A new route for total synthesis of (±) dephospho Form B of molybdenum cofactor by direct one step thiophene annulation from suitable pterin alkynes

Article abstract of DOI:10.1016/j.tetlet.2013.02.090

A new convenient total synthesis of (±) dephospho Form B of molybdenum cofactor has been described. The development of direct thiophene annulation onto pterin from a simple direct thiophene annulation at the alkyne moiety at 6-position of pterin is successfully achieved by using excess of sodium sulfide. This novel method conveniently led to the synthesis of a series of pterin fused α-substituted thiophenes (9-11, respectively).

Full text of DOI:10.1016/j.tetlet.2013.02.090

Tetrahedron Letters 54 (2013) 2373–2376
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Tetrahedron Letters
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A new route for total synthesis of ( ) dephospho Form B of molybdenum
cofactor by direct one step thiophene annulation from suitable pterin alkynes
⇑
Shyamaprosad Goswami , Annada C. Maity , Shampa Chakraborty, Manas Kumar Das,
Bhaswati Goswami
Department of Chemistry, Bengal Engineering and Science University, Shibpur, Howrah 711 103, West Bengal, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A new convenient total synthesis of ( ) dephospho Form B of molybdenum cofactor has been described.
The development of direct thiophene annulation onto pterin from a simple direct thiophene annulation at
the alkyne moiety at 6-position of pterin is successfully achieved by using excess of sodium sulfide. This
Received 30 November 2012
Revised 23 February 2013
Accepted 27 February 2013
Available online 7 March 2013
novel method conveniently led to the synthesis of a series of pterin fused
a-substituted thiophenes
(9–11, respectively).
Ó 2013 Elsevier Ltd. All rights reserved.
Dedicated to Professor Edward C. Taylor of
Princeton University, USA on his 90th
birthday for his well-known contribution in
heterocyclic, medicinal, and thallium
chemistry
Keywords:
Pterins
Dephospho Form B
Pterin alkynes
Thiophene annulation
All oxomolybdoenzymes appear to require a common cofactor
(molybdenum cofactor) as their catalytic center.1,2 Molybdenum
cofactor (Moco) is essential for the function of the human enzymes
for example sulfite oxidase, xanthine dehydrogenase/oxidase, and
aldehyde oxidase. The presence of pterin moiety is the reason of
blue fluorescence in the degradation products of the Moco. The
common organic cofactor for the activity of the enzymes has been
termed molybdopterin. The dephospho derivatives Form A, Form B,
urothione, and camMPT (carboxamidomethyl derivative) have
the pterin ring in camMPT, Form B, and urothione and the
formation of these compounds is consistent with the decomposi-
tion of proposed 1,2-ene-dithiolate structure of Moco. The
decomposition of Moco-containing molybdoenzymes was carried
out in the absence of iodine and a degradation product dephospho
Form B was obtained which contains only one of the two sulfur
atoms present in molybdopterin itself, for which the structure
(thieno[3,2-g]pterin) is shown as compound 12.23 In 1984, Form
B
structure was proposed by Johnson and Rajagopalan and
been isolated from molybdenum cofactor 1 (Moco) (Fig. 1).1–5
A
co-workers based upon proton NMR and mass spectral analysis.24
Thus Form B and urothione are the two thiophene fused pterin
metabolites which have been isolated from Moco from human
urine. Taylor et al. have reported13–15,22,24,26 the chemical synthesis
of the dephospho Form A (asymmetric and racemic synthesis),
Form B, deoxyurothione, and urothione. We describe in this Letter
a novel, concise, and convenient total synthesis of ( ) dephospho
Form B (12) (from the pivaloyl and silyl protected thienopterin
11) by a simple elegant direct thiophene annulation method onto
a pre-formed suitable pterin alkyne developed by us. We also re-
port here that this method conveniently led to the synthesis of a
significant number of review articles have been published to
discuss the biochemistry, spectroscopy, mechanism, and recent
advances of the Moco containing enzymes and the Moco struc-
ture.6–11 Several oxidative degradation products of molybdopterin
have been synthesized.12–23 The suggested ligation of sulfur li-
gands of molybdopterin is in accordance with the results of EXAFS
(extended X-ray absorption fine structure) studies.5a Sulfur func-
tionalities are attached with the
a and b unsaturated carbons to
⇑
Corresponding author. Fax: +91 33 2668 2916.
series of pterin fused
respectively).
a-substituted thiophenes (9, 10, and 11
E-mail address: spgoswamical@yahoo.com (S. Goswami).
