Effect of a hydrogen bonding carboxamide group on universal bases

Article abstract of DOI:10.1135/cccc20060899

A number of aromatic universal base analogues have been described in the literature, but most are non-hydrogen bonding. We have examined the effect of introducing hydrogen bonding carboxamide groups onto the pyrrole ring of 5-nitroindole. The modified ana

Full text of DOI:10.1135/cccc20060899

Effect of a Hydrogen Bonding
899
EFFECT OF A HYDROGEN BONDING CARBOXAMIDE GROUP ON
UNIVERSAL BASES
1
2
3
4,
Kathleen TOO , Daniel M. BROWN , Philipp HOLLIGER and David LOAKES *
Medical Research Council, Laboratory of Molecular Biology,
1
Hills Road, Cambridge, CB2 2QH, UK; e-mail: katt@mrc-lmb.cam.ac.uk,
2
3
4
jer@mrc-lmb.cam.ac.uk, ph1@mrc-lmb.cam.ac.uk, dml@mrc-lmb.cam.ac.uk
Received February 27, 2006
Accepted April 26, 2006
Dedicated to Professor Antonín Holý, on the occasion of his 70th birthday, in recognition of his
outstanding contributions to the area of nucleic acid chemistry.
A n um ber of arom atic un iversal base an alogues h ave been described in th e literature, but
m ost are n on -h ydrogen bon din g. We h ave exam in ed th e effect of in troducin g h ydrogen
bon din g carboxam ide groups on to th e pyrrole rin g of 5-n itroin dole. Th e m odified an alogues
retain un iversal base features, but th ere are n o overall effects on duplex stability. Th is leads
to th e suggestion th at th e n itro group is with in th e h ydrogen bon din g face of th e duplex,
an d th e h ydrogen bon din g carboxam ide group is in th e duplex m ajor groove.
Keyw ord s: Nucleosides; Un iversal n ucleobases; 5-Nitroin dole; In doles; Hydrogen bon din g;
Stackin g en ergies; Duplex stability; Oligon ucleotides; DNA duplexes.
Un iversal bases h ave th e ability to pair with each of th e n atural bases foun d
in DNA or RNA with out distin guish in g between th em to a greater or lesser
degree. Wh ilst th ere h ave been m an y such an alogues described, th ey each
h ave a defin ed set of features, n am ely th at th ey are arom atic, plan ar, h y-
droph obic an d n on -h ydrogen bon din g1,2. As yet th ere are n o un iversal
bases described th at fun ction th rough h ydrogen bon din g to th e n atural
bases an d still retain th e property of n ot sh owin g an y preferen ce to an y
particular n ucleobase, th ough Seela et al. h ave described an 8-aza-7-deaza-
aden in e derivative th at m ay m eet som e of th ese requirem en ts2. A n um ber
of an alogues, such as azolecarboxam ides, h ave been sh own to pair with th e
n atural DNA bases by h ydrogen bon din g. Th ese an alogues exh ibit two al-
tern ative h ydrogen bon din g faces by rotation about th e am ide bon d. In so
doin g, azolecarboxam ides presen t base pairin g properties th at, in prin ciple,
allow th em to h ydrogen bon d to all four DNA bases. However, in practice
th ese an alogues always sh ow base pairin g preferen ces3,4
.
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 6, pp. 899–911
© 2006 Institute of Organic Chemistry and Biochemistry
doi:10.1135/cccc20060899

900
Too, Brown, Holliger, Loakes:
As un iversal bases do n ot possess h ydrogen bon din g features, th ey stabi-
lise duplexes with DNA (or RNA) by en h an ced stackin g in teraction s. Th e
stability of th e base pair with a n atural n ucleobase in a duplex is related to
th e surface area of th e un iversal base, larger surface areas resultin g in
greater stability. Th e m ost widely used un iversal base an alogues are 3-n itro-
pyrrole an d 5-n itroin dole, with th e larger 5-n itroin dole form in g m ore sta-
ble duplexes wh en in corporated in to oligodeoxyn ucleotides5–7. A recen t
report by Klewer et al. discussed th e solution structure of a duplex con tain -
in g 3-n itropyrrole sh owin g th at th e 3-n itropyrrole base h ad reduced π–π
stackin g in teraction s with its adjacen t n ucleobases. Th e lower stackin g in -
teraction of th e 3-n itropyrrole group was due to its reduced size com pared
to a n atural DNA base. In terestin gly, it was sh own th at th e n itro group pro-
trudes in to th e m ajor groove in order to adopt an orien tation reducin g
crowdin g with in th e duplex structure8,9
.
