,- (‘0,-l<+

\
\
\~‘_., ()11
‘(It1 110
(III 011
I
(‘II:,
I
 (il SONDHEIMEK, COVITZ .&Xl) M.~RQUISEE

art also a iiumher of papers demonstrating and

that cafkine can affect, rates of enzymatic
rcact~ions; cxamplcs arc amine oxidase ((i),
muscle phosphorylnsP (i), c*holilv&ernsc (8),
and lipases (9). Whcthcr or not, the type of where f’.k = initial concentration of A, Cflnll =
complexing dealt with here also accounts for concentration of total B in t,he :queous phase. and
some of the rnzymic tft’ects of &feint: is not Cc = concentration of the complex. The partition
kno\v11. However, the possibilit,y that t,hese coefficient k = CBz;,,/C’~orr: , where CR;<, = 1m-
int~cractions nrr prwaltiitj among many wmplcscd 13 in the aqueous phase anti C’J+,~~ =
naturally occurring compounds and may 1~ concentration of B in the organic phase. Thrrc-
forr, (‘c = (“I~;,<, - , and
importnnt in chemical and enzymic reac&s PHi<, = fk,,,, - kC,,,r,

strrsscs the need for further investigations.

This wport deals with a study of the solu-
tion properties of the chlorogcnic widk~af-
feint associatioll, pH effects, and a dctcr- Thrls, 15~1. (3) permits the c:dcul:~tiotl of K from
the init,i:tl concentration of A and 13, the partition
minntion of the role of the different, portions
coefficient, and :L dctcrminntion of ~~~~~
of the chlorogrnic acid and caffcinc molcclilcs
in c~nnplt~x format,ion.
When :L romples is formed bet WPCII two mm-
ponerits in solution, a bnt~hochromic shift is frc-
quently observed. If this shift OCCII~S in :L region
where nrithcr of the components absorbs or whcrc
If a two-ph:w;e solvent system is chosen so that only one absorbs, it is frequently possible to use
both components and the complex arc soluble in spcct rophotometric data to calculate t.he equilib-
the :rqurow ph:tsr but only one of the componentjs, rium constants, rut.io of the components. and the
designated component B, is distributable to a molar absorptivit)- of the complex. hIany of the
mcasurcd extent in both phases, then the decrease combinations tested here have rshit)itrd batho-
in the :tmount of the cstract~ahlr component in chromic shifts in a region where only one of the
the organic ph:rsc is :I measure of the extent of components xbsorbed, and it. was therefore pas:-
complex formation. This decrease can bc usrd to siblr to we these spectral shifts for the ywntitn~
evalrxttc the relative complexing abilit,y of se- tive stutly of complrsing.
lccted test compounds, under standardized con- h general method for evaluating acid-base
ditions, without rcqlCring nny wsumptions as to equilibria from spect,roscopic data has been pre-
the nmntwr of complexes present in solution or sented l)y Itosc xnd lktgo (10) in which the only
the ratio of the components. WC have cxpresscd :wsumption made w:ts that t,hcre are at most two
this decrease as the amount of component B which absorbing species which obey Beer’s law in the
can be extracted under standardized conditions, concentration range cmployrd. Their trcxtment
from thr :q\Ieous phase in the presence of test yields l<ki. (1).
rompo~lnd. divided by the amounl that can br
extr:ic*ted in the absence of test compound. This
ratio is called the atraction coefficient hcrc. A
v:dur of one indicates the absence of detecl:~ble
complex formation, while a decrease in the CX- \VhPW

t,rnction coefficient below one shows increasing (1.4 = init,i:tl concentration of A, i.e. the com-
amountr of complrs. ponent which absorbs near the complex
If it is known that only one complex is present, (‘13 = init,ial concent,r:ttion of I3
in solution and that the components unite in :L ec = molar :ibsorptivity of the complex
1:l ratio, then thr partition data can be llscd to CA = molar :tbsorptivity of A
c:rlcul:cte the equilibrium const,:lnt K. The treat- 1) = ((‘.a - CC) c.4 + C‘~ec for :i l-cm. light path
ment used here is essrnti:tlly that of Higwhi and .Dk = f”.kt,, for a l-cm. light path
‘hck (2n). If components ,2 and B form complrs An = 11 - 1j.i = (‘r.le
C, then At = E(‘ - t:,
For those cases where CD >> (I, , one may often
A + B .-- C, (1) make the assumption that C”B - Cc = CB This
Ic:~dh lo I~;(l. (5) for ilt(~ cqrtilil)rilun pq~ressiotl
for :, 1 : 1 corn],1cx.
 2.4

