Synthesis of N1-and N8-(γ-L-Glutamyl) spermidines and (γ-L-Glutamyl) putrescine

Article abstract of DOI:10.2174/157017811794557813

(γ-L-Glutamyl)putrescine and N¹- and N⁸- (γ-L-glutamyl)spermidines, possible regioisomeric products of the polyamine glutamyl transferase of bacteria such as Escherichia coli and Pseudomonas aeruginosa, were synthesized from N-Boc-protected putrescine and 1,3-propanediamine.

Full text of DOI:10.2174/157017811794557813

Letters in Organic Chemistry, 2011, 8, 1-4
1
Synthesis of N1- and N8-(ꢀ-L-Glutamyl)spermidines and (ꢀ-L-Glutamyl)
putrescine
Kazuya Toho1, Naoki Abe*,1, Teiko Yamada2, Yoshifumi Ito#,1, Hiroyuki Konno3,
Shigefumi Kuwahara2 and Hiromasa Kiyota*,2
2
1Lab. of Applied Microbiology; Lab. of Applied Bioorganic Chemistry, Graduate School of Agricultural Science,
Tohoku University, Aoba-ku, Sendai, 981-8555 Japan; 3Department of Chemistry and Chemical Engineering, Graduate
School of Science and Engineering, Yamagata University, Jonan, Yonezawa 992-8510, Japan
Received January 19, 2010: Revised June 02, 2010: Accepted June 14, 2010
Abstract: (ꢀ-L-Glutamyl)putrescine and N1- and N8-(ꢀ-L-glutamyl)spermidines, possible regioisomeric products of the
polyamine glutamyl transferase of bacteria such as Escherichia coli and Pseudomonas aeruginosa, were synthesized from
N-Boc-protected putrescine and 1,3-propanediamine.
Keywords: glutamylspermidine, glutamylputrescine, Pseudomonas aeruginosa, polyamine biosynthesis, polyamine glutamyl
transferase.
INTRODUCTION
(2) and (ꢀ-L-glutamyl)putrescine (3) were prepared from
N1,N4-bis(Boc)-spermidine (7) [12] and N-Boc-putrescine (9)
Polyamines (spermine, spermidine, putrescine etc.) are a
group of ubiquitous polycations necessary for optimal cell
growth of procalyotic and eukaryotic cells [1], and thus it is
important to elucidate their metabolic pathways. Recently, ꢀ-
glutamylation was found to be a key reaction in the
metabolism. N1-(ꢀ-L-Glutamyl)spermidine (1, Fig. 1) and N8-
1
[13], respectively. Most of the H and 13C NMR resonances
(Table 1) of the target compounds were assigned by the HH-
COSY, HMQC and HMBC spectra. Positions of each amide
1
bond were confirmed by the observation of H-13C three
bond correlations from H(1) to C(5’) for 1 and from H(8) to
C(5’) for 2 in the HMBC spectra.
(ꢀ-L-glutamyl)spermidine
(2),
plausible
metabolic
O
O
intermediates, were firstly reported by Folk as unnatural
compounds prepared by enzymatic procedure [2]. Later, (ꢀ-
L-glutamyl)spermidine conjugates were identified in vitro as
a major protein secreted from the rat seminal vesicles [3].
Folk et al. also showed their ion-exchange chromatogram
using HPLC method [2, 4]. ꢀ-L-Glutamylputrescine (3), one
of the key compounds in polyamine metabolism, was found
in mammalian brain [5], and recently in Escherichia coli K-
12 [6, 7]. We report here the synthesis and spectroscopic
data of three ꢀ-L-glutamylated polyamines (1, 2 and 3) as
standards for the metabolic studies.
8
7
5
3
1
3'
4
1
2'
H2N
1'
5'
N
H
N
H
OH
4'
8
6
2
NH2
N1-(ꢀ-
L-glutamyl)spermidine (1)
NH2
7
5
3
1
H
N
8
4
1
HO
3'
5'
1'
2'
4'
N
NH2
8
6
2
H
O
O
N8-(ꢀ-
L
-glutamyl)spermidine (2)
O
O
4
3
1
3'
4
1
RESULTS AND DISCUSSION
2'
H2N
1'
5'
N
OH
4'
2
H
The synthesis of the compounds is shown in Scheme 1.
