential synthesis of Quercetin phosphate derivatives
With the OH group at positions 5 and 4’, we anticipated that using Silverberg treatment15 (1 equivalent of
dibenzyl phospite) will preferentially avail 4’-Quercetin phosphate Intermediate (4’-QPI compound 8) as

6
Tetrahedron
the only phosphate intermediate. Though we isolated 4’-QPI as yellow oily compound we also isolated a
diphosphate compound as yellow oily compound as a minor product from the reaction which turned out to
be 5, 4’-Quercetin Diphosphate Intermediate (5, 4’-QDPI compound 7). The proton NMR for 5, 4’- QDPI
and 4’-QPI confirmed the identity of the compounds. From the NMR data the number of methylene (CH2)
protons was found to be 14, the benzylic protons was 35 and the aromatic proton being 5, totaling to 40
protons for 5, 4’- QDPI. On the other hand the numbers of protons for methylene, benzylic and aromatic
protons were found to be 12, 30 and 5 respectively yielding a total of 35 protons.
Scheme 4: Synthesis of Apigenin Triphosphate Intermediate (14) and Apigenin Triphosphate (15)
31P NMR as a structural tool for identification in the field of organophosphorus chemistry is strongly
influenced by the P chemical shifts to the structural environment.29, 30 Chemical shifts provide valuable
31
information about the nearest neighbours of the P atom in the molecule. 29 Since organophosphorus became
useful in many industrial, agricultural and household products, several studies have been undertaken in
31
structural elucidation techniques. However, no unified theory of factors affecting P chemical shifts and
coupling constants has been reached.29-31 A theoretical quantum-mechanical calculation proposes that
electronegativity differences in the P-X bond, changes in the π– electron overlap, and changes in the bond
angle play significant role.31,32 Recently, Pedrosa et al reported a correlation of ab-initio calculation of
chemical shifts with experimental data of substituted aryl dialkyl phosphates and concluded that δ 31P NMR
chemical shifts were mainly governed by symmetry of electron cloud on the phosphorus atom and that is

7
directly related to the electronegativity of the substituent. Their finding implicates the effect of back-
bonding from phosphoryl oxygen to phosphorus atom33, and was further confirmed by Koo’s density
29
functional theory (DFT) studies. Flavonoid modified with phosphate groups and their intermediates are
similar to aryl phosphates studied by Pedrosa. They are tetracoordinate phosphorus compound and the
benzyl protected intermediate will differ from its acid counterparts by the enhanced d-orbital occupancy in
the former33, a character that will enhance back-bonding from the phosphoryl oxygen atom to phosphorus.30
31
A review by Wigfield found that P NMR signal of dibenzyl phosphate resonates at +1.1 ppm (Chemical
19,
shifts with respect to 85% H3PO4)34, however, recent experimental reports
35-36, and theoretical
31
predictions 33 have indicated that the numbers could be more downfield. Our P chemical shifts for QPPI
(2), ATRPI (14), 4’-QPI (10) and 5, 4’-QDPI (9) are as shown in Figures 2, 3 and S1-S3. Pedrosa reported
that the chemical shift of phosphorus in the aryl phosphate resonates around -7ppm and vary depending on
the magnitude of electronegativity of substituents on the aryl ring.33 The P with proton decoupling and
31
without proton decoupling (Figure 3, 4 and S1) confirmed that phosphate was successfully installed giving
2 peaks and 1 peak for 5, 4’-QDPI and 4’-QPI respectively.
Characteristic of benzyl protected phosphate is the signature quintet splitting pattern in the 31P peaks as
shown in Figure 1. The presence of 4 H three bonds away, splits the P nucleus signal into 5 peaks with
considerable J coupling constants a factor likely to be attributed to magnetic properties of phosphorus.29
We speculate that for 5, 4’- QDPI the -7.27 ppm peak may be at 4’ unlike the 5 which could be at -5.86
regions. The differences in the phosphates on catecholic hydroxyls 4’ and 5 in δ ppm could be due to pπ-dπ
bonding interaction between aryl oxygen and phosphorus; and this effect results from the substitution at
para and meta positions of the phenyl ring. 29, 31 Position 4’ lies directly para to the bond projecting to ring
C of the flavone structure and therefore decreases the d orbital occupation on phosphorus and shifts the 31P
signal to lower fields 29,30
.