Un iversal base an alogues h ave sh own value in a n um ber of application s
in volvin g th eir h ybridisation properties. Th us th e prin ciple uses of th ese
an alogues are in probes an d prim ers. However, despite a n um ber of differ-
en t an alogues h avin g been assayed in polym erase-based reaction s, n on e
h ave yet been sh own to be good substrates for an y DNA polym erase. In
gen eral th ey act as blockin g lesion s eith er wh en presen t in th e tem plate or
wh en in corporated as a 5′-triph osph ate. Wh en th ey are presen t in a tem -
plate th ey gen erally direct th e in corporation of dAMP, wh ich is presum ably
m ore a fun ction of th e polym erase exten dase activity th an of an y property
of th e un iversal base.
In th is work we wan ted to assess th e action of in corporatin g a h ydrogen
bon din g fun ction al group on to a un iversal base. We h ave previously exam -
in ed th e effect of in dole n ucleosides, wh ich h ad h ydrogen bon din g poten -
tial10. Reduction of 5-n itroin dole-5′-triph osph ate gave th e 5-am in oin dole
derivative 1, wh ich was also con verted to its form am ido derivative 2. Th e
in corporation properties of th ese were exam in ed, wh ere it was foun d th at
th e form am idoin dole 2 beh aved essen tially as a un iversal base, th ough like
5-n itroin dole 3 it acted as a ch ain term in ator. However, th e 5-am in oin dole
derivative 1 was n o lon ger a un iversal base, in corporatin g preferen tially
opposite aden in e, but un der th ese con dition s th e prim er could be ch ain ex-
ten ded. To furth er exam in e un iversal base an alogues with h ydrogen bon d-
in g fun ction ality we ch ose to syn th esise an alogues of 5-n itroin dole bearin g
a carboxam ide group at th e 3-position (4 an d 5) (Ch art 1). If th e carbox-
am ide group of such an an alogue were to be directed towards th e h ydrogen
bon din g face in a duplex th en it m igh t be expected th at its un iversal base
properties would be altered. It m igh t be an ticipated th at such an an alogue
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 6, pp. 899–911

Effect of a Hydrogen Bonding
901
would still possess stabilisin g stackin g in teraction s, but th e added h ydrogen
bon din g fun ction ality m ay alter its un iversal base properties. Th is m igh t, in
turn , m ake such an an alogue a better substrate for polym erase recogn ition .
O
H
O
RHN
NO2
NO2
NH
NH2
N
N
N
N
HO
HO
HO
HO
O
O
O
O
OH
OH
OH
OH
2
4
5
R = H
R = Me
3
1
CHART 1
RESULTS
C3-substituted 5-n itroin dole was prepared by action of 5-n itroin dole 6 with
oxalyl ch loride yieldin g th e glyoxalyl ch loride derivative 7. Th is was con -
verted to th e C3-substituted carboxylic acid derivative 8 by first alkalin e
h ydrolysis followed by an oxidative decarboxylation with h ydrogen perox-
ide11. Th e resultan t 3-carboxylic acid was esterified by refluxin g in m eth a-
n ol in th e presen ce of sulfuric acid. Glycosylation usin g th e sam e m eth od
used for th e syn th esis of 5-n itroin dole n ucleoside 10 proceeded in greater
th an 70% yield, givin g on ly th e desired β-an om er of th e m eth yl ester.
Deprotection to th e free n ucleoside 11 was carried out by refluxin g in
m eth an ol, in th e presen ce of trieth ylam in e. For ch aracterisation , an d to
study th e con version to th e correspon din g am ide, portion s of th e ester 11
were treated with con cen trated am m on ia an d 40% aqueous m eth ylam in e
at 50 °C to gen erate th e am ide 4 an d th e N-m eth ylam ide 5, respectively
(Sch em e 1). It was observed th at th ese reaction s required lon ger th an 24 h
for com plete con version to th e correspon din g am ide, an d th erefore for
oligon ucleotide syn th esis a 48-h deprotection would be required. Fin ally,
con version to th e ph osph oram idite derivative 13 was carried out in th e
usual m an n er.
Th e ph osph oram idite was in corporated in to oligodeoxyn ucleotides usin g
stan dard ph osph oram idite DNA syn th esis ch em istry. Con version of th e
m eth yl 5-n itroin dole-3-carboxylate con tain in g oligon ucleotides to th e cor-
respon din g carboxam ide 4 was carried out in am m on ia, an d to th e N-
m eth ylcarboxam ide 5 usin g 40% aqueous m eth ylam in e. Oligon ucleotides
(ODNs) were th en purified by HPLC an d an alysed by MALDI-TOF m ass
spectrom etry. It was observed in m ass spectrom etry th at, as with 5-n itro-
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 6, pp. 899–911

902
Too, Brown, Holliger, Loakes:
in dole 3 con tain in g ODNs, th e m olecular ion often sh owed loss of oxygen
from th e n itro group.
In order to determ in e wh eth er th e 3-carboxam ide group substitution
on to 5-n itroin dole h ad an y effect on duplex stability, th erm al m eltin g (Tm )
studies were con ducted. Duplexes were prepared con tain in g 5-n itroin dole
3, th e 3-carboxam ide 4 an d th e 3-(N-m eth ylcarboxam ide) 5 derivatives.
