Conformationally restrained chiral analogues of spermine: Chemical synthesis and improvements in DNA triplex stability

Article abstract of DOI:10.1021/jo970414j

The synthesis of novel chiral analogues of spermine, 11 (2R,4S) and 14 (2S,4R), is reported starting from trans-4-hydroxy-L-proline 3. These cyclic analogues are generated from linear, achiral spermine by incorporating a pyrrolidine ring on the backbone t

Full text of DOI:10.1021/jo970414j

J . Org. Chem. 1997, 62, 5169-5173
5169
Con for m a tion a lly Restr a in ed Ch ir a l An a logu es of Sp er m in e:
Ch em ica l Syn th esis a n d Im p r ovem en ts in DNA Tr ip lex Sta bility†
Kallanthottathil G. Rajeev, Gangadhar J . Sanjayan, and Krishna N. Ganesh*
Organic Chemistry Division (Synthesis), National Chemical Laboratory, Pune 411008, India
Received March 5, 1997X
The synthesis of novel chiral analogues of spermine, 11 (2R,4S) and 14 (2S,4R), is reported starting
from trans-4-hydroxy-L-proline 3. These cyclic analogues are generated from linear, achiral spermine
by incorporating a pyrrolidine ring on the backbone to effect conformational rigidity, with
simultaneous creation of two asymmetric centers. The chiral analogues bind both AT and CG rich
DNA duplexes as effectively as spermine and exhibit even better association with DNA triplex
than spermine. The newer analogues have features for structural elaboration to novel molecular
entities of potential importance in therapeutics and material design.
The linear polyaminessputrescine, spermidine, and
spermine (1)sare biological cations ubiquitously found
in all cells with a diverse role in physiological processes.1
These range from stabilization/modulation of membrane
transformed cells has led to design of their analogues as
inhibitors of polyamine biosynthesis enzymes such as
ornithine decarboxylase and S-adenosylmethionine de-
carboxylase.11 The modulatory activity of spermine on
the N-methyl-D-glutamate (NMDA) receptor has poten-
1
function and mitochondria and facilitation of DNA
2
transfection by phage to regulation of cell growth and
tial for application of polyamine analogues in therapy of
differentiation.3 They play an important role in prolif-
erative processes, in particular, neoplastic growth and
chemical carcinogenesis.4 The binding of spermine to
DNA stabilizes the duplex, causes conformational changes
in DNA such as B-Z transition, and induces bending of
the helix axis at specific sequences which has importance
in chromatic condensation and regulation of transcrip-
tion.5 Spermine is known to promote triplex stabilization
neurological diseases such as epilepsy.
12
Polyamine
analogues are also emerging as serious therapeutic
options for development of potent new antidiarrheals, in
particular, AIDS-related diarrhea.13 The wide therapeu-
tic potential of polyamines has led to design, synthesis,
and biological evaluation of a large number of their
analogues toward understanding structure-activity re-
14
lationships.
These studies have pointed out that
6
when externally added, as well as upon covalent conju-
polyamine analogues at the tetramine level must be
charged to be recognized by the cell and analogues with
low pK s that are poorly protonated at pH 7.0 do not
a
gation to sugar7 or nucleobase residues.8 In vitro,
polyamines affect many enzymatic systems such as
phosphatidylinositol-G and adenylate cyclase-G protein
pathways and post-translational modification of proteins
compete efficiently with biogenic amines for uptake. The
several structural modifications made to the polyamine
backbone include variation in the number and distance
9
by transglutaminases and also inhibit voltage-activated
ion channels.10 The high level of polyamines noticed in
between nitrogens, terminal N-substitutions of varying
15
sizes,13 and rigidification via interconnection of secondary
amines (cyclopolyamines) based on the cycloputrescine
core.16 Most of these chemical modifications are through
N-substitutions and are achiral.
†
NCL Communication No. 6390.
*
Author to whom all correspondence should be addressed. Fax: 91
2
12 335153. E-mail: kng@ems.ncl.res.in.
X
Abstract published in Advance ACS Abstracts, J une 15, 1997.
(
1) (a) Tabor, C. W.; Tabor, H. Annu. Rev. Biochem. 1984, 53, 749-
90. (b) Schuber, F. Biochem. J . 1989, 260, 1-10.
2) Henner, W. D.; Kleber, I.; Benzinger, R. J . Virol. 1973, 12, 741-
47.
3) (a) Pegg, A. E. Cancer Res. 1988, 48, 759-774. (b) Pegg, A. E.
Biochem. J . 1986, 234, 249-262.
4) (a) Heby, O.; J anne, J . In Polyamines in Biology and Medicine;
Since many of the polyamine receptor sites (nucleic
acids, membranes) are chiral in nature, we reasoned that
7
7
(
(
(8) (a) Nara, H.; One, A.; Matsuda, A. Bioconjugate Chem. 1995, 6,
54-61. (b) Prakash, T. P.; Barawkar, D. A.; Kumar, V. A.; Ganesh, K.
N. BioMed. Chem. Lett. 1994, 4, 1733-1738. (c) Barawkar, D. A.;
Kumar, V. A.; Ganesh, K. N. Biochem. Biophys. Res. Commun. 1994,
205, 1665-1670. (d) Barawkar, D. A.; Rajeev, K. G.; Kumar, V. A.;
Ganesh, K. N. Nucleic Acids Res. 1996, 24, 1229-1237. (e) Pallan, P.
