Synthesis of desmosine-d4: Improvement of isotopic purity by D-H exchange of amino groups

Article abstract of DOI:10.1016/j.tetlet.2017.02.018

Desmosine is a crosslinking pyridinium amino acid of elastin, which is a useful biomarker for the diagnosis of chronic obstructive pulmonary disease (COPD) by LC–MS/MS analysis. We previously reported a synthesis of desmosine-d₄, which is useful as an internal standard for quantitative LC–MS/MS analysis of desmosines, by deuterogenation of an alkyne group; however, the isotopic purity of the desmosine-d₄was only ca. 50%. The present report describes a new synthesis of desmosine-d₄that improves the isotopic purity to ca. 90% by exchanging the protons of the amino groups to deuterium using deuterogenation.

Full text of DOI:10.1016/j.tetlet.2017.02.018

Accepted Manuscript
Synthesis of desmosine-d : Improvement of isotopic purity by D-H exchange
4
of amino groups
Daisuke Watanabe, Rina Suzuki, Toyonobu Usuki
PII:
S0040-4039(17)30189-2
DOI:
http://dx.doi.org/10.1016/j.tetlet.2017.02.018
TETL 48629
Reference:
To appear in:
Tetrahedron Letters
Received Date:
Revised Date:
Accepted Date:
27 December 2016
2 February 2017
8 February 2017
Please cite this article as: Watanabe, D., Suzuki, R., Usuki, T., Synthesis of desmosine-d : Improvement of isotopic
4
purity by D-H exchange of amino groups, Tetrahedron Letters (2017), doi: http://dx.doi.org/10.1016/j.tetlet.
2
017.02.018
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Graphical Abstract
Synthesis of desmosine-d
4
: Improvement of
Leave this area blank for abstract info.
isotopic purity by D-H exchange of amino
groups
Daisuke Watanabe, Rina Suzuki, and Toyonobu Usuki*
NRBoc
NH2
CO2tBu
CO2H
D
D
CO2tBu
CO2tBu
NRBoc
CO2tBu
CO2H D
NH2
CO2H
D
deuterogenation
deprotection
BocRN
Boc)2N
H2N
H2N
N
CO2H
N
d4 = 88%
d3 = 12%
(
R = H
R = D
desmosine-d4
D-H exchange

1
Tetrahedron Letters
Synthesis of desmosine-d : Improvement of isotopic purity by D-H exchange of
4
amino groups
Daisuke Watanabe, Rina Suzuki, and Toyonobu Usuki *
Department of Materials and Life Sciences, Faculty of Science and Technology, Sophia University, 7-1 Kioicho, Chiyoda-ku, Tokyo 102-8554, Japan
ARTICLE INFO
ABSTRACT
Article history:
Received
Desmosine is a crosslinking pyridinium amino acid of elastin, which is a useful biomarker for
the diagnosis of chronic obstructive pulmonary disease (COPD) by LC-MS/MS analysis. We
Received in revised form
Accepted
Available online
previously reported a synthesis of desmosine-d
quantitative LC-MS/MS analysis of desmosines, by deuterogenation of an alkyne group;
however, the isotopic purity of the desmosine-d was only ca. 50%. The present report describes
a new synthesis of desmosine-d that improves the isotopic purity to ca. 90% by exchanging the
protons of the amino groups to deuterium using deuterogenation.
4
, which is useful as an internal standard for
4
4
Keywords:
Desmosine
Biomarker
2
009 Elsevier Ltd. All rights reserved.
Total synthesis
Deuterium
Deuterogenation
6
b
1
. Introduction
deuterogenation of the alkyne. In addition, MS analysis
confirmed that the isotopic purity of 1-d was d = 51%, d
9%, and d = 10%, respectively, although low deuteration ratios
4
4
3
=
Desmosine (1, Figure 1) and isodesmosine are
3
2
tetrasubstituted pyridinium amino acids that exist only in the
elastin matrix. Elastin is a major protein of elastic fiber and
4d
do not affect LC-MS/MS results.
