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Conformation of bilirubin amides from circular dichroism spectroscopy
OF BILIRUBI~ AMIDES FEOX CIECULM
ASTIR
DICHEOISh SPEClROSCOPY
DAVID A. LICK*
and HAN-tEM WANG
istry. Rsno,
lewda
of
Univsrsfty
89557-0020
Nevada
USA
The first optically active umider of bilirubln-IXa with amino Abwtract. acids lhow bisi%n@ze circular dichtoism Coeton effects (CEs) ln the vicinity of the pigment’s long uwelength W-visible absorption, e.g.. for the bisamide utth L-alurine methyl eeter in acetonttrtie A#:$! - -47, Aa:;: - +68 the dioster from S-f-)-tactic acid In marked contrast, and e:$; - 33,800. - -2.6 and e;;: - 32.800 in acetoethyl eeter exhibits AC::: - +l.O, At;;; The CEs of the mono-amides are generally raaller than those of the nftrile. are generaIly larger rh#n those bfs-amlde6: whereas, the CEs of mono-esters of the dlestsrs.
The yellow-oxnn.Be, duced abundantly d&ion
and
connection
phototherapy The latter
carboxylic
bonding tion
(Fig.
acid
have
has
neonatal been
and lactam
or C,, -C,,
ptgments
that
Jaundice long
been
attributed
of
to
a 3.dimsnsionrl linked
which involves,
fnter
double
without
alla,
is
pro-
glucuronirecently
in
its unusal hydrophobic
structurs3s6
through
reeort
for
especially of
In which
fntrrmolecular
the
hydrogen
configurational
the aseociatcd
bonds,
by the liver
(BR-IX),
the liver
because
are
carbon-carbon
to
inttrcst,2
and also
jaundice3
functionalitfes
can be excreted
(42,152)-billrubin-IXo
of hame and transported
structure for
And in phototherapy,
1).7
at the C, -C,
iaomeric
pi&ment of
by catebolisn
Its
excretion.1 with
propertiea.4 polar
cytoeoxic
in maarrls
isomerfr~-
H-bond breekln%
yields
to conju&ation.1*3
NWRE
1. Interconvsrtin&, folded and intramolecularly hydro&enbonded conformational lnantiomers Conof (42,132)-bilirubin-EXa, former X has the negative chirrlity orientation of the pyrromethonone 1onB wavelength electric transition dipolan. Conformer I, has the posttive chfraIfty orientation. (See ref. I2a)
(p)
(M) BR-IX can fold SiOI-XS
of
8
-18
receric
solutions
md
into
either
are stabilized
kcal/mole
And a rata
nixturo
of
are
optlcelly
as e*rum aIbumfnlD*ll
of
NO enantiosorlc
by intramolecular of
7.2
conformational
f0.4
sec‘i
enantiowrs,
inactive;
howavor,
and optically
active
conformationsI~S
H-bonds
yet
that
fnterconvert
(-38*C).5a*b*9 I( ? P (Fig.
in the presence
As such, 1). of
a chiral
static
ropul-
an activation it
In Isotropic
aaims, 12*13 the sotutions 4679
mfnirite
with
ten be viewed agent,
circular
le
e
BR-IX
rolvents.
comptcxin& exhibit
energy
euch
dichro-
4480
D. A. LIGHINEIR and M.-H.
1x9
(CD) for
origin
the
the
of
lnducad
in the comploxos Although
lxuplas
there
of
A#:;;
t9.9
are
*R-IX -
for
its
CD
In the currant (ax unxeparatcd
distal
(BR-IX
BR-IX
tha
of
and In the C-pigment
Synthds
ity
and covalently
alcohol
procaads
of the starting Cfrcular CD Cotton
effects
md
ln Tablax
Tables
xolvmtx
moLcculcs
vlth
intans* pl.x.*IO*IL folded,
lxupla
methyl
the
to
lxter
(BR-IX ME)
and S-
BR-IX BAA.
pigment
ix
optically
glucuronldas
(*star)
or uldc).
by coupling as outlined
the chlral
amino to BR-IX
earliar, for
except
plgmants
dlmcthyl-
vhosa
solubil-
from the p-toluoncsulfonata
conflguratlon
of
tha alcohol
the R-configuration
of ethyl sa1t.24*27
component;
and not
lo.
tha
tha S-conflguratlon
(1)
dilution and
large
blsignate and ulna
chlral t1.3
mochanlxn
and large (x-x*)
CEs of
transltlon
CDs are
har llttla not
(R).a
alxo
other
aolvcnts
that
Chs,
but
The bir-ml&s
associated
tend
with
tha orlgln
blxlgnato the pigment
for
the amino
of
amides
of
A<:;; the optfcal are
of
Flg.
only
solvcntx lxhlbit
to
-
for
dialactrlc
la-got-
lnherantly
CEx than from an
-1.3
for
lctlvlty.
the monomer in form).?’
In fact, of
having
3-dlmnrional
both
blchromophorlc llttla
inter-
xtructure.
hot~roarxoclation
to asxuma a moda-•tely The tendency
lymat-
pyrromothonono
H-bonded dlmcrs
xtructuras
1.
lmarga
as H-recapIn H-bonding
actlvo
charact*rlstlc
blllrubln-protein
1s thought
thoxc
can sarva
veakar,
points
salf-assochtfon.
to the plycnt’s
characterlstlc
whare the pigment
lnteraxtlng
on the CD, ax expectad
moments and veil-defined
of
of
lntanso
surprisingly large 28 optically e.g.,
phenethylulde:
the alanyl
akln to llthar
lxhlblt
than tha CEa in lov
In CHCL, where lntarmolecularly for
vork
absorption
H-&nor
or no affect
They are a clue
o~crlap.~’ xalts,12
reduced
perturbation,
acid -
At::;
this qulta
Saveral
to or even larger
1 would bo considcrod
ramota
conformation
opposlta.
(2)
on.
of W-v18
ara ganarally
Hlgh dlclcctric
and &Cl,).
a differant
vavalongth
llkyl amino CE magnitudes are gcnarally
CEs, comparable
Tablo
of
long
xhow signlflcantly
C,H,
-3.1,
p-orbital
chiral
eater
on tha
lxpacted
are
and diextorr:
as in bilirubln
(ester
argu-
tha 4-xubstltuantx
athyl
The amide darlvatiwx the
tha CEs lx
(xanthoblllrublc
nature
chromophorlc
acid
and
tha mono--l&s
BR-IX bix-tetra-n-butylmroniua
The CE intcnsltla
tha molecule
of
-
xuggast
of
large
vith
and amides
Ila,SO and AaTff tha bislgnate
of have
tha corraxpondlng
Hotwaver.
The CE magnItudea
chromophoros
near
1 and 2:
(cf.
proporty
The data
for
(!la,SO)
do tha mono-amidox.
asters
otters
t6.
-2.4.
tha ltaroochamlcal
S- (+)-alanina
wax more Bultabla using
-
-
mada from S-(+)-2-
and L2-ixomerx
blr-amides
were prepared
BR-IX
Actif
Act;;
CD of
and
of
tha 8.
propared
2-butanol
Cmformtlon.
progression
H-bonding
lntrlnslc
of
1 and 2).
(t4aOH) shov unoxpoctadly non-polar
Vera
The esters
cantared
whoraax,
xlgnad
in
BR-IX fro=
proccduro 25)
an SN2 inverxlon
(CEx)
from tha data of tars
vork
(HSA),
t3.1.
The
(2)
alcohol.
derlvatfvo;
and tha
of
bound HSA-bilirubin
the pigment
Dlcbzof~
(xuuarfxcd acid
via
of
or&ring
group,
-
howover,
In lach
a chlral
darlvatlws.
been raportcd;
BR-IX estcrx
the
S-(-)-lactic
This modification
lx low.
componentx
of
(Shloirl
and the mcthancsulfonata
Tha reaction
in
and a fw
cyclodaxtrlrul’
BR-TX with N-•catyl-L-cyxtalne
on the preparation
respactlvaly.
this
wax usod ax solvent.
in dlmathylfo-lda
lactate
rlc
of axIda
of
and (5)
as the corresponding
attachment
blllrubln
At;;;
Iha
tha plgmont.
BR-IX10*15
to human serum albumlnx
adductx
YC report
CD of
haw
tha 6-plgment.20
rcportad;23
(BR-IX NM).
The uidos
.
active
450 IN.
of I( and P apaclw of
amlncs. 12.13.16
no CD xpactra
and magnitudes)
DBE,
induce
actfvo
prwlously,12*17~18a
as well
dlphenylphoxphoryl
to
C-8 and C-12 laomars;
of
CD signs
remote
populations
group, (3)
near
transformation14
optically
on differencea
BR-IX DLE and BR-IX
by virtue
sulfoxfde
&pend
ME).
of vhlch
acid
yet
in non-equal swving
tha uo-vinyl
mixturax
sm.
(BR-IX
BBA.
using
(4)
ra not
l
S-(+)-2-butylamtna
MM),
(+)-2-butanol active
propionic
absorption
optically
no rxamplrs
and ax noted
rings.
caxcntlally
with for
W-visible
asyrotrlc
of proteins
Investigation
work do not
lactu
glw
order
at pH 4.5-7.5;lg
which
and eaters
thlx
almort
r3
butano1.24 ments of
can ba xa.n
flrxt
l
cxuplas
buffar;21
pH 7.3
glutathlonc.22
wavelength
glucuronl&s.l
through
Ae:ft,
in
activity
about by uny
thare
covalontly m -0.6.
long
CD coming from BR-IX
(1)
include:
linked
optical
are
lnducod
A,--= .,,
blllrubln
brought
and rni~~ll~x,~* last
lntwua
ZHANG
CO.-
wall-defined
of BR-IX to adopt
1. Circular Dichrofum (CD) and Ultraviolet-audible (W) SpectralData for 1.0-1.6x lo-' tlSolution8of Billrubin Mono- and Bis-Amides Fornad froa S-(+)-Alanine Hethyl Uteri
%bie
W
CD Amide
Solvents
423
-11.9 (401) -19.9 (395)
49.000 56.600
14469 (443)
423 623
+17.5 (448) +34.7 (444)
49,000 60,000
(446) (442)
-33.3 (403) -49.5 (395)
423 427
+49.7 (451) +8L.9 (645)
46.500 59,000
(440) (442)
-26.5 (402) -54.3 (395)
421 415
GO.7 (650) i83.8 (442)
SO.0~ 38,600
(438) (437)
MeOH
-13.6 (401) -41.4 (403)
423 623
+24.0 (452) 60.2 (452)
46,500 57,100
(446) (443)
n4,
* 2.4 (405) * 2.3 (399)
424 417
+ 6.0 (458) +x1.1 (452)
50,200 38,900
(453) (4%)
4i&N
Df HOZtO
425
+27.5 (452) G7.9 (450)
Di UOnO
-20.2 (402) -40.0 (401)
(A* I=)
Dl EfO!%O
hr..,(&)
c&t
e_
ns, co
t4OllO
AZ at At-0
Aa_
so
Di
(refo. 25,26) of 3R-IX with L-aIenine methyl ester. Spectra V~(I run at 21-C within 0.5 hre. of solutionpreparationusing the correspondingamides of D.L-alaninamethyl aster as the (Ar - 0) baseline. h C,% - benzene, CH,Cl, - diehloroaathme, Hs,CO - acetone, HeCN - acetonicriIe, HezSO dI~tbyl~lfox~d~. P At 2.5 x 10m6 !f the va‘luerare: (Hono) Aa:;; - -32.8,A@:;; - +42.1, Ae - 0 at 427 M, (:I; - 33,800; (Di) A#:;;,- -44.8,A<:;: - t66.0,Ar - 0 At 421 im <:$I,- 52.000. A Prepared by Shioiri coupling
Irbl8 2. CircularDlchroisa (CD) and Ultraviolet-Visible (W) SpectralData for 1.2-2.5x 10" ffSoluclonrof Bilirubin-IXaMono. and Bir-AmLdesLof S-(+)-2-Butyllains at 21-C.
CEH,
+ 2.0 (415) + 1.2 (405)
439 424
* 1.0 (470) -16.3 (455)
66,900 61,200
(453) (453)
CH,Cl,
+ 2.9 (410) + 3.1 (410)
429 427
- 7.0 (460) - 8.8 (460)
57,100 63,700
(448) (448)
non0 Df
Hc,CO
‘ 0.1 + 5.1 (405)
*._
- 2.6 (460)
426
-12.4 (455)
64,000 55,900
(4439 (440)
Non0 Di
l4tCN
. 0.1 + 6.2 (405)
__* 420
. 0.1 -14.7 (455)
55,600 59.500
(445) (435)
- 7.6 (463) -27.3 (455)
50,900 32,600
(447) (446)
- 0.9 (465) - 3.4 (460)
65,600 52.300
(433) (450)
Mono
Di lfOM,
D1
Hono SDi
MeOH
Kono
i-fezso
835
+ 5.7 (tlsr +13.6 (405)
423
. 0.1
Di
mm.
430
+ 2.0 (410)
L Preparedby the procedureof Shioirl et al.
(rat-s. 25,26). Spectrawere run within 0.5 hrs. of solution preparation uefng the correeponding &aider of rmxmic 2-uinobutene aa th* (A( 0) bemlfns.
k &H+ - banzone, C%Cla - dichloroatthana,Ke,CO a acetone. M&N - acetonitrils,Na,SO diwthylsulfoxlde. k With added urea (0.18n), the CD veluas axe reducedto A?,,, - *I2.7 (410)sA< - 0 et 430 ma, c - 51,300 (446 ma). A%., - -22.9 (457).W:
e folded conformation II governed by factor8
1% sharply lowers the eonfo~t~o~l Even
partial,
H-bonding
propionic ecid
cm
otbr
thm
lntrmolacular
H-bondfng.8 but H-bond-
global enot~ minima, adding a powerful strbilfaatton.
be beneflelel,
grcupe are deprotonated,
u
es with mono-estsr~~~~ and urfd.8, 26 or when the in the bis-i~opropylamoniua32and him-tetra-n-
lIkylunonium rrlta.33 So vhen ths anidea of M-IX are formed from prfaary mains&, on* ml&t view conforutlonal stabilizationas taking phc4 throughl proptonufde N-H @'lg. 2) in place of the propionic ectd O-H (Ffg. 1).
