Aqueous diastereoselective hydrogenation of folic acid to tetrahydrofolic acid in the presence of water-soluble Rh and Ir diphosphine complexes

Article abstract of DOI:10.1016/j.tetasy.2005.12.034

Rhodium and iridium catalysts with chiral, water soluble diphosphine ligands, were used for the diastereoselective hydrogenation of folic acid disodium salt in water. Using a modified Rh/Josiphos type at 30 °C, l-tetrahydrofolic acid, a relevant pharmaceutical intermediate, was obtained with a selectivity of up to 49% de; at 70 °C turnover numbers of up to 2800 were achieved, albeit with lower selectivity. These results define the state of the art for this reaction.

Full text of DOI:10.1016/j.tetasy.2005.12.034

Tetrahedron: Asymmetry 17 (2006) 544–549
Aqueous diastereoselective hydrogenation of folic acid
to tetrahydrofolic acid in the presence of water-soluble
Rh and Ir diphosphine complexes
a,
b
b
a
*
Beno ˆı t Pugin, Viola Groehn, Rudolf Moser and Hans-Ulrich Blaser
a
Solvias AG, PO Box, CH-4002 Basel, Switzerland
Merck Eprova AG, Im Laternenacker 5, CH-8200 Schaffhausen, Switzerland
b
Received 16 November 2005; accepted 26 December 2005
Available online 21 February 2006
Dedicated to Jack Halpern on the occasion of his 80th birthday
Abstract—Rhodium and iridium catalysts with chiral, water soluble diphosphine ligands, were used for the diastereoselective hydro-
genation of folic acid disodium salt in water. Using a modified Rh/Josiphos type at 30 ꢀC, L-tetrahydrofolic acid, a relevant pharma-
ceutical intermediate, was obtained with a selectivity of up to 49% de; at 70 ꢀC turnover numbers of up to 2800 were achieved, albeit
with lower selectivity. These results define the state of the art for this reaction.
ꢁ
2006 Elsevier Ltd. All rights reserved.
1. Introduction
the physiological enzymatic reduction by dihydrofolate
reductase is stereoselective, leading to the natural,
7
L-Tetrahydrofolic acid 1a ([6S,aS]-tetrahydrofolic acid)
enantiomerically pure [6S,aS]-tetrahydrofolic acid 1a,
conventional chemical reductions always afford an equi-
molar mixture of diastereomers. [6S,aS]-Tetrahydrofolic
acid 1a can then be isolated, for example, as a sulfonate
salt by repeated fractional crystallization. With this pro-
cedure at least half of the starting material is lost while
the numerous crystallizations result in a low overall
plays a central role in cell metabolism as a carrier of
1
one carbon units of different oxidation state. Tetra-
hydrofolates serve as cofactors in a variety of enzymatic
reactions involved in nucleotide and amino acid biosyn-
thesis and several tetrahydrofolic acid derivatives are
manufactured for pharmaceutical applications. For
example, L-leucovorin 3 ([6S,aS]-5-formyltetrahydro-
folic acid, folinic acid) is utilized in cancer chemo-
therapy as it enhances the antitumor activity of 5-
8
yield of 28% of L-tetrahydrofolic acid sulfonate salt.
A stereoselective hydrogenation, which preferentially
affords the desired [6S,aS]-diastereomer 1a would,
therefore, be a significant improvement of the synthesis.
The development of such a stereoselective process is an
ambitious task for two major reasons. First, most effec-
tive enantioselective hydrogenation catalysts are Rh,
Ru, and Ir diphosphine complexes, which are not solu-
ble in water and, second, only very few stereoselective
reductions of heteroaromatic rings are known and in
most cases, selectivities and/or catalytic activity are
2
fluorouracil. L-Mefolinate 4 ([6S,aS]-5-methyltetra-
hydrofolic acid), which is an active metabolite of folic acid,
3
is effective in lowering elevated homocysteine levels
reported to increase the risk of cardiovascular diseases.
4
As shown in Scheme 1, L-tetrahydrofolic acid 1a is a key
intermediate in the conventional industrial four-step
synthesis of Ca-L-leucovorin 3 and Ca-L-mefolinate 4.