S.; Ganesh, K. N. Biochem. Biophys. Res. Commun. 1996, 222, 416-
420.
(9) (a) Seiler, N. Prog. Drug Res. 1991, 37, 107-160. (b) Cordella-
Miele, E.; Miele, L.; Beninati, S.; Mukherjee, A. J . Biochem. 1993, 164-
173.
(10) Scott, R. H.; Sutton, K. G.; Dolphin, A. C. Trends Neurosci.
1993, 16, 153-160.
(
Morris, D. R., Morton, L. J ., Eds.; Dekker: New York, 1981. (b) Behr,
J . P. Acc. Chem. Res. 1993, 26, 274-278.
(
5) (a) Ghosule, L. C.; Schellman, J . A. J . Mol. Biol. 1978, 121, 311-
3
1
26. (b) Chattoraj, D. K.; Gosule, L. C.; Schellman, J . A. J . Mol. Biol.
978, 121, 327-337. (c) Behe, M.; Felsenfeld, G. Proc. Natl. Acad. Sci.
U.S.A. 1981, 78, 1619-1623. (d) Majumder, K.; Brahmachari, S. K.
Biochem Int. 1989, 18, 455-465. (e) Feurstein, B. G.; Pattibhiraman,
N.; Marton, L. J . Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 5948-5952.
(
2
f) J ain, S.; Zon, G.; Sundaralingam, M. Biochemistry 1989, 28, 2360-
364. (g) Feurstein, B. G.; Pattibhiraman, N.; Marton, L. J . Nucleic
Acids Res. 1989, 17, 6883-6892. (h) Feurstein, B. G.; Pattibhiraman,
N.; Marton, L. J . Nucleic Acids Res. 1990, 18, 1271-1282. (i) Delcros,
J .-G.; Strukenboom, C. J . M.; Basu, H. S.; Shafer, R. H.; Szollosi, J .;
Feuerstein, B. G.; Marton, L. J . Biochem. J . 1993, 291, 269-274. (j)
Esposito, D.; Vecchio, P. D.; Barone, G. J . Am. Chem. Soc. 1997, 119,
(11) Flink, I.; Pettijohn, D. E. Nature 1975, 253, 62-63.
(12) (a) J ohnson, T. D. Trends Pharmacol. Sci. 1996, 17, 22-27. (b)
Williams, K. Neurosci. Lett. 1995, 184, 181-184. (c) Bergeron, R. J .;
Weimar, W. R.; Wu, Q.; Austin, J . K., J r.; McManis, J . S. J . Med. Chem.
1995, 38, 425-428.
(13) Bergeron, R. J .; Yao, G. W.; Yao, H.; Weimar, W. R.; Sninsky,
R. B.; Feng, Y.; Wu, Q.; Gao, F. J . Med. Chem. 1996, 39, 2461-2471.
(14) Bergeron, R. J .; McManis, J .; Weimar, W. R.; Schreier, S.; Gao,
F.; Wu, Q.; Ortiz-Ocasio, J .; Luchetta, G. R.; Porter, J . R.; Vinson, T.
J . Med. Chem. 1995, 38, 2278-2285.
(15) (a) Ganem, B. Acc. Chem. Res. 1982, 15, 290-298. (b) Bergeron,
R. J . Acc. Chem. Res. 1986, 19, 105-113.
(16) Brand, G.; Hosseini, M. W.; Ruppert, R. Tetrahedron 1994, 35,
8609-8612.
2
606-2614.
(
6) (a) Hampel, K. J .; Crosson, P.; Lee, J . S. Biochemistry 1991, 30,
4
1
1
455-4459. (b) Thomas, T.; Thomas, T. J . Biochemistry 1993, 32,
4068-14074. (c) Volker, J .; Klump, H. H. Biochemistry 1994, 33,
3502-13508.
(
7) (a) Tung, C. H.; Breslauer, K. J .; Stein, S. Nucleic Acids Res.
1
993, 21, 5489-5494. (b) Bigey, P.; Prativel, G.; Meunier, B. J . Chem.
Soc., Chem. Commun. 1995, 181-182. (c) Sund, C.; Puri, N.; Chatto-
padhyaya, J . Tetrahedron 1996, 52, 12275-12290.
S0022-3263(97)00414-3 CCC: $14.00 © 1997 American Chemical Society

5
170 J . Org. Chem., Vol. 62, No. 15, 1997
Rajeev et al.
Sch em e 1
Sch em e 2
introduction of asymmetry into the polyamine backbone
may be beneficial to their activity. This and our own
studies on stabilization of DNA triplexes at physiological
8
b-d
conditions through covalent linking of spermine
and
use of bisguanidine analogues of spermine8 prompted us
to synthesize chiral spermine analogues and study their
interaction with nucleic acids.
e
the expected one, and on the basis of spectral data (NMR,
mass), it was identified as the bicyclic compound (1S,4S)-
5
-(3-aminopropyl)-2-(tert-butoxycarbonyl)-2,5-diaza-
bicyclo[2.2.1]heptane (6a ). This product possibly arises
from the intermediate 7 resulting from an initial (faster)
displacement of primary mesylate at C2 in 4c by the
diamine.20 A trans relationship of the 4-mesylate with
the C2 substituent in 7 subsequently leads to a stereo-
chemically favored intramolecular S 2 attack by the
N
secondary amino group of the C2 side chain on C4. The
identity of the resulting bicyclic product 6a was sup-
Resu lts a n d Discu ssion
Ch em ica l Syn th esis of P yr r olid yl P olya m in es.