1
provides elasticity for organs such as alveoli, skin, ligamentum,
and blood vessels. The irreversible degradation of elastin-
NHBoc
CO2Bn
NH2
2
CO H
2
1
) D2, Pd/C
MeOH-d4, rt, 6 d
D
14
13
D
CO2H
CO2H
NH2
containing tissues occurs in diseases such as chronic obstructive
pulmonary disease (COPD) and aortic aneurysm, which result in
the excretion of desmosines. Based on liquid chromatography
mass spectrometry (LC-MS or LC-MS/MS) analysis, levels of 1
and isodesmosine in clinical samples of COPD patients tend to be
CO2Bn
NHBoc
CO2Bn
D
D
H2N
H2N
CO2H
BocHN
Boc)2N
2
) TFA, H2O
rt, 2 h
quant (2 steps)
N
d4: 51%
d3: 39%
d2: 10%
CO2tBu
N
(
desmosine-d4 (1-d4)
3,4
greater than those of healthy controls. Therefore, desmosine 1
and isodesmosine are considered useful biomarkers for COPD.
To accurately quantify desmosines using LC-MS/MS, a stable
isotopically labeled internal standard is needed.
4 4
Scheme 1. Previous synthesis of desmosine-d (1-d ).
4
In the previous synthesis of 1-d , benzyl (Bn) groups on the
carboxylic acids were removed to generate free carboxyl groups
during the deuterogenation step (Scheme 1). Deuterogenation in
NH2
6b
17
15
16 CO2H
NH2
18
21'
the presence of carboxyl groups might have caused the
1
4
CO2H1
8'
13
4
8
21
H2N 20'
20
19
CO2H
insufficient isotopic purity. Another cause was likely the amino
1
9'
5
6
3
12
2
CO2H
N
7
or free carboxyl group protons in the molecule. Therefore, a
synthetic intermediate (2, Scheme 2), which had tert-butyl (tBu)
groups as the protecting group on carboxyl groups instead of Bn
groups, was designed. The tBu groups cannot be removed under
the conditions used for deuterogenation and can be removed by
trifluoroacetic acid (TFA) along with tert-butoxycarbonyl (Boc)
during the final step. The presence of amino protons in the
substrates was also a concern during the deuterogenation step.
Thus, the goal of the present study was to improve the
11
1
0
8
9
H2N
desmosine (1)
Figure 1. Structure of desmosine (1).
Recently, we reported a total synthesis of desmosine 1 via
5,6
palladium-catalyzed cross-coupling reactions along with the
synthesis of 13,14-tetradeuterated desmosine (desmosine-d , 1-d
which allowed precise quantitative analysis of
desmosines. The synthesis of 1-d relied on stepwise and
regioselective Sonogashira and Negishi cross-coupling reactions
between the pyridine core and corresponding alkyne, followed by
4
4
,
6
b,7
deuteration ratio of tetradeuterated desmosine-d
synthesis of desmosine-d was achieved by changing the
carboxylic acid protecting group from Bn to tBu and by
4 4
1-d . Successful
Scheme 1),
4
d
4
4
*
Corresponding author. Tel.: +81 3 3238 3446; fax: +81 3 3238 3361; e-mail address: t-usuki@sophia.ac.jp (T. Usuki)

2
Tetrahedron Letters
exchanging the amino protons to deuterium during
deuterogenation.
and -iodoalkylated L-glycine 4, prepared from tert-butyl-2-(S)-
14
[bis-(tert-butoxycarbonyl)-amino]-6-hydroxyhexanoate
via
), then afforded 2 in
and Pd/C in MeOH-d
followed by removal of the Boc and tBu groups using TFA,
successfully produced crude 1-d . Purification by reversed-phase
high-performance liquid chromatography (RP-HPLC) afforded
pure desmosine-d 1-d in 38% yield over two steps.
Unfortunately, the deuterated ratio of the end product was d
5%, d = 37%, and d = 8%, as determined by MS analysis. This
tendency was similar to that of the previously reported synthetic
15
Appel reaction in nitromethane (MeNO
4% yield. Deuterogenation with D
2
9
2
4
,
2. Results and discussion
4
Compound 1-d can be prepared via deuterogenation and
4
deprotection of tetrasubstituted pyridine 2, as shown in Scheme 2.
Compound 2 is formed from 3,4,5-trisubstituted pyridine 3 and
iodo amino acid (-iodobutyl L-glycine) derivative 4 through N-
alkylation. Synthesis of 3 then involves Negishi cross-coupling
between 4-alkynyl-3,5-dibromopyridine 5, which had been
4
4
4
=
5
3
2
4d,6b
product.