Cl
O
O
OMe
O
OH
O2N
O
O2N
O2N
(d)
O2N
(b), (c)
(a)
N
H
N
H
N
H
N
H
9
6
7
8
(e)
O
N
MeO
O
O
H2N
MeO
NO2
NO2
NO2
(g)
(f)
N
HO
N
HO
O
TolO
O
O
OH
11
(i)
OH
OTol
4
(h)
10
Tol = 4-methylbenzoyl
O
MeHN
O
NO2
MeO
NO2
N
O
HO
MeO
N
O
DMTO
NO2
O
OH
N
(j)
OH
5
DMTO
O
O
12
O
P
CN
N
13
Reaction con dition s: (a) (COCl)2, Et2O, 0 °C, 24 h , 88 %. (b) KOH, H2O, 90 °C, 1 h . (c) H2O2
(30 % wt in H2O), reflux, 64 % from 7. (d) H2SO4, MeOH, reflux, 6 h , 67 %. (e) NaH,
3,5-di-O-p-toluoyl-2-deoxy-α-ribofuran osyl ch loride, MeCN, RT, 2 h , 73 %. (f) MeOH, NEt3,
reflux, 16 h , 90 %. (g) aq. NH3 (d = 0.88), 50 °C, 16 h , 46 %. (h ) MeNH2 (40 % aq. solution ),
50 °C, 16 h , 48 %. (i) Dim eth oxytrityl ch loride, pyridin e, RT, 87 %. (j) 2-Cyan oeth yl
N,N-diisopropylch loroph osph oram idite, N,N-Diisopropyleth ylam in e, DCM, RT, 1 h , 72 %.
SCHEME 1
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 6, pp. 899–911

Effect of a Hydrogen Bonding
903
Th e Tm s of th ese duplexes were m easured (Table I) an d from th ese th e
stackin g en ergy (∆H) calculated5. It can be seen th at each an alogue beh aved
as a un iversal base as expected, th ough th e effect of in corporation of a h y-
drogen bon din g group was n egligible. Th e 3-carboxam ide group 4 actually
h ad a sligh t destabilisin g effect com pared to 5-n itroin dole 3, an d th e 3-(N-
m eth ylcarboxam ide) 5 derivative caused a furth er sligh t destabilisation .
An alysis of th e stackin g en ergies of th e duplexes sh owed greater varia-
tion . 5-Nitroin dole sh owed a h igh er stackin g en ergy opposed to aden in e
an d lowest again st th ym in e. Th is could be explain ed by th e fact th at ade-
n in e h as a larger surface area th an th ym in e. However, th e carboxam ide de-
rivatives sh owed h igh er stackin g en ergies wh en opposed to pyrim idin es
th an purin es in con trast to th at of th e paren t 5-n itroin dole.
TABLE I
Meltin g tem peratures an d stackin g en ergy of duplexes con tain in g 5-n itroin dole 3 an d its
3-carboxam ide 4 an d 3-(N-m eth ylcarboxam ide) 5 derivatives
T
m , °C
–∆H, kJ/m ol
GTAAAACGA3GGCCAGT
CATTTTGCTGCCGGTCA
---------A-------
---------C-------
---------T-------
64
64
65
64
311.87
329.32
318.34
304.39
GTAAAACGA4GGCCAGT
CATTTTGCTGCCGGTCA
---------A-------
---------C-------
---------T-------
64
63
63
62
315.35
303.17
327.35
334.38
GTAAAACGA5GGCCAGT
CATTTTGCTGCCGGTCA
---------A-------
---------C-------
---------T-------
63
302.87
309.38
317.81
315.44
62.5
62.5
62
CGCGAAXTTCGCG
GCGCTTXAAGCGC
X = 5-n itroin dole
62
n da
n da
X = 5-n itroin dole-3-carboxam ide
63.5
a
n d = n ot determ in ed.
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 6, pp. 899–911

904
Too, Brown, Holliger, Loakes:
From th is in itial data it appears th at th e presen ce of th e h ydrogen bon d-
in g carboxam ide group h as a sligh t destabilisin g effect on duplex stability
com pared to 5-n itroin dole 3. In order to exam in e th e effect of self-pair 3
an d its carboxam ide derivative 4, each was in corporated in to a tridecam er
at th e cen tre of an oth erwise self-com plem en tary duplex (Table I). Un der
th ese con dition s it was observed th at th e 5-n itroin dolecarboxam ide self-
pair was sligh tly m ore stable th an th e 5-n itroin dole self-pair12
.
A furth er feature of un iversal bases th at we h ad observed is th at con tigu-
ous substitution s m ain tain duplex stability12, an d th e decrease in stability
due to th e loss of furth er h ydrogen bon din g is off-set by en h an ced stackin g
in teraction s. Th is was also observed for th e 3-carboxam ide derivative 4
(Table II) usin g a differen t sequen ce con text. However, th e 3-(N-m eth yl-
carboxam ide) 5 sh owed a sligh t decrease in duplex stability wh en two con -
tiguous substitution s are m ade, an d th is is reflected in a lowerin g of its
stackin g en ergy.
