A novel synthetic method for preparation of some folates

Article abstract of DOI:10.1007/s11164-012-0751-2

An improved method was developed for preparation of 5,6,7,8-tetrahydrofolic acid (THF) and calcium-5-methyltetrahydrofolate (5-MTHF-Ca) by reduction of folic acid using KBH₄ catalyzed by Pb(NO₃)₂. The yields of THF and 5-MTHF-Ca were 56.5 and 42.7 %, respectively. A convenient method for measurement of THF and 5-MTHF-Ca using liquid chromatography-mass spectrometry (LC-MS) was also established, enabling analysis of those folates within 10 min without application of gradient elution.

Full text of DOI:10.1007/s11164-012-0751-2

Res Chem Intermed (2013) 39:2211–2218
DOI 10.1007/s11164-012-0751-2
A novel synthetic method for preparation of some
folates
•
Minghao Zhou Mei Hong Guomin Xiao
•
Received: 4 July 2012 / Accepted: 26 July 2012 / Published online: 18 August 2012
Ó Springer Science+Business Media B.V. 2012
Abstract An improved method was developed for preparation of 5,6,7,8-tetra-
hydrofolic acid (THF) and calcium-5-methyltetrahydrofolate (5-MTHF-Ca) by
reduction of folic acid using KBH4 catalyzed by Pb(NO3)2. The yields of THF and
5-MTHF-Ca were 56.5 and 42.7 %, respectively. A convenient method for mea-
surement of THF and 5-MTHF-Ca using liquid chromatography–mass spectrometry
(LC–MS) was also established, enabling analysis of those folates within 10 min
without application of gradient elution.
Keywords Calcium-5-methyltetrahydrofolate Á Gradient elution Á Reduction Á
LC–MS Á 5,6,7,8-Tetrahydrofolic acid
Introduction
5,6,7,8-Tetrahydrofolic acid and calcium-5-methyltetrahydrofolate are widely used
as both food additives and drug additives, being of great importance to human
health [1–5]. 5,6,7,8-Tetrahydrofolic acid, mainly produced by reduction of folic
acid or by catalytic hydrogenation with H2/PtO2 or other noble-metal catalysts such
as Rh [6–8], can serve as a cofactor in a variety of enzymatic reactions in our body
involved in nucleotide and amino acid biosynthesis. Moreover, several 5,6,7,8-
tetrahydrofolic acid derivatives, such as calcium-L-5-formyltetrahydrofolate, (6S)-5-
methyltetrahydrofolic acid, and calcium-L-5-methyltetrahydrofolate, are manufac-
tured for medical and pharmaceutical applications; for example, (6S)-tetrahydro-
folic acid plays a crucial role in cell metabolism as a carrier of one-carbon units of
different oxidation state [9]. Calcium-L-5-formyltetrahydrofolate is broadly utilized
M. Zhou Á M. Hong Á G. Xiao (&)
School of Chemistry and Chemical Engineering, Southeast University, Nanjing 210094,
People’s Republic of China
e-mail: xiaogm@seu.edu.cn
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M. Zhou et al.
in anemia treatment and cancer chemotherapy, and (6S)-5-methyltetrahydrofolic
acid, which is an active metabolite of folic acid, is suitable for prevention of neural
tube defects in the fetus and treatment of arteriosclerosis. Calcium-L-5-methyltet-
rahydrofolate, mostly used as a nutrient supplement, is the predominant, naturally
occurring form of folate in the human body.
Herein, we report some results on synthesis of 5,6,7,8-tetrahydrofolic acid and
calcium-5-methyltetrahydrofolate. An effective method using KBH4 catalyzed by
Pb(NO3)2 for reduction of folic acid in water without use of a noble-metal catalyst was
developed (Scheme 1). To date, application of Pb(NO3)2 as a catalyst in the reaction to
produce 5,6,7,8-tetrahydrofolic acid has not been reported in the open literature.
Moreover, the optimum reaction condition has been studied through single-factor and
orthogonal experiments. For detection of folates, a direct and sensitive method using
liquid chromatography–mass spectrometry (LC–MS) was established, which can give
precise detection results without any pretreatment and is superior to other reported
methods [10–14].