It was thus found that the generation of free carboxyl
9
prepared previously, and the iodo amino acid segment 6.
groups on the deuterogenation step did not affect the distribution
of the deuterated ratio.
NHBoc
NH2
CO2tBu
CO2H
NHBoc
D
D
CO2H
CO2H
NH2
CO2H
deuterogenation
deprotection
CO tBu
NHBoc
I
CO2tBu
6
D
D
2
NHBoc
CO2tBu
NHBoc
CO2tBu
NHBoc
CO2tBu
H2N
H2N
BocHN
2
2
CO tBu
Zn, TMSCl, DMF
rt, 1 h
N
2
CO tBu
N
CO2tBu
NHBoc
CO2tBu
NHBoc
CO2tBu
(Boc) N
then,
Pd-PEPPSI-IPr
Br
Br
Br
1
-d4
2
BocHN
(
20 mol%)
N
DMF, 60 °C, 1.5 h
N
N
N-alkylation
5
3 (59%)
14 (31%)
NHBoc
CO2tBu
NHBoc
CO2tBu
I
NHBoc
CO2tBu
N(Boc)2
CO tBu
Negishi
cross-coupling
CO2tBu
NHBoc
CO2tBu
I
NHBoc
CO2tBu
2
2
1) D , Pd/C
MeOH-d4, rt, 6 d
Br
Br
BocHN
4
CO tBu
NHBoc
I
tBuO2C
2
N(Boc)2
N
N
MeNO2
BocHN
CO tBu 2) TFA, H2O
2
6
2
0 °C to 65 °C
d, 94%
rt, 3 h
38% (2 steps)
5
6
3
4
2
CO tBu
N
(Boc)2N
2
Scheme 2. Retrosynthesis of desmosine-d
4
1-d
4
.
NH2
CO2H
D
D
This synthesis began with the stereoselective preparation of
iodo amino acid 6 starting from L-aspartic acid 7 (Scheme 3).
Methyl esterification of 7 using thionyl chloride (SOCl ) was
2
CO2H
CO2H
D
D
NH2
CO2H
H2N
H2N
N
d4: 55%
d3: 37%
d2: 8%
1-d4
SOCl2, MeOH
rt, 25 min
Boc2O, Na2CO3
NH2
CO2H
NH3Cl
CO2H
NHBoc
CO2H
HO2C
MeO2C
dioxane, 40 °C
MeO2C
18 h, 81% (2 steps)
L-aspartic acid 7
8
9
4 4
Scheme 4. Synthesis of desmosine-d 1-d .
tBuBr, K2CO3
BnEt3NCl
NHBoc
CO2tBu
2N NaOH
NHBoc
CO2tBu
MeO2C
HO2C
MeCN, 50 °C
MeOH, rt, 3 h
94%
The protons of pyridinium salt 2 were assumed to cause the
distribution of incorporation of deuterium. Concretely, the
protons of the amino groups at the C16, C20, and C20’ positions
24 h, 63%
1
0
11
NMM, EtOCOCl
THF, -15 °C, 1 h
NHBoc
CO2tBu
PPh3, I2, imidazole
CH2Cl2, 0 °C, 2 h
NHBoc
CO2tBu
iodo amino acid 6
of
2
probably reduced the ratio of 1-d
4
during the
then, NaBH4, H2O
HO
I
0
°C, 30 min, 81%
7
5%
deuterogenation step. Replacement of the protons of the amino
groups on pyridinium salt 2 with deuterium to form 2’ was thus
performed by dissolving 2 in MeOH-d and removing the solvent
4
1
1
2
0
conducted to give 8, followed by Boc protection using di-tert-
butyl dicarbonate (Boc
afford 9 in 81% yield in two steps. Treatment of 9 with Boc
and N,N-dimethylaminopyridine (DMAP) gave 10 in 29%
yield. When 9 was treated with tert-butylbromide (tBuBr),
potassium carbonate (K CO ), and benzyltriethylammounium
chloride (BnEt NCl), desired 10 was obtained in 63% yield.
O) and sodium carbonate (Na CO ) to
2 2 3
11
five times (Scheme 5). Replacement of the protons with
2
O
1
deuterium was confirmed by H NMR spectrum. Compound 2’
12
was then subjected to deuterogenation and deprotection. As a
result, deuteration of the 1-d
improved to d = 88%, d = 12%.