Present address: Department of Bioinspired Science, Department of Chemistry
and NanoScience, Center for Biomimetic Systems, Ewha Womans University, Seoul
120-750, Republic of Korea.
The key to success of our direct thiophene annulation onto pter-
in having suitable alkyne moiety at 6-position was the anticipation
0040-4039/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2013.02.090

2374
S. Goswami et al. / Tetrahedron Letters 54 (2013) 2373–2376
O
N
O
SMe
N
N
_
3
N
N
HN
H N
HN
H N
OPO
OH
H
H
S
OH
S
OH
2
N
2
Form B
Urothione
O
O
H N
NH
2
2
H
OH
_
3
CH CH
2
2
O
H
N
SH
O
OPO
O
N
O
N
H
N
S
S
SH
N
_
3
HN
H N
HN
H N
OPO
HN
H N
CHOHCH
_
3
2
OPO
N
N
H
N
2
2
N
H
2
Form A
camMPT
MOLYBDOPTERIN
O
OH
O
O
P
H
N
O
HN
O
S
S
H N
2
N
N
H
Mo
(X = O or S)
O
X
MOLYBDENUM COFACTOR
1
Figure 1. Structures of the Moco, MPT, and four degradation compounds of MPT.
that sulfide ion (S2ꢀ) is nucleophilic enough to attack the acetylenic
carbon attached with electron withdrawing pterin system (Michael
addition) and then attack further in an intramolecular way the
electron deficient imine carbon at 7-position of pterin for cycliza-
tion which really happened to our satisfaction. Thus one step direct
thiophene annulation onto pterin from suitable pterin alkynes was
successfully achieved simply in one step by sulfide ion (a very
strong nucleophile, nucleophilicity order is 3.3).
Now we describe the synthesis of thienopterins and hence Form
B (dephospho). Taylor and Sabb13 have reported a method for gener-
ating the highly functionalized thieno[2,3-b]pyrazine which was la-
ter applied to the synthesis of thieno[3,2-g]pterin. In our case, we
used the pterin alkynes (6, 7 and 8) for the thiophene annulation
onto pterin by treating simply with sodium sulfide leading to the
synthesis of thiophene fused pterins 9, 10, and 11 respectively of
which 11 was converted into target Form B (12). The details of the
synthesis are delineated in Scheme 2 (see also Supplementary data).
Thus thienopterins 9 and 10 have been readily accessible from
alkynylpterins 6 and 7, respectively by the reaction with excess so-
dium sulfide in THF. Similarly, treatment of 8 with sodium sulfide
in THF gave the pivaloyl derivative 11 in 69% yield. The progress of
the thiophene annulation reaction was monitored by TLC which
showed the appearance of a more polar spot compared to the start-
ing material and within a few hours it showed disappearance of the
starting material.
To follow the above synthetic strategy, we wanted to synthe-
size the suitable alkyne substituted pterins to reach the target
thienopterins. While a broad range of N-pivaloylated-C(6)-al-
kyne substituted pterins (PPt–C„C–CHRR1 where PPt is
2-N-pivaloylpterin)15,17 can be prepared following the procedure
15,24,25
reported.
Thus the alkyne functionality in compounds 6 and 8 were
obtained by a palladium catalyzed coupling reaction of the corre-
sponding terminal alkynes with C(6)-chloro-N-pivaloylpterin
according to the procedure reported by Taylor et al.15b,26a
(Scheme 1).