Since the emida N-H hydrogen is lees acidic tlun the acid
O-H, the effectiveness of H-bonding fr probably IeBeened, but such secondary eaide derivatives
of
ZING
D. A. LIOHTN~~ and M.-H.
4682
bllirubln
aster*
offor
constdorably
l
or tortiaq
groxear
potential
for
stabilixlng
conformations
than
do
amides.
FTWRG 2. Interconverting, intramolecularly hydrogen-bonded lnantlomeric conforutlons of BRIX (left) bls-ribs fron 1’ amino* and (right) mono-•atar#. (Only th* c, mono-•stor is Vhan tha R group containa a chlral center, ahown. ) as in the amidea and astars of this work.
the interconvarting l tructurex tfonr appear to bo onorgy-miniThe mono- and bls-amidax
pairs
of interconverting
rho
two pyrromthenona
the
concentration
reamer
a
ara
rough
the M’ + P’
uidsx
(Tabla
amides
tha
stronger
1)
CEs probably
to
of
the
eaters
sponding
exhibit
larger It
dfaxtaroomcr factors
the
CEx of
Hera and with
of
configuration
that
one diastarsomsr
Although
acrier
(S),
large
tha alanine through
and tha amino acid,
amide CD‘.35
that
the alanina
guy be noted
srter.34
an
in diaxta-
a pure
However,
unclear.
the pyrroaathanona
lbrolute
propionic
vavclength goncrxlly
(Table
absorption
whatawr
1 vs.
Table
Importance
In fact
4).
also
acid
a
akin to that
the amidax of Table6 the
inversion
a
3).
But unlike
carboethoxy
the axtar
ester
th*
data
of
or
(Fig.
of
the
that
carbomothoxy
acid
Tablax
esters,
CD CEs for
mono-uide,
do not
hero
And like
bisignate
the L-alanyl
carbomethoxy
lactic
H-bonding,
2).27*31
exhibits
in aolvsnts
the amino acid’s
to tha CDs of
intramolecular
group
ethyl
to be Iargcrt
influonca 2),
acid
acid
(Table
tonding
partial
retain
carboxylic
from L-lactic
weaker,
tha
his-amides, Although
*stars.
do not
achlavc
this
ing
solvent
further
tends
to dimorira
to
pigment
Our understanding
with
of
show l much graxtcr
tions
are
rtabilixatlon
(amino acid)
sama (amino)
cm-l.
for
algnificxntly
methyl
ACj’(P’),
l
vi11
of
itx the
the mono-
interfere
vlth
H-
group might have on the
group36
apparently
dosa
3 and 4 are vary
similar.
not
sac-butyl
cf. the counterpart
and the signad
of the CEs is not reversed.
Unlike
ovar
between
biliverdinoid
tha mono-eater
(Table
2)
a greater
mono-amides , mono-esters
assume a similar
order
interaction for
Furthermore,
CDs
reflect
(Table
xolutionx
valuer
as
diapoaitfon
the CEs is unexplained.
long
CEs are
amides
the
the amides of L-tryptophan
amide analog.
amida
gonerats
may thus be vlwad
Large diffarencw
with
IAel - 260 K’
to
conforu-
have AC/‘(H’)
and the
diastsreomar.
sac-butyl
the
bonding.
limlt,12* xo u
?‘,
CEs,
ptgant 8).
have an snantiomerlc
typically
of
large
for
the underivatizad
pigment’s
ylsld
the
to account
Like through
ester
theoretical
that
the pradomlnant to
that
dfarteroorrs to
vs.
large
order
Since
2)
obxawad
1 and 2 belong signed
of
Fig.
?‘,
be aqua1
expected
lquflibrium
dipole-dipole
suggastad
not
charactorixtic
affect
CEs are alro
will
BR-TX MBA, BR-IX BAA. BR-IX BM)
(H’
chromophoras .
ll’
concsntratlon
approaching
(BR-IX HM,
diastcrsomws
of
lctlvlty
optical
are conformational dlaxtersolerr. The folded structurox even without the H-bonding (ref.
the
range
xs those
diexter my
CEs are
through
tha CD of
2.
If, v.
of
intermolwular
l nsuma
that
than of
those
of
Figs.
1 and 2,B thay
and
in non-polar
pyrrorthanonc-pyrromathanono
than
their
complicated.
do mono-esters,
corre-
H-bond-
aolventr
tha
H-bonds.6a137-39 Since
mono.amldes
CD may not be derived
tha
In &SO.
stabiliratlon.
H-bonding,6c
is corraxpondingly
dapcndsnce
vaaksr
conformationa
H-bonding
full
intramolecular
dieters
concantrxtion
considerably
x~sume the folded
the added benefit
dcxtabilixax
10e’-10V7 in Fig.
the
diesters
entirely
the dl*at@r or
CEs
his-amides
from conforma-
of bilirubia
Confonnrtion
(W) Spactrd formed from
Clrcul~ Dichroism (CD) lnd Ultrwiolot-Visible )I Solutiolv of Billrubin Nono- md Di-Estws Estma
Tab18 3.
Aa_
4683
xmidu
CD A1 xt At-O
W (1,
(453) (450)
50,600 52 ( 300
(442) (398)
-43.1 - 3.9
(455) (450)
SB,200 57,400
(430) (396)
428 412
-15.3 - 2.1
(453) (450)
57,100 58,200
(436) (402)
(405) (400)
426 420
- 6.9 - 2.6
(455) (450)
51,200 52,800
(415) (394)
(403)
424 ___
- 6.7 - 3.7
(453) (455)
63,100 53,200
(450) (458)
_-_--
- 3.1 - 4.3
(455) (455)
50 ( 200 57.000
(452) (450)
AC,,(&)
Solvm*
non0 Di
C,H,
+13.9 t 2.1
(405) (400)
428 420
-18.1 - 3.5
bSOll0
%%
t30.9 + 2.1
(405) (405)
428 417
co
+10.3 + 1.0
(405) (400)
Mono Di
n*CN
t 5.2 t 1.0
l4OllO
MeOH
+ 3.0 l 0.1
ne, so
‘ 0.1 a 0.1
IhI
no,
Di
Di Mono Di
1.7-3.9 x loAcid Ethyl
SIrnI
0 1)
Ester
Di
kta for S-(-)-Lctic
m)
L Prepared by SN2 romztion of BR-IX his-tmtra-n-butyluoniur salt with the p-toluenwulfomto of S-(-)-lactic mid *thy1 *star. Spetra were run at 20-C within 0.5 bra. of solutior, prepmation using the corresponding estera of racoric lactic acid ethyl lstor as tha (At - 0) bmallne. CH,Cl, - dichloromthum, HeCN - ~cctonitrll~. MoOH - methmol. Ha,SO k CoH, - bmumm, dlmthylsulfoxid~.
T&lo
4.
Dichroism (CD) ld of the Bilirubin-I&a
Circular Solutloru
(W)
Ultrwlolet-Vlsibla Nono- ld Dl-esters
Spectral Data for 2-5 formed from S-(+)-2-Butmola
CD X, at At-0
Solv*ntk
Mono Di
C,H,
+12.6 t 1.1
(405) (400)
424 424
-21.2 - 1.5
Mono Di
%CIl
+10.2 + 1.3
(405) (400)
424 421
-15.7 - 1.7
Mono Di
nb, co
t 8.2 t 0.6
(405) (400)
423 420
non0 Di
nmCN
t 3.4 . 0.1
(405)
HmOH
+ 1.3 t 1.0 = 0.1 + 1.4
Mono Di
Me, so
I Prepared by SN2 ramtion S-(+)-2-butmol. Spctra cormspondinS lstora of -Cl, c C,H, - bmxmw. diwthylsulfoxid~. - tl.1 (500), Ar ’ At..=
Excfton
lqulwlent
Electrortatic m lxciton
48,000 34,500
(438) (402)
(450) (450)
54,300 33) 700
(430) (398)
-12.2 (450) - 0.86(450)
60,000 34,500
(438) (406)
422 ___
- 5.2 = 0.1
(450)
35.500 37,700
(420) (400)
(400) (410)
423 ___
- 2.2 . 0.1
(450)
51,400 35 (400
(448) (450)
(415)
___ ___
56, SO0 32,400
(454) (453)
Absoluta
&nffgur8tron.
the
heteramsociation
uinee12
that
act
of the
the
sltution
wtth
comploxo~
of BR-IX with
chiral
lgonta
alao
comploxing
pigmmt hold
lxcitmd
uy
in
l
chlml
llmtric stat*
bm viewed
to
mono-•tors.
attached
pyrromthmono
molmculm
Iho
ia covalmtly
as chiral
chromophoros
intwmtlon of
porturber
thm bichromophoric
pyrrouthmnono
splitting
0.1 = 0.1 l
0 at 460 IU.
of
cm*,
A*..=(&)
of BR-IX bia-tetra-n-butyluonim salt with thm m~thmm~ulfon~to of were run at 20-C within 0.5 bra. of solution propw&lon using the racomic P-butmol ls the (be - 0) baseline. - dichloromothmm, MoCN - metonitrile, MaOH - rthmol, k,SO -
chiral
ad
two
(450) (450)
the
In llthm
taining
1 .\I&
whom to tit
.lbwfnlo*ll mmt.
uxf
couplfiy
and bls-uidm,
,_._
A*..= 0,)
n
W
Eater
Mono Dl
x 10”
the
md
dipole
trmuition
in two long
mono-ui&a uy
agents induce
as . moloculm
orlontation,
resulting
pignnt
l
s
In
bc seen
such CD of
l
thm plS-
.xciton30 Fig*.
mommts46 wmwlongth
m
se-
con-
1 ld
2.
leads
to
llectronic
4684
D. A. L~o~nrar md
ihANG
in ltmrgy lnd on* lover in anorgy, with the lw*r*tion
tr*nsitions, one hi&*r rol*tiv* oriont*tion
M.-H.
*nd ltrongth of
th* tr*nrition mom*nta.
Accordi-
Qpondont
oxciton tr*naltiotU llw*yr hav* oppo*it*ly-*ign*d CD CE*. typic*lly fl*nklng the W-vi* *itfon(*),12s30 l* la ob**rv*d (Pig. 3). t*r*onr
can b* predicted
The *b*olut* confiyr*tion
tr*ruitlon mollsnt.l**
Thus, BR-IX MM
*nd BR-IX BAA lr* predicted
to hav* *n *xc*** of the pi&ment with ? h*nd*dn***, vhsr***, BR-IX HBA, BR-IX BM, HBE are
tr*n-
of th* prodominant dl*a-
from oxciton coupling theory *nd * knovl*dg* of th* pyrrom*th*none
long v*v*lonlth W-vi*lbl*
lnd BR-IX
on the
to th*~ry,~O the tvo
prodlctsd
to h*v* ln *xc*** of th* pign*nt with II h*nd*dno**.
lnt*ruity of th* CE *nd using th* c*lcul*t*d CE* for P or lf,l** w* l*tiut*
BR-IX HLE From th*
* dl*at*r*om*rlc
*xc*** of -138 for BR-IX HAA in MsCN and 268 for BR-IX BAA in !foCN. In contr**t, BR-IX MBA
lhov* e**sntl*lly no dlut*riom*ric
*xc***, lnd BR-IX BM,
BR-IX NLK and BR-IX blBE show only
-28 di*rt*r*om*ric *XC*** in XoCN lolvont.
FIUIRE 3. Circul*r dichroi*m *nd W-vi* lp*ctr* of 1.5 x 1O-5 W lolutiona of BR-IX mono-amida of L(t)-al*nin* m4thyl e*t*r (-). th* bl*-amida (- . . .). BR-IX monol*t*r from L-(-)-l*ctlc *cld *thy1 **tsr(- - - -) lnd dl**t*r (-e-.) in Iho mono-•*t*r M&N run lt 20-C. The sCUN* I* ugntfled x 3. lh*p*d CD curve* ltr*ddl* th* Ae - 0 lln*, which corr**pond* to th* b***line run on ‘amplo* uda from D,Lll*niru mothyl l*t*r *nd D.L-l*ctic rho w-vi* lp.clcld ethyl l*t*r. tr* *how l v*lru* lncr**ring from ths bottom of the Figure, the t-O b*rellne.
A. “rn
lith*r of
to l**-
BR-IX ton&
two folded.
lntramol*cul*rly H-bonded conforutlona
th*t
interconvert by br**king *nd ramakin& lll 6 H-bond* (Fig. 1) lnd luy thu, bs l**n ** * r*c*alc mlxtur*.
Uh*n th* conformation-d*t*rmlnlng
l*con&ry
mid**,
lntruolscul*rly
propionic lcld c*rboxyl &roup* lr* &rlv*tir*d
H-bonded conform*tlon* may *till be r*t*inod
l*
(Fig. 2), but
when the amide amln* mol*ty PO******* l chlr*l c*nter. the *r*tvhlls anantiomerr become dl*rt*r*om*r*, lnd th* mixturo is no longer r*c*nic. ruco*r*rlly
l:l,
[H'] +
nor* importantly, the squillbrlum i* no long*r
[P'], *nd th* optic*1 lctlvlty **socl*t*d wlth
th* mixture
c*n bs
expsctod to *how bi*ign*t* CD CEa *r*ocl*t*d with sxciton coupling of th* colpon*nt pyrromcth*th* intriruic CEa lr* typlc*lly v*ry l*rso for ths snantlom*rlc
non* chromophoro*.
Since
conformor*. modor*t*
dl*pl*c*montr
lr* obrerved
from
a
1:l lquillbrium will r**ult in lub*tantl*l Cl!*, l*
for th* bi*-amide* with L-*l*nin* methyl **tar.
at111 r**ult in *lgnlflc*nt CE*. ** *r* ob*ow*d l*rly, mono-ami&*
Ev*n *m*ll dl*pl*c*m*nt* will
for th* bl*-amid** vith 2-amlnobut*nr.
Slmi-
*nd mono-•*ter*, which *till r*t*in lt l***t 501 of th* lntruol*cul*r
H-
bonda through th* undorfv*tlred proplonic *cld c*rboxyl group, c*n lxhlbit conformational di*lt*r*om*ri*m vh*n the amino or *lcohol portion cont*lns l chlr*l conter.
tlons such
u
those
of
Fig.
Hero. too, conforu-
2 lppe*r to b* l**onti*l in undar*t*ndlng th* orl;ln of th* bi*lg-
rat* CD*.