4
’ obtained was successfully
2
3
13
4
3
3
Hydrolysis of 10 produced 11 in 94% yield. Treatment of 11 with
N-methylmorpholine (NMM) and ethylchloroformate (EtOCOCl)
followed by sodium borohydride (NaBH ) with H O afforded
4 2
alcohol 12 in 81% yield. The Appel reaction was then performed
to give iodo amino acid 6 in 75% yield. Thus, the synthesis of 6
in 29% yield was accomplished over six steps starting from 7.
NHBoc
NDBoc
16
CO2tBu
16
CO2tBu
NHBoc
0'CO2tBu
CO2tBu
20'CO2tBu
BocDN
NDBoc
2
MeOH-d4
BocHN
20 CO2tBu
20 CO2tBu
dissolving/
removal
N
CO2tBu N
(Boc) N
2
2
(Boc) N
2
2'
Scheme 3. Synthesis of iodo amino acid 6.
NH2
CO2H
D
1
) D2, Pd/C
MeOH-d4, rt, 6 d
D
CO2H
H2N
NH2
D
D
4
Synthesis of 1-d was conducted as shown in Scheme 4.
CO2H
2
) TFA, H2O
rt, 3 h
quant (2 steps)
Negishi cross-coupling reaction using pyridine-enhanced
precatalyst preparation stabilization and initiation (PEPPSI-IPr)
2
CO H
N
d4: 88%
d3: 12%
H2N
1-d4'
was attempted between obtained
6
and 4-alkynyl-3,5-
dibromopyridine derivative 5, which was prepared via
9
Sonogashira cross-coupling reaction. The desired trisubstituted
4 4
Scheme 5. Synthesis of desmosine-d 1-d ’ after D-H exchange.
pyridine 3 was obtained in 59% yield, along with disubstituted
product 14 in 31% yield. Formation of the pyridinium salt of 3

3
3. Conclusion
Acknowledgments
We thank Dr. Yong Y. Lin (Mount Sinai School of Medicine)
and Prof. Gerard M. Turino (Mount Sinai School of Medicine)
for valuable discussions, Ms. Miho Tanigawa (Sophia
University) for the preparation of compound 4. This work was
supported by a Grant-in-Aid for Young Scientists (B) from the
Japan Society for the Promotion of Science (JSPS; KAKENHI
Grant No. 25750388).
In summary, synthesis of desmosine-d
successfully achieved with good isotopic purity (d
4
1-d
: 88%, d
2%) by exchanging the amino protons with deuterium during
4
’
was
3
:
4
1
deuterogenation. This compound can act as an internal standard
for LC-MS/MS analysis of desmosine 1 and isodesmosine in
clinical samples, which are derived from elastin degradation in
4d
diseases such as COPD.
6
.
Sonogashira and Negishi cross-coupling route, see: (a) Yanuma,
H.; Usuki, T. Tetrahedron Lett. 2012, 53, 5920-5922. (b) Suzuki,
R.; Yanuma, H.; Hayashi, T.; Yamada, H.; Usuki, T. Tetrahedron
2015, 71, 1851-1862.
References and notes
1
2
3
.
.
.
(a) Partridge, S. M.; Elsden, D. F.; Thomas, J. Nature 1963, 197,
297-1298. (b) Thomas, J.; Elsden, D. F.; Partridge, S. M.;
Bedford, G. R.; Katritzey, A. R. Nature 1963, 200, 651-652.
(a) Rosenbloom, J.; Abrams, W. R.; Mecham, R. FASEB J. 1993,
1
3
15
1
7.
3 1
As an alternative internal standard, isodemosine- C , N was also
prepared via Chichibabin pyridine synthesis, see: Tanigawa, T.;
Komatsu, A.; Usuki, T. Bioorg. Med. Chem. Lett. 2015, 25, 2046-
2049.
Kurita, T.; Aoki, F.; Mizumoto, T.; Maejima, T.; Esaki, H.;
Megawa, T.; Monguchi, Y.; Sajiki, H. Chem. Eur. J. 2008, 14,
3371-3379.
7
, 1208-1218. (b) Debelle, L.; Tamburro, A. M. Int. J. Biochem.
Cell. Biol. 1999, 31, 261-272.
8.
9.