The material 11 as isolated was shown by mass spectroscopy to
have a molecular ion at m/e 602 (MH+) and its 1H NMR spectrum
was consistent with the expected structure 11. We have thus
O
HC
OTBDMS
OTBDMS
a)
H
N
N
3
O
N
O
N
N
H
N
6
N
N
Cl
HN
O
N
H
2
Me
OTBDPS
Me
O
2-pivalolyl-6-chloropterin
OTBDPS
HC
H
N
OTBDPS
4
O
N
HC
b)
c)
N
H
N
N
OH
5
7
OH
O
N
OTBDPS
H
N
N
O
N
N
H
8
Scheme 1. Synthesis of alkyne substituted pterins 6–8. Reagents and conditions: (a) Pd(OAc)2, CuI, PPh3, Et3N, MeCN, 60 °C, 4 h, 76%; (b) Pd(PPh3)4, CuI, Et3N, MeCN, 60 °C,
3 h, 72%; (c) Pd(OAc)2, (o-tolyl)3P, CuI, MeCN, 100 °C, 3.5 h, 62%.

S. Goswami et al. / Tetrahedron Letters 54 (2013) 2373–2376
2375
O
N
H
N
N
O
N
OTBDMS
S
N
H
9
a)
R = CH2OTBDMS
6
O
N
H
N
N
O
N
R
O
N
OTBDPS
Me
b)
H
N
O
N
S
N
H
R = CH(Me)OTBDPS
10
N
H
N
7
c)
O
R = CH(OH)CH2OTBDPS
8
O
_
+
H
N
H
N
N
i) (C4H9)4N F, 18 h.
O
N
N
OTBDPS
OH
S
N
ii) 1.0 N NaOH, 24 h.
N
H
N
S
H2N
( )
N
OH
22
OH
11
Form B (dephospho) 12
Scheme 2. Synthesis of thienopterins 9–11 and dephospho ( ) Form B (12). Reagents and conditions: (a) excess Na2S, dry THF, rt, 1 h, 63%; (b) excess Na2S, dry THF, rt, 4 h,
65%; (c) excess Na2S, dry THF, rt, 4 h, 47%.
successfully and conclusively demonstrated that the thieno[3,2-
g]pterin ring system can be formed from alkynylpterin. The
remaining part of the synthesis of Form B was straightforward.
The thienopterin 11 was treated with tetrabutylammonium fluo-
ride to cleave the TBDPS group and then hydrolysis with 1.0 N
NaOH in aqueous solution at room temperature gently removed
the pivaloyl group to give the desired ( ) Form B (dephospho) 12
the structure of which was confirmed by comparison of spectro-
scopic data of the reported ( ) Form B (dephospho) 12 isolated
from human urine.24c,23d
appearance of thiophene H-30 of 9, 10, and 11 at d 7.34, 7.37, and
7.38, respectively. The NMR of dephospho Form B shows H-30 of
the thiophene ring at d 7.37 (s) (Supplementary data).
The possible mechanism for thiophene annulation is explained
in Scheme 3. The first Michael attack of nucleophile S2ꢀ to the al-
kyne possibly results in an allene (A) which is resonance stabilized
(B) which follows an intramolecular attack of thiolate ion (B) to
give rise to thiophene fused dihydropterin system (9X) which on
aerial oxidation for facile aromatization affords the target thienop-
terin 9.
The thiophene annulation is confirmed by the NMR spectral
data which show the absence of pterin H-7 peak of the pyrazine
ring of 6, 7, and 8 at d 8.63, 8.80, and 8.77, respectively and the
Thus, we have successfully utilized the advantages of conju-
gated acetylene moiety at the 6-position of the pterin system; (1)
easy conversion of the corresponding pterin anion (negative charge
+
O
Na
O
N
_
OTBDMS
N
OTBDMS
N
O
HN
SNa
2
O
HN
S
+
2Na
N
H
N
N
N
H
N
A
6
+
Na
O
N
+
O
N
_
Na
N
_
O
HN
OTBDMS
N
_
HN
O
+
OTBDMS
S
Na
_
N
N
N
B
S
N
H
H
H
+
Na
9X
Possible protonation from excess thiols used or from
aqueous work-up to dihydrothienopterin followed by aerial
oxidation (facile aromatisation of dihydropterin to pterin system)
O
N
O
HN
OTBDMS
S
N
H
N
N
9
Scheme 3. Plausible mechanism for the formation of thienopterin 9.