Aci.l¶oulodg-t. U* thank th* N*tional In*titut** of H**lth (HD-17779) for &*n*rour *upport of thi* work.
X-H. Zhans vu
i*try, Ac*&ml*
* vi*ltlng *chol*r on le*ve from th* Inrtituto of Photosr*phic Ch*m-
Slnic*, B*ijing.
4685
All tircul~r dichroisr (CD) epactrs vere run on 8 JASCO J-40 inatrumant lquippad vitb calerd: and all w-as spectra var. run on a cary 219 lpectropBotomat*r. All photoelastic modulator, HI(R spectra vare run on m 13~ RR-8DAF spectrometer in either de-dinathylaulfoxide (Aldrich, All IR spectra vet. obtainad on . Perkin99.98 de) or deutoriochlorofora (Y9.9r. Aldrich). Optical rotations, [a],,, , waxsoobtained on a Perkln-Elner model Elmer model 599 iwtnnt. RoIting points were datermined on a Nel-Toap capillary unit. Combus141 rpectropolaxi*tar. Analytical thin layer tlon sticr~a~lysu vere obtainad from Dasort Analytics, Tucson. AE. chroastography (TLC) vaa carried out using Analtech Unlplatar (ailMa, ZSOr); preparativa TLC Column chromatography vaa was carried out on Analtach Uniplates (silica, 500~ or 1000~). HPLC analyses were carrted out achieved using neutral alurinur oxide (activity II-III. Uoela). on a Per-kin-Elmer Sories 3 liquid chroaatograph equipped with an Altex 5~ ODS C-18 ultcasphera ion psiring column (25 cm) win& 0.1 W di-n-octylaaine ncetate in St aq. methanol as eluant The retentfon times (min.) found (flow rate 1 .O al/s&n) and a column temperature of 23’C.‘” BR-IX. 20; BR-IX BM, S; BR-IX NBA. 9; BR-IX BAA, 4.3; BR-IX t&A, 8; BR-IX BI)E, 9; B&-IX were: ffBE. 11; BR-IX BLE, 5.5; B&k-I&a KLE, 9. Biiirubin-IXo vas from Porphyrin Products, Logan, WI, and contained lerr than Sq of the 1110 and XIIIo isomers ae determined by HPLC. S-f-)-Iactate acid ethyl eeter, [a]:* -12’ (naat), diphenyl phosphoryl azida, oethenesulfonyl chloride, pterra-n-butylammonium hydroxide and triethylamlne were from Aldrich. toluanesul fonyl chloride, [a],+13.6’ (neat) were from Norse S-(t)-2.Aminobutane, [a]s+7.4” (neat) and S-(+)-2-butanol, S-(t)-alanine methyl eatar hydrochlorida vas from Sigma. Pyridine was dried by dfstilLabs. lation from KrCO, snd di~thylauIfoxf& was dried by distillation froar 3k molecular sieves. Bilirubin_IXa (BR-m. BilirubinS-(+)-alanine aethyl ester hydrochloride (14 mg, b-1 moles), IXa (29.2 ng, 0.05 amoles), diphenylphosphoryl lzlde (11 ag, 0.1 amoles) and triethylrmlne (20 mg, 0.2 mmoles) were stirred In 1 IL of dry MeaS in a 3 mL glass vial at room temparsture in the dark under nitrogen for 1 h. An additional 14 mg of almfne methyl ester hydrochlorida, 11 ag of diphaylphosphoryl astirred at side snd 10 mg of triethyl amina were then added, and the mixtura was continuously roofs temperature in the dark under nitrogen for 3 h. HPLC malysfs indicated that 40% of the mono-amida and 46* of the his-amids vere formed and that 141 of FIR-IX was unreactad. Ihree milliliters of dichlororthana was then added snd the solution vas vsshad vith cold water (3x1 Then tha dichloronethene solution was applied to a column (10 cm long mL) and drled (NasSO,). x 2 co intarnal diemeter) of neutral alumina and aluted with dichloro=ethanc (200 paL) to remove BR-IX-BAA vas then eluted with dfchloro~ethsne/~tha~l unresctad arfde and axcesa aminas. After evaporation of the solvent. 5 ng of the his-aaide (IS% yield) was (2O:l v/v) (200 SL). obtained. The l lwina column was then cluted with dichloromethane/methanol/aswonium hydroxide (3Ot aq.) (80:20:2 v/v/v) until an orange brnd of the mono-•mldt scparrted from a small amount of unrescted BR-IX, left at the top of the column. The alumina vas extruded and the mono-amide band was excised and extracted to give 11 mg (30t yield) of BR.IX HAA. It had dec. >2OO’C; [a];;, - +420* (c 0.02, CRsC1,); IR (KEr) V: 3430. 3350. 2930, 1750, 1690, 1625, 1450, 1260, 1000, 710 ca-1; ‘II (12H. 4xCH,), 1.20 (1.55) fd, 3H, CR, J - 7 Hz), 1.95-2.12 (CDCI,) 6: 3.51 (3.75) (a. 3X, CR)), 4.02 (s, 2X, CHt), 4.17 (4.26) 2.6-2.78 (n, 8H, 4x (q. Hi, CR, Y - 7 Hz), 5.24-6.78 &IH, -CR), 9.23 (d, lH, O-C-NB-, J c 7 Hz), 9.34 (s, IH. pyrrole NH), 10.06 (9.91) (s, lH, pyrrole NH), 10.69 (s. lH, lactam NH), 10.79 (s. 1H. lactam NH) ppn. AD&. Calcd for C,,H,,N,O, (669.8): C, 66.35: H. 6.47: N, 10.66. Found : C. 65.96; H, 6.51: N, 10.14. The b&s-amide had a.p. 137-14O’C; [a]$:, - +370’ (c 0.02, CH,Cl,); IR (KBr) Y: 3430, 2930, 1750, 1680. 1660, 1640, 1550, 1460, 1260. 1220, 1175. 1000, 710 cm‘;; IH-NHR (CDCL,) 6: 1.38 (d, 6H, 2xCH, J . 7 Hz), 1.73-1.97-2.10 (12H, 4xCH,), 2.99-2.83 (P, BH, 4x(&). 3.72 (s. 6H. (in. 8H, -CH). 10.13 (d. 2H, 2x+ ) , 4.12 (s, 2H, CH,). 4.66 (qs 2H. 2xCH, J - 7 Hz). 5.20-6.77 O-C-N!?). 10.40 (s. lN, pyrrole), 10.70 (s. 1H. pyrrole NH), 11.03 (s, 1H lactam NH) ppn. &l&. Calcd for C,,Hr,N,O, (7S4.9): C, 65.24; H, 6.68; N, 11.13. Fwnd: C. 64.88; H, 6.62; N, 10.75. -t -XX t%dQ: Bilirubin IXo (29.2 mg. 0.05 moles), S-(+)-2aminobutane (7.; mg, 0.1 Imoles). diphenylphoaphoryl axide (11.6 ng, 0.1 maoles) and triethylamine (10.1 mg, 0.1 moles) vere stirred in 1 mL of dried Re,SO in a 3 mL gloss vial at room tcmperrture under nitrogen in the dark for 1 h. Another 7.3 mg of 2-aainobutane and 11.6 mg of dlphenylphosphoryi azfde vcre then added and the nfxture wes stirred continuously at ro01p tamperdture under nttrogen in the dark for 5 hrs, HPLC analysis indicated that 38% of the 1w)noamide end 44* of the bfs-aide was formed and that 18% of BR-I&a was unreactad. Three ailliliters of dichlorowthans vas than added. solution vaa vashed with cold vater (3x1 ML) and dried over sodium sulfate. Ihsn the dichloroaethanc solution was lppllad to a column (10 C= long x 2 cm lntcrual diameter) of neutral alumina and eluted with dichloromethana (200 mt) to remove atide end axcess amine. A small amount of BR-IX MA was elutad with dlchloromathane/ncthanol (20~1 v/v. 200 sL). Elution was continued vith dfchlorocw:thans/rthinol/~nius hydroxide (3Ot aq.1 (80:20:2 v/v/v) until an orange band of the mono-amide vas sapareted froa a small band of uaructed BR-IX AC the top of column, Tha alumina was then extruded from the coluan, and the band corresponding to BR-XX NBA vas excised. The purified &no.-amide vas vashed froa the alumina with dichloromethen~plethanol/gleeial lcetlc ecid (98:l:l v/r/p). The acidic dichloromthane eluate wss vashed vith water to pH=7, dried over sodium sulfate and evaporated at room temperature to give the mono-amide, 10 mg (27% yield) with rsp, 180-3’~ (dec.), (Caraful daoxygenation of all of solvents and exclusion of light Era the chrovatography rstardad product isorserization.) BR-IX KM had [a]j;s ; -366’ (CHsCl~); IR (Wr) V: 3410, 3340. 29B0, 2920, 1680, 1645, 1615. 144S, 1250, 995 cm ; H-NHR (CDCl,) 6: 0.93 (t, 3H, CH, .I - 6 Hz). 1.24 (d, 3H, CH, J - 6 Hz). 1.54 (q. 2H. CH,, J - 6 Hz). 1.96-2.16 (MI. 4xCR,),
4686
2.32-2.79 (m, 8H, 4xCH,), 3.44 0.5 H, O-C-NR-,cfs, J - 6 Hz), NH), 10.26 (6. lH; pyrrole NH), &&. Calcd for C,,H,,N,Os
D. A. LKMTNLTR and M.-H. ZHANG
(m, lH, CH) 4.04 (a. 2H. CH,). 5.27-6.78 (a, 8H, -CH), 8.76 (d, 8.84 (d, 0.5 H, O-C-NH-,crans, J - 6 Hz), 9.39 (s, 1H. pyrrole 10.71 (a. lH, lecternNH), 10.82 (s. lH, lecternNH) ppm. (639.8): C, 69.46; H, 7.10; N, 10.95. Found: C, 69.45; H, 6.70; N, 10.68.
IXa Bis _2 _ku$&Ag& (BR-IX m Bilirubin-IXa (29.2 mg, 0.05 molea) S-(+)-2aminobutane (36.7 mg. .0.5 moles), diphenylphosphoryl uide (58 mg. 0.5 -10s) and'triethylamine (50.5 mg. 0.5 moles) vere stirred in 1 EL of dried Ue,SO in l 3 mL of glees vie1 et room temporeture in the derk under nitrogen for 24 hre. .HPLC enalyeis indicated thet the BRA-IXwee complstely,,convertod to the bie-amide. Three milliliters of dichlororsthane was then ldded, and the solution was vashed with cold water (5x1 mL). dried over sodium sulfate ld evaporated to drynoes. The residue wee washed with diethyl other (anhydroue) (5x5 mL) to extract the yellow-orange BR-IX BRA, 17.6 mg (508 yield), m.p. 204-206'C (dec). It bed [o]lip - -512' (CH,Cl,); IR (RBr) Y: 3340, 2980, 2920, 1680, 1635, 1445, 1255, 1170. 995 cm-l; 'H-N?IR (CDCl,) 6: 0.93 (t, 6H, 2xCH,, J - 6 Hz, 1.24 (d, 6H, 2xCH, J - 6 Hz) 1.50 (q, 4H, 2xCHI, J - 6 Hz), 1.96-2.16 (12H, 4xCHs). 2.32-2.79 (n. 8H, 4xCHs) 3.41 (m, 2H. 2xCH). 4.02 (a, 2H, CH,) 5.216.66 (m. 8H, -cm), 8.7 (d, lH, &C-WH-,cfs. J - 6 Hz) 8.8 (d, lH, O-C-NX-,trans, J - 6 Hz). 10.35 (s, LH, pyrrole NH). 10.35 (II, 1H. Lactam), 10.87 (s, LH, pyrrole NH). 11.19 (s. lH, lecternNH) ppa. u. Calcd for C,,H,,N,O, (694.9): C. 70.86: H, 7.83: N, 12.09. Found: C, 70.56; H, 8.14; N. 12.18. IXn plpllp-end Df-es ters (BR-w and BR-IX pBEz: BilirubfnIXo (29.2 mg, 0.05 -10s) end 1% lq. tetre-n-butylemonium hydroxide (2.6 mL, 0.10 rmoles) were stirred in l glsss vial at room tempereture in dark under nitrogen for 30 min. The re*ction mixture vu then evaporated to dryness et 40-C. Then S-(+)-P-butenol aethoneaulfonate (30 ng, 0.2 mmolce) and 3 mL of dichloromethane vere added to the residue of BR-IXp bis-tetra-nbutylmonium propionate salt, and the dichloromethane solution wee heated et reflux for 14 h. HPLC enelysie indicated thst 178 of dieeter. 73% of monoester and 101 unknown impurity were formed. The dichloremethene solution vas cooled, washed with veter (3x1 mL). then dried over sodium eulfste. The dry dichloromethane solution was then applied to (t column (10 cm long x2 cm internal diameter) of neutrel alumfna with dichloromethane as aluant. Unreacted methanesulfonate was eluted first. BR-IX DIE van eluted next vith dichloromethene/methanol (2O:l v/v) and purified by preparative TLC on silica gel (1 rm layer) by developing the chromatogram with dichlorowthane/osthanol/glaciaL acetic acid (98:l:l V/V/V). Ihe purified diester (R, -0.3) was eluted from the silica gel with dichloromethane/methanol (2O:l v/v) to give -2 qg (yield -68). To isolate the mono-ester, the alumina column was continuously eluted with dichloromethane/mathanol/-nium hydroxide (308 aq.) (80:20:2 v/v/v) until an orange band of BR-IX MBE was separated from a small bend of unrcected BR-LX bis salt, which remained at the top of column. The aluaina was then extruded from the column, and the band corresponding to the mono-ester was excised. The purified mono-ester was washed from the alumina vith dichloromethane/methanol/glacial acetic acid (98:l:l v/v/v) and the acidic dichloronethane eluate was washed with wster (2x5 mL) and dried over sodium sulfate. The solvent vas evaporated at room temperature to give BR-IX RBE, 14 mg (438 yield). (Pigment isowrixation was retarded by excluding Light.) BR-IX XBB had mp 169-171'C; [a]$ip - -692' (CH,OH); IR (XBr) Y: 3400, 3340. 