TABLE II
Effect of con tiguous substitution s on duplex stability
Tm , °C
–∆H, kJ/m ol
CATTTTGCTGCCGGTCA
GT3AAACGACGGCCAGT
--33-------------
--4--------------
--44-------------
--5--------------
--55-------------
72
72
71
70
71
69
326.64
339.07
318.20
323.15
330.00
314.16
Th e fin al sequen ce con texts th at we exam in ed are n ear-en d effects. Wh en
each of th e 5-n itroin dole an alogues was in corporated at th e en d of th e du-
plex, th ey still acted as a un iversal base with th e 3-(N-m eth ylcarboxam ide)
derivative 5 again sh owin g th e lowest stability. Stackin g en ergy of th ese
duplexes was n ot so clear, th ough th e 3-carboxam ide derivative 4 sh owed
greatest stackin g en ergy with cytosin e again , an d th e 3-(N-m eth ylcarbox-
am ide) derivative 5 with aden in e an d cytosin e (Table III).
Kool et al. h ave studied th e effect of dan glin g bases an d sh own th at a sin -
gle addition al n ucleotide at th e en d of a duplex aids stabilisation , described
as actin g as an en d-cap13. We studied th e effect of addin g th e 5-n itroin dole
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 6, pp. 899–911

Effect of a Hydrogen Bonding
905
derivatives as a dan glin g base an d th e 3-carboxam ide derivative sh owed th e
h igh est stabilisation , an d th is is reflected in th e h igh est stackin g en ergy
(Table IV).
TABLE III
Near-en d effects on duplex stability
Tm , °C
–∆H, kJ/m ol
GTAAAACGACGGCCAG3
CATTTTGCTGCCGGTCA
----------------T
----------------C
----------------G
75
375.32
408.17
447.49
450.40
74.5
75
75.5
GTAAAACGACGGCCAG4
CATTTTGCTGCCGGTCA
----------------T
----------------C
----------------G
75
406.54
378.54
439.34
413.54
76.5
75
76.5
GTAAAACGACGGCCAG5
CATTTTGCTGCCGGTCA
----------------T
----------------C
----------------G
74
446.56
373.19
403.06
320.78
74
72.5
74
TABLE IV
Effect of dan glin g bases on duplex stability
T
m , °C
–∆H, kJ/m ol
CATTTTGCTGCCGGTC
GTAAAACGACGGCCAG
---------------3
---------------4
---------------5
69.5
71.5
72.5
71
435.13
400.75
491.22
399.59
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 6, pp. 899–911

906
Too, Brown, Holliger, Loakes:
DISCUSSION
Gen erally, n on -h ydrogen bon din g un iversal bases cause som e duplex in sta-
bility, th ough larger residues (e.g. pyren e) can raise th e Tm above th at ob-
served for a fully n ative DNA duplex13. Th e in stability caused by such
un iversal bases is due to a n um ber of factors, th e m ajor on e bein g th e loss
of h ydrogen bon din g stabilisation , but oth er stabilisin g factors, such as sol-
vation , are in volved13. However, th ese losses are in part m ade up for by en -
h an ced stackin g in teraction s, an d th is is gen erally proportion al to th e
surface area of th e un iversal base. We were in terested to kn ow wh at would
be th e effect of in troducin g a h ydrogen bon din g group on to a un iversal
base, to kn ow wh eth er it could lead to furth er duplex stability. We ch ose
th e carboxam ide group as th ere h ave been m an y azolecarboxam ides stud-
ied as poten tial un iversal bases14. Th e presen ce of th e carboxam ide group
can , in th eory, presen t two altern ative h ydrogen -bon din g faces allowin g
base pairin g to each of th e n ative DNA/RNA n ucleosides.
In th e presen t study we decided to in troduce a carboxam ide group on to
th e pyrrole rin g of 5-n itroin dole. Klewer et al. h ave sh own by NMR th at th e
n itro group of th e un iversal base 3-n itropyrrole protrudes in to th e m ajor
groove, away from th e h ydrogen bon din g face8. If th e 5-n itroin dole residue
adopts a sim ilar con form ation , th en th e n itro group would protrude in to
th e m ajor groove leavin g th e n ew carboxam ide group in th e duplex h ydro-
gen bon din g face. In such a con form ation th e carboxam ide group m igh t
give addition al stability by form in g h ydrogen bon ds to th e n ative base in
th e com plem en tary stran d. Th e altern ative con form ation would direct th e
carboxam ide group in to th e m ajor groove leavin g th e n itro group with in
th e h ydrogen bon din g region . Due to th e in creased size of th e in dole deriv-
atives, com pared to n ative bases, we assum e th at th e in dole bases would in -
tercalate in to th e opposin g stran d. Th e N-m eth ylcarboxam ide derivative 5
was prepared as th e m eth yl group m ay alter th is n ew in teraction by in tro-
ducin g steric bulk wh ich m igh t in terfere with an y h ydrogen bon din g in ter-
action . It was an ticipated th at th e experim en ts described in th is paper
m igh t lead us to in fer wh ich con form ation th e in dole base adopts with in a
duplex.
