..,
~
t]fI'If)
Associationof +)-catechin and catechin-(4a ~ 8)-catechin with oligopeptides
Tsutomu Hatanot and Richard W. Hemingway.
SouthernResearchStation.USDAForestService.2500 ShreveportHighway. Pineville.LA 7/360. USA
NOE studieson the complexationof (poly)flavanoidswith
peptidescontaining proline residuesin aqueoussolutions
reveal site specific approach directed by hydrophobic
interactionof the aromatic rings of catechinand its dimer,
catechin-(4CJ.
-+
8)-catechin,to conformationallyaccessible
regionsof peptideswithout strongpreferencefor interaction
with proline residues.
Recentfindings on phannacologicalactivities such as antiviral
and antitumour properties of tannins and related polyphenols'
have prompted renewed studies on the fundamental mechanisms of polyphenol interaction with proteins in aqueous
solutions}.3 Murray4 showed that. in the interactions of
pentagalloylglucosewith proline rich peptides, the interaction
was selective to proline and neighbouring residues.Changesin
NMR chemical shifts were used to predict sites of complexation. In studies of the interactions of polyphenols with some
oligopeptides including bradykinin, Haslam3 stressed the
importance of hydrophobic interactions. The phannacological
activities of polyphenolsseemto d~pendon their stnlctures,and
some specificity among the polyphenols was shown.s.6Our
investigations reveal specificity in the locations of association
of both oligopeptides and of flavanoids related to condensed
tannins.
(+)-Catechin (I), a representative of the monomeric constituentsof condensedtannins,hasbeenconsideredtoo small to
causeprecipitation of proteins.7However, -.yhena small amount
of catechin was added to an aqueous solution:!: of polY(-Lproline) (m.w. 10<XX>-30
<XX»(9 mmol dm-3 for catechin and
120 mmol dm-3 for a monomeric proline residue), a white
precipitate was fonned that showedonly the polyproline signals
at the same chemical shifts as polyproline alone. This result
indicates that even low proportions of catechin to prolyl
residues fonn a complex so insoluble as to obscure NMR
resonancesdue to the complex, although the interaction of
catechin with a local portion of d1e polypeptide chain was
proposed.8
When catechin was added to m~ soluble peptides, precipitates did not occur and d1echanges in chemical shifts4 that
might be due to complex formation were extremely small.
Duplication or broadening of 'H and 'JC signals due to
equilibration between the free and complexed fonns were not
observed.This suggeststhat the associationis not strongenough
to result in changes in d1e chemical shifts or that the
equilibration is too fast to see changes. Therefore, NOE
experiments might offer a better approach to study these
complexes.
Catechin showed intennolecular NOEs with either monomeric proline or hydroxyproline in experiments using the
NOESYHG pulsesequence.9When catechinand L-proline were
dissolved in water:!:(50 mmol dm-3 each) no precipitate was
observedand the NOESYHG experiment showed a cross peak
for correlation betweencatechin B-ring protons and the proline
Cy protons (Fig. 1). The ~ andCbprotonsof prolinealsohave
weakerinteractionswith aromatic protonsof both the A- and Brings of catechin.
The NOESYHG spectnlm of the mixture of catechinand cis4-hydroxY-L-proline in water:!:(50 mmol dm-3 each) showed
significant cross peaksbetweenthe Cb protons of hydroxypro-
line and B-ring protons of catechin. Correlations between a
hydroxyproline C6 proton (a-oriented proton) to catechin H-6A
and H-SAalso showedcrosspeaks.However. presenceof strong
hydrogen bonding effects was not shown by comparisonof IJC
chemical shifts. In the presenceof catechin.noneof the carbons
of theseamino acids (including carbonyl carbons)shifted from
their frequency in spectrarecorded in the absenceof catechin
(changeswere within 0.02 ppm). Likewise. noneof the catechin
carbons (including those bearing a hydroxy group) moved.
These results pointed to the importance of hydrophobic
interaCtion of the aromatic A and B rings with the aliphatic
regions of proline and hydroxyproline.
Although polyphenolshavebeenconsideredto preferentially
attack. proline residues in peptides. catechin showed intermolecular NOEs with the other amino acid residues when we
usedthe following dimeric peptidesas targetsfor complexation.
Gly-Pro showed two sets of IH and 13Csignals. wbich are
attributable to the isomers of different configuration at the
nitrogen of imino group of proline. The major isomer. with a 2H
singlet-like signal for glycine Ca protons. was assignedto the
trans-isomer. The minor isomer. with two doublets of the
corresponding protons are separateddue to the neighbouring
proline carbonyl carbon. was assignedto the cis-isomer. For
Gly-Pro. the NOESY spectrumshowed significant cross peaks
betweenthe catechin H-6A and H-SA and the glycine protons.
Among them. the cis-isomer showed strong cross peaks
whereasthe trans-isomer showed only very weak cross peaks
with the B-ring protons of catechin.
proton
In
mixtures
showed
of
NOE
catechin
with
the glycine
with
Ca protons.
Pro-Gly.
the
catechin
Pro-Val
H-8
showedNOE betweenmethyl signals of the valine residueand
the catechin H-SA as well as the H-28 proton. Pro-Pheshowed
NOE between the catechin B-ring and the phenyl ring of the
peptide. These results indicate that the two aromatic rings of
catechin molecule preferentially associate with hydrophobic
substituents other than the proline residues. Catechin also
exhibits strong self-association1owhen dissolved in water (50
mmol dm-3). even in competition with oligopeptides. due to
hydrophobic interaction as shown by NOEs between H-28 to
H-SA.
Experiments using the following two oligopeptides showed
that not onJy the hydrophobicity. but also the molecular
~
{
coo
H OC.- 'N'
H..jl
I'*'
\
co
H~NH-OC
H
H-}-NH,
H_H.
~
JOH
~;~~OH
OH
Fig. Ilnrennolecular
NOEs suggesting die preferred sires in the associatIon
of ( oj-)-catechin and Gly-Pro-Gly-Gly
Chem.Commun..1996
confonnationsof both die polyphenol and peptideare imponant
f~tors for specificity in their complexation. Bradykinin (ArgPro-Pro-Gly-Phe-Ser-Pro-