3250. 2940, 2910, 1725, 1695, 1655, 1625, 1450, 1360, 1250, 1170, 990, 690. cm-‘; 'H-NNR (CDCl,) 6: 0.89 (t, 3H, CHS, J - 6 Hz), 1.01 (d. 2H, CH,, J - 6 Hz), 1.25 (n. 2H, CH,, J - 6 Hz), 1.95-2.12 (12H, 4x'+), 2.59-2.77 (m, 8H, 4xCH2), 3.97 (s, 2H, CH,), 4.84 (m, H. CH), 5.42-6.80 (m. 8H, 9.27 (s. lH, pyrrole NH), 9.30 (8, LH, pyrrole NH), 10.70 (8, lH, lactam NH), 10.80 (s, -w. lH, lactam NH) ppa. m. Calcd for C,,H,,N,Os*HsO (658.8): C, 67.45: H, 7.04; N, 8.50. Found: C, 67.76; H, 6.62; N, 8.08. BR-IX DBE had mp 123-127-C; [o]:;, - -995' (CH,Cl,); IR (XBr) Y: 3340, 2980, 2930, 1730, 1670, 1630, 1450, 1370, 1260, 1170, 990, 690 cm-i; 'H-NHR (CDCL,) 6: 0.89 (t, 6H, 2xCH, J - 6 Hz). 1.20 (d, 6H, 2xCH, J - 6 Hz), 1.50 (q, 4H. 2xCH, J - 6 Hz), 1.76-1.98-2.09 (12H 4xCH,), 2.31-2.81 (m, 8H 4x%), 4.18 (s. W, CH,), 4.88 (n, W, 2xCH), 5.32-6.54 (m, 8H. -CH), 10.10 (s, LH, Loctam NH), 10.11 (s, LH, pyrrole NH), 10.45 (6, 1H. pyrrole NH), 11.18 (8, lH, lactam NH) pp=. w. Calcd for C,lH,zN,Os (696.90): C. 70.66; H. 7.52: N, 8.04. Found: C, 70.38; H, 7.22; N. 7.93. and w (BP-v. Bilirubin-IXe (29.2 py, 0.05 -10s) and la lq. tetra-n-butylarmn im hydroxide (2.6 mL, 0.10 -10s) were stirred in a glass vial at room tenperature in dark under nitrogen for 30 min. The reaction mixture was then evaporated to dryness at 40-C. Then the tosylate of S-(-)-lactic acid ethyl eater (54.4 mg. 0.2 moles) and 3 mL of acetone were added to the residue of BR-1%~ his-tetra-n-butyl -niw propionate salt, which was not isolated and purified, and the acetone solution was heated at reflux for 12 h. HPLC anelysls indicate that 46% of di-ester, 44t of mono-ester, LO1 of unreacted BR-I&a bis-salt vete formed. The acetone vae evaporated. and 3 mL of dichlorolpethane VU then added, and the solution v.ss washed vith water (3x1 mL) then dried over sodim sulfete. The dry dichloromathane solution contained mono-, his-ester and BR-. IX bls salt, which could be separated and purified by column chromatography on alumina, followed by preparative TU on stlioa gel as described above to give 7 mg of pure BR-IX DLlI (18% yield). It had mp 78-82-C; [e]$is - -355' (CHsCl,); IR (RBr) Y: 3420, 3270, 2930, 1750, 1700, 1665. 1635. 1450, 1260. 1000, 710 cm-'; 'H-NNR (CDCl,) 6: 1.26 (t, 6H, 2xCH, J - 7 Hz), 1.47 (d, 6H. 2xCHs J - 7 HZ), 1.73-2.08 (L2H, 4xCH,), 2.53-2.83 (I, 8H, 4xCHs). 4.15 (6, 2H, CHI). 4.20 (q, 4H. 2xCH,, J - 7 Hz), 4.92 (q. 2H, 2xCH, J - 7 Hz), 5.2-6.72 (n, 8H. -CH). 10.11 (6,
Conformatian
of bin
4687
amides
1H. lactaa RR). 10.14 (a. l.R, pyrrolo NH), 10.44 (s, 1H. pyrrola NH), 11.15 (a, lH, lactu) PP.. hpaz. Calcd for C,,H,,N,O,, (784.9): C, 65.80; H, 6.68; N, 7.14. Found: C, 65.78: H. 6.81; N, 7.43. It had rep llO'-113'C; [a]::, The purified BR-IX KLE (10 qg) was isolatad (29r yield). ;; 3400, 3000, 2930, 1750. 1680, 1660, 1640, 1460, 1270, LIOO, 1000, I.23 (d. 3H. CH,, J - 7 Hz), 1.47 (t. 3H. CR,. J - 7 Hz). 1.972.13-2.43 (12W, IxM,), 2.81 (m, BH, 4xORe), 3.98 (=. 2H, CR,), 4.02 (q, 2H, CH,, J - 7 Hz), 4.93 (a. lH, CH, 3 - 7 Rr), 5.27-6.79 (o, 8H, -CH), 8.04 (8.14) (6, LQI, pyrrole RR), t.89 (8.89) (a, 1H. pyrrole RH). 9.10 (9.10) (8, LH, lactam NW), 10.69 (10.78) (1, lH, lactam RR) PPm. &J& Calcd for CI,H,,N,OI (684.8): C, 66.65: H, 6.48: N, 8.18. Found: C, 66.45; H, 6.80; N. 8.68. ;~2~h_~q~cRLa)&It"~C~~;
-._ &~cfd4~ Ethyl S-(-)-lactate (4.00 I, 0.034 moles) vas dissolvad .in pyrLdina (40 mL) and eoolsd tn an iea-bath to -5'C. p-~ol~na~lf~~l ehlorida (8.00 8, 0.042 wlrs) was added tn small portions over a period of 15 min. and the raaultfng light yellow aolutlon was kapt for 24 h. at -20'C. Small portions of ica (1 g, 2 g, 2 g) vare then added, vith a period of 15 oin. betvaan additfona, insuring that tha tmperature remained near 0-C. 'The solution vaa then poured into ica vatar (100 ML) and extracted with chloroform (3x25 rL). The chloroform extracts vara dried over anhydrous sodium sulfate end evaporatad. l'ba syrupy residue was taken up in hexane-dichloromath~~ (lO:l, 80 mL) and crystalliaed by rapid cooling in a dry ice-acetone bath to yield 7g (7% yield) the tosylate. Recryatallir.ation from haxane-dlchloromathane (10:lf at -2O'C afforded colorlesa needles, 5 g (54% yiald), mp 30-32'C; [a]:il - -49.5' (CH,Cl,); 'H-RhR (CDCl,) 6: L.21 (t. 3H, CR,) 1.50 (d, 3H. CH,) 2.46 (a, 3H. CH,) 4.10 (q, 2H. CR,). 7.30 (d, 2H. Ar), 7.78 (d. 2H, Ar) ppm. S-(+)-2--:43 S-(t)-2.butyi alcohol (0.74 g, 0.01 moles) uaa dissolved in cold dichloromethana (10 ot) and cooled in an ice bath (O'C). Pyrtdfna (1.62 sL, 0.02 molas) was then added. followed by the addition of nethanaaulfonyl chloride (1.72 g. 0.015 moles) in small portions with constant stirring at the ice bath (
1. D.A. Lightnar and A.F. HcDona8h. Accounts Chem. 2, A.P.
HcDonagh in The (L979), pp 293-491.
Vol. VI
Porphyrins.
3. A.F. McDonagh and D.A, Ligbtner, Pedfatrics 4. R. Brodersrn in Bile Pigments York (1986), pp 157-181. 5.
(a)
It;
cc>
fn Bilfmbtn, Racon, FL (1982), pp L-58. (a) (b) (c)
(b)
Press, New York
75, 443-455 (1985). (3.D. Ostrow,
VoJ
I
hares1 Dekkar. Inc., Nav
ed.),
(K.P.R. Helrwegh and S.B. Broun, eda.), CRC Press, Boca
Cham.
Sot.
Cheat.
Cw.
122-123 (1976).
Loading references, sea G. Blauer. Isr. J. Chem.
11. D.A. Lightnar, tI. Raisfngtr and C.L. Landen, J. 12. (a)
ad.), Academic
H. Falk and N. Rilller, Honatsh. Chem. 112. 1325-1322 (1981). H. FaLk and N. hOllcr, Tetrahedron 39, 1875-1885 (1983). R.V. Parson and D.A. LiRhtner, unpublished calculations.
9. P. Nanltto and D. Xonti. J.
10. For
(D. Dolphin,
For leading refercncas sea D. Kaplan and C. Navon, Irr. J. Chem. 23, 177-186 (1983). G. Navon. S. Frank and D. Kaplan, J. Chem. Sot. Parkfn Trans. 11, 1145-1149 (1984). D. Kaplan and G. Navon, Blochem. J. 201, 605-613 (1982).
7. D.A. Li@ner
8.
Jaundice
17, 417-424 (1986)
R. Bennett, J.E. Davies, N.B. Hursthouae and G.U. Shsldrick, Proc. R. Sot. London Ser. 1202, 249-268 (1978). G. LeBas, A. Allsgret Y. Maugutn, C. D&an&o and K. BaiLly, Acta CrystrlIogr. B36, 3007-3011 (1980).
(b) 6.
and
Res.
BfoJ
23. 201-209 (1983). Chm.
261.
D.A. Ltghtner, J.K. Cavrohski and W.R.D. Wifekoon, J. (1987). D.A.
Lightner and J.Y. An, Tetrahedron
43.
4287-4296
6034-6038 Am.
(1987).
Chsra.
(1986). Sot.
109, 6248-6259
D. A. Lw#fmm and M.-H.&ANC
4688 13. D. Harr-Laizy,K. Lahlrl and
P.
Balm-u, Inc. J. PeptfdsProtalnRON. 25. 290-296 (1985).
14. 16.X.Turner and K.H. Ikretz,Quart. Rev, Chu. Soo., 299-330 (1947). 15. G. Blzuzr.Arch. Bfoche~.Bioph~s.661, 602-604 (1986). 16. That BR-IX can &ve zn ICD in the proxenca of (+) or (-)-a-phonylethyluinain orSznic lolvmtz haz boon cited by Dlawr tn ref. 10 and again in l dlrcuzrionfollowIn&z lecture by G. Blzuer. and E. Lavla in Pmtofn-Lfgmd Imc~r8ctfons(X. Sund and G. Blauar, eds.). pp 399-416, W. de Cruyter, KY (1975), for work of 0. Kayer, Ph.D. thoafs, Univ. Konstam, (1979). 17. D.A. Lightnrr,J.K. Czweohrkiand K. Cmohzkz, J. Am. Cham. Sot. 107, 2456-2461(1985). 18. (z) For loading referoncas,me H. RsizinRerznd D.A. Lightner,J. Inclus.Phuzom. 3, 479. 485 (1985). (b) X.L. Furman end P. Balarum,Pmt. IndirnAcad. Scf. @hr. Scf.) 98, 453-468 (1987). 19. C.C. Kuonzlo,N. Citrolun-Cumerumy and K.J. Wilson,J. Mol. Chem. 251. 801-807 (1976). 20. A.P. NeDon@, L.A. Palm, J.J. Lauff and T-W. Uu, J. Clfn. Imost. 74. 763-770 (1984)and reforancm therefn. 21. M. Raisinger.T-W. Wu and D. A. L&htner, unpublizhadwork. 22. P. Umitto and D. lfonti, &xporluxia 28, 379-380 (1972). 23. U.B.D. Ufjokoon and D.A. Ltghtner.fn prrpzrztlon. 24. D.A. Lightnor.H-H. Zhmg and P.R. Trull, T8trrhsdronLet%. 28, 4033-4036(1987). 25. T. Shfoiri,K. Hinomiyzznd S. Yzudz, J. Am. Chen. Sot. 94, 6203-6205(1972). 26. D.A. Lightnor,T.C. Adacu and J-S. Ha, rremhedron 21, 4253-4260(1984). 27. D.A. Lightner,F.R. Tiull end N-H. Zhang, TetrahedronLett. 28, 1047-1055(1987). 28. C.W. Dautzcho, 448 (1969).
D.A. Lfghtner,R.W. Woody and A.
Morcowitz,
Amu.
Rev.
Phys. Chm. 20. 407.
29. D.A. Lightnet,H. Ralzingetznd U.K.D. Wijskoon,J. Org. Chem. 52, 5391-5395(1987). 30. W. Her&a and K. Nakanishf,Circular Dfchroic Spectroscopy- Excieon Coupling tn Organic Stereochm!izery, UniversityScienceBooke, Hill Valley, CA (1983). 31. D.A. Lfghtnarand J-S. UA, Spsctrorc.Lett. 17, 317-327 (1984). 32. A. Hugnoli,P. Kanftto and D. Xontf, Acta Cryrtallogr.Sec. CM. 1287-1291(1983). 33. D.A. Lightnor, J-S. Ka and X-X. Vu, SpectroscopyLett. 19, 311-320 (1986). 34. l'homono-mida of L-tryptophm methyl ezter zhov CD CEs: AC::f -12.3, A r:;; t20.8 (C#H,);Actif -46.6,A#:;; +93.3 (MoCN);Ac:cf -29.5,Art3$ +49.5 (HeOH):znd Acrf;';-19.0, Act:; t27.5 (Ne,SO). 35. K. Grubmzyrand il.Falk, !Jonxt&Chesr.118, 837-843 (1987). 36. The esters of L-lzctlcacid wthyl
l zter
ahow CD CEe within 10% of the values of Table 3.
37. A.R. Holzwzrth, t. La&or, H. Lehner znd K. Schzffner,Phocochem.PhocobioI. 32. 17-26 (1980). 38. H. Polk, T. Schledererand P. Uohlmhmn, &mtdz.
Chea. il2, 199-207 (1981).
39. D.A. Lightnorand P.R. Trull, SpectroscopyLate. 16. 785-803 (1983). 40. (4) G. Alzuer znd G. LlzmiAre.J. Am. Chew Sot. 97. 1949-1954(1975). ib) H. Folk, K. Crubm~r, G: NIllbzchzr,L. Hofe;, A. LeoQlt.r,eF. Ieuffngerl and J. Rib+, nomesh Chm. 108, 1113-1130(1977). 41. A.F. 8!cDonagh, L.A. Palm. P.R. Trull and D.A. Lightnar,J. An. Checn.Sot. IO*, 6860-6867 (1902). 42. K.N. SLessor and B.D. Johnmton,Can. J. Chem. 57. 233-235 (1979). 43. R.S. Bly znd R-A. Kaesr, J. Org. Chem. 38, 1518-1527(1973).