2.2-

2.0-

I.0 -

1.6 -

0”
UJ i.o-
3
w 1.4- \

0.6-

0.4 -
(‘1ILOl~O(IEXIC .lCII)-C.bFFl!XX\‘E COMPLKS
68 SONDHEIMER, COVITZ A&-D MAI~QU~SEE

TABLE: I pound is obtained which is comparahlc in

COiVPARISOs OF THE F~TRACTIOS
AT 30” OF CAFFEINE WITH
TEST COMIYJI-XIX
‘L All acids were nrut,txlizrd
KOH.
diffcrcnt, moieties of the chlorogenic acid
molecule in complcxing wit,h raffcinc, a
number of simpler, related compounds wrc
tested with the partition procedure. These
results are summarized in Table I. Chloro-
gcnatc and its isomers, compounds l-4,
complex st,rongly with cnffeinc. On the other
hand, quinatc is completely inert. E’rom the
extraction coefkients of compounds G-9 it
is seen t,hat, a progressive increase in the
complcxing abilit,y rcsu1t.s from the addition
of a conjugated double bond and phcnolic
grollps to henzost,e until in caffcat’c a com-
COEFFICIENTS complcsing ability to that of chlorogenutc.
V.4RIOvS Compounds 12-14 show that methylation
of the phcnolic groups does not cn~w:r ap-
prrciahle alteratlion in the cxtraction cwcffi-
cients, and it, can therefore be argued that
H-bonding of the phrnolk groups with raf-
feint does not, rontrihlltr to cwmplrs stnhil-
ity. Confirmation of the significance of the
conjugat,cd double bond in t,hc enhancement
of t#hc complexing ahilit,y was ohtaiwcl by
t’esting the dihydrocinnamic acids, corn-
0.91 polmds 15-21. In every case the wmplcxing
0.80 ability was drast,icully lower than that of the
0.72 respective parcntJ compound.
0. 59 Potassium chlorogenatc will associate
0.59 weakly with 1 ,3-dimcthylura~il, cst~rwtion
0.54 cwrfficient = 0.90, hut not with 2 ,l-dicar-
bosamido - :I’ - methylimidazolc. There also
0.73
0.5’
was no observable association hetwrn 3,3-
0.5p dimttho?iyrinnarnat[~ and N-methylimiduz-
ok. lcrom this it is suggested that the
0.89 pyrimidinc portion of caffeine is csstntinl for
0.84 wmplrx formation and that an, as yet, IU~-
0.84 identified portion of the imidnzolc moict#y
0.X% iiiwrascs the extent of the associatioll.
0.X0
0.84
Spectral studies t)y Cbvalicri c’t al. (1 I)
0 .i(i
showed t,hat the ultraviolet uhsorptioll apec-
t,rum of caffeine remains un:tltcrcd in the pH
to pII 5.5M;.O with range of 1.18-12. Since caft’eine has no
enolizablc N-H groups, t.his result is readily
account,cd for, and it \vas roncludcd that in
this pII region the stru&xr of cb!fc~ine is
unchanged. Caffeine does exhibit vhangcs in
the absorption spcc~trum in the region of pH
0 kx:alw of proton addition. WC have f’o\md
that the partition cw4hcient of caticilw also
remains constant twtn-ccll pII 2 a11d I2 and
is changed only ill the region where protona-
tioll occurs. Therefore, c&ftinc eomplrxes
can be studied over a wide pH range by
cithrr t hr partition or the spectrophoto-
metric method. l’rom Table II it may be
seen that chlorogrnic acid will complex with
caffeine to thr samr t>xt,ent, as potassiltm
chlorogrnatr. If, on thr other hand, the pH
of t,he sollltion is raised ahove 6, a pro-
noun& drcrcasr in the rxtmt of wmplex-
ing oe(*,lrs. ‘l’his drcrease was also obstrvrd
with dihydrovhlorogrnatr and p-hydrosy-
 predict with some SIICCCSSwhat, typw of
cwmpounds will form strong complcxrs with
wffeinc. The basis for this sekkion was a
I<xtraction rcquircnicnt~ for u suhxtit’lltcd aromatic ring
coefficient
and a conjItgut~cd donblc bond. Thus it, was
fo~uid that call’eiiic forms 1 : 1 asso&it~ioiis in
:q~~cous solution u-it11 coumarin, isocngcnol,
indolwwtic~ acid, and thr allt~hocyunidin
~~clzLrgonidill~hl~)ride (;3,3,7 ,~‘-tet,r:Lhydlos?
flar$ium chloride). Thr latter, I<25o = 76
liters molcsP1 is of particular int~rrestj hcc21w
the pronounwd l)nthochromic shift, I’ig. 4,
shows a striking similarity to the?“cwpigmcnt
vffwt” dcswihcd hy Rkinson and Robinson
(12). It ~tls ol~srrvc~tl 1))~thcw authors that
wrtaiti canpounds, 11ota1,ly %hydrosv-
sallthonc, rutitl, and qncrc~itrin (YUISC:I shfft,
ton-ad higher \vavrlwgths in the ahsorpt ion
spec%r:l of anthoqwlilw ill acidic solution.
They hc~lic~vc~tlthat, complex formation \\-a::
ill\-ol\-cd sitlw the bathochromica shift tlc-
cwascd at clc\.atcd tcmpcwtiuc and w-
tlwwd to Ilormal again on cooling. WP for~ld
this also to hc tlitt ~asc with the pclargoiiiditl--
thcrcforc