2376
S. Goswami et al. / Tetrahedron Letters 54 (2013) 2373–2376
2. Nason, A. D.; Antoine, P. A.; Ketchum, W. A.; Fraizer, D. K.; Lee, D. Proc. Natl.
Acad. Sci. U.S.A. 1970, 65, 137.
3. Cramer, S. P.; Gray, H. B.; Rajagopalan, K. V. J. Am. Chem. Soc. 1979, 101, 2772.
4. Berg, J. M.; Hodgson, K. O.; Cramer, S. P.; Corbin, J. L.; Elsberry, A.; Pariyadath,
N.; Stiefel, E. I. J. Am. Chem. Soc. 1979, 101, 2774.
5. (a) Garrett, R. M.; Rajagopalan, K. V. J. Biol. Chem 1996, 271, 7387; (b) Llamas, A.;
Mendel, R. R.; Schwarz, G. J. Biol. Chem. 2004, 279, 55246.
6. (a) Basu, P.; Burgmayer, S. J. N. Coord. Chem. Rev. 2011, 255, 1016;
(b)Molybdenum and Molybdenum-Containing Enzymes; Coughlan, M. P., Ed.;
Pergamon Press: London, 1980.
at N5) formed by nucleophilic attack by sulfide to the triple bond
or subsequently produced dihydropterin to aromatized pterin by
oxidation27 and (2) the superior nucleophilicity of naked sulfide
ion to result in direct thiophene annulation onto pterin.
Thus our strategy provides the first one step synthesis of thie-
nopterin 9, from pterin-substituted acetylene 6 (Scheme 2). Previ-
ously Joule et al. reported28 thiophene annulation on quinoxaline
from dibromo derivative of 2-alkynylquinoxalines with dipotas-
sium trithiocarbonate.
We thus report here a convenient and concise total synthesis of
thienopterins and ( ) Form B (dephospho) 12. The synthesis of
thienopterins and ultimately Form B has evolved from a simple no-
vel method of efficient formation of dithiolenes and thienopterin
from the suitable acetylene moiety attached with pterin ring
system. The specific thiophene annulation onto pterins is thus
demonstrated by the direct synthesis of thienopterins 9–11 in good
yield.
7. Holm, R. H. Coord. Chem. Rev. 1990, 100, 183.
8. (a) Leimkühler, S.; Wuebbens, M. M.; Rajagopalan, K. V. Coord. Chem. Rev. 2011,
255, 1129; (b) Rajagopalan, K. V. In Advances in Enzymology and Related Areas of
Molecular Biology, Meister, A., Ed.;Wiley Publishers:1991; Vol. 64, p 215.
9. Pilato, R. S.; Stiefel, E. I. Bioinorganic Catalysis, Reedijk, J., Dekker, M., Eds.;
Marcel Dekkar Inc.: New York, 1993, p 131.
10. Goswami, S. Heterocycles 1993, 35, 1551.
11. (a) Hine, F. J.; Taylor, A. J.; Garner, C. D. Coord. Chem. Rev. 2010, 254, 1570; (b)
Collison, D.; Garner, C. D.; Joule, J. A. Chem. Soc. Res. 1996, 25.
12. Johnson, J. L.; Waud, W. R.; Rajagopalan, K. V.; Duran, M.; Beemer, F. A.;
Wadman, S. K. Proc. Natl. Acad. Sci. U.S.A. 1980, 77, 3715.
13. (a) Taylor, E. C.; Sabb, A. L. J. Org. Chem. 1988, 53, 5839; (b) Taylor, E. C.;
Darwish, I. S. Adv. Exp. Med. Biol. 1993, 338, 363.