It was produced by the reduction of folic acid 2 with
5
6
9
NaBH4 or by catalytic hydrogenation with H /PtO
low. In principle, there are two approaches for solving
2
2
in aqueous solutions. The reduction of the pyrazine ring
produces a new stereogenic center in position 6. While
these problems. One can either adapt the substrate to
existing catalysts or modify existing catalysts to be able
to work in water. In fact, both approaches have been de-
scribed in the literature. An early attempt by Boyle and
Keating to hydrogenate a silylated folic acid derivative
in benzene using a Rh–DIOP complex failed, because no
1
0
*
Corresponding author. Tel.: +41 (0)61 686 63 35; fax: +41 (0)61 686
3 11; e-mail: benoit.pugin@solvias.com
6
0957-4166/$ - see front matter ꢁ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2005.12.034

B. Pugin et al. / Tetrahedron: Asymmetry 17 (2006) 544–549
545
O
O
H
OH
O
N
N
H
αS
N
N
OH
HN
N
folic acid
H
O
2
H N
2
NaBH4
or
H /PtO
2
2
O
O
O
H
O
H
OH
OH
OH
O
N
N
α
S
O
N
N α
S
H 6
R
H
H
N
N
6S
H
OH +
N
H
HN
HN
H N
N
H
O
O
H N
N
H
2
N
H
2
1a
1b
[
6S,αS
]-tetrahydrofolic acid = L-tetrahydrofolic acid
[6
R,αS
]-tetrahydrofolic acid = D-tetrahydrofolic acid
O
O
H
R
N
O
O
2
Ca
+
waste
O
N
N α
S
6S
H
O
HN
N
H
H N
N
H
2
3
4
: R= CHO = L-5-formyltetrahydrofolic acid calcium salt = Ca-L-Leucovorin
: R= CH3 = L-5-methyltetrahydrofolic acid calcium salt= Ca-L-mefolinate
Scheme 1.
conversion was obtained. Recently we demonstrated
that such an approach is actually feasible and that folic
acid dimethylester benzenesulfonate can be hydro-
genated in organic solvents with up to 60% de and turn-
2. Results and discussion
The disodium salt of folic acid was obtained by suspend-
ing folic acid in water and adjusting the pH to 7.5 with
30% sodium hydroxide solution followed by the addi-
tion of ethanol. The isolated folic acid disodium salt
was used as a substrate for all the hydrogenation experi-
ments described herein.
1
1
over numbers up to 400. Using a Rh/BPPM complex
1
2
adsorbed on silica gel Brunner et al. were able to
hydrogenate folic acid in aqueous buffer solutions with
up to 42% de but low activity and catalyst productivity.
This case also demonstrates a third difficulty, namely,
1
2a
analysis of the reaction products. In the first paper,
Although chiral water soluble diphosphine ligands have
1
3
up to 90% des were claimed, which later turned out to
be an artifact due to the enrichment of one diastereomer
during the preparation of the analytical sample, which
been known for many years, their diversity is still very
small compared to that of classical chiral diphosphine
ligands and, to the best of our knowledge, no such ligand
is commercially available. For catalyst screening, espe-
cially for such a ‘difficult’ substrate, a major challenge
was to gain access to as many water soluble ligands as
possible. In recent years we have developed a modular
toolbox that allows tethering functionalized diphos-
1
2c
required product derivatization.
Brunner’s approach to adsorb conventional catalysts on
an inorganic support is very elegant since it requires no
synthetic modification of the chiral ligand. On the other
hand, the use of porous solids may, among other things,
lead to poor mass transport. In fact, turnover numbers
1
4
phine ligands to various solid supports. The same
functionalized ligands were made water soluble by
attaching them to water soluble vehicles, using a concept
(
ton) as well as turnover frequencies (tof) as published
1
5
by Brunner were very low and far from practical inter-
est. Herein we report the use of water soluble Rh and
Ir complexes for the stereoselective hydrogenation of
folic acid disodium salt in water with up to 49% de and
originally developed by Malmstr o¨ m and Andersson.
They tethered chiral diphosphine ligands containing an
N–H group to non-cross linked polyacrylic acid via
amide bonds. While in many cases this concept worked
well, it was also shown that the attachment of several
complexes to one polymer string can give rise to a high
local catalyst concentration. Depending on the immobi-
lized complex, the resulting interactions between active
1
1a
turnover numbers up to 2800. We also report a direct
HPLC method to determine the diastereomeric excess,
which does not require any product derivatization or
work-up before analysis.