Meltin g tem perature studies of th e two 5-n itroin dole-3-carboxam ide de-
rivatives dem on strated th at th ey still retain ed un iversal base h ybridisation
features. Com pared to 5-n itroin dole itself, h owever, th ey were sligh tly de-
stabilisin g in m ost of th e sequen ce con texts exam in ed. Th e N-m eth yl-
carboxam ide group in 5 sh owed lower stability th an th e carboxam ide
group in 4, an d th is m ay th en be due to steric effects rath er th an reducin g
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 6, pp. 899–911

Effect of a Hydrogen Bonding
907
h ydrogen bon din g capability of th e an alogue. An alysis of th e m eltin g data
provides furth er in form ation about stackin g in teraction s. As n oted above,
5-n itroin dole exh ibits greatest stackin g en ergy wh en opposed to aden in e,
presum ably as a result of en h an ced stackin g in teraction s with it. However,
th e two carboxam ide derivatives sh ow en h an ced stackin g in teraction s
wh en opposed to a pyrim idin e. Th is suggests th at th e carboxam ide group is
n ot in volved in th e h ydrogen bon din g face an d m ay be protrudin g in to th e
m ajor groove of th e duplex, with th e n itro group directed towards th e com -
plem en tary stran d, an d en d-cap experim en ts, wh ere in tercalation can n ot
be in volved, supports th is con clusion .
On e of th e requirem en ts for a un iversal base is th at it sh ould be able to
form “base pairs” with each of th e n atural bases in polym erase reaction s.
Th is would allow th e syn th esis of libraries of oligon ucleotides from a sin gle
un iversal base-con tain in g oligon ucleotide12. 5-Nitroin dole can beh ave as a
un iversal base by addin g, as its 5′-triph osph ate, opposite each of th e n atu-
ral tem plate bases, an d can direct th e in corporation of all four n atural
dNTPs wh en in a tem plate. However, polym erase reaction s in volvin g
5-n itroin dole are very in efficien t, an d it usually acts as a term in ator on ce
in corporation h as occurred. Work is un derway to determ in e wh eth er th e
3-carboxam ide derivative is a better polym erase substrate, an d will be re-
ported in due course.
EXPERIMENTAL
1H an d 31P NMR spectra were obtain ed on a Bruker DRX 300 spectrom eter, an d spectra were
obtain ed in DMSO-d6, un less oth erwise stated. 31P NMR spectra are extern ally referen ced
to ph osph oric acid. Ch em ical sh ifts are given in ppm (δ-scale), couplin g con stan ts (J) in Hz.
Ultraviolet spectra (λ in n m ) were recorded on a Perkin Elm er Lam bda 2 or Lam bda 40
spectroph otom eter dissolved in 10% aqueous m eth an ol un less oth erwise stated. Meltin g
poin ts were m easured with a Stuart Scien tific SMP3 m eltin g poin t apparatus an d are un cor-
rected. Mass spectra were recorded on a Bruker FTICR Bioapex II. TLC was carried out on
pre-coated F254 silica plates an d colum n ch rom atograph y with Merck kieselgel 60. Un less
oth erwise stated, reaction s were worked up as follows: th e solven t was rem oved by evapora-
tion an d th e product dissolved in ch loroform an d wash ed with saturated aqueous sodium
h ydrogen carbon ate. Th e ch loroform fraction s were dried over an h ydrous sodium sulfate, fil-
tered an d evaporated to dryn ess. Oligon ucleotides were syn th esised on an Applied Biosystem
380B syn th esiser with th e n orm al syn th esis cycle. 5-Nitroin dole ph osph oram idite was pur-
ch ased from Glen Research . 5-Nitroin dole-3-carboxam ide n ucleotides were prepared by de-
protectin g with am m on ia (d = 0.91) at 80 °C for 2 days. N-Meth ylcarboxam ide oligon ucleo-
tides were prepared by deprotection with 40% aqueous m eth ylam in e at 80 °C for 2 days.
Oligon ucleotides were purified by HPLC on a Dion ex Nucleopac PA100 ion exch an ge col-
um n usin g a gradien t of 1 to 400 m M sodium perch lorate con tain in g 0.02 M Tris (pH 6.8)
an d 25% aqueous form am ide, followed by dialysis an d con cen tration . MALDI-TOF m ass
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 6, pp. 899–911

908
Too, Brown, Holliger, Loakes:
spectroscopy was carried out on a Hewlett–Packard G205A spectrom eter usin g n egative po-
larity, in a m atrix of 2-cyan o-3-(4-h ydroxyph en yl)propen oic acid in aceton itrile/water (1:1)
with 3% trifluoroacetic acid.