Materials and methods
Materials and devices
All chemicals were from commercial sources and used without further purification. All
reagents were of analytical grade apart from those used as mobile phase for high-
performance liquid chromatography (HPLC), such as methanol, acetonitrile, and acetic
acid, which were chromatographically pure. HPLC experiments were performed on
LCQ Deca XP plus, consisting of a reversed C18 column, ø4.6 9 150 mm. MS was
performed on an electrospray mass spectrometer (Finnigan LCQ Deca XP plus, USA).
Scheme 1 Process for preparation of THF and 5-MTHF-Ca
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A novel synthetic method for preparation of some folates
2213
Preparation of 5,6,7,8-tetrahydrofolic acid
A mixture of folic acid (4.5 g) and Pb(NO)2 (0.1 g) in degassed water was stirred at
6 °C under nitrogen for 30 min, then aqueous NaOH (20 %) was added in a 250-mL
four-necked flask equipped with thermometer and water bath. KBH4 aqueous was
added dropwise with citric acid used to adjust pH at about 8–10. Then, the reaction
mixture was warmed to 30 °C and stirred for another 4 h. After the reaction, the
mixture was cooled to 5 °C. Then, 18 % hydrochloric acid was added dropwise
until the mixture had a pH value of 2.6. During the acidification, the 5,6,7,
8-tetrahydrofolic acid began to precipitate. The precipitated 5,6,7,8-tetrahydrofolic
acid was isolated by means of filtration, then washed with degassed and deionized
water for three times or more. The so-obtained white or yellowish solid was dried
under reduced pressure at drying temperature no higher than 30 °C.
Preparation of calcium-5-methyltetrahydrofolate
In a 250-mL four-necked flask, 5,6,7,8-tetrahydrofolic acid (3 g) was added to a
stirred solution containing sodium hydroxide with pH of 9. Then, formaldehyde
(1.7 g) was added to the reaction mixture. In 30 min, potassium borohydride
solution was added dropwise. All these were carried out at 15 °C and the reaction
condition maintained for 4 h. Then, the reaction mixture was warmed to 55 °C,
stirred for 30 min. Finally, the mixture was cooled to 5 °C. Eighteen percent
hydrochloric acid was added dropwise until the mixture had pH value 6–7, then
solid calcium chloride (1.5 g) was added to give salt of 5-methyltetrahydrofolic
acid. The precipitated 5-MTHF-Ca was isolated by means of filtration, then washed
with degassed and deionized water for three times or more. The so-obtained white
solid was dried under reduced pressure.
Results and discussion
Process optimization of 5,6,7,8-tetrahydrofolic acid
To identify the optimum reaction conditions, we took the following factors into
consideration: mole ratio of raw materials, reaction temperature, and reaction time.
We employed several different kinds of catalysts in the preparation of 5,6,7,
8-tetrahydrofolic acid, such as Ni(NO3)2, Cu(NO3)2, Zn(NO3)2, and Pb(NO3)2. In
comparison, Pb(NO3)2 gave the best yield among those catalysts (Table 1). Then,
we identified the best reaction condition through single-factor and orthogonal
Table 1 Influence of different catalysts on the THF yield
Catalyst
None
20.6
Ni(NO3)2
31.5
Cu(NO3)2
29.2
Zn(NO3)2
38.4
Pb(NO3)2
56.5
Yield (%)
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M. Zhou et al.
Fig. 1 Influence of different factors on the yield of THF: a mole ratio, b reaction time, c catalyst amount,
and d reaction temperature
experiments considering the factors mentioned above. All the experimental results
are presented in Fig. 1.
From the four curves showing the influence of mole ratio, reaction time, catalyst
amount, and reaction temperature, there was a distinct salient point in each figure.
The yield of THF first increased and then decreased in the four curves. From these
results, we can see that, when the reaction was carried out under mole ratio
(KBH4:folic acid) of 4, it gave the best yield of THF. Also, when the amount of
catalyst used was up to 0.1 g, we could get the best yield, maybe due to a proper
amount of catalytic active sites. Regarding the influence of reaction time and
reaction temperature, we speculate that prolonged reaction time and higher reaction
temperature would lead to deterioration of folic acid and the product THF which
was thermally unstable, then give rise to some side-reactions, and finally reduce the
yield of THF. Based on all the results above, we identified the best reaction
conditions for preparation of 5,6,7,8-tetrahydrofolic acid to be: optimum reaction
temperature 30 °C, reaction time 4 h, catalyst amount 0.1 g Pb(NO)2, and mole
ratio of reactants 4:1 (KBH4:folic acid). The yield of THF reached 56.5 %.