546
B. Pugin et al. / Tetrahedron: Asymmetry 17 (2006) 544–549
R P
EtOOC
EtOOC
2
EtOOC
EtOOC
NH
O
O
R P
O
O
2
PR2
O
N
N
O
NH2
+
N
N
N
NH
O
H
O
PR2
EtOOC
EtOOC
1
) KOH / H O
2
2) HCl
HOOC
HOOC
O
O
PR2
PR2
O
N
NH
O
H
HOOC
Scheme 2.
centers can lead to negative effects on their catalytic
performance. To avoid this problem, we also used a well
defined dendrimer fragment with carboxylic acid groups
as water soluble vehicles, which can be attached to func-
The first series of hydrogenations was performed with
catalysts formed in situ from [Rh(NBD) ]BF and the
2
4
ligands depicted in Figure 1. The results are listed in
Table 1, entries 1–10. Whereas the diastereoselectivities
ranged from 18% (Pyrphos 10) to 47% for PPM-de 5b
and 46% de for BIPHEMP 8 (entries 2 and 6), the catal-
ytic activity varied much more. It is interesting to note
that the dendrimer vehicle gave catalysts with signifi-
cantly better activities and selectivities than the poly-
acrylic acid vehicle with PPM (entries 1 and 2).
Similarly, the dendritic Josiphos 7b reached a very
1
6
tionalized ligands (see Scheme 2).
The water soluble ligands used herein are depicted in
1
7
Figure 1. PPM and functionalized Josiphos ligands
1
5
were attached to polyacrylic acid, as well as to the
dendritic vehicle (ligands 5–7). The BIPHEMP and
1
6
18
Pyrphos moieties were only attached to the dendrimer
À1
(
ligands 8 and 10). Additionally, a sulfonated biaryl
remarkable tof of 184 h (entry 5), almost twice as high
ligand 9 was obtained from Roche.
as that of the corresponding Josiphos-pa 7a (entry 4).
PR'2
Ph P
PR'2
PPh2
2
Fe PPh2
PPh2
Fe
N
NH
O
R
O
R
7
BPPM
Josiphos
5
a: R = polyacrylic acid
b: R = HN(CH)(CH OCH CH COOH)
6: R' = cyclohexyl; R = polyacrylic acid
7: R' = 3,5-xylyl
5
2
2
2
3
a: R' = 3,5-xylyl; R= polyacrylic acid
2
2
2
3
Ph P
PPh2
2
Ph P
2
Ph P
NaO S
N
2
O
3
O
O
P
P
2
2
O
NH
O
NH
O
NaO S
O
O
3
O
O
O
COOH
COOH
COOH
COOH
COOH
COOH
8: BIPHEMP-dendrimer
9: MeOBIPHEP-SO Na
3
10: Pyrphos-dendrimer
Figure 1. Water soluble diphosphine ligands.

B. Pugin et al. / Tetrahedron: Asymmetry 17 (2006) 544–549
Table 1. Asymmetric hydrogenation of folic acid disodium salt in water
547
a
À1
)
Entry
Catalyst
t (h)
Conv. (%)
de (%)
tof (h
Comments
1
2
3
4
5
6
7
8
9
Rh/PPM-pa 5a
Rh/PPM-de 5b
18.5
4
79
87
61
90
92
89
87
82
2
28
47
36
30
31
46
33
18
9
4
22
3
100
184
27
6
41
0.1
0.3
Rh/Josiphos-pa 6
Rh/Josiphos-pa 7a
Rh/Josiphos-de 7b
Rh/BIPHEMP-de 8
Rh/MeOBIPHEP-SO
Rh/Pyrphos-de 10
Ir/Josiphos-de 7b
Ir/Josiphos-pa 7a
17.5
0.9
0.5
3.2
14
2
23
20
3
Na 9
13.4 equiv NaI/Ir
1320 equiv NaI/Ir
1
0
6
1
Reaction conditions: p = 80 bar, T = 70 ꢀC, substrate/catalyst ratio (s/c) 100, substrate concentration 0.13 mol/l, ligand/metal molar ratio = 1.25,
pH = 7 (phosphate buffer).
a
[
2 4 2
Rh(NBD) ]BF or [Ir(COD)Cl] (NBD = norbornadiene, COD 1,5-cyclooctadiene).