5-Nitroin dole-3-carboxylic Acid (8)11
To a solution of 5-n itroin dole (3 g, 18.5 m m ol) in eth er (100 cm 3) at 0 °C, oxalyl ch loride
(8 cm 3, 92 m m ol) was added dropwise, an d stirrin g con tin ued at 0 °C for 24 h . Th e solid
was filtered an d dried. Yield 4.1 g (88%). Th e acid ch loride 7 was suspen ded in water
(100 cm 3) an d potassium h ydroxide (1.1 g, 19.6 m m ol) added an d th e solution h eated at
90 °C for 1 h . Th e solution was cooled, acidified an d th e yellow solid filtered. Th e solid was
resuspen ded in h ydrogen peroxide solution (30%, 50 cm 3) an d th e solution h eated at reflux
for 3 h . After coolin g th e solid was filtered, dried an d recrystallised from aqueous eth an ol to
give a green ish -yellow solid. Yield 2.45 g (64%), m .p. 285–287 °C. 1H NMR: 7.64 (1 H, d, J =
9, H-7), 8.07 (1 H, dd, J1 = 9, J2 = 2.2, H-6), 8.27 (1 H, s, H-2), 8.88 (1 H, d, J = 2.2, H-4),
12.44 (2 H, br s, NH, CO2H).
Meth yl-5-n itroin dole-3-carboxylate (9)
To a solution of 5-n itroin dole-3-carboxylic acid (2.45 g, 12 m m ol) in m eth an ol (50 cm 3),
sulfuric acid (1 cm 3) was added an d th e solution stirred at reflux for 6 h . Th e solution was
allowed to cool an d th en poured on to ice-water, n eutralised with sodium h ydrogen -
carbon ate an d th e product filtered to give a yellow solid, wh ich was recrystallised from
m eth an ol as an off-wh ite solid. Yield 1.76 g (67%), m .p. 282–284 °C. 1H NMR: 3.85 (3 H, s,
OCH3), 7.65 (1 H, d, J = 9, H-7), 8.08 (1 H, dd, J1 = 9, J2 = 2.3, H-6), 8.35 (1 H, s, H-2), 8.83
(1 H, d, J = 2.3, H-4), 8.5 (1 H, s, NH). UV: λm ax 320 (15 000), 251 (32 450), λm in 284, 213;
pH 12 λm ax 366 (13 100), 271 (31 500), 213 (48 100). MS, m/z: 243.1 (M + Na)+. Accurate
m ass m easurem en t on (M + Na)+ C10H8N2NaO4 243.0396, deviation –5.73 ppm .
Meth yl 1-[2-Deoxy-3,5-bis(4-m eth ylben zoyl)-β-D-ribofuran osyl]-
5-n itroin dole-3-carboxylate (10)
To a solution of m eth yl 5-n itroin dole-3-carboxylate (1.22 g, 5.5 m m ol) in aceton itrile
(50 cm 3), sodium h ydride (60%, 0.27 g, 7 m m ol) was added an d th e solution stirred at room
tem perature for 30 m in . To th is, 3,5-di-O-p-toluoyl-2-deoxy-α-ribofuran osyl ch loride (2.6 g,
6.7 m m ol) was th en added an d stirrin g con tin ued for 2 h . Th e solven t was evaporated an d
th e product worked up to give a brown foam , wh ich was ch rom atograph ed (CH2Cl2/0–2%
MeOH) to give a pale yellow foam . Yield 2.25 g (73%). 1H NMR: 2.36 (3 H, s, toluoyl-CH3),
2.40 (3 H, s, toluoyl-CH3), 2.80–2.87 (1 H, m , H-2′), 2.99–3.08 (1 H, m , H-2′′), 3.82 (3 H, s,
OCH3), 4.44–4.67 (3 H, m , H-4′, H-5′, H-5′′), 5.71–5.73 (1 H, m , H-3′), 6.72 (1 H, t, J = 6.7,
H-1′), 7.26–7.39 (4 H, m , toluoyl-CH), 7.78–8.03 (6 H, m , 4 × toluoyl-CH, H-7, H-6), 8.57
(1 H, s, H-2), 8.81 (1 H, d, J = 1.9, H-4). UV: λm ax 316 (9600), 246 (32 300), λm in 302, 222;
pH 1 λm ax 274 (28 400), 253 (29 800), λm in 268, 225; pH 12 λm ax 316 (13 700), 229 (34 900),
λm in 306, 232, 209. MS, m/z: 595.1 (M
31H28N2NaO9 595.16750, deviation –2.99 ppm .