ASTIR
DICHEOISh SPEClROSCOPY
DAVID A. LICK*
and HAN-tEM WANG
istry. Rsno,
lewda
of
Univsrsfty
89557-0020
Nevada
USA
The first optically active umider of bilirubln-IXa with amino Abwtract. acids lhow bisi%n@ze circular dichtoism Coeton effects (CEs) ln the vicinity of the pigment’s long uwelength W-visible absorption, e.g.. for the bisamide utth L-alurine methyl eeter in acetonttrtie A#:$! - -47, Aa:;: - +68 the dioster from S-f-)-tactic acid In marked contrast, and e:$; - 33,800. - -2.6 and e;;: - 32.800 in acetoethyl eeter exhibits AC::: - +l.O, At;;; The CEs of the mono-amides are generally raaller than those of the nftrile. are generaIly larger rh#n those bfs-amlde6: whereas, the CEs of mono-esters of the dlestsrs.
The yellow-oxnn.Be, duced abundantly d&ion
and
connection
phototherapy The latter
carboxylic
bonding tion
(Fig.
acid
have
has
neonatal been
and lactam
or C,, -C,,
ptgments
that
Jaundice long
been
attributed
of
to
a 3.dimsnsionrl linked
which involves,
fnter
double
without
alla,
is
pro-
glucuronirecently
in
its unusal hydrophobic
structurs3s6
through
reeort
for
especially of
In which
fntrrmolecular
the
hydrogen
configurational
the aseociatcd
bonds,
by the liver
(BR-IX),
the liver
because
are
carbon-carbon
to
inttrcst,2
and also
jaundice3
functionalitfes
can be excreted
(42,152)-billrubin-IXo
of hame and transported
structure for
And in phototherapy,
1).7
at the C, -C,
iaomeric
pi&ment of
by catebolisn
Its
excretion.1 with
propertiea.4 polar
cytoeoxic
in maarrls
isomerfr~-
H-bond breekln%
yields
to conju&ation.1*3
NWRE
1. Interconvsrtin&, folded and intramolecularly hydro&enbonded conformational lnantiomers Conof (42,132)-bilirubin-EXa, former X has the negative chirrlity orientation of the pyrromethonone 1onB wavelength electric transition dipolan. Conformer I, has the posttive chfraIfty orientation. (See ref. I2a)
(p)
(M) BR-IX can fold SiOI-XS
of
8
-18
receric
solutions
md
into
either
are stabilized
kcal/mole
And a rata
nixturo
of
are
optlcelly
as e*rum aIbumfnlD*ll
of
NO enantiosorlc
by intramolecular of
7.2
conformational
f0.4
sec‘i
enantiowrs,
inactive;
howavor,
and optically
active
conformationsI~S
H-bonds
yet
that
fnterconvert
(-38*C).5a*b*9 I( ? P (Fig.
in the presence
As such, 1). of
a chiral
static
ropul-
an activation it
In Isotropic
aaims, 12*13 the sotutions 4679
mfnirite
with
ten be viewed agent,
circular
le
e
BR-IX
rolvents.
comptcxin& exhibit
energy
euch
dichro-
4480
D. A. LIGHINEIR and M.-H.
1x9
(CD) for
origin
the
the
of
lnducad
in the comploxos Although
lxuplas
there
of
A#:;;
t9.9
are
*R-IX -
for
its
CD
In the currant (ax unxeparatcd
distal
(BR-IX
BR-IX
tha
of
and In the C-pigment
Synthds
ity
and covalently
alcohol
procaads
of the starting Cfrcular CD Cotton
effects
md
ln Tablax
Tables
xolvmtx
moLcculcs
vlth
intans* pl.x.*IO*IL folded,
lxupla
methyl
the
to
lxter
(BR-IX ME)
and S-
BR-IX BAA.
pigment
ix
optically
glucuronldas
(*star)
or uldc).
by coupling as outlined
the chlral
amino to BR-IX
earliar, for
except
plgmants
dlmcthyl-
vhosa
solubil-
from the p-toluoncsulfonata
conflguratlon
of
tha alcohol
the R-configuration
of ethyl sa1t.24*27
component;
and not
lo.
tha
tha S-conflguratlon
(1)
dilution and
large
blsignate and ulna
chlral t1.3
mochanlxn
and large (x-x*)
CEs of
transltlon
CDs are
har llttla not
(R).a
alxo
other
aolvcnts
that
Chs,
but
The bir-ml&s
associated
tend
with
tha orlgln
blxlgnato the pigment
for
the amino
of
amides
of
A<:;; the optfcal are
of
Flg.
only
solvcntx lxhlbit
to
-
for
dialactrlc
la-got-
lnherantly
CEx than from an
-1.3
for
lctlvlty.
the monomer in form).?’
In fact, of
having
3-dlmnrional
both
blchromophorlc llttla
inter-
xtructure.
hot~roarxoclation
to asxuma a moda-•tely The tendency
lymat-
pyrromothonono
H-bonded dlmcrs
xtructuras
1.
lmarga
as H-recapIn H-bonding
actlvo
charact*rlstlc
blllrubln-protein
1s thought
thoxc
can sarva
veakar,
points
salf-assochtfon.
to the plycnt’s
characterlstlc
whare the pigment
lnteraxtlng
on the CD, ax expectad
moments and veil-defined
of
of
lntanso
surprisingly large 28 optically e.g.,
phenethylulde:
the alanyl
akln to llthar
lxhlblt
than tha CEa in lov
In CHCL, where lntarmolecularly for
vork
absorption
H-&nor
or no affect
They are a clue
o~crlap.~’ xalts,12
reduced
perturbation,
acid -
At::;
this qulta
Saveral
to or even larger
1 would bo considcrod
ramota
conformation
opposlta.
(2)
on.
of W-v18
ara ganarally
Hlgh dlclcctric
and &Cl,).
a differant
vavalongth
llkyl amino CE magnitudes are gcnarally
CEs, comparable
Tablo
of
long
xhow signlflcantly
C,H,
-3.1,
p-orbital
chiral
eater
on tha
lxpacted
are
and diextorr:
as in bilirubln
(ester
argu-
tha 4-xubstltuantx
athyl
The amide darlvatiwx the
tha CEs lx
(xanthoblllrublc
nature
chromophorlc
acid
and
tha mono--l&s
BR-IX bix-tetra-n-butylmroniua
The CE intcnsltla
tha molecule
of
-
xuggast
of
large
vith
and amides
Ila,SO and AaTff tha bislgnate
of have
tha corraxpondlng
Hotwaver.
The CE magnItudea
chromophoros
near
1 and 2:
(cf.
proporty
The data
for
(!la,SO)
do tha mono-amidox.
asters
otters
t6.
-2.4.
tha ltaroochamlcal
S- (+)-alanina
wax more Bultabla using
-
-
mada from S-(+)-2-
and L2-ixomerx
blr-amides
were prepared
BR-IX
Actif
Act;;
CD of
and
of
tha 8.
propared
2-butanol
Cmformtlon.
progression
H-bonding
lntrlnslc
of
1 and 2).
(t4aOH) shov unoxpoctadly non-polar
Vera
The esters
cantared
whoraax,
xlgnad
in
BR-IX fro=
proccduro 25)
an SN2 inverxlon
(CEx)
from tha data of tars
vork
(HSA),
t3.1.
The
(2)
alcohol.
derlvatfvo;
and tha
of
bound HSA-bilirubin
the pigment
Dlcbzof~
(xuuarfxcd acid
via
of
or&ring
group,
-
howover,
In lach
a chlral
darlvatlws.
been raportcd;
BR-IX estcrx
the
S-(-)-lactic
This modification
lx low.
componentx
of
(Shloirl
and the mcthancsulfonata
Tha reaction
in
and a fw
cyclodaxtrlrul’
BR-TX with N-•catyl-L-cyxtalne
on the preparation
respactlvaly.
this
wax usod ax solvent.
in dlmathylfo-lda
lactate
rlc
of axIda
of
and (5)
as the corresponding
attachment
blllrubln
At;;;
Iha
tha plgmont.
BR-IX10*15
to human serum albumlnx
adductx
YC report
CD of
haw
tha 6-plgment.20
rcportad;23
(BR-IX NM).
The uidos
.
active
450 IN.
of I( and P apaclw of
amlncs. 12.13.16
no CD xpactra
and magnitudes)
DBE,
induce
actfvo
prwlously,12*17~18a
as well
dlphenylphoxphoryl
to
C-8 and C-12 laomars;
of
CD signs
remote
populations
group, (3)
near
transformation14
optically
on differencea
BR-IX DLE and BR-IX
by virtue
sulfoxfde
&pend
ME).
of vhlch
acid
yet
in non-equal swving
tha uo-vinyl
mixturax
sm.
(BR-IX
BBA.
using
(4)
ra not
l
S-(+)-2-butylamtna
MM),
(+)-2-butanol active
propionic
absorption
optically
no rxamplrs
and ax noted
rings.
caxcntlally
with for
W-visible
asyrotrlc
of proteins
Investigation
work do not
lactu
glw
order
at pH 4.5-7.5;lg
which
and eaters
thlx
almort
r3
butano1.24 ments of
can ba xa.n
flrxt
l
cxuplas
buffar;21
pH 7.3
glutathlonc.22
wavelength
glucuronl&s.l
through
Ae:ft,
in
activity
about by uny
thare
covalontly m -0.6.
long
CD coming from BR-IX
(1)
include:
linked
optical
are
lnducod
A,--= .,,
blllrubln
brought
and rni~~ll~x,~* last
lntwua
ZHANG
CO.-
wall-defined
of BR-IX to adopt
1. Circular Dichrofum (CD) and Ultraviolet-audible (W) SpectralData for 1.0-1.6x lo-' tlSolution8of Billrubin Mono- and Bis-Amides Fornad froa S-(+)-Alanine Hethyl Uteri
%bie
W
CD Amide
Solvents
423
-11.9 (401) -19.9 (395)
49.000 56.600
14469 (443)
423 623
+17.5 (448) +34.7 (444)
49,000 60,000
(446) (442)
-33.3 (403) -49.5 (395)
423 427
+49.7 (451) +8L.9 (645)
46.500 59,000
(440) (442)
-26.5 (402) -54.3 (395)
421 415
GO.7 (650) i83.8 (442)
SO.0~ 38,600
(438) (437)
MeOH
-13.6 (401) -41.4 (403)
423 623
+24.0 (452) 60.2 (452)
46,500 57,100
(446) (443)
n4,
* 2.4 (405) * 2.3 (399)
424 417
+ 6.0 (458) +x1.1 (452)
50,200 38,900
(453) (4%)
4i&N
Df HOZtO
425
+27.5 (452) G7.9 (450)
Di UOnO
-20.2 (402) -40.0 (401)
(A* I=)
Dl EfO!%O
hr..,(&)
c&t
e_
ns, co
t4OllO
AZ at At-0
Aa_
so
Di
(refo. 25,26) of 3R-IX with L-aIenine methyl ester. Spectra V~(I run at 21-C within 0.5 hre. of solutionpreparationusing the correspondingamides of D.L-alaninamethyl aster as the (Ar - 0) baseline. h C,% - benzene, CH,Cl, - diehloroaathme, Hs,CO - acetone, HeCN - acetonicriIe, HezSO dI~tbyl~lfox~d~. P At 2.5 x 10m6 !f the va‘luerare: (Hono) Aa:;; - -32.8,A@:;; - +42.1, Ae - 0 at 427 M, (:I; - 33,800; (Di) A#:;;,- -44.8,A<:;: - t66.0,Ar - 0 At 421 im <:$I,- 52.000. A Prepared by Shioiri coupling
Irbl8 2. CircularDlchroisa (CD) and Ultraviolet-Visible (W) SpectralData for 1.2-2.5x 10" ffSoluclonrof Bilirubin-IXaMono. and Bir-AmLdesLof S-(+)-2-Butyllains at 21-C.
CEH,
+ 2.0 (415) + 1.2 (405)
439 424
* 1.0 (470) -16.3 (455)
66,900 61,200
(453) (453)
CH,Cl,
+ 2.9 (410) + 3.1 (410)
429 427
- 7.0 (460) - 8.8 (460)
57,100 63,700
(448) (448)
non0 Df
Hc,CO
‘ 0.1 + 5.1 (405)
*._
- 2.6 (460)
426
-12.4 (455)
64,000 55,900
(4439 (440)
Non0 Di
l4tCN
. 0.1 + 6.2 (405)
__* 420
. 0.1 -14.7 (455)
55,600 59.500
(445) (435)
- 7.6 (463) -27.3 (455)
50,900 32,600
(447) (446)
- 0.9 (465) - 3.4 (460)
65,600 52.300
(433) (450)
Mono
Di lfOM,
D1
Hono SDi
MeOH
Kono
i-fezso
835
+ 5.7 (tlsr +13.6 (405)
423
. 0.1
Di
mm.
430
+ 2.0 (410)
L Preparedby the procedureof Shioirl et al.
(rat-s. 25,26). Spectrawere run within 0.5 hrs. of solution preparation uefng the correeponding &aider of rmxmic 2-uinobutene aa th* (A( 0) bemlfns.
k &H+ - banzone, C%Cla - dichloroatthana,Ke,CO a acetone. M&N - acetonitrils,Na,SO diwthylsulfoxlde. k With added urea (0.18n), the CD veluas axe reducedto A?,,, - *I2.7 (410)sA< - 0 et 430 ma, c - 51,300 (446 ma). A%., - -22.9 (457).W:
e folded conformation II governed by factor8
1% sharply lowers the eonfo~t~o~l Even
partial,
H-bonding
propionic ecid
cm
otbr
thm
lntrmolacular
H-bondfng.8 but H-bond-
global enot~ minima, adding a powerful strbilfaatton.
be beneflelel,
grcupe are deprotonated,
u
es with mono-estsr~~~~ and urfd.8, 26 or when the in the bis-i~opropylamoniua32and him-tetra-n-
lIkylunonium rrlta.33 So vhen ths anidea of M-IX are formed from prfaary mains&, on* ml&t view conforutlonal stabilizationas taking phc4 throughl proptonufde N-H @'lg. 2) in place of the propionic ectd O-H (Ffg. 1).