These analogues are derived from spermine (1) by
introducing a bridge of CH -NH between the carbon
2
atoms R and γ of the central tetramethylene fragment
as shown in Scheme 1 to generate a five-membered
pyrrolidine ring (2). This makes the molecule chiral with
creation of two asymmetric centers (C2 and C4) and
hence two pairs of diastereomers (cis 2S,4S, 2R,4R and
trans 2S,4R, 2R,4S) with a simultaneous introduction of
one additional nitrogen atom. We report here the chemi-
cal synthesis of two of the four possible stereoisomers
ported by the presence of a methylene triplet due to the
1
CH
2
NH
2
in its H NMR spectrum and by converting 6a
into its monotrifluoroacetamide derivative 6b. This also
ruled out the formation of the isomeric product 8 that
lacks a CH NH group and forms a ditrifluoroacetamide
2 2
(
trans 2S,4R and 2R,4S) of the designed newer analogues
upon derivatization. The synthesis of 2,4-trans-polyamines
11 (2R,4S) and 14 (2S,4R) could be achieved alternatively
from 3 by selective inversion of configuration at C2 and
C4. The epimerization of trans-4-hydroxy-L-proline (3)
of spermine and demonstrate that these analogues are
better than spermine in selectively stabilizing the DNA
triple helix.
The spermine analogues 2 were synthesized from the
commercially available and versatile chiral starting block
trans-4-hydroxy-L-proline17 (3) which was treated with
thionyl chloride in methanol to yield the 2-carboxymethyl
ester that was subsequently reacted with tert-butoxy
carbazide for protection of the ring N to give trans-4-
1
7
at C2 by acetic anhydride/acetic acid treatment followed
by reflux in 2 N HCl gave the cis-4-hydroxy-D-proline (9)
13
whose optical integrity was confirmed by C NMR
spectrum and optical rotation. The N-Boc-C2-methyl
ester 10a , obtained from 9, upon reduction with lithium
borohydride gave the cis-4-hydroxy-D-prolinol (10b) which
was derivatized to the cis-dimesylate 10c. This on
treatment with 1,3-diaminopropane gave the expected
product 11a unlike in the case of the trans-dimesylate
4c. The cis configuration of the C4 mesylate with the
C2 side chain amine in the intermediate 10d does not
favor an intramolecular substitution, and as a result, the
attack of a second molecule of diamine at C4 eventually
occurs, leading to the chiral polyamine 11a . The suc-
cessful isolation of the highly water soluble amine 11a
in 95-97% yield was achieved under nonaqueous workup
using ethyl acetate (see the Experimental Section for
details). The N-t-Boc function of 11a was deprotected
1
8
hydroxy-N-(tert-butoxycarbonyl)-L-proline methyl ester
(
4a ). This was reduced with lithium borohydride19 to
trans-4-hydroxy-L-prolinol (4b) and converted to the
corresponding dimesylate 4c by reaction with mesyl
chloride in pyridine. The 2,4-trans-dimesylate 4c was
reacted with neat 1,3-diaminopropane with the expecta-
tion of obtaining precursor 5 (2S,4S; Y ) Boc) for the cis
isomer, through a simple displacement of both mesylates
by the diamine with a simultaneous inversion at C4.
Although the reaction led to a single product, it was not
(
17) Remuzon, P. Tetrahedron 1996, 52, 13803-13835.
(18) Kaname, M.; Yoshifuji, S. Tetrahedron Lett. 1992, 33, 8103-
8
104.
(
19) Baker, G. L.; Fritschel, S. J .; Stille, J . R.; Stille, J . K. J . Org.
(20) J ordis, U.; Sauter, F.; Siddiqi, S. M.; Kuenburg, B.; Bhatta-
charya, K. Synthesis 1990, 925-930.
Chem. 1981, 46, 2954-2960.

Synthesis of Novel Chiral Spermine Analogues
J . Org. Chem., Vol. 62, No. 15, 1997 5171
using 5 N HCl to obtain the 2,4-trans (2R,4S) analogue
as its pentahydrochloride salt 11b.
Ta ble 1. UV-tm (°C) of DNA Du p lex/Tr ip lex in th e
a
P r esen ce of Sp er m in e a n d Its An a logu es
duplex/
no.
triplex
control
1
11a
11b
14a
14b
1
2
3
15
16:17
17*18:19
50
43
nd
56
50
30
53.0
49.5
nd
58.0
51.5
42.0
55.0
48.5
nd
57.0
51.0
45.0
b
b
b
a
For conditions, see Experimental Section; [amine] ) 1 mM,
no Mg2
+ b
.
Not detectable.