+
Na)+. Accurate m ass m easurem en t on
C
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Effect of a Hydrogen Bonding
909
Meth yl 1-(2-Deoxy-β-D-ribofuran osyl)-5-n itroin dole-3-carboxylate (11)
A solution of m eth yl 1-(2-deoxy-3,5-bis(4-m eth ylben zoyl)-β-D-ribofuran osyl)-5-n itroin dole-
3-carboxylate (2.3 g, 4 m m ol) in m eth an ol (100 cm 3) con tain in g trieth ylam in e (5 cm 3) was
h eated at reflux overn igh t. Th e solution was evaporated an d th e product ch rom atograph ed
(CH2Cl2/0–5% MeOH) to give a pale yellow solid, wh ich recrystallised from eth an ol to give
off-wh ite n eedles. Yield 1.22 g (90%), m .p. 162–164 °C. For C15H16N2O7 (336.1) calculated:
53.57% C, 4.80% H, 8.33% N; foun d: 53.41% C, 4.83% H, 8.27% N. 1H NMR: 2.31–2.39
(1 H, m , H-2′), 2.48–2.56 (1 H, m , H-2′′), 3.51–3.63 (2 H, m , H-5′, H-5′′), 3.30–3.38 (1 H, m ,
H-4′), 3.33 (3 H, s, CH3), 4.35–4.40 (1 H, m , H-3′), 5.04 (1 H, t, J = 5.1, 5′-OH), 5.36 (1 H, d,
J = 4.1, 3′-OH), 6.50 (1 H, t, J = 6.2, H-1′), 7.96 (1 H, d, J = 9.2, H-7), 8.13 (1 H, dd, J1 = 9.1,
J2 = 2.3, H-6), 8.62 (1 H, s, H-2), 8.84 (1 H, d, J = 2.2, H-4). 13C NMR: 163.7, 142.9, 138.8,
135.5, 125.7, 118.1, 117.0, 112.5, 108.7, 87.9, 85.8, 70.4, 61.3, 51.4, 40.3. UV: λm ax 318
(9100), 266 (24 300), λm in 290, 216. MS, m/z: 359.1 (M + Na)+. Accurate m ass m easurem en t
on C15H16N2NaO7 359.08480, deviation –2.09 ppm .
1-(2-Deoxy-β-D-ribofuran osyl)-5-n itroin dole-3-carboxam ide (4)
A solution of th e above ester (300 m g, 0.89 m m ol) in am m on ia solution (d = 0.91; 10 cm 3)
was stirred at 50 °C overn igh t. Th e solution was evaporated an d th e product crystallised
from eth an ol to give a pale green solid. Yield 133 m g (46%), m .p. 220–222 °C. For
C
15H16N3O7 (350.3) calculated: 52.34% C, 4.71% H, 13.08% N; foun d: 52.15% C, 4.75% H,
12.87% N. 1H NMR: 2.34–2.49 (2 H, m , H-2′, H-2′′), 3.46–3.59 (2 H, m , H-5′, H-5′′),
3.85–3.89 (1 H, m , H-4′), 4.39 (1 H, br s, H-3′), 4.89 (1 H, t, J = 5.3, 5′-OH), 5.38 (1 H, d, J =
4.2, 3′-OH), 6.47 (1 H, t, H-1′), 7.16, 7.70 (2 × br s, CONH2), 7.86 (1 H, d, J = 9, H-7), 8.08
(1 H, dd, J1 = 9, J2 = 2, H-6), 8.48 (1 H, s, H-2), 9.06 (1 H, d, J = 2, H-4). 13C NMR: 165.2,
142.3, 139.0, 131.2, 126.5, 118.1, 117.7, 112.8, 111.7, 87.7, 85.0, 70.5, 61.8, 40.2. UV: λm ax
320 (9700), 267 (24 600), λm in 291, 223. ε260 (µm ol/l) = 22.7. MS, m/z: 344.1 (M + Na)+.
Accurate m ass m easurem en t on C14H15N3NaO6 344.08470, deviation –3.45 ppm .
1-(2-Deoxy-β-D-ribofuran osyl)-N-m eth yl-5-n itroin dole-3-carboxam ide (5)
A solution of m eth yl ester (200 m g, 0.59 m m ol) in 40% aqueous m eth ylam in e solution
(10 cm 3) was stirred at 50 °C overn igh t. Th e solution was evaporated an d th e product crys-
tallised from eth an ol to give a yellow solid. Yield 96 m g (48%), m .p. 248–250 °C. For
C15H17N3O7·0.5H2O (359.3) calculated: 52.32% C, 5.26% H, 12.20% N; foun d: 52.51% C,
5.07% H, 12.03% N. 1H NMR: 2.33–2.46 (2 H, m , H-2′, H-2′′), 2.79 (3 H, d, J = 4.4, NHCH3),
3.46–3.57 (2 H, m , H-5′, H-5′′), 3.85–3.89 (1 H, m , H-4′), 4.39 (1 H, br s, H-3′), 4.91 (1 H,
br s, 5′-OH), 5.39 (1 H, d, J = 3, 3′-OH), 6.47 (1 H, t, J = 6.5, H-1′), 7.86 (1 H, d, J = 9, H-7),
8.08 (1 H, dd, J1 = 9.1, J2 = 2.2, H-6), 8.19 (1 H, br, NH), 8.41 (1 H, s, H-2), 9.06 (1 H, d,
J = 2, H-4). 13C NMR: 163.7, 142.3, 138.8, 130.3, 126.3, 118.0, 117.7, 113.1, 111.7, 87.7,
85.0, 70.5, 61.8, 40.2, 25.6. UV: λm ax 321 (8300), 267 (20 700), 203 (25 800), λm in 291, 222.