Since the emida N-H hydrogen is lees acidic tlun the acid
O-H, the effectiveness of H-bonding fr probably IeBeened, but such secondary eaide derivatives
of
ZING
D. A. LIOHTN~~ and M.-H.
4682
bllirubln
aster*
offor
constdorably
l
or tortiaq
groxear
potential
for
stabilixlng
conformations
than
do
amides.
FTWRG 2. Interconverting, intramolecularly hydrogen-bonded lnantlomeric conforutlons of BRIX (left) bls-ribs fron 1’ amino* and (right) mono-•atar#. (Only th* c, mono-•stor is Vhan tha R group containa a chlral center, ahown. ) as in the amidea and astars of this work.
the interconvarting l tructurex tfonr appear to bo onorgy-miniThe mono- and bls-amidax
pairs
of interconverting
rho
two pyrromthenona
the
concentration
reamer
a
ara
rough
the M’ + P’
uidsx
(Tabla
amides
tha
stronger
1)
CEs probably
to
of
the
eaters
sponding
exhibit
larger It
dfaxtaroomcr factors
the
CEx of
Hera and with
of
configuration
that
one diastarsomsr
Although
acrier
(S),
large
tha alanine through
and tha amino acid,
amide CD‘.35
that
the alanina
guy be noted
srter.34
an
in diaxta-
a pure
However,
unclear.
the pyrroaathanona
lbrolute
propionic
vavclength goncrxlly
(Table
absorption
whatawr
1 vs.
Table
Importance
In fact
4).
also
acid
a
akin to that
the amidax of Table6 the
inversion
a
3).
But unlike
carboethoxy
the axtar
ester
th*
data
of
or
(Fig.
of
the
that
carbomothoxy
acid
Tablax
esters,
CD CEs for
mono-uide,
do not
hero
And like
bisignate
the L-alanyl
carbomethoxy
lactic
H-bonding,
2).27*31
exhibits
in aolvsnts
the amino acid’s
to tha CDs of
intramolecular
group
ethyl
to be Iargcrt
influonca 2),
acid
acid
(Table
tonding
partial
retain
carboxylic
from L-lactic
weaker,
tha
his-amides, Although
*stars.
do not
achlavc
this
ing
solvent
further
tends
to dimorira
to
pigment
Our understanding
with
of
show l much graxtcr
tions
are
rtabilixatlon
(amino acid)
sama (amino)
cm-l.
for
algnificxntly
methyl
ACj’(P’),
l
vi11
of
itx the
the mono-
interfere
vlth
H-
group might have on the
group36
apparently
dosa
3 and 4 are vary
similar.
not
sac-butyl
cf. the counterpart
and the signad
of the CEs is not reversed.
Unlike
ovar
between
biliverdinoid
tha mono-eater
(Table
2)
a greater
mono-amides , mono-esters
assume a similar
order
interaction for
Furthermore,
CDs
reflect
(Table
xolutionx
valuer
as
diapoaitfon
the CEs is unexplained.
long
CEs are
amides
the
the amides of L-tryptophan
amide analog.
amida
gonerats
may thus be vlwad
Large diffarencw
with
IAel - 260 K’
to
conforu-
have AC/‘(H’)
and the
diastsreomar.
sac-butyl
the
bonding.
limlt,12* xo u
?‘,
CEs,
ptgant 8).
have an snantiomerlc
typically
of
large
for
the underivatizad
pigment’s
ylsld
the
to account
Like through
ester
theoretical
that
the pradomlnant to
that
dfarteroorrs to
vs.
large
order
Since
2)
obxawad
1 and 2 belong signed
of
Fig.
?‘,
be aqua1
expected
lquflibrium
dipole-dipole
suggastad
not
charactorixtic
affect
CEs are alro
will
BR-TX MBA, BR-IX BAA. BR-IX BM)
(H’
chromophoras .
ll’
concsntratlon
approaching
(BR-IX HM,
diastcrsomws
of
lctlvlty
optical
are conformational dlaxtersolerr. The folded structurox even without the H-bonding (ref.
the
range
xs those
diexter my
CEs are
through
tha CD of
2.
If, v.
of
intermolwular
l nsuma
that
than of
those
of
Figs.
1 and 2,B thay
and
in non-polar
pyrrorthanonc-pyrromathanono
than
their
complicated.
do mono-esters,
corre-
H-bond-
aolventr
tha
H-bonds.6a137-39 Since
mono.amldes
CD may not be derived
tha
In &SO.
stabiliratlon.
H-bonding,6c
is corraxpondingly
dapcndsnce
vaaksr
conformationa
H-bonding
full
intramolecular
dieters
concantrxtion
considerably
x~sume the folded
the added benefit
dcxtabilixax
10e’-10V7 in Fig.
the
diesters
entirely
the dl*at@r or
CEs
his-amides
from conforma-
of bilirubia
Confonnrtion
(W) Spactrd formed from
Clrcul~ Dichroism (CD) lnd Ultrwiolot-Visible )I Solutiolv of Billrubin Nono- md Di-Estws Estma
Tab18 3.
Aa_
4683
xmidu
CD A1 xt At-O
W (1,
(453) (450)
50,600 52 ( 300
(442) (398)
-43.1 - 3.9
(455) (450)
SB,200 57,400
(430) (396)
428 412
-15.3 - 2.1
(453) (450)
57,100 58,200
(436) (402)
(405) (400)
426 420
- 6.9 - 2.6
(455) (450)
51,200 52,800
(415) (394)
(403)
424 ___
- 6.7 - 3.7
(453) (455)
63,100 53,200
(450) (458)
_-_--
- 3.1 - 4.3
(455) (455)
50 ( 200 57.000
(452) (450)
AC,,(&)
Solvm*
non0 Di
C,H,
+13.9 t 2.1
(405) (400)
428 420
-18.1 - 3.5
bSOll0
%%
t30.9 + 2.1
(405) (405)
428 417
co
+10.3 + 1.0
(405) (400)
Mono Di
n*CN
t 5.2 t 1.0
l4OllO
MeOH
+ 3.0 l 0.1
ne, so
‘ 0.1 a 0.1
IhI
no,
Di
Di Mono Di
1.7-3.9 x loAcid Ethyl
SIrnI
0 1)
Ester
Di
kta for S-(-)-Lctic
m)
L Prepared by SN2 romztion of BR-IX his-tmtra-n-butyluoniur salt with the p-toluenwulfomto of S-(-)-lactic mid *thy1 *star. Spetra were run at 20-C within 0.5 bra. of solutior, prepmation using the corresponding estera of racoric lactic acid ethyl lstor as tha (At - 0) bmallne. CH,Cl, - dichloromthum, HeCN - ~cctonitrll~. MoOH - methmol. Ha,SO k CoH, - bmumm, dlmthylsulfoxid~.
T&lo
4.
Dichroism (CD) ld of the Bilirubin-I&a
Circular Solutloru
(W)
Ultrwlolet-Vlsibla Nono- ld Dl-esters
Spectral Data for 2-5 formed from S-(+)-2-Butmola
CD X, at At-0
Solv*ntk
Mono Di
C,H,
+12.6 t 1.1
(405) (400)
424 424
-21.2 - 1.5
Mono Di
%CIl
+10.2 + 1.3
(405) (400)
424 421
-15.7 - 1.7
Mono Di
nb, co
t 8.2 t 0.6
(405) (400)
423 420
non0 Di
nmCN
t 3.4 . 0.1
(405)
HmOH
+ 1.3 t 1.0 = 0.1 + 1.4
Mono Di
Me, so
I Prepared by SN2 ramtion S-(+)-2-butmol. Spctra cormspondinS lstora of -Cl, c C,H, - bmxmw. diwthylsulfoxid~. - tl.1 (500), Ar ’ At..=
Excfton
lqulwlent
Electrortatic m lxciton
48,000 34,500
(438) (402)
(450) (450)
54,300 33) 700
(430) (398)
-12.2 (450) - 0.86(450)
60,000 34,500
(438) (406)
422 ___
- 5.2 = 0.1
(450)
35.500 37,700
(420) (400)
(400) (410)
423 ___
- 2.2 . 0.1
(450)
51,400 35 (400
(448) (450)
(415)
___ ___
56, SO0 32,400
(454) (453)
Absoluta
&nffgur8tron.
the
heteramsociation
uinee12
that
act
of the
the
sltution
wtth
comploxo~
of BR-IX with
chiral
lgonta
alao
comploxing
pigmmt hold
lxcitmd
uy
in
l
chlml
llmtric stat*
bm viewed
to
mono-•tors.
attached
pyrromthmono
molmculm
Iho
ia covalmtly
as chiral
chromophoros
intwmtlon of
porturber
thm bichromophoric
pyrrouthmnono
splitting
0.1 = 0.1 l
0 at 460 IU.
of
cm*,
A*..=(&)
of BR-IX bia-tetra-n-butyluonim salt with thm m~thmm~ulfon~to of were run at 20-C within 0.5 bra. of solution propw&lon using the racomic P-butmol ls the (be - 0) baseline. - dichloromothmm, MoCN - metonitrile, MaOH - rthmol, k,SO -
chiral
ad
two
(450) (450)
the
In llthm
taining
1 .\I&
whom to tit
.lbwfnlo*ll mmt.
uxf
couplfiy
and bls-uidm,
,_._
A*..= 0,)
n
W
Eater
Mono Dl
x 10”
the
md
dipole
trmuition
in two long
mono-ui&a uy
agents induce
as . moloculm
orlontation,
resulting
pignnt
l
s
In
bc seen
such CD of
l
thm plS-
.xciton30 Fig*.
mommts46 wmwlongth
m
se-
con-
1 ld
2.
leads
to
llectronic
4684
D. A. L~o~nrar md
ihANG
in ltmrgy lnd on* lover in anorgy, with the lw*r*tion
tr*nsitions, one hi&*r rol*tiv* oriont*tion
M.-H.
*nd ltrongth of
th* tr*nrition mom*nta.
Accordi-
Qpondont
oxciton tr*naltiotU llw*yr hav* oppo*it*ly-*ign*d CD CE*. typic*lly fl*nklng the W-vi* *itfon(*),12s30 l* la ob**rv*d (Pig. 3). t*r*onr
can b* predicted
The *b*olut* confiyr*tion
tr*ruitlon mollsnt.l**
Thus, BR-IX MM
*nd BR-IX BAA lr* predicted
to hav* *n *xc*** of the pi&ment with ? h*nd*dn***, vhsr***, BR-IX HBA, BR-IX BM, HBE are
tr*n-
of th* prodominant dl*a-
from oxciton coupling theory *nd * knovl*dg* of th* pyrrom*th*none
long v*v*lonlth W-vi*lbl*
lnd BR-IX
on the
to th*~ry,~O the tvo
prodlctsd
to h*v* ln *xc*** of th* pign*nt with II h*nd*dno**.
lnt*ruity of th* CE *nd using th* c*lcul*t*d CE* for P or lf,l** w* l*tiut*
BR-IX HLE From th*
* dl*at*r*om*rlc
*xc*** of -138 for BR-IX HAA in MsCN and 268 for BR-IX BAA in !foCN. In contr**t, BR-IX MBA
lhov* e**sntl*lly no dlut*riom*ric
*xc***, lnd BR-IX BM,
BR-IX NLK and BR-IX blBE show only
-28 di*rt*r*om*ric *XC*** in XoCN lolvont.
FIUIRE 3. Circul*r dichroi*m *nd W-vi* lp*ctr* of 1.5 x 1O-5 W lolutiona of BR-IX mono-amida of L(t)-al*nin* m4thyl e*t*r (-). th* bl*-amida (- . . .). BR-IX monol*t*r from L-(-)-l*ctlc *cld *thy1 **tsr(- - - -) lnd dl**t*r (-e-.) in Iho mono-•*t*r M&N run lt 20-C. The sCUN* I* ugntfled x 3. lh*p*d CD curve* ltr*ddl* th* Ae - 0 lln*, which corr**pond* to th* b***line run on ‘amplo* uda from D,Lll*niru mothyl l*t*r *nd D.L-l*ctic rho w-vi* lp.clcld ethyl l*t*r. tr* *how l v*lru* lncr**ring from ths bottom of the Figure, the t-O b*rellne.
A. “rn
lith*r of
to l**-
BR-IX ton&
two folded.
lntramol*cul*rly H-bonded conforutlona
th*t
interconvert by br**king *nd ramakin& lll 6 H-bond* (Fig. 1) lnd luy thu, bs l**n ** * r*c*alc mlxtur*.
Uh*n th* conformation-d*t*rmlnlng
l*con&ry
mid**,
lntruolscul*rly
propionic lcld c*rboxyl &roup* lr* &rlv*tir*d
H-bonded conform*tlon* may *till be r*t*inod
l*
(Fig. 2), but
when the amide amln* mol*ty PO******* l chlr*l c*nter. the *r*tvhlls anantiomerr become dl*rt*r*om*r*, lnd th* mixturo is no longer r*c*nic. ruco*r*rlly
l:l,
[H'] +
nor* importantly, the squillbrlum i* no long*r
[P'], *nd th* optic*1 lctlvlty **socl*t*d wlth
th* mixture
c*n bs
expsctod to *how bi*ign*t* CD CEa *r*ocl*t*d with sxciton coupling of th* colpon*nt pyrromcth*th* intriruic CEa lr* typlc*lly v*ry l*rso for ths snantlom*rlc
non* chromophoro*.
Since
conformor*. modor*t*
dl*pl*c*montr
lr* obrerved
from
a
1:l lquillbrium will r**ult in lub*tantl*l Cl!*, l*
for th* bi*-amide* with L-*l*nin* methyl **tar.
at111 r**ult in *lgnlflc*nt CE*. ** *r* ob*ow*d l*rly, mono-ami&*
Ev*n *m*ll dl*pl*c*m*nt* will
for th* bl*-amid** vith 2-amlnobut*nr.
Slmi-
*nd mono-•*ter*, which *till r*t*in lt l***t 501 of th* lntruol*cul*r
H-
bonda through th* undorfv*tlred proplonic *cld c*rboxyl group, c*n lxhlbit conformational di*lt*r*om*ri*m vh*n the amino or *lcohol portion cont*lns l chlr*l conter.
tlons such
u
those
of
Fig.
Hero. too, conforu-
2 lppe*r to b* l**onti*l in undar*t*ndlng th* orl;ln of th* bi*lg-
rat* CD*.
Aci.l¶oulodg-t. U* thank th* N*tional In*titut** of H**lth (HD-17779) for &*n*rour *upport of thi* work.