The inversion of configuration of hydroxyl at C4 in 4
was achieved via Mitsunobu reaction21 with benzoic acid
affording the cis-4-O-benzoyl-N-(tert-butoxycarbonyl)-L-
proline methyl ester (12). This ester on reduction with
lithium borohydride in dry THF gave the cis-diol 13a that
was mesylated to the cis-dimesylate 13b followed by
reaction with neat 1,3-diaminopropane to give the
polyamine 14a . The removal of t-Boc protection in 14a
was achieved by treatment with 5 N HCl to furnish 2,4-
trans (2S,4R) polyamine 14b, isolated as its pentahydro-
chloride salt.
F igu r e 1. UV-melting profiles of triplex 17*18:19 in TRIS
buffer containing 0.1 M NaCl at pH 7.3, in the presence of 1
mM (a) spermine (1), (b) 11b, and (c) 11a . Dotted curve is the
first derivative of profile b.
It is seen from the experimental data that the self-
complementary CG rich Dickerson 12-mer duplex 15 is
stabilized by spermine 1 (∆t
amine analogues 11b and 14b (∆t
to the control (entry 1). Although no significant differ-
ences were noticed among the free amines, the N-Boc
derivatives 11a and 14a exhibited a relatively lower
m
≈ 6 °C), as well as the free
m
≈ 7-8 °C) compared
DNA Du p lex a n d Tr ip lex Sta biliza tion w ith Sp er -
m in e An a logu es 11b a n d 14b. Spermine is well known
to stabilize DNA duplexes and triplexes,5
e-j,22
although
stabilizing effect (∆t
hindrance by the Boc substituent upon complex formation
with DNA double/triple helices or lack of protonation on
m
≈ 3-5 °C) arising from either steric
the exact molecular details are not yet clear. The
terminal ammonium cations may bind to anionic phos-
phates of DNA, while the internal amino moieties may
be involved in specific hydrogen bonding with nucleo-
bases in the major groove, with a cross groove binding
for A:T base pairs and a down groove mode among G:C
base pairs.22 In the case of triplexes, stabilization by
cationic polyamines is ascribed to proceed via electro-
static neutralization of large negative charge density on
triplexes.6 Spermine, a linear aliphatic chain, enjoys
enormous conformational freedom, and theoretical cal-
culations have suggested a preferential occurrence of
trans/ gauche conformations at C-N and N-C bonds.23
In the presently designed analogues, conformational
constrain imposed by the five-membered chiral pyrroli-
dine ring (2R,4S and 2S,4R) on the spermine backbone
may influence its interaction with DNA, and to examine
2 m
the ring N. The addition of 20 mM MgCl raised the t
by 3-5 °C, in the case of control and duplex containing
Boc derivatives 11a and 14a (data not shown) as ex-
pected, but with spermine 1 or its analogues 11b and
14b, the increase was not appreciable. The non-self-
complementary AT rich duplex (16:17) showed a similar
trend of stabilization with spermine and its analogues
(Table 1, entry 2). Thus the constrained spermine
analogues are as good as spermine in effecting duplex
stability of both AT and CG rich sequences, and struc-
tural specificity effects are minimal on duplex stabiliza-
tion which is largely governed by the number of positive
charges.
c
such structural effects, we carried out UV-t
m
DNA
melting studies of DNA duplexes and triplexes in the
presence of these analogues at 1 mM concentration; the
results are presented in Table 1. Since Mg2 is well
known to stabilize duplexes and triplexes, all melting
+
In comparison, considerable specificity differences were
noticed among spermine and its constrained analogues
in their interaction with the triplex (17*18:19). In the
absence of Mg2 , while no triplex was detected with the
control (Table 1, entry 3), spermine and its analogues 11b
and 14b exhibited well-recognizable triplex formation
experiments were carried out under identical conditions,
in both the presence and absence of Mg2
+
.
+
(
21) (a) Mitsunobu, O. Synthesis 1981, 1-28. (b) Williams, M. A.;
Rapoport, H. J . Org. Chem. 1994, 59, 3616-3625.
22) Haworth, I. S.; Rodger, A.; Richards, W. G. Proc. R. Soc. London
B 1994, 244, 107-116.
23) Laquori, A. M.; Constantino, L.; Crescenzi, V. J . Mol. Biol. 1967,
4, 113-122.
(
(Figure 1) with the lower transition corresponding to
(
2
triplex S duplex and the higher one to duplex melting.

5
172 J . Org. Chem., Vol. 62, No. 15, 1997
Rajeev et al.