8
Tetrahedron
Figure 1: Shows representative NMR of dibenzyl phosphate signature splitting patterns (a) structure of
dibenzyl protection of phosphorus center (b) Quintet splitting pattern of phosphorus in 31P NMR (c)
splitting pattern of -CH2 proton in 1H NMR.
27, 28
Studies
have shown that the 5 position of Quercetin or any other flavonoids cannot be benzylated
easily in the conditions stated in the synthesis since the 5-OH is less acidic than the rest of the phenolic
moieties. This fact is attributed by an acid weakening effect of an intramolecular H-bond between the 5-
hydroxyl group and the 4-keto group. Therefore 4’- Quercetin monophosphate intermediate is preferred to
5- Quercetin monophosphate. The debenzylation of the intermediates was carried out by using TMSBr. The
31P with proton decoupling confirmed the product with 4’-QP giving one peak and 5, 4’-QDP showing two
peaks.

9
Figure 2: 31P with proton decoupling of 5, 4’-Quercetin Diphosphate (QDP)
Figure 3: 31P with Proton Decoupling of 4’-Quercetin Monophosphate (QP)
From reports 29-31 33, 34, we can reiterate that the more upfield -4.98 in QPPI is due to significant effect
arising from electronegativity. The carbonyl group at position 4 which lies in conjugation with C – C
double bond at position 2 and 3 deprives the oxygen at position 3 electrons reducing the back-bonding
ability which is consistent with Bent rule of electron withdrawing substituent requiring more p-character in
the bonding orbital.37-38 Similarly, the enol ether oxygen at position 1 further complicates the
electronegativity contribution and therefore the phosphorus at position 3 on ring C will be more upfield as
seen in QPPI 31P NMR. On the other hand, the splitting pattern of proton signals in the 1H NMR resonating

10 Tetrahedron
around 5ppm indicates the presence of the two methylene groups on the benzyl ether groups attached to
phosphorus center. Ordinarily, these germinal protons are not expected to have any splitting since there is
no vicinal proton neighbor or stereochemistry in the molecule, but due to the presence of the phosphorus
nucleus at three bonds away, signals a powerful internuclear interaction as depicted in Figure 1.
However, the pattern in the 1H spectrum quickly degenerate into complexity with increase in the number of
the benzyl protected phosphates groups as seen in QPPI. This comes from superimposition of peaks and
possible remote interaction of neighboring groups. We were therefore able to account for 20 methylene
protons and 55 aromatic protons for QPPI (2) by integration corresponding to the claimed structure.
Debenzylation of QPPI (2) yielded QPP (3) in 56% yield.
In summary, we have successfully synthesized four intermediates 2, 8, 9 en-routes to Quercetin phosphate
analogs with a yield of 47%, 59.84 % and 19.65 % respectively from a reaction starting from Quercetin to
give final products of compounds 3, 9 and 10 giving a yield of 56%, 60.5% and 85% respectively. On the
other hand Apigenin triphosphate intermediate (ATRPI 14) and Apigenin Triphosphate (ATRP 15) gave a
yield of 56.5% and 99% respectively.
6. Biasutto, L.; Marotta,E.; Mattarei, A.; Beltramello, S.; Caliceti, P.;
Salmaso, S.; Bernkop-Schnϋrch, A.;Garbisa,S.; Zoratti, M.;
Paradisi, C. Absorption and metabolism of resveratrol
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CALCULATIONS OF 31P NMR CHEMICAL SHIFTS OF

12
Tetrahedron
Highlights
• The first sequential
phosphorylation of quercetin
• Structural characterization
• High yield