X-H. Zhans vu
i*try, Ac*&ml*
* vi*ltlng *chol*r on le*ve from th* Inrtituto of Photosr*phic Ch*m-
Slnic*, B*ijing.
4685
All tircul~r dichroisr (CD) epactrs vere run on 8 JASCO J-40 inatrumant lquippad vitb calerd: and all w-as spectra var. run on a cary 219 lpectropBotomat*r. All photoelastic modulator, HI(R spectra vare run on m 13~ RR-8DAF spectrometer in either de-dinathylaulfoxide (Aldrich, All IR spectra vet. obtainad on . Perkin99.98 de) or deutoriochlorofora (Y9.9r. Aldrich). Optical rotations, [a],,, , waxsoobtained on a Perkln-Elner model Elmer model 599 iwtnnt. RoIting points were datermined on a Nel-Toap capillary unit. Combus141 rpectropolaxi*tar. Analytical thin layer tlon sticr~a~lysu vere obtainad from Dasort Analytics, Tucson. AE. chroastography (TLC) vaa carried out using Analtech Unlplatar (ailMa, ZSOr); preparativa TLC Column chromatography vaa was carried out on Analtach Uniplates (silica, 500~ or 1000~). HPLC analyses were carrted out achieved using neutral alurinur oxide (activity II-III. Uoela). on a Per-kin-Elmer Sories 3 liquid chroaatograph equipped with an Altex 5~ ODS C-18 ultcasphera ion psiring column (25 cm) win& 0.1 W di-n-octylaaine ncetate in St aq. methanol as eluant The retentfon times (min.) found (flow rate 1 .O al/s&n) and a column temperature of 23’C.‘” BR-IX. 20; BR-IX BM, S; BR-IX NBA. 9; BR-IX BAA, 4.3; BR-IX t&A, 8; BR-IX BI)E, 9; B&-IX were: ffBE. 11; BR-IX BLE, 5.5; B&k-I&a KLE, 9. Biiirubin-IXo vas from Porphyrin Products, Logan, WI, and contained lerr than Sq of the 1110 and XIIIo isomers ae determined by HPLC. S-f-)-Iactate acid ethyl eeter, [a]:* -12’ (naat), diphenyl phosphoryl azida, oethenesulfonyl chloride, pterra-n-butylammonium hydroxide and triethylamlne were from Aldrich. toluanesul fonyl chloride, [a],+13.6’ (neat) were from Norse S-(t)-2.Aminobutane, [a]s+7.4” (neat) and S-(+)-2-butanol, S-(t)-alanine methyl eatar hydrochlorida vas from Sigma. Pyridine was dried by dfstilLabs. lation from KrCO, snd di~thylauIfoxf& was dried by distillation froar 3k molecular sieves. Bilirubin_IXa (BR-m. BilirubinS-(+)-alanine aethyl ester hydrochloride (14 mg, b-1 moles), IXa (29.2 ng, 0.05 amoles), diphenylphosphoryl lzlde (11 ag, 0.1 amoles) and triethylrmlne (20 mg, 0.2 mmoles) were stirred In 1 IL of dry MeaS in a 3 mL glass vial at room temparsture in the dark under nitrogen for 1 h. An additional 14 mg of almfne methyl ester hydrochlorida, 11 ag of diphaylphosphoryl astirred at side snd 10 mg of triethyl amina were then added, and the mixtura was continuously roofs temperature in the dark under nitrogen for 3 h. HPLC malysfs indicated that 40% of the mono-amida and 46* of the his-amids vere formed and that 141 of FIR-IX was unreactad. Ihree milliliters of dichlororthana was then added snd the solution vas vsshad vith cold water (3x1 Then tha dichloronethene solution was applied to a column (10 cm long mL) and drled (NasSO,). x 2 co intarnal diemeter) of neutral alumina and aluted with dichloro=ethanc (200 paL) to remove BR-IX-BAA vas then eluted with dfchloro~ethsne/~tha~l unresctad arfde and axcesa aminas. After evaporation of the solvent. 5 ng of the his-aaide (IS% yield) was (2O:l v/v) (200 SL). obtained. The l lwina column was then cluted with dichloromethane/methanol/aswonium hydroxide (3Ot aq.) (80:20:2 v/v/v) until an orange brnd of the mono-•mldt scparrted from a small amount of unrescted BR-IX, left at the top of the column. The alumina vas extruded and the mono-amide band was excised and extracted to give 11 mg (30t yield) of BR.IX HAA. It had dec. >2OO’C; [a];;, - +420* (c 0.02, CRsC1,); IR (KEr) V: 3430. 3350. 2930, 1750, 1690, 1625, 1450, 1260, 1000, 710 ca-1; ‘II (12H. 4xCH,), 1.20 (1.55) fd, 3H, CR, J - 7 Hz), 1.95-2.12 (CDCI,) 6: 3.51 (3.75) (a. 3X, CR)), 4.02 (s, 2X, CHt), 4.17 (4.26) 2.6-2.78 (n, 8H, 4x (q. Hi, CR, Y - 7 Hz), 5.24-6.78 &IH, -CR), 9.23 (d, lH, O-C-NB-, J c 7 Hz), 9.34 (s, IH. pyrrole NH), 10.06 (9.91) (s, lH, pyrrole NH), 10.69 (s. lH, lactam NH), 10.79 (s. 1H. lactam NH) ppn. AD&. Calcd for C,,H,,N,O, (669.8): C, 66.35: H. 6.47: N, 10.66. Found : C. 65.96; H, 6.51: N, 10.14. The b&s-amide had a.p. 137-14O’C; [a]$:, - +370’ (c 0.02, CH,Cl,); IR (KBr) Y: 3430, 2930, 1750, 1680. 1660, 1640, 1550, 1460, 1260. 1220, 1175. 1000, 710 cm‘;; IH-NHR (CDCL,) 6: 1.38 (d, 6H, 2xCH, J . 7 Hz), 1.73-1.97-2.10 (12H, 4xCH,), 2.99-2.83 (P, BH, 4x(&). 3.72 (s. 6H. (in. 8H, -CH). 10.13 (d. 2H, 2x+ ) , 4.12 (s, 2H, CH,). 4.66 (qs 2H. 2xCH, J - 7 Hz). 5.20-6.77 O-C-N!?). 10.40 (s. lN, pyrrole), 10.70 (s. 1H. pyrrole NH), 11.03 (s, 1H lactam NH) ppn. &l&. Calcd for C,,Hr,N,O, (7S4.9): C, 65.24; H, 6.68; N, 11.13. Fwnd: C. 64.88; H, 6.62; N, 10.75. -t -XX t%dQ: Bilirubin IXo (29.2 mg. 0.05 moles), S-(+)-2aminobutane (7.; mg, 0.1 Imoles). diphenylphoaphoryl axide (11.6 ng, 0.1 maoles) and triethylamine (10.1 mg, 0.1 moles) vere stirred in 1 mL of dried Re,SO in a 3 mL gloss vial at room tcmperrture under nitrogen in the dark for 1 h. Another 7.3 mg of 2-aainobutane and 11.6 mg of dlphenylphosphoryi azfde vcre then added and the nfxture wes stirred continuously at ro01p tamperdture under nttrogen in the dark for 5 hrs, HPLC analysis indicated that 38% of the 1w)noamide end 44* of the bfs-aide was formed and that 18% of BR-I&a was unreactad. Three ailliliters of dichlorowthans vas than added. solution vaa vashed with cold vater (3x1 ML) and dried over sodium sulfate. Ihsn the dichloroaethanc solution was lppllad to a column (10 C= long x 2 cm lntcrual diameter) of neutral alumina and eluted with dichloromethana (200 mt) to remove atide end axcess amine. A small amount of BR-IX MA was elutad with dlchloromathane/ncthanol (20~1 v/v. 200 sL). Elution was continued vith dfchlorocw:thans/rthinol/~nius hydroxide (3Ot aq.1 (80:20:2 v/v/v) until an orange band of the mono-amide vas sapareted froa a small band of uaructed BR-IX AC the top of column, Tha alumina was then extruded from the coluan, and the band corresponding to BR-XX NBA vas excised. The purified &no.-amide vas vashed froa the alumina with dichloromethen~plethanol/gleeial lcetlc ecid (98:l:l v/r/p). The acidic dichloromthane eluate wss vashed vith water to pH=7, dried over sodium sulfate and evaporated at room temperature to give the mono-amide, 10 mg (27% yield) with rsp, 180-3’~ (dec.), (Caraful daoxygenation of all of solvents and exclusion of light Era the chrovatography rstardad product isorserization.) BR-IX KM had [a]j;s ; -366’ (CHsCl~); IR (Wr) V: 3410, 3340. 29B0, 2920, 1680, 1645, 1615. 144S, 1250, 995 cm ; H-NHR (CDCl,) 6: 0.93 (t, 3H, CH, .I - 6 Hz). 1.24 (d, 3H, CH, J - 6 Hz). 1.54 (q. 2H. CH,, J - 6 Hz). 1.96-2.16 (MI. 4xCR,),
4686
2.32-2.79 (m, 8H, 4xCH,), 3.44 0.5 H, O-C-NR-,cfs, J - 6 Hz), NH), 10.26 (6. lH; pyrrole NH), &&. Calcd for C,,H,,N,Os
D. A. LKMTNLTR and M.-H. ZHANG
(m, lH, CH) 4.04 (a. 2H. CH,). 5.27-6.78 (a, 8H, -CH), 8.76 (d, 8.84 (d, 0.5 H, O-C-NH-,crans, J - 6 Hz), 9.39 (s, 1H. pyrrole 10.71 (a. lH, lecternNH), 10.82 (s. lH, lecternNH) ppm. (639.8): C, 69.46; H, 7.10; N, 10.95. Found: C, 69.45; H, 6.70; N, 10.68.
IXa Bis _2 _ku$&Ag& (BR-IX m Bilirubin-IXa (29.2 mg, 0.05 molea) S-(+)-2aminobutane (36.7 mg. .0.5 moles), diphenylphosphoryl uide (58 mg. 0.5 -10s) and'triethylamine (50.5 mg. 0.5 moles) vere stirred in 1 EL of dried Ue,SO in l 3 mL of glees vie1 et room temporeture in the derk under nitrogen for 24 hre. .HPLC enalyeis indicated thet the BRA-IXwee complstely,,convertod to the bie-amide. Three milliliters of dichlororsthane was then ldded, and the solution was vashed with cold water (5x1 mL). dried over sodium sulfate ld evaporated to drynoes. The residue wee washed with diethyl other (anhydroue) (5x5 mL) to extract the yellow-orange BR-IX BRA, 17.6 mg (508 yield), m.p. 204-206'C (dec). It bed [o]lip - -512' (CH,Cl,); IR (RBr) Y: 3340, 2980, 2920, 1680, 1635, 1445, 1255, 1170. 995 cm-l; 'H-N?IR (CDCl,) 6: 0.93 (t, 6H, 2xCH,, J - 6 Hz, 1.24 (d, 6H, 2xCH, J - 6 Hz) 1.50 (q, 4H, 2xCHI, J - 6 Hz), 1.96-2.16 (12H, 4xCHs). 2.32-2.79 (n. 8H, 4xCHs) 3.41 (m, 2H. 2xCH). 4.02 (a, 2H, CH,) 5.216.66 (m. 8H, -cm), 8.7 (d, lH, &C-WH-,cfs. J - 6 Hz) 8.8 (d, lH, O-C-NX-,trans, J - 6 Hz). 10.35 (s, LH, pyrrole NH). 10.35 (II, 1H. Lactam), 10.87 (s, LH, pyrrole NH). 11.19 (s. lH, lecternNH) ppa. u. Calcd for C,,H,,N,O, (694.9): C. 70.86: H, 7.83: N, 12.09. Found: C, 70.56; H, 8.14; N. 12.18. IXn plpllp-end Df-es ters (BR-w and BR-IX pBEz: BilirubfnIXo (29.2 mg, 0.05 -10s) end 1% lq. tetre-n-butylemonium hydroxide (2.6 mL, 0.10 rmoles) were stirred in l glsss vial at room tempereture in dark under nitrogen for 30 min. The re*ction mixture vu then evaporated to dryness et 40-C. Then S-(+)-P-butenol aethoneaulfonate (30 ng, 0.2 mmolce) and 3 mL of dichloromethane vere added to the residue of BR-IXp bis-tetra-nbutylmonium propionate salt, and the dichloromethane solution wee heated et reflux for 14 h. HPLC enelysie indicated thst 178 of dieeter. 73% of monoester and 101 unknown impurity were formed. The dichloremethene solution vas cooled, washed with veter (3x1 mL). then dried over sodium eulfste. The dry dichloromethane solution was then applied to (t column (10 cm long x2 cm internal diameter) of neutrel alumfna with dichloromethane as aluant. Unreacted methanesulfonate was eluted first. BR-IX DIE van eluted next vith dichloromethene/methanol (2O:l v/v) and purified by preparative TLC on silica gel (1 rm layer) by developing the chromatogram with dichlorowthane/osthanol/glaciaL acetic acid (98:l:l V/V/V). Ihe purified diester (R, -0.3) was eluted from the silica gel with dichloromethane/methanol (2O:l v/v) to give -2 qg (yield -68). To isolate the mono-ester, the alumina column was continuously eluted with dichloromethane/mathanol/-nium hydroxide (308 aq.) (80:20:2 v/v/v) until an orange band of BR-IX MBE was separated from a small bend of unrcected BR-LX bis salt, which remained at the top of column. The aluaina was then extruded from the column, and the band corresponding to the mono-ester was excised. The purified mono-ester was washed from the alumina vith dichloromethane/methanol/glacial acetic acid (98:l:l v/v/v) and the acidic dichloronethane eluate was washed with wster (2x5 mL) and dried over sodium sulfate. The solvent vas evaporated at room temperature to give BR-IX RBE, 14 mg (438 yield). (Pigment isowrixation was retarded by excluding Light.) BR-IX XBB had mp 169-171'C; [a]$ip - -692' (CH,OH); IR (XBr) Y: 3400, 3340. 