Further, the analogues showed remarkably improved
stabilization of triplexes (∆t
≈ 12-15 °C) over that by
determined from the midpoint of the first transition from the
plot of fraction absorbance change versus temperature and was
further confirmed by differential [(dA/dT)260 nm vs T] curves.
m
spermine, with (2S,4R)-14b isomer being slightly better
than (2R,4S)-11b isomer. Interestingly, the N-Boc ana-
logues 11a and 14a failed to promote triplexation in the
absence of Mg2 . As expected, all including control and
Boc derivatives, showed the triplex in the presence of
Mg2 (not shown). These results indicate that the
constrained spermine analogues 11b and 14b, while
stabilizing duplexes as effectively as spermine, are
significantly better than spermine in selectively stabiliz-
ing DNA triplexes. The topology of polyamine-DNA
interactions in duplexes differs considerably from that
in triplexes where polyamine structural effects (number
and distribution of positive charges) have profound
consequences on stability.6b The magnitude of observed
triplex stability with 11b and 14b cannot be solely
attributed to the effect of an additional positive charge
in them as compared to spermine. It is more likely that
the rigid conformations of the constrained analogues 11
and 14 are more suitable for triplex binding than the
The t values are accurate to (0.5 °C over the reported values.
m
tr a n s-4-H yd r oxy-N-(ter t-b u t oxyca r b on yl)-L-p r olin ol
4b). To an ice cold solution of 4a (1.2 g, 5.0 mmol) in THF
20 mL) was added lithium borohydride (0.45 g, 21 mmol), and
(
(
+
the mixture was stirred at 5-10 °C for 4 h, continued at
ambient temperature for 36 h. The reaction was quenched
by addition of saturated ammonium chloride solution (10 mL)
at 0 °C with stirring for a further 2 h. It was concentrated to
+
a pasty mass under reduced pressure and extracted into CHCl
3
followed by evaporation to give a colorless viscous liquid. This
was purified by column chromatography on silica gel (eluent:
dichloromethane/EtOAc, 1:1) to yield the diol 4b as a colorless
-1
1
oil (1 g, 94%). IR (CHCl
3
): 3500-3300, 1670 cm
.
H NMR
δ (CDCl + D O): 4.33 (bs, 1H), 4.12-4.05 (m, 1H), 3.62-3.35
3
2
(m, 4H), 2.08-1.99 (bm, 1H), 1.75-1.59 (bs, 1H), 1.45 (s, 9H).
2
5
25
20
[
)
R]
D
) -56.2 ( 0.5 (c ) 0.56, CHCl
1.01, EtOH)].
cis-4-Hydr oxy-N-(ter t-bu toxycar bon yl)-D-pr olin ol (10b).
3
) [lit. [R]
D
) -58.87 (c
The reduction of 10a (1.25 g, 5.1 mmol) using lithium boro-
hydride (0.8 g, 37 mmol) in THF (20 mL) as in the case of 4a
afforded 10b (0.8 g, 72%) as a white solid, mp ) 91-93 °C. IR
2
3
-1
1
flexible conformation of spermine.
(Nujol): 3300-3100, 2920-2850, 1670 cm
.
H NMR δ
O): 4.28 (bt, 1H), 4.05-3.99 (bm, 2H), 3.55-3.44
bm, 3H), 2.37-2.26 (bm, 1H), 1.94-1.80 (bm, 1H), 1.46 (s,
In summary, novel chiral analogues of spermine,
incorporating a pyrrolidine ring on its backbone with
consequential conformational restraint, have been syn-
thesized from trans-4-hydroxy-L-proline, and these are
shown to substantially improve DNA triplex stability
over that by spermine. Further studies are aimed at
rational fine tuning of stereochemistry in the polyamine
analogues through molecular modeling and derivatiza-
(CDCl
3
+ D
2
(
1
3
9
6
0
H). C NMR δ (CDCl
3.2, 58.3, 56.3, 37.7, 37.0, 28.3. [R]
.16, CHCl ). Anal. for C10 Calcd: C, 55.29; H, 8.75;
3
): 155.5, 154.7, 79.8, 69.4, 68.7, 63.7,
2
5
D
) +46.3 ( 0.5 (c )
3
H19NO
4
N, 6.45. Found: C, 55.65; H, 8.39; N, 6.77.
cis-4-Hydr oxy-N-(ter t-bu toxycar bon yl)-L-pr olin ol (13a).
The reduction of 12 (1.8 g, 5.2 mmol) with lithium borohydride
(0.75 g, 34 mmol) in THF (25 mL) followed by silica gel column
chromatography as in the case of 4b yielded 13a (0.65 g, 58%)
as a white solid, mp ) 88-90 °C. IR (Nujol): 3300-3200,
8
e
tion (e.g., guanidylation ) to develop more efficient and
selective DNA-binding agents. The newer spermine
analogues reported here have features for extension to
branched structures through the ring N, and such new
entities add to an expanding library of polyamines
required for drug and novel material designs.
-
1
1
2
1
1
1
2
920-2860, 1685 cm
.
H NMR δ (CDCl
3
2
+ D O): 4.29 (bs,
H), 4.08-4.01 (bm, 2H), 3.61-3.45 (bm, 3H), 2.37-2.26 (bm,
13
H), 1.95-1.75 (bm, 1H), 1.46 (s, 9H). C NMR δ (CDCl
3
):
55.7, 154.9, 80.0, 69.6, 68.9, 64.0, 63.4, 58.5, 56.6, 38.0, 37.3,
25
8.5. [R]
D
) -44.6 ( 0.5 (c ) 0.4, CHCl
Calcd: C, 55.29; H, 8.75; N, 6.45. Found: C, 55.53; H,
.51; N, 6.47. FAB MS: 218 (M + 1, 32), 188 (100).
tr a n s-4-H yd r oxy-N-(ter t-b u t oxyca r b on yl)-L-p r olin ol
3 19
). Anal. for C10H -
NO
8
4
+
Exp er im en ta l Section
All the melting points are uncorrected. All solvents used
were purified according to literature procedures. trans-4-
Hydroxy-L-proline, tert-butyl carbazate, DEAD, and 1,3-di-
Dim eth a n esu lfon a te (4c). To a precooled, stirred solution
of diol 4b (0.95 g, 4.5 mmol) in pyridine (10 mL) was added
methanesulfonyl chloride (1 mL, 12.9 mmol) dropwise under
nitrogen. The stirring was continued at 0 °C for 4 h. The
solvent was removed under vacuum at 30 °C; the residue was
extracted into ethyl acetate (20 mL) and upon workup yielded
4c as a yellow solid (1.5 g, 91%). This solid was used directly
2
4
1
aminopropane were obtained form Aldrich Chemical Co.