ε260 (µm ol/l)
15H17N3NaO6 358.10120, deviation –0.94 ppm .
= 19.2. MS, m/z: 358.1 (M +
Na)+. Accurate m ass m easurem en t on
C
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910
Too, Brown, Holliger, Loakes:
Meth yl 1-[2-Deoxy-5-(4,4′-dim eth oxytrityl)-β-D-ribofuran osyl]-
5-n itroin dole-3-carboxylate (12)
To a solution of th e above ester (0.86 g, 2.55 m m ol) in pyridin e (25 cm 3), 4,4′-dim eth oxy-
trityl ch loride (0.95 g, 2.8 m m ol) was added an d th e solution stirred at room tem perature
overn igh t. Furth er portion s of dim eth oxytrityl ch loride were th en added un til th e reaction
was com plete. Th e solution was evaporated an d th e reaction worked up an d ch rom atog-
raph ed (CH2Cl2/1% MeOH) to give a yellow foam . Yield 1.42 g (87%). 1H NMR: 2.43–2.49
(1 H, m , H-2′), 2.73–2.77 (1 H, m , H-2′′), 3.31 (2 H, br s, H-5′, H-5′′), 3.69 (6 H, s, 2 ×
OCH3), 3.80 (3 H, s, CO2CH3), 3.99–4.01 (1 H, m , H-4′), 4.47–4.51 (1 H, m , H-3′), 5.43 (1 H,
d, J = 4.6, 3′-OH), 6.53 (1 H, t, J = 5.9, H-1′), 6.73–6.84, 7.13–7.26 (13 H, m , Ar-H), 7.97
(1 H, d, J = 9.1, H-7), 8.07 (1 H, dd, J1 = 9.1, J2 = 2, H-6), 8.49 (1 H, s, H-2), 8.85 (1 H, d, J =
1.9, H-4). UV: λm ax 318 (6600), 265 (22 800), 237 (34 400), λm in 298, 228. MS, m/z: 661.2
(M + Na)+. Accurate m ass m easurem en t on C36H34N2NaO9 661.21560, deviation –0.95 ppm .
Meth yl-1-(3-O-{[(2-cyan oeth oxy)(diisopropylam in o)ph osph on yl]oxy}-
2-deoxy-5-O-(4,4′-dim eth oxytrityl)-β-D-ribofuran osyl)-5-n itroin dole-3-carboxylate (13)
To a solution of th e dim eth oxytritylated n ucleoside (0.6 g, 0.9 m m ol) in dich lorom eth an e
(10 cm 3), eth yl(diisopropyl)am in e (0.49 cm 3, 2.8 m m ol) followed by 2-cyan oeth yl N,N-diiso-
propylph osph oram idoch loridite (0.31 cm 3, 1.39 m m ol) was added, an d th e solution stirred
at room tem perature for 1 h . Th e reaction was quen ch ed with m eth an ol (100 µl), an d th e so-
lution diluted with eth yl acetate (100 cm 3) an d th en wash ed (5% sodium carbon ate solution
(2 × 25 cm 3) followed by saturated brin e (2 × 25 cm 3)), dried an d evaporated to a gum . Th is
was ch rom atograph ed (eth yl acetate/h exan e/trieth ylam in e 10:10:1) to give an off-wh ite
foam wh ich was th en precipitated from an ice-cold solution of h exan e. Yield 0.57 g (72%).
31P NMR: 146.95, 147.59.
Th erm al Dissociation (Tm ) Measurem en ts
Ultraviolet spectra were recorded on a Perkin Elm er Lam bda 2 or Lam bda 40 spectroph oto-
m eter fitted with a Peltier cell. Meltin g tran sition s were m easured at 260 n m in 6 × SSC
(0.9 M sodium ch loride, 0.09 M sodium citrate, pH 7) at an oligom er stran d con cen tration of
approxim ately 2 µm ol/l. Absorban ce versus tem perature for each duplex was obtain ed at a
h eatin g an d coolin g rate of 0.5 °C/m in , an d m eltin g tem peratures (Tm ) were determ in ed as
th e m axim a, or m in im a, of th e differen tial curves, with an error of ±0.5 °C.
We wish to acknowledge the use of the EPSRC’s Chemical Database Service at Daresbury.
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