2 4
Table 2. Asymmetric hydrogenation of folic acid disodium salt in water catalyzed by [Rh(NBD) ]BF /Josiphos-de 7b
À1
Entry
p
T
s/c
100
100
100
100
100
100
100
100
t (h)
Conv. (%)
de (%)
tof (h
)
Comments
1
2
3
4
5
6
7
8
9
80
80
80
20
80
80
80
80
80
80
70
70
70
70
30
30
70
70
70
70
0.5
23.5
8
92
23
90
94
97
26
92
100
89
91
31
21
26
26
49
38
26
23
30
26
184
1
11
47
8
Standard
Josiphos 7
[Rh(COD)Cl] /Josiphos 7/SiO
2 2
2
12
16.5
0.3
1
4
8
2
pH = 6
100 equiv NaBr/Rh
20 equiv KI/Rh
Substrate = 0.28 M
Substrate = 0.24 M
307
100
223
341
1000
3000
a
10
Reaction conditions: pH 7, phosphate buffer, substrate concentration 0.13 mol/l, ligand/metal molar ratio = 1.25.
a
Substrate was purified by stirring over Rh/C before use.
Since iridium diphosphine complexes in the presence of
iodide are known to be highly efficient catalysts for the
hydrogenation of imines, a few experiments were car-
ried out with selected Ir complexes (entries 9 and 10).
However, the diastereoselectivities as well as activities
were very low.
tof decreased strongly. At 0 ꢀC no conversion was
observed any more. Changing the pH from 7 to 6
decreased the de somewhat but slowed the reaction even
further, probably because under these conditions, the
starting material was not completely dissolved (entry
6). The addition of additives, such as NaBr or KI, went
along with a slight decrease of diastereoselectivity but
the presence of NaBr was beneficial for the catalytic
activity (entries 7 and 8). While it was possible to run
the reaction at an s/c ratio of 1000 within a reasonable
reaction time (entry 9), a further increase to 3000 was
only possible with an additional purification of the sub-
strate. When the folic acid disodium salt was stirred over
Rh/C before use, a conversion of >90% was reached
after 8 h (entry 10). Without this pre-treatment there
was no conversion, which indicates that the substrate
contains a catalyst poison that is adsorbed on Rh/C.
1
9
The dendritic Rh/Josiphos-de 7b catalyst was chosen for
further investigations. The reason for this choice were
the very high activity observed and the fact that Josi-
phos ligands are available on large scale, which is not
the case for the PPM and the functionalized BIPHEMP
ligands used in this investigation. In order to check
whether it was really necessary to have water soluble
catalysts, we carried out experiments with the unfunc-
tionalized Josiphos 7, as well as with Josiphos adsorbed
on a silica gel and we investigated the effect of various
reaction parameters. The results of these studies are
compiled in Table 2. A comparison of entries 2 and 3
with the standard experiment (entry 1) clearly shows
that the unfunctionalized or adsorbed Rh complexes
have very low activity, even though the stereoselectivity
is not affected so much. This clearly demonstrates that it
is essential to have a catalyst, which is soluble in the
reaction medium. The stereoselectivity was not much af-
fected by the reaction pressure (entry 4) but depended
strongly on the reaction temperature. Decreasing the
reaction temperature from 70 to 30 ꢀC resulted in an
increase of the de from 31% to 49% (entry 5), but as
expected the reaction times became longer and the
3. Conclusion
We have demonstrated that a modular approach based
on attaching different functionalized ligands to various
water soluble groups is feasible for screening a broad
range of water soluble homogeneous catalysts for a
commercially relevant application. We also found that
folic acid disodium salt can be hydrogenated stereoselec-
tively in aqueous solution with water soluble Rh diphos-
phine complexes. Diastereoselectivities of up to 47–49%
were obtained with Rh/BIPHEMP 8, Rh/PPM-de 5b,

548
B. Pugin et al. / Tetrahedron: Asymmetry 17 (2006) 544–549
and with Rh/Josiphos-de 7b (at 30 ꢀC), which is a slight
for ligand 7a. The preparation of the dendrimer bound
ligands 5b, 7b, 8, and 10 is described in Ref. 16.