H
1
3
NMR spectra were recorded at 200 MHz and C NMR at 50
MHz, and all the chemical shifts are referred to internal TMS
or 1,4-dioxane. Silica gel column chromatographic separations
were followed by TLC analysis using precoated silica gel TLC
plates (Merck Kieselgel 60 F254) and visualized by ninhydrin
spray.
1
for the next reaction without further purification. H NMR δ
(CDCl ): 5.29-5.26 (m, 1H), 4.70-4.55 (bm, 1H), 4.40-4.28
3
(m, 2H), 3.88-3.76 (m, 1H), 3.62-3.48 (bm, 1H), 3.08 (s, 3H),
3.05 (s, 3H), 2.47-2.28 (m, 2H), 1.48 (s, 9H).
Oligon u cleotid e Syn th esis a n d UV-Meltin g Exp er i-
m en ts. All oligonucleotides were synthesized on 1.3 µmol
scale on a Pharmacia GA Plus DNA synthesizer and purified
ci s-4-H yd r ox y-N -(t er t -b u t o xy c a r b o n y l)-D -p r olin ol
Dim eth a n esu lfon a te (10c). The reaction of compound 10b
(0.5 g, 2.5 mmol) with mesyl chloride (0.6 mL, 7.8 mmol) in
pyridine (5 mL) gave 10c (0.85 g, 99%) as a pale yellow solid.
1
2c,d
according to literature procedure.
Duplex and triplex
melting experiments were carried out in TRIS (25 mM) buffer
at pH 7.3, containing NaCl (100 mM) in the presence or
This solid was used for the next reaction without further
1
absence of MgCl
2
(20 mM). Appropriate oligonucleotides, each
purification. H NMR δ (CDCl
3
): 5.32-5.28 (m, 1H), 4.46-
at a strand concentration of 1 µmol based on UV absorbance
at 260 nm calculated using molar extinction coefficients of
respective nucleosides, were mixed in the buffer containing
polyamines (1 mM), heated at 80 °C for 3 min, and cooled to
room temperature followed by overnight storage at 4 °C. A260
values at various temperatures were recorded using a Perkin
Elmer Lambda 15 UV/vis spectrophotometer, fitted with a
water jacketed 5-cell holder and J ulabo temperature program-
mer with a heating rate of 0.5 °C/min over the range 5-75
4.44 (m, 1H), 4.42-4.16 (bm, 2H), 3.71-3.69 (m, 2H), 3.10 (s,
3H), 3.06 (s, 3H), 2.47-2.37 (bm, 2H), 1.48 (s, 9H).
ci s-4-H y d r o x y -N -(t er t -b u t o x y c a r b o n y l)-L -p r o lin o l
Dim eth a n esu lfon a te (13b). The compound 13a (0.55 g, 2.5
mmol) was mesylated using mesyl chloride (0.6 mL, 8 mmol)
1
in dry pyridine (5 mL) to yield 13b (0.94 g, 99%). H NMR δ
(CDCl ): 5.31-5.26 (m, 1H), 4.47-4.43 (bm, 1H), 4.36-4.14
3
(bm, 2H), 3.70 (bs, 2H), 3.08 (s, 3H), 3.04 (s, 3H), 2.41-2.31
(bm, 2H), 1.48 (s, 9H).
°
C. Dry nitrogen gas was flushed in the spectrophotometer
chamber to prevent moisture condensation at temperatures
below 15 °C. The triplex dissociation temperature (t ) was
(1S,4S)-5-(3-Am in op r op yl)-2-(ter t-bu toxyca r bon yl)-2,5-
d ia za bicyclo[2.2.1]h ep ta n e (6a ): P r oced u r e 1. The dime-
sylate 4c (0.8 g, 2 mmol) was stirred in neat 1,3-diaminopro-
m
(
24) Perrin, D. D.; Armarego, W. L. F. Purification of Laboratory
(25) Ojima, I.; Kogure, T.; Yoda, N. J . Org. Chem. 1980, 45, 4728-
4739.
Chemicals; Pergamon Press: New York, 1988.