350 370 390 410 430 450 470 490 510 530 550 570 590 610
WAVELENGTH mp
serm dwirablr to rcinr-c&gate the “wpig-
mtwt’ rfkct” with the qnantit,atirc nict,hods
dcscribcd here :JJJdto nscc>rtniJJwhcthcr this
iiitcrartion fits iJit0 :L more gc~rcral pattern.
That complex fornJnt,ioJJ of c*lJlorogc~JJic:
acid iS JlOt limited tcox:uJthiJJr or pyrimidincl
derivatives was dcmonstmtrd by work with
riboflakl phosphntc~. TlJiS cwmporJJJd warts
tested bc~:Ju.S~~ of its strwtmxl Similaritks to
cnffcine and bccausc~Harbury and Foley (13)
and Isc~nberg aud Szcnt-GyGrgyi (1-k) lJ:~w
Show1 that, this flaviJJ c~omplex~s with
trypt’ophan. ‘l’hc r:tt,io of rlJlorogt~JJ:Jt(~to
riboflakl in t,hc co~l~plcs is I : 1, and t,hc
complex has :I wd color, I\‘,+ = 10 litcrs
molcs~l.
THE XATITI~J~: 01: TIIE ;\TTI{.~(‘TIvI~; I~OICC~ES
IS (:OMPLE:S lcOllMATION

Complex formatio~i iii SolutioJJ is t,hc rcwdt

of il Jict :IttractivC forw bc~twwii the sprcirs
making up tlJc>c~omplcs. PosSiblc s011rws of
this attrwt~iw might’ be II-bouding or
charge-t,raiisftr iJJtcr:wtioJJS. It scrms \-cry
unlikely that T-I-boJJdiJJg iS rcspoJlsiblc foJ
Ihc nx~lccwlar ;woriations dtwribcd 1Jcre
siiiw :3,3-diJJJctho~~c,iJJJJ~J~J~~t c ~~~n~plrxcs
\\.it,tl c’:LffViJK :Jt 1WSt :LS StJYJJl& :I3 :5,-k-
tlilJSdrosy(:ilJrJ:tnlntci, aJJd iSocvlgc~JJo1:~lso
interacts with caffrinc or riboflaGn. ‘l’hcrc-
few, II-bondiJJg IJctwcvJJ raffcinc nJJd
phenolic hydrosyl groups or wrboxylir acids
is not n rcvliJiSitc> for c~omplcx formatiolr.
The hypot,hcsis that ~~Jmplcs formation
belw-~n c3hlorogcxJJica :wid alit1 c2affeiiicxis due
to ckwgc-t raiisfri~ iJitcJwtioJlS :tlso swms
quwtioJJablc. C’hargr-transfer ~oJJJplcsc~arc:
JJSJJallyiclciitifiiablc from t,lJcbappcWYUJWOf n
Jww, and wry t)road, abrorption band \\-hich
is Jiot c~har:rc*teristic~of oithcr cYJlJJp0llcJlt~
(I *j). The compoJJcJJtsarc generally foulrd iJJ
1 : 1 r:Ltio aiid 0IJv of thvJJJ is un clcc~t.roJl
donor and the other ;LIJ c~ltc*troJJacwptor.
l;amili:lr vs:~mplcs of donors :x1’ :wonJatic
hydrocnrboiis :~iJd of wccptors s!~wtriiJitro-
bcnzwle, tct~r:Jc,yaJJocth~lcllc, or iodine. The
iJJt,crac.t,ioJJis SJJbjwt to stcriv hiJJdraJJw :LS
~~srmplificd by thv f:rc*t that hcsamcthyl-
bcnzclJJc>forrJJS nJJJc*h Stronger complexes
Jvith trtr:lc~~-:lrloc~th~~l(~J~cth:tJJ d(J(lS h(>s:L-
&y1h1mwc (l(i).
111the c~mJpl~w~sdcscribvd htw IIO 11(v
baJJdS were dctwtcd ; rathw the obsclJ.v(ld
1;.

‘1.
‘7)
_I.
‘3.