Recently, Jeitner et al. reported the synthesis of HCl salts of
1 and 3 in a similar manner as we describe here [8]. The free
N1 amino group of N4,N8-bis(Boc)-spermidine (4) [9, 10] was
condensed with N-Boc-O-t-Bu-L-glutamic acid (5) [11] using
water-soluble carbodiimide (EDCI) in 55% yield to give 6
[8]. Finally, acidic deprotection of the three Boc and one t-
Bu group afforded 1. Similarly, N8-(ꢀ-L-glutamyl)spermidine
NH2
N-(ꢀ-L-glutamyl)putrescine (3)
Fig. (1). N1- and N8-ꢀ-L-glutamylspermidines, and (ꢀ-L-glutamyl)
putrescine.
EXPERIMENTAL
General
*Address correspondence to these authors at the Lab. of Applied Bioorganic
Chemistry, Graduate School of Agricultural Science, Tohoku University,
Aoba-ku, Sendai, 981-8555 Japan; Tel/Fax: +81-22-717-8785;
E-mail: kiyota@biochem.tohoku.ac.jp
Lab. of Applied Microbiology, Graduate School of Agricultural Science,
Tohoku University, Aoba-ku, Sendai, 981-8555 Japan; Tel: +81-22-717-
8781; Fax: +81-22-717-8780; ; E-mail: nabe@biochem.tohoku.ac.jp
#The late.
Optical rotation: Horiba Sepa-300 apparatus. FT-IR:
1
Jasco 4100 apparatus (ATR, Zn-Se); in cm–1. H and 13C
1
NMR: Varian Inova 600 (600 MHz for H and 150 MHz for
1
13C) and Inova 500 apparatuses (500 MHz for H and 126
MHz for 13C); rel. to Me4Si (= 0 ppm). Mass spectra: Jeol
JMS–700; in m/z.
1570-1786/11 $58.00+.00
© 2011 Bentham Science Publishers Ltd.

2
Letters in Organic Chemistry, 2011, Vol. 8, No. 1
Toho et al.
BocHN
BocHN
N
Boc
NH2
NH2
lit. [9,10]
9
4
(55%)
(35%)
O
a)
a)
O
O
O
BocHN
BocHN
N
N
H
Ot-Bu
N
H
Ot-Bu
Boc
NHBoc
NHBoc
6
10
b)
(quant.)
b)
(55%)
N1-(ꢀ-
L
-glutamyl)spermidine
N-(ꢀ-
L
-glutamyl)putrescine
(3)
(1)
H2N
O
O
N
NHBoc
Boc
HO
Ot-Bu
7
NHBoc
5
a)
(40%)
NHBoc
b)
H
N
N8-(ꢀ-
L
-glutamyl)spermidine
t-BuO
N
NHBoc
(2)
Boc
O
O
(94%)
8
Scheme 1. Synthesis of N1- and N8-ꢀ-L-glutamylspermidines, and (ꢀ-L-glutamyl)putrescine.
a) N-Boc-O-t-Bu-L-Glu (5), EDCI, CH2Cl2. b) CF3CO2H, CH2Cl2, 0°C.
Table 1. 1H and 13C NMR Spectral Data of 1, 2 and 3 (600 MHz for 1H and 150 MHz for 13C in D2O)
1
2
3
No
1H
mult. (Hz)
13C
1H
mult. (Hz)
13C
1H
mult. (Hz)
13C
1
2
3.21
1.82
2.98
t (6.5)
quint (7.3)
m
36.26
25.99*
45.23
3.09*
2.03
t (7.8)
quint (7.3)
t (8.7)
36.65
23.81
45.54
3.20
1.56
1.65
2.98
t (6.5)
quint (7.0)
quint (7.3)
t (7.3)
38.78
25.47
24.26
39.22
3
3.04*
4
5
2.99
1.690
1.679
2.97
-
m
m
m
m
47.03
22.86
23.99
38.88
171.49
52.30
25.65*
3.02
1.64
1.52
3.16
-
t (8.1)
quint (7.3)
quint (7.3)
t (6.9)
47.46
25.50
23.07
38.69
171.45
52.54
25.78
6
7
8
1’
2’
3’
-
171.82*
52.62
4.03
2.13
2.17
2.40
2.44
-
t (6.5)
4.04
2.14
2.19
2.41
2.44
-
t (6.6)
4.04
2.19
m
m
dt (15.0, 7.2)
dt (15.0, 6.6)
dt (15.5, 7.6)
dt (15.5, 7.3)
dt (15.0, 7.3)
dt (15.0, 7.3)
dt (15.3, 7.6)
dt (15.5, 7.6)
25.89
4’
31.11
31.24
2.46
m
31.32
-
-
5’
174.56
174.19
174.24*
NH
7.57
8.14
8.30
10.8
br
Unable to redefine the ambiguous assignments (*) due to the overlapping and/or lack of the key HMBC/HMQC correlation.