14. Taylor, E. C.; Reiter, L. A. J. Am. Chem. Soc. 1989, 111, 285.
15. (a) Taylor, E. C.; Ray, P. S.; Darwish, I. S.; Johnson, J. L.; Rajagopalan, K. V. J. Am.
Chem. Soc. 1989, 111, 7664; (b) Taylor, E. C.; Goswami, S. P. Tetrahedron Lett.
1991, 32, 7357; (c) Chinchilla, R.; Najera, C. Chem. Rev. 2007, 107, 874.
16. Koschara, W. Z. Phys. Chem. 1940, 263, 78. 1943, 277, 284; 1943, 279, 44.
17. Goto, M.; Sakurai, A.; Yamakami, H. Nippon Kagaku Zasshi 1967, 88, 897. Chem.
Abstr. 1968, 69, 52107.
18. Goto, M.; Sakurai, A.; Ohta, K.; Yamakami, H. Tetrahedron Lett. 1967, 4507.
19. Goto, M.; Sakurai, A.; Ohta, K.; Yamakami, H. J. Biol. Chem. 1969, 65, 611.
20. Sakurai, A.; Goto, M. Tetrahedron Lett. 1968, 2941.
Our method of synthesis is significant to establish a synthetic
correlation between Form A derivative (pterin alkyne with a suit-
able substituent at the alkyne moiety) and Form B, which are de-
rived as decomposition products from Moco.
Acknowledgments
We thank DST, Govt. of India (SR/S1/0C-58/2010) for the finan-
cial support. A.C.M. thanks UGC, Govt. of India for a research fel-
lowship. And we also appreciate mass spectral help from
Professor Thomas Schrader, Universitätsstrasse 5, 45117 Essen,
Germany.
21. Sakurai, A.; Goto, M. J. Biochem. (Tokyo) 1969, 65, 755.
22. Taylor, E. C.; Reiter, L. A. J. Org. Chem. 1982, 47, 528.
23. (a) Johnson, J. L.; Hainline, B. E.; Rajagopalan, K. V. J. Biol. Chem. 1980, 255,
1783; (b) Johnson, J. L.; Waud, W. R.; Rajagopalan, K. V.; Duran, M.; Beemer, F.
A.; Wadman, S. K. Proc. Natl. Acad. Sci. U.S.A. 1980, 77, 3715; (c) Johnson, J. L.;
Hainline, B. E.; Rajagopalan, K. V.; Arison, B. H. J. Biol. Chem. 1984, 259, 5414; (d)
Ushio, K.; Ishizuka, M.; Kogushi, M.; Fukui, S.; Toraya, T. Biochem. Biophys. Res.
Commun. 1986, 135, 256; (e) Kramer, S. P.; Johnson, J. L.; Ribeiro, A. A.;
Millington, D. S.; Rajagopalan, K. V. J. Biol. Chem. 1987, 262, 16357.
24. Taylor, E. C.; Ray, P. S. J. Org. Chem. 1987, 52, 3997.
Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.tetlet.2013.
02.090.
25. Bradshaw, B.; Collison, D.; Garner, C. D.; Joule, J. A. Org. Biomol. Chem. 2003, 1,
129. Chem. Commun. 1998, 417.
26. (a) Pilato, R. S.; Eriksen, K. E.; Greaney, M. A.; Stiefel, E. I.; Goswami, S.;
Kilpatrick, L.; Spiro, T. G.; Taylor, E. C.; Rheingold, R. J. Am. Chem. Soc. 1991, 113,
9372; (b) Taylor, E. C.; Dotzer, R. J. Org. Chem. 1991, 56, 1816.
27. Inui, Y.; Miyazaki, S.; Ohkubo, K.; Shunichi, F.; Kojima, T. Angew. Chem., Int. Ed.
2012, 51, 4623.
References and notes
1. Pateman, J. A.; Cove, D. J.; Rever, B. M.; Roberts, D. B. Nature 1964, 58, 201.
28. Joule, J. A.; Armengel, M. J. Chem. Soc., Perkin Trans. 1 2001, 154.