Process optimization for calcium-5-methyltetrahydrofolate
Attempting to obtain calcium-5-methyltetrahydrofolate, we tried two different
kinds of reaction system, i.e., methylation in the HCHO/KBH4 and HCHO/HCOOH
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Fig. 2 Influence of different factors on the yield of 5-MTHF-Ca: a mole ratio of THF/HCHO, b mole
ratio of HCHO/KBH4, c reaction time, and d reaction temperature
system, respectively. In comparison, methylation in the HCHO/KBH4 system seemed
to give better results, while the products obtained through methylation by HCHO/
HCOOH contained lots of impurities. Also, we considered the following factors:
reaction time, mole ratio of raw materials, and reaction temperature. Then we
identified the best reaction conditions through single-factor analysis considering the
impact factors mentioned above. All the experimental results are presented in Fig. 2.
In the curves showing the influence of mole ratio (THF:HCHO:KBH4) and
reaction time, there was a peak value, after which the yield began to decrease. The
yield of 5-MTHF-Ca decreased with increasing reaction temperature, as the reactant
THF is thermolabile, converting to some other chemical compound at higher
reaction temperature. Also, prolonged reaction would lead to lower yield due to the
presence of unknown byproduct, so finally the reaction was carried out for 4 h in
order to reduce the amount of byproduct. From all the results above, we identified
the best reaction condition for the preparation of calcium-5-methyltetrahydrofolate
to be: optimum reaction temperature 5–15 °C, reaction time 4 h, and mole ratio of
reactants 1:3:9 (THF:HCHO:KBH4). The yield of 5-MTHF-Ca reached 42.7 %.
HPLC–MS analysis of THF and 5-MTHF-Ca
Direct injection of THF and 5-MTHF-Ca using electrospray ionization (ESI) in
positive ion mode was chosen because greater signal-to-noise ratios were obtained
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Fig. 3 Total scan chromatogram of THF (a) and 5-MTHF-Ca (b)
Fig. 4 MS spectra of THF (a) and 5-MTHF-Ca (b)
compared with atmospheric-pressure chemical ionization (APCI). Figures 3 and 4
show the spectra obtained for THF and 5-MTHF-Ca. The retention times of THF
and 5-MTHF-Ca were 5.66 and 4.43 min, respectively, as shown in Fig. 3. The
structure of THF and 5-MTHF-Ca can be confirmed from the fragments of the MS
spectra, as marked in Fig. 4. All the fragment information supports the presence of
THF and 5-MTHF-Ca (Table 2).
Conclusions
Based on the results presented, it was speculated that Pb(NO3)2 benefits production
of the target THF. Feasible synthetic routes have been established for synthesis of
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Table 2 Fragmentation spectra
Folate
Suggested ion
of THF and 5-MTHF-Ca
THF
483.99 [M ? K]?
468.02 [M ? Na]?
446.05 [M ? H]?
299.01
OH
H
N
O
C
N
N
H
N
N
H
H2N
5-MTHF-Ca
498.26 [M ? K]?
482.42 [5-MTHF ? Na]?
460.43 [5-MTHF ? H]?
313.47
OH
N
CH3
N
O
C
N
N
H
N
H
H2N
180.16
OH
N
CH3
N
N
N
H
H2N
THF and 5-MTHF-Ca. Additionally, an efficient and easy method using HPLC–MS
was established for qualitative and quantitative analysis of THF and 5-MTHF-Ca
without application of gradient elution. Quantitative analysis of THF and 5-MTHF-
Ca was carried out using an external standard method. Judging from the conditions
employed, the presented method has great prospects for use in applications,
although further work is also needed.
Acknowledgments The authors are grateful for the financial support from the National Natural Science
Foundation of China (no. 21076044) and the National Basic Research Program of China (973 Program)
(grants 2010CB732206 and 2012CB215306).
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