Synthesis of N1- and N8-(g-L-Glutamyl)spermidines
Letters in Organic Chemistry, 2011, Vol. 8, No. 1
3
N4,N8-Bis(tert-butoxycarbonyl)-N1-(N-tert-butoxycarbonyl-
O-tert-butyl-ꢀ-L-glutamyl)spermidine (6) [8]
N8-(ꢀ-L-Glutamyl)spermidine (2)
In a similar manner as described for 1, 8 (112 mg, 0.178
mmol) was converted to 2 (FW: 274.36, 46.0 mg, 0.168
mmol, 94%) as a colorless oil, [ꢀ]D +3.0° (c 1.1, MeOH).
Rf 0.05 (CHCl3/MeOH/aq. NH3 = 2:2:1). IR: 3384s (N–H),
2948s, 1736m (C=O), 1634m (C=O), 1210m, 1048m. FAB-
MS: 273 ([M–H]–), 195, 155, 127, 97. HR-FAB-MS:
273.1925 ([M–H]–, C12H25O3N4; calc. 273.1927).
To a solution of amine 4 (FW: 345.48, 80 mg, 0.22
mmol), 5 (FW: 303.35, 64 mg, 0.21 mmol) and i-Pr2NEt
(FW: 129.24, 39 mg, 0.23 mmol) in dry CH2Cl2 (2 ml) was
27
added
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride (EDCI•HCl, FW: 191.71, 50 mg, 0.26 mmol)
at 0°C and the mixture was stirred at room temp. for 12 h.
Then the mixture was diluted with EtOAc, washed three
times with water, dried with MgSO4 and concd. in vacuo.
The residue was chromatographed on silica gel
(CHCl3/MeOH = 100:1) to give 6 (FW: 630.81, 78 mg, 0.12
N4-(tert-Butoxycarbonyl)-N1-(N-tert-butoxycarbonyl-O-
tert-butyl-ꢀ-L-glutamyl)putrescine (10)
In a similar manner as described for 6, 9 (FW: 188.27, 80
mg, 0.43 mmol) was converted to 10 (FW: 473.60, 71 mg,
0.15 mmol, 35%) as a colorless oil, [ꢀ]D +1.2° (c 0.70,
22
mmol, 55%) as a colorless oil, [ꢀ]D +1.6° (c 3.9, CHCl3).
27
Rf 0.40 (CHCl3/MeOH = 20:1). IR: 3350m (N–H), 1699s
CHCl3). Rf 0.54 (CHCl3/MeOH = 20:1). IR: 3341m (N–H),
2977m, 2934m, 1698s (C=O), 1652m (C=O), 1524m, 1366m,
1251s, 1172s. 1H NMR (CDCl3, 500 MHz) ꢁ: 1.44 (s, t-Bu),
1.45 (s, t-Bu), 1.46 (s, t-Bu), 1.54 (m, CH2(2, 3)), 1.85 (m, 1
H-(3’)), 2.15 (m, 1 H-(3’)), 2.25 (t, J = 7.1, CH2(4’)), 3.14
(br s, CH2(NH)), 3.28 (m, CH2(NH)), 4.14 (m, 1 H-(N)), 4.65
(br s, 1 H-(N)), 5.25 (d, J = 8.0, 1 H-(2’)), 6.43 (br s, 1 H-
(N)). FAB-MS: 474 ([M+H]+), 374 ([M+2H–(t-
BuOC=O)]+), 318 ([M+3H–(t-BuOC=O)–t-Bu]+), 262, 244,
1
(C=O), 1679s (C=O), 1366s, 1258s, 1152s, 749vs. H NMR
(CDCl3, 500 MHz) ꢁ: 1.44 (s, 2 x t-Bu), 1.454 (s, t-Bu),
1.462 (s, t-Bu), 1.4-1.6 (m, CH2(6, 7)), 1.65 (br s, CH2(2)),
1.91 (br s, 1 H-(3’)), 2.15 (m, 1 H-(3’)), 2.27 (m, CH2(4’)),
3.12 & 3.14 (br s, CH2(5, 8)), 3.20 (br s, CH2(1)), 3.26 (br s,
CH2(3)), 4.14 (br s, 1 H, H-(2’)), 4.57 (br, 0.75 H, H-(N8)),
4.68 (br, 0.25 H-(N8)), 5.27 (pseudo d, J = 7.3, H-(NCHC2)),
6.41 (br, 0.75 H -(N1)), 6.89 (br, 0.75 H-(N1)). 13C NMR
(CDCl3, 126 MHz) ꢁ: 27.9, 28.3, 28.4, 32.7, 35.7, 40.0, 43.3,
46.6, 53.6, 53.8, 79.1, 79.7, 81.9, 82.2, 155.6, 155.9, 171.5,
172.0. FAB-MS: 631 ([M+H]+), 531 ([M+2H–t-
BuOC=O]+), 475, 275. HR-FAB-MS: 631.4286 ([M+H]+,
C31H59O9N4; calc. 631.4282).