Synthesis of Novel Chiral Spermine Analogues
J . Org. Chem., Vol. 62, No. 15, 1997 5173
pane (5 mL) at ambient temperature for 4 h followed by
stirring at 60 °C for 24 h. The excess diamine was removed
under reduced pressure at 50 °C. The residue was dissolved
in dichloromethane (30 mL) followed by washing with water
phenylphosphine (2.8 g, 10.5 mmol), and benzoic acid (1.3 g,
10.5 mmol) in acetonitrile (30 mL) at 0 °C was added
diisopropyl azodicarboxylate (2.15 g, 10.6 mmol) as a neat
liquid. The reaction mixture was stirred at 0 °C for 2 h and
then at ambient temperature for 24 h. After addition of
methanol (1 mL) the reaction mixture was concentrated to a
pasty mass which was dissolved in diethyl ether (30 mL),
triturated with petroleum ether (40-60 °C), and kept at 5 °C
overnight. The triphenylphosphine oxide formed was filtered
off, the filtrate was concentrated to dryness, and the residue
was chromatographed over silica gel using petroleum ether/
(20 mL). The dichloromethane extract was dried over anhy-
drous sodium sulfate and concentrated to yield a pale yellow
oil of 6a (0.45 g, 40%).
P r oced u r e 2. The dimesylate 4c (0.95 g, 2.5 mmol) was
stirred in neat diaminopropane (6 mL) under argon atmo-
sphere at ambient temperature for 4 h and then at 60 °C for
8
4
h. The excess diamine was removed under low pressure at
0 °C, the residue was dried with anhydrous sodium sulfate
dichloromethane (2:3; R
) 0.4) as eluent to obtain 12 (2.2 g,
f
(
1
10 g), and the product was extracted into ethyl acetate (3 ×
77%) as a white solid, crystallized from EtOAc/petroleum ether
0 mL) and filtered. Evaporation of the filtrate under vacuum
(40-60 °C), mp 85-88 °C. IR (Nujol): 2950-2850, 1745, 1710,
1
-1
1
gave 6a as a pale yellow oil (0.9 g, 100%). H NMR δ (CDCl
3
1690 cm
.
H NMR δ (CDCl ): 7.98 (d, 2H), 7.61-7.40 (m,
3
+
3
2
(
4
D
2
O): 4.34 (bs, 0.5H), 4.21 (bs, 0.5H), 3.55-3.43 (bt, 2H),
3H), 5.54 (bm, 1H), 4.64-4.58 (m, 0.5H), 4.51-4.46 (m, 0.5H),
13
.16-3.11 (bd, 1H), 2.95-2.86 (t, 1H), 2.79-2.73 (t, 2H), 2.64-
3.87-3.68 (m, 5H), 2.59-2.42 (m, 2H), 1.47 (d, 9H). C NMR
δ (CDCl ): 172.4, 172.1, 165.7, 154.1, 153.7, 133.3, 129.7, 128.4,
80.3, 73.5, 72.4, 57.9, 57.6, 52.5, 52.1, 36.6, 35.7, 28.3. [R]
). Anal. for C18 Calcd:
1
3
.43 (m, 3H), 1.79-1.52 (bm, 4H), 1.45 (s, 9H). C NMR δ
3
2
5
CDCl
3
): 153.7, 78.6, 60.9, 60.3, 59.6, 57.3, 56.3, 51.8, 51.4,
D
2
5
9.2, 48.4, 40.0, 35.7, 35.0, 32.1, 28.1. [R]
D
) -47.5 ( 0.5 (c
) -28.0 ( 0.5 (c ) 0.5, CHCl
3
H23NO
6
)
0.64, MeOH). FAB MS: 256 (M+ + 1, 100).
C, 61.86; H, 6.64; N, 4.01. Found: C, 61.78; H, 6.76; N, 4.01.
+
(
2R,4S)-2-[[N-(3-Am in op r op yl)a m in o]m eth yl]-4-[N-(3-
FAB MS: 350 (M + 1, 18), 250 (100).
a m in op r op yl)a m in o]-1-(t er t -b u t oxyca r b on yl)p yr r oli-
d in e (11a ). Compound 10c (0.8 g, 2.1 mmol) was treated with
neat 1,3-diaminopropane (6 mL) at 60 °C for 24 h and worked
(2R,4S)-2-[[N-(3-Am in op r op yl)a m in o]m eth yl]-4-[N-(3-
a m in op r op yl)a m in o]p yr r olid in e P en t a h yd r och lor id e
(11b). Compound 11a (0.2 g, 0.6 mmol) was treated with 5 N
HCl (10 mL) at ambient temperature for 8 h. Water and HCl
were removed under vacuum, and the residue was dried over
up according to procedure 2 for the preparation of 6a to get
1
1
D
2
1
5
1a (0.6 g, 85%) as a pale yellow oil. H NMR δ (CDCl
2
3
+
O): 3.97 (bs, 1H), 3.53-3.48 (m, 1H), 3.47-3.06 (bm, 2H),
anhydrous P
ride salt 11b (0.2 g, 80%) as a white hygroscopic solid.
NMR δ (D O): 4.40-4.25 (m, 2H), 4.14-4.03 (m, 1H), 3.73-
2 5
O under vacuum to obtain the pentahydrochlo-
1
.79-2.58 (m, 10H), 2.09-1.99 (bm, 1H), 1.80-1.47 (m, 5H),
H
1
3
.46 (s, 9H). C NMR δ (CDCl
3
): 154.3, 78.6, 77.5, 55.8, 55.5,
2
2.7, 51.8, 47.2, 45.6, 39.7, 39.1, 36.4, 36.3, 33.1, 32.1, 27.9.