(a) Kaga, N.; Soma, S.; Fujimura, T.; Seyama, K.; Fukuchi, Y.;
Murayama, K. Anal. Biochem. 2003, 318, 25-29. (b) Boutin, M.;
Berthelette, C.; Gervais, F. G.; Scholand, M.-B.; Hoidal, J.;
Leppert, M. F.; Bateman, K. P.; Thibault, P. Anal. Chem. 2009,
Murakami, Y.; Yanuma, H.; Usuki, T. Tetrahedron: Asymmetry
2012, 23, 1557-1563.
8
1, 1881-1887. (c) Shiraishi, K.; Matsuzaki, K.; Matsumoto, A.;
10. Cox, R. J.; Wang, P. S. H. J. Chem. Soc., Perkin Trans 1 2001, 1,
2022-2034.
11. Oliver, A.; Goubert, M.; Trusun, A.; Canet, I.; Sinibaldi, M.
ARKIVOC, 2010, 9, 108-126.
12. Takeda, K.; Akiyama, A.; Nakamura, H.; Takizawa, S.; Mizuno,
Y.; Takayanagi, H.; Harigaya, Y. Synthesis 1994, 1063-1066.
13. (a) Chevallet, P.; Garrouste, P.; Malawska, B.; Martinez, J.
Tetrahedron Lett. 1993, 34, 7409-7412. (b) Anderson, J. T.;
Toogood, P. L.; Marsh, E. N. G. Org. Lett. 2002, 4, 4281-4283.
14. Usuki, T.; Sugimura, T.; Komatsu, A.; Koseki, Y. Org. Lett. 2014,
16, 1672-1675.
Hashimoto, Y.; Iba, K. J. Oleo Sci. 2010, 59, 431-439. (d)
Miliotis, T.; Lindberg, C.; Semb, K. F.; van Geest, M.; Kjellstrom,
S. J. Chromatogr. A 2013, 1308, 73-78. (e) Albarbarawi, O.;
Barton, A.; Miller, D.; McSharry, C.; Chaudhuri, R.; Thomson, N.
C.; Palmer, C. N. A.; Devereux, G.; Huang, J. T.-J. Bioanal. 2013,
5
, 1991-2001. (f) Lamerz, J.; Friedlein, A.; Soder, N.; Cutler, P.;
Dobeli, H. Anal. Biochem. 2013, 436, 127-136. (g) Ongay, S.;
Hendriks, G.; Hermans, J.; van den Berge, M.; ten Hacken, N.
H.T.; van de Merbel, N. C.; Bischoff, R. J. Chromatogr. A 2014,
1
326, 13-19.
4
5
.
.
(a) Ma, S.; Lieberman, S.; Turino, G. M.; Lin, Y. Y. Proc. Natl.
Acad. Sci., U.S.A. 2003, 100, 12941-12943. (b) Ma, S.; Lin, Y. Y.;
Turino, G. M. Chest 2007, 131, 1363-1371. (c) Ma, S.; Turino, G.
M.; Lin, Y. Y. J. Chromatogr. B 2011, 879, 1893-1898. (d) Ma.
S.; Turino, G. M.; Hayashi, T.; Yanuma, H.; Usuki, T.; Lin, Y. Y.
Anal. Biochem. 2013, 440, 158-165. (e) Murakami, Y.; Suzuki, R.;
Yanuma, H.; He, J.; Ma. S.; Turino, G. M.; Lin, Y. Y.; Usuki, T.
Org. Biomol. Chem. 2014, 12, 9887-9894.
15. Synthesis of compound 4 will be published elsewhere.
Supplementary Material
1
13
H and C NMR spectra, supplementary data associated with
this article can be found in the online version, at http://
Sonogashira cross-coupling route, see: (a) Usuki, T.; Yamada, H.;
Hayashi, T.; Yanuma, H.; Koseki, Y.; Suzuki, N.; Masuyama, Y.;
Lin, Y. Y. Chem. Commun. 2012, 48, 3233-3235. (b) Yamada, H.;
Hayashi, T.; Usuki, T. Bull. Chem. Soc. Jpn. 2015, 88, 673-683.
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4
Tetrahedron
Highlights



Synthesis of desmosine-d
was conducted.
The protons of amino groups were exchanged
to deuterium.
4
via deuterogenation
Isotopic purity of newly synthetic desmosine-
4
d was improved to ca. 90%.