218
([M+4H–2(t-BuOC=O)–t-Bu]+).
HR-FAB-MS:
474.3186 ([M+H]+, C23H44O7N3; calc. 474.3179).
N-(ꢀ-L-Glutamyl)putrescine (3)
In a similar manner as described for 1, 10 (70 mg, 0.15
mmol) was converted to 3 (FW: 217.27, 38 mg, 0.12 mmol,
55%) as an amorphous solid, [ꢀ]D +4.62° (c 2.25, MeOH).
N1-(ꢀ-L-Glutamyl)spermidine (1)
28
A solution of 6 (FW: 630.81, 70 mg, 0.11 mmol) in
CF3CO2H (2.8 ml) and CH2Cl2 (1.7 ml)) was stirred at 40°C
for 12 h. Then the mixture was concd. in vacuo and the
Rf 0.3 (CHCl3/MeOH/aq. NH3 = 2:2:1). IR: 3330m (N–H),
3190br vs (O–H), 1736m (C=O), 1634s (C=O), 1561m,
1509m, 1399s (C–O), 1228w, 1160w, 1048w. FAB-MS (in
D2O): 225 ([M–6H+7D]+) 176, 156, 154, 138, 136, 108, 89,
78; (in H2O): 216 ([M–H]–), 188, 151. HR-FAB-MS:
225.1952 (in D2O, [M–6H+7D]+, C9H13D7O3N3; calc.
225.1944), 216.1347 (in H2O, [M–H]–, C9H18O3N3; calc.
216.1348).
residue
was
chromatographed
on
silica
gel
(CHCl3/MeOH/aq. NH3 = 2:2:1) to give 1 (FW: 274.36, 38
mg, 0.12 mmol, quant.) as a colorless oil, [ꢀ]D22 +6.1° (c 1.3,
MeOH). Rf 0.05 (CHCl3/MeOH/aq. NH3 = 2:2:1). IR:
3330m, (N–H), 3190m (N–H), 2958s, 1696m, (C=O), 1625s
(C=O), 1235m, 1153w. FAB-MS: 297 ([M+Na]+), 275
([M+H]+), 180, 153, 135, 115, 72, 61. HR-FAB-MS:
275.2080 ([M+H]+, C12H27O3N4; calc. 275.2083).
ACKNOWLEDGEMENT
N1,N4-Bis(tert-butoxycarbonyl)-N8-(N-tert-butoxycarbonyl-
O-tert-butyl-ꢀ-L-glutamyl)spermidine (8)
We appreciate the financial supported from the Grant-in-
aid from Japan Society for the Promotion of Science
(19580120).
In a similar manner as described for 6, 7 (FW: 345.48,
205 mg, 0.593 mmol) was converted to 8 (FW: 630.81, 151
27
REFERENCES
mg, 0.239 mmol, 40.3%) as a colorless oil, [ꢀ]D +1.7° (c
1.1, CHCl3). Rf 0.40 (CHCl3/MeOH = 20:1). IR: 3330m (N–
H), 1692vs (C=O), 1678vs (C=O), 1365s, 1249s, 1151vs,
752vs. 1H NMR (CDCl3, 500 MHz) ꢁ: 1.438 (s, t-Bu), 1.443
(s, t-Bu), 1.461 (s, 2 x t-Bu), 1.50 (m, 2 H), 1.56 (m, 2 H),
1.65 (br s, 2 H), 1.95 (br s, 1 H), 2.15 (m, 1 H), 2.26 (t, J =
6.8, 2 H), 3.10 (br s, 2 H), 3.6 (m, 2 H), 3.18-3.31 (m, 4 H),
4.13 (br s, 1 H-(2’)), 4.82 (br s, 0.5 H), 5.27 (br d, J = 6.8, 1
H), 5.32 (br s, 0.5 H), 6.43 (br s, 0.5 H), 6.54 (br s, 0.5 H).