1
2c
improvement in comparison with Brunner’s best de of
0%. However, good catalytic activity could only be
4
achieved with Rh/Josiphos-de 7b with tons up to
4.3. Procedures for the hydrogenation of folic acid
disodium salt
À1
3
34 h at 70 ꢀC. However, under these conditions, ste-
reoselectivity varies between 26% and 31% de. Turnover
numbers of >2700 were obtained (with substrate stirred
over Rh/C before use), which is a huge improvement
4
.3.1. Hydrogenations with Rh catalysts. To 25 lmol
water soluble diphosphine ligand were added 5 ml water
1
2
compared to Brunner’s best tons of 40. While catalyst
activity and productivity are very close to being techni-
cally viable, the diastereoselectivities are still too low to
make this an alternative to the classical process. Further
efforts are needed to identify a catalytic system with
similar activities and improved diastereoselectivities in
order to make this new method an alternative to the
classical process.
and 0.5 ml buffer solution of pH 7. By the addition of
0
.1 M NaOH (about 1 ml), the carboxyl groups of the
ligand were deprotonated. The resulting clear solution
was added to 20 lmol [Rh(NBD) ]BF and stirring con-
2
4
tinued until a clear solution was obtained. This catalyst-
solution was added to a solution of 2 mmol folic acid
disodium salt in 11 ml water and 1.5 ml buffer solution
of pH 7. At this point additives were added to the reac-
tion mixture if required or the pH was adjusted to pH 6
by the addition of 4 ml of 1 M KH PO solution. The
2
4
4. Experimental
mixed solution was added via a capillary into a 50 ml
autoclave flushed with nitrogen. The autoclave was
sealed and the hydrogenation started by stirring at
4
.1. General
7
0 ꢀC with constant hydrogen pressure of 80 bar unless
All manipulations with oxygen and moisture sensitive
materials were performed under an argon atmosphere.
All solvents, which were used for preparing catalysts
and for the hydrogenation reactions, were degassed at
a vacuum line and flushed with argon. The buffer solu-
tion of pH 7, which was added to the reaction mixtures
was prepared by dissolving 0.041 mol Na HPO and
otherwise is stated. The reactions were run until the
hydrogen uptake stopped. After hydrogenation, the
reaction mixtures were set under argon and samples
for HPLC analysis of tetrahydrofolic acid taken. The
hydrogenations with high s/c were performed in a 5–10
mmol substrate scale.
2
4
0
.028 mmol KH PO in 1 l of water.
2 4
4.3.2. Hydrogenations with Ir catalysts. To 25 lmol
diphosphine ligand and 12.5 lmol [Ir(COD)Cl] , 5 ml
2
The hydrogenations were run in steel autoclaves,
equipped with sensors for temperature and pressure.
The reaction pressure was kept constant by continuous
supply of hydrogen from a reservoir. Hydrogen con-
sumption was monitored indirectly by measurement of
the pressure decrease in the reservoir. Hydrogenation
solutions were stirred with a magnetic stirring bar. Folic
acid dihydrate was purchased from Fluka (47620). Folic
acid disodium salt had a purity of 95% and contained
methanol was added. After stirring for 10 min, the meth-
anol was evaporated and a solution of 1.25 g (2 mmol)
folic acid disodium salt in 25 ml of water and 2 ml of
buffer solution added. By addition of 0.1 M NaOH, a
clear solution was obtained. At this point, additives were
added if required. The solution was transferred via
a narrow tube into a 50 ml autoclave flushed with
nitrogen. The hydrogenations were then performed as
described above.
5
% water.
4
.3.3. Pre-treatment of folic acid disodium salt. Rh/C
4
4
.2. Preparation of the water soluble ligands
(600 mg) (Engelhard 4806) was washed three times with
distilled water and added to a solution of 6 mmol folic
acid disodium salt in 20 ml of water and 3.5 ml of buffer
solution. After stirring under an argon atmosphere for
.2.1. Functionalized ligands. (À)-(2S,4S)-2-Diphenyl-
phosphinomethyl-4-diphenylphosphino-pyrrolidine was
purchased from Aldrich (32,677-1). (3R,4R)-Bis(di-
phenylphosphino)pyrrolidine was prepared as described
by Nagel et al., the Josiphos- and Xyliphos-ligands
with an aminomethyl function at the bottom cyclopen-
tadienyl ring were prepared as described in Ref. 17
and the BIPHEMP ligand with the OH function was
synthesized as described in Ref. 18.