) +45.3 ( 0.5 (c ) 0.64, MeOH).
2S,4R)-2-[[N-(3-Am in op r op yl)a m in o]m eth yl]-4-[N-(3-
3.55 (m, 3H), 3.31-3.08 (m, 8H), 2.73-2.68 (m, 1H), 2.59-
2
5
13
[R]
D
2
2.48 (m, 1H), 2.21-2.06 (m, 4H). C NMR δ (D O): 56.4, 56.2,
(
48.6, 48.2, 46.7, 45.2, 37.6, 32.5, 24.8, 24.6.
a m in op r op yl)a m in o]-1-(t er t -b u t oxyca r b on yl)p yr r oli-
d in e (14a ). Compound 13b (0.9 g, 2.4 mmol) was treated with
neat 1,3-diaminopropane (6 mL) at 60 °C for 24 h and worked
(2S,4R)-2-[[N-(3-Am in op r op yl)a m in o]m eth yl]-4-[N-(3-
a m in op r op yl)a m in o]p yr r olid in e P en t a h yd r och lor id e
(14b). Compound 14a (0.2 g, 0.6 mmol) was treated with 5 N
HCl at ambient temperature for 8 h. Workup as above yielded
the pentahydrochloride salt 14b (0.18 g, 73%) as a white
up according to procedure 2 for the preparation of 6a as above
1
to get 14a (0.66 g, 83%) as a pale yellow oil. H NMR δ (CDCl
3
+
D
2
O): 3.95 (bs, 1H), 3.55-3.48 (m, 1H), 3.38-3.10 (bm, 2H),
hygroscopic solid. 1H NMR δ (D
O): 4.44-4.18 (m, 2H), 4.13-
2
2
1
5
.81-2.50 (m, 10H), 2.1-1.99 (bm, 1H), 1.83-1.57 (m, 5H),
4.02 (m, 1H), 3.70-3.53 (m, 3H), 3.30-2.96 (m, 8H), 2.74-2.64
1
3
13
.46 (s, 9H). C NMR δ (CDCl
3
): 154.4, 78.7, 77.6, 55.9, 55.5,
(m, 1H), 2.58-2.45 (m, 1H), 2.20-2.05 (m, 4H). C NMR δ
2
5
2.9, 52.0, 47.3, 45.7, 39.8, 39.2, 36.1, 33.1, 33.0, 28.0. [R]
-45.9 ( 0.5 (c ) 0.64, MeOH). FAB MS: 330 (M + 1, 100).
1S,4S)-5-[3-(Tr iflu or oa cet a m id o)p r op yl]-2-(ter t-b u -
D
2
(D O): 56.1, 55.9, 48.3, 47.9, 46.4, 44.9, 37.4, 32.3, 24.6, 24.4.
+
)
(
Ack n ow led gm en t. K.G.R. and G.J .S. thank CSIR,
toxyca r bon yl)-2,5-d ia za bicyclo[2.2.1]h ep ta n e (6b). The
compound 6a (0.75 g) was treated with ethyl trifluoroacetate
New Delhi, for financial support. K.N.G. acknowledges
J awaharlal Nehru Centre for Advanced Scientific Re-
search, Bangalore, of which he is a Honorary Senior
Fellow.
(0.5 mL) in ethanol (10 mL) followed by standard work up and
column chromatography on silica gel (elution: dichloromethane/
ethyl acetate, 4:1) to yield the trifluoroacetamide derivative
1
6
9
4
2
1
1
5
0
b as a yellow oil (1.1 g, 67.6%). H NMR δ (CDCl
3
): 9.67-
Su p p or tin g In for m a tion Ava ila ble: 1H and 13C NMR
.58 (bd, 1H), 4.39 (bs, 0.6H), 4.25 (bs, 0.4H), 3.58-3.35 (bm,
H), 3.21 (d, 0.6H, J ) 2.0 Hz), 3.16 (d, 0.4H, J ) 2.0 Hz),
.95-2.79 (m, 3.5H), 2.52-2.47 (bd, 0.5H), 1.76-1.65 (m, 4H),
3
): 157.4, 156.9, 156.5, 154.0,
21.7, 117.9, 114.1, 79.4, 61.7, 61.2, 59.6, 57.3, 56.3, 54.5, 53.6,
spectra for compounds 6a , 12, and 14b, mass spectra for
compounds 6a , 12, and 14a , and UV-melting profile for duplex
1
6:17 in the presence of 1 and 14a ,b (7 pages). This material
1
3
.46 (s, 9H). C NMR δ (CDCl
is contained in libraries on microfiche, immediately follows this
article in the microfilm version of the journal, and can be
ordered from the ACS; see any current masthead page for
ordering information.
2
5
3.3, 49.9, 48.4, 40.8, 36.4, 35.9, 34.7, 28.2. [R]
D
) -28.8 (
+
.5 (c ) 0.84, MeOH). FAB MS: 352 (M , 100).
cis-4-O-Ben zoyl-1-(ter t-bu toxycar bon yl)-L-pr olin e Meth -
yl E st er (12). To a solution of 4 (2.0 g, 8 mmol), tri-
J O970414J