13C NMR (CDCl3, 126 MHz) ꢁ: 28.0, 28.3, 28.5, 32.8, 37.4,
37.7, 39.2, 43.7, 44.3, 46.1, 46.6, 53.4, 78.9, 79.2, 79.6, 79.9,
80.0, 82.2, 82.4, 155.6, 156.0, 156.2, 171.4, 172.2. FAB-MS:
631 ([M+H]+), 531 ([M+2H–t-BuOC=O]+), 475, 275. HR-
FAB-MS: 631.4282 ([M+H]+, C31H59O9N4; calc. 631.4282).
[1]
Glansdorff, N. In: Escherichia coli and Salmonella. Cellular and
Molecular Biology; Neidhardt, F. C., Ed.; Am. Soc. Microbiol.,
Washington, DC, 1996, pp. 408-433.
[2]
Folk, J.E. Synthesis of N1-(ꢂ-glutamyl)spermidine, N8-(ꢂ-
glutamyl)spermidine, N1,N8-bis(ꢂ-glutamyl)spermidine, N1-(ꢂ-
glutamyl)spermine, N1,N12-bis(ꢂ-glutamyl)spermine, and N1,N4-
bis(ꢂ-glutamyl)putrescine. Methods Enzymol., 1983, 94, 451-457.
Porta, R.; Esposito, C.; Metafora, S.; Malorni, A.; Pucci P.; Marino,
G. Purification and structural characterization of in vitro
synthesized (ꢂ-glutamyl)spermidine conjugates of a major protein
secreted from the rat seminal vesicles. Adv. Exp. Med. Biol., 1988,
250, 403-409.
[3]
[4]
Beninati, S.; Martinet, N.; Fold, J.E. High-performance liquid
chromatographic method for the determination of ꢃ-(ꢂ-
glutamyl)lysine and mono- and bis-ꢂ-glutamyl derivatives of
putrescine and spermidine. J. Chromatog., 1988, 443, 329-335.

4
Letters in Organic Chemistry, 2011, Vol. 8, No. 1
Toho et al.
[5]
Nakajima, T.; Kakimoto, Y.; Tsuji, M.; Konishi, H. Occurrence
and formation of ꢀ-glutamylputrescine in mammalian brain. J.
Neurochem., 1976, 26, 115-118.
Kurihara, S.; Oda, S.; Kato, K.; Kim, H.-G.; Koyanagi, T.;
Kumagai, H.; Suzuki, H. A novel putrescine utilization pathway
involves ꢀ-glutamylated intermediates of Escherichia coli K-12. J.
Biol. Chem., 2005, 280, 4602-4608.
Kurihara, S.; Oda, S.; Tsuboi, Y.; Kim, H.-G.; Oshida, M.;
Kumagai, H.; Suzuki, H. ꢀ-Glutamylputrescine synthetase in the
putrescine utilization pathway of Escherichia coli K-12. J. Biol.
Chem., 2008, 283, 19981-19990.
Jeitner, T.M.; Matson, W.R.; Folk, J.E.; Blass, J.P.; Cooper, A.J.L.
Increased levels of ꢀ-glutamylamines in Huntington disease CSF. J.
Neurochem., 2008, 106, 37-44.
propyl)-N,N'-di-t-butyloxycarbonyl-l,4-diaminobutane. Polish J.
Chem., 1978, 52, 1167-1172.
Gardner, R.A.; Kinkad, R.; Wang, C.; Phanstiel IV, O. Total
synthesis of petrobactin and its homologues as potential growth
stimuli for Marinobacter hydrocarbonoclasticus, an oil-degrading
bacteria. J. Org. Chem., 2004, 69, 3530-3537.
Han, G.; Tamaki, M.; Hruby, V.J. Fast, efficient and selective
deprotection of the tert-butoxycarbonyl (Boc) group using
HCl/dioxane (4 M). J. Peptide Res., 2001, 58, 338-341.
Lochner, M.; Geneste, H.; Hesse, M. Allgemeiner Zugang zu
Polyaminen mit Ethan-1,2-diamin-Einheiten: Synthese von nicht
natürlich vorkommenden homologen und isomeren N1,4-Di(4-
cumaroyl)sperminen. Helv. Chim. Acta, 1998, 81, 2270-2281.
Krapcho, A.P.; Kuell, C.S. Mono-protected diamines. N-tert-
[10]
[6]
[7]
[11]
[12]
[8]
[9]
[13]
butoxycarbonyl-ꢁ,ꢂ-alkanediamines from ꢁ,ꢂ-alkanediamines.
Synth. Commun., 1990, 20, 2559-2564.
Andruszkiewicz, R.; Wojciechowska, H.; Borowski, E. Syntheses
of edeine antibiotics and their analogs. 1. Synthesis of N-(3-amino-