1
h, the Rh/C was filtered off and washed with 1 ml of
water. The aqueous substrate solution was then used
for the hydrogenation.
2
0
4
acid
.4. HPLC methods for the analysis of tetrahydrofolic
The content of tetrahydrofolic acid was determined
8
4
.2.2. Water soluble ligands. The polyacrylic acid
by using the following HPLC method. Na HPO
2
4
attached ligand 5a was prepared as described in Ref. 14
while ligands 6a and 7a were prepared in an analogous
way, as described in Ref. 17. Polyacrylic acid with an
average MW of 2000 was used and was purchased as a
(0.03 mol) and KH PO (0.03 mol) were dissolved in
2 4
1 l of water to give solvent A. Solvent B was composed
of 800 ml solvent A and 200 ml methanol. By addition
of phosphoric acid the pH was adjusted to pH 7.8. A
solution of tetrahydrofolic acid in solvent A (1 mg/ml)
was analyzed on an ODS column (Macherey and Nagel,
Shandon-Hypersil, 250 · 4 mm) by using a linear gradi-
6
0% aqueous solution from Aldrich (19,202-3). The
polymer loadings were 0.67 mmol ligand/g for ligand
a, 0.54 mmol ligand/g for ligand 6a, and 0.22 mmol/g
5

B. Pugin et al. / Tetrahedron: Asymmetry 17 (2006) 544–549
549
ent (flow rate 1 ml/min) from 0% to 85% solvent B with-
in 28 min and UV detection at 280 nm. To protect tetra-
hydrofolic acid from oxidative degradation, all eluents
were continuously flushed with argon.
7. Uwajima, T.; Oshiro, T.; Eguchi, T.; Kuge, Y.; Horiguchi,
A.; Igarashi, A.; Mochida, M.; Iwakura, K. Biochem.
Biophys. Res. Commun. 1990, 171, 684–689.
8
. M u¨ ller, H. R.; Ulmann, M.; Conti, J.; M u¨ rdel, G. EP
495204, 1991 (assigned to Eprova AG).
0
9
. For a recent overview see: Blaser, H. U.; Malan, Ch.;
Pugin, B.; Spindler, F.; Steiner, H.; Studer, M. Adv. Synth.
Catal. 2003, 345, 103–151.
The diastereoisomers of tetrahydrofolic acid were
determined by using the following HPLC method.
The eluent was prepared by mixing 960 ml water,
8
1
0. Boyle, P. H.; Keating, M. T. J. Chem. Soc., Chem.
Commun. 1974, 375–376.
4
0 ml acetonitrile, 8 g b-cyclodextrine, and 10 ml tri-
ethylamine. By adding of acetic acid, the pH was
adjusted to pH 7.0 at which point 274 ll 37%
formaldehyde was added. A solution of tetrahydrofolic
acid in the eluent (0.5 mg/ml) was analyzed (flow rate
11. (a) M u¨ ller, H. R.; Moser, R.; Groehn, V.; Pugin, B. WO
01/04120, 2001 (assigned to Eprova AG); (b) Groehn, V.;
Moser, R.; Pugin, B. Adv. Synth. Catal. 2005, 347, 1855–
1
862.
2. (a) Brunner, H.; Huber, C. Chem. Ber. 1992, 125, 2085–
093; (b) Brunner, H.; Bublak, P.; Helget, M. Chem. Ber./
1
1
ml/min) on an ODS column (Macherey and Nagel
2
Nucleosil/CART, 120-5, 250 · 4 mm) with UV-detec-
tion at 280 nm.
Recl. 1997, 130, 55–61; (c) Brunner, H.; Rosenboem, S.
Monatsh. Chem. 2000, 131, 1371–1382.
1
1
3. Sinou, D. Adv. Synth. Catal. 2002, 344, 221–237.
4. Pugin, B.; Landert, H.; Spindler, F.; Blaser, H. U. Adv.
Synth. Catal. 2002, 344, 974–979; Pugin, B. Chimia 2001,
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