3250. 2940, 2910, 1725, 1695, 1655, 1625, 1450, 1360, 1250, 1170, 990, 690. cm-‘; 'H-NNR (CDCl,) 6: 0.89 (t, 3H, CHS, J - 6 Hz), 1.01 (d. 2H, CH,, J - 6 Hz), 1.25 (n. 2H, CH,, J - 6 Hz), 1.95-2.12 (12H, 4x'+), 2.59-2.77 (m, 8H, 4xCH2), 3.97 (s, 2H, CH,), 4.84 (m, H. CH), 5.42-6.80 (m. 8H, 9.27 (s. lH, pyrrole NH), 9.30 (8, LH, pyrrole NH), 10.70 (8, lH, lactam NH), 10.80 (s, -w. lH, lactam NH) ppa. m. Calcd for C,,H,,N,Os*HsO (658.8): C, 67.45: H, 7.04; N, 8.50. Found: C, 67.76; H, 6.62; N, 8.08. BR-IX DBE had mp 123-127-C; [o]:;, - -995' (CH,Cl,); IR (XBr) Y: 3340, 2980, 2930, 1730, 1670, 1630, 1450, 1370, 1260, 1170, 990, 690 cm-i; 'H-NHR (CDCL,) 6: 0.89 (t, 6H, 2xCH, J - 6 Hz). 1.20 (d, 6H, 2xCH, J - 6 Hz), 1.50 (q, 4H. 2xCH, J - 6 Hz), 1.76-1.98-2.09 (12H 4xCH,), 2.31-2.81 (m, 8H 4x%), 4.18 (s. W, CH,), 4.88 (n, W, 2xCH), 5.32-6.54 (m, 8H. -CH), 10.10 (s, LH, Loctam NH), 10.11 (s, LH, pyrrole NH), 10.45 (6, 1H. pyrrole NH), 11.18 (8, lH, lactam NH) pp=. w. Calcd for C,lH,zN,Os (696.90): C. 70.66; H. 7.52: N, 8.04. Found: C, 70.38; H, 7.22; N. 7.93. and w (BP-v. Bilirubin-IXe (29.2 py, 0.05 -10s) and la lq. tetra-n-butylarmn im hydroxide (2.6 mL, 0.10 -10s) were stirred in a glass vial at room tenperature in dark under nitrogen for 30 min. The reaction mixture was then evaporated to dryness at 40-C. Then the tosylate of S-(-)-lactic acid ethyl eater (54.4 mg. 0.2 moles) and 3 mL of acetone were added to the residue of BR-1%~ his-tetra-n-butyl -niw propionate salt, which was not isolated and purified, and the acetone solution was heated at reflux for 12 h. HPLC anelysls indicate that 46% of di-ester, 44t of mono-ester, LO1 of unreacted BR-I&a bis-salt vete formed. The acetone vae evaporated. and 3 mL of dichlorolpethane VU then added, and the solution v.ss washed vith water (3x1 mL) then dried over sodim sulfete. The dry dichloromathane solution contained mono-, his-ester and BR-. IX bls salt, which could be separated and purified by column chromatography on alumina, followed by preparative TU on stlioa gel as described above to give 7 mg of pure BR-IX DLlI (18% yield). It had mp 78-82-C; [e]$is - -355' (CHsCl,); IR (RBr) Y: 3420, 3270, 2930, 1750, 1700, 1665. 1635. 1450, 1260. 1000, 710 cm-'; 'H-NNR (CDCl,) 6: 1.26 (t, 6H, 2xCH, J - 7 Hz), 1.47 (d, 6H. 2xCHs J - 7 HZ), 1.73-2.08 (L2H, 4xCH,), 2.53-2.83 (I, 8H, 4xCHs). 4.15 (6, 2H, CHI). 4.20 (q, 4H. 2xCH,, J - 7 Hz), 4.92 (q. 2H, 2xCH, J - 7 Hz), 5.2-6.72 (n, 8H. -CH). 10.11 (6,
Conformatian
of bin
4687
amides
1H. lactaa RR). 10.14 (a. l.R, pyrrolo NH), 10.44 (s, 1H. pyrrola NH), 11.15 (a, lH, lactu) PP.. hpaz. Calcd for C,,H,,N,O,, (784.9): C, 65.80; H, 6.68; N, 7.14. Found: C, 65.78: H. 6.81; N, 7.43. It had rep llO'-113'C; [a]::, The purified BR-IX KLE (10 qg) was isolatad (29r yield). ;; 3400, 3000, 2930, 1750. 1680, 1660, 1640, 1460, 1270, LIOO, 1000, I.23 (d. 3H. CH,, J - 7 Hz), 1.47 (t. 3H. CR,. J - 7 Hz). 1.972.13-2.43 (12W, IxM,), 2.81 (m, BH, 4xORe), 3.98 (=. 2H, CR,), 4.02 (q, 2H, CH,, J - 7 Hz), 4.93 (a. lH, CH, 3 - 7 Rr), 5.27-6.79 (o, 8H, -CH), 8.04 (8.14) (6, LQI, pyrrole RR), t.89 (8.89) (a, 1H. pyrrole RH). 9.10 (9.10) (8, LH, lactam NW), 10.69 (10.78) (1, lH, lactam RR) PPm. &J& Calcd for CI,H,,N,OI (684.8): C, 66.65: H, 6.48: N, 8.18. Found: C, 66.45; H, 6.80; N. 8.68. ;~2~h_~q~cRLa)&It"~C~~;
-._ &~cfd4~ Ethyl S-(-)-lactate (4.00 I, 0.034 moles) vas dissolvad .in pyrLdina (40 mL) and eoolsd tn an iea-bath to -5'C. p-~ol~na~lf~~l ehlorida (8.00 8, 0.042 wlrs) was added tn small portions over a period of 15 min. and the raaultfng light yellow aolutlon was kapt for 24 h. at -20'C. Small portions of ica (1 g, 2 g, 2 g) vare then added, vith a period of 15 oin. betvaan additfona, insuring that tha tmperature remained near 0-C. 'The solution vaa then poured into ica vatar (100 ML) and extracted with chloroform (3x25 rL). The chloroform extracts vara dried over anhydrous sodium sulfate end evaporatad. l'ba syrupy residue was taken up in hexane-dichloromath~~ (lO:l, 80 mL) and crystalliaed by rapid cooling in a dry ice-acetone bath to yield 7g (7% yield) the tosylate. Recryatallir.ation from haxane-dlchloromathane (10:lf at -2O'C afforded colorlesa needles, 5 g (54% yiald), mp 30-32'C; [a]:il - -49.5' (CH,Cl,); 'H-RhR (CDCl,) 6: L.21 (t. 3H, CR,) 1.50 (d, 3H. CH,) 2.46 (a, 3H. CH,) 4.10 (q, 2H. CR,). 7.30 (d, 2H. Ar), 7.78 (d. 2H, Ar) ppm. S-(+)-2--:43 S-(t)-2.butyi alcohol (0.74 g, 0.01 moles) uaa dissolved in cold dichloromethana (10 ot) and cooled in an ice bath (O'C). Pyrtdfna (1.62 sL, 0.02 molas) was then added. followed by the addition of nethanaaulfonyl chloride (1.72 g. 0.015 moles) in small portions with constant stirring at the ice bath (
1. D.A. Lightnar and A.F. HcDona8h. Accounts Chem. 2, A.P.
HcDonagh in The (L979), pp 293-491.
Vol. VI
Porphyrins.
3. A.F. McDonagh and D.A, Ligbtner, Pedfatrics 4. R. Brodersrn in Bile Pigments York (1986), pp 157-181. 5.
(a)
It;
cc>
fn Bilfmbtn, Racon, FL (1982), pp L-58. (a) (b) (c)
(b)
Press, New York
75, 443-455 (1985). (3.D. Ostrow,
VoJ
I
hares1 Dekkar. Inc., Nav
ed.),
(K.P.R. Helrwegh and S.B. Broun, eda.), CRC Press, Boca
Cham.
Sot.
Cheat.
Cw.
122-123 (1976).
Loading references, sea G. Blauer. Isr. J. Chem.
11. D.A. Lightnar, tI. Raisfngtr and C.L. Landen, J. 12. (a)
ad.), Academic
H. Falk and N. Rilller, Honatsh. Chem. 112. 1325-1322 (1981). H. FaLk and N. hOllcr, Tetrahedron 39, 1875-1885 (1983). R.V. Parson and D.A. LiRhtner, unpublished calculations.
9. P. Nanltto and D. Xonti. J.
10. For
(D. Dolphin,
For leading refercncas sea D. Kaplan and C. Navon, Irr. J. Chem. 23, 177-186 (1983). G. Navon. S. Frank and D. Kaplan, J. Chem. Sot. Parkfn Trans. 11, 1145-1149 (1984). D. Kaplan and G. Navon, Blochem. J. 201, 605-613 (1982).
7. D.A. Li@ner
8.
Jaundice
17, 417-424 (1986)
R. Bennett, J.E. Davies, N.B. Hursthouae and G.U. Shsldrick, Proc. R. Sot. London Ser. 1202, 249-268 (1978). G. LeBas, A. Allsgret Y. Maugutn, C. D&an&o and K. BaiLly, Acta CrystrlIogr. B36, 3007-3011 (1980).
(b) 6.
and
Res.
BfoJ
23. 201-209 (1983). Chm.
261.
D.A. Ltghtner, J.K. Cavrohski and W.R.D. Wifekoon, J. (1987). D.A.
Lightner and J.Y. An, Tetrahedron
43.
4287-4296
6034-6038 Am.
(1987).
Chsra.
(1986). Sot.
109, 6248-6259
D. A. Lw#fmm and M.-H.&ANC
4688 13. D. Harr-Laizy,K. Lahlrl and
P.
Balm-u, Inc. J. PeptfdsProtalnRON. 25. 290-296 (1985).
14. 16.X.Turner and K.H. Ikretz,Quart. Rev, Chu. Soo., 299-330 (1947). 15. G. Blzuzr.Arch. Bfoche~.Bioph~s.661, 602-604 (1986). 16. That BR-IX can &ve zn ICD in the proxenca of (+) or (-)-a-phonylethyluinain orSznic lolvmtz haz boon cited by Dlawr tn ref. 10 and again in l dlrcuzrionfollowIn&z lecture by G. Blzuer. and E. Lavla in Pmtofn-Lfgmd Imc~r8ctfons(X. Sund and G. Blauar, eds.). pp 399-416, W. de Cruyter, KY (1975), for work of 0. Kayer, Ph.D. thoafs, Univ. Konstam, (1979). 17. D.A. Lightnrr,J.K. Czweohrkiand K. Cmohzkz, J. Am. Cham. Sot. 107, 2456-2461(1985). 18. (z) For loading referoncas,me H. RsizinRerznd D.A. Lightner,J. Inclus.Phuzom. 3, 479. 485 (1985). (b) X.L. Furman end P. Balarum,Pmt. IndirnAcad. Scf. @hr. Scf.) 98, 453-468 (1987). 19. C.C. Kuonzlo,N. Citrolun-Cumerumy and K.J. Wilson,J. Mol. Chem. 251. 801-807 (1976). 20. A.P. NeDon@, L.A. Palm, J.J. Lauff and T-W. Uu, J. Clfn. Imost. 74. 763-770 (1984)and reforancm therefn. 21. M. Raisinger.T-W. Wu and D. A. L&htner, unpublizhadwork. 22. P. Umitto and D. lfonti, &xporluxia 28, 379-380 (1972). 23. U.B.D. Ufjokoon and D.A. Ltghtner.fn prrpzrztlon. 24. D.A. Lightnor.H-H. Zhmg and P.R. Trull, T8trrhsdronLet%. 28, 4033-4036(1987). 25. T. Shfoiri,K. Hinomiyzznd S. Yzudz, J. Am. Chen. Sot. 94, 6203-6205(1972). 26. D.A. Lightnor,T.C. Adacu and J-S. Ha, rremhedron 21, 4253-4260(1984). 27. D.A. Lightner,F.R. Tiull end N-H. Zhang, TetrahedronLett. 28, 1047-1055(1987). 28. C.W. Dautzcho, 448 (1969).
D.A. Lfghtner,R.W. Woody and A.
Morcowitz,
Amu.
Rev.
Phys. Chm. 20. 407.
29. D.A. Lightnet,H. Ralzingetznd U.K.D. Wijskoon,J. Org. Chem. 52, 5391-5395(1987). 30. W. Her&a and K. Nakanishf,Circular Dfchroic Spectroscopy- Excieon Coupling tn Organic Stereochm!izery, UniversityScienceBooke, Hill Valley, CA (1983). 31. D.A. Lfghtnarand J-S. UA, Spsctrorc.Lett. 17, 317-327 (1984). 32. A. Hugnoli,P. Kanftto and D. Xontf, Acta Cryrtallogr.Sec. CM. 1287-1291(1983). 33. D.A. Lightnor, J-S. Ka and X-X. Vu, SpectroscopyLett. 19, 311-320 (1986). 34. l'homono-mida of L-tryptophm methyl ezter zhov CD CEs: AC::f -12.3, A r:;; t20.8 (C#H,);Actif -46.6,A#:;; +93.3 (MoCN);Ac:cf -29.5,Art3$ +49.5 (HeOH):znd Acrf;';-19.0, Act:; t27.5 (Ne,SO). 35. K. Grubmzyrand il.Falk, !Jonxt&Chesr.118, 837-843 (1987). 36. The esters of L-lzctlcacid wthyl
l zter
ahow CD CEe within 10% of the values of Table 3.
37. A.R. Holzwzrth, t. La&or, H. Lehner znd K. Schzffner,Phocochem.PhocobioI. 32. 17-26 (1980). 38. H. Polk, T. Schledererand P. Uohlmhmn, &mtdz.
Chea. il2, 199-207 (1981).
39. D.A. Lightnorand P.R. Trull, SpectroscopyLate. 16. 785-803 (1983). 40. (4) G. Alzuer znd G. LlzmiAre.J. Am. Chew Sot. 97. 1949-1954(1975). ib) H. Folk, K. Crubm~r, G: NIllbzchzr,L. Hofe;, A. LeoQlt.r,eF. Ieuffngerl and J. Rib+, nomesh Chm. 108, 1113-1130(1977). 41. A.F. 8!cDonagh, L.A. Palm. P.R. Trull and D.A. Lightnar,J. An. Checn.Sot. IO*, 6860-6867 (1902). 42. K.N. SLessor and B.D. Johnmton,Can. J. Chem. 57. 233-235 (1979). 43. R.S. Bly znd R-A. Kaesr, J. Org. Chem. 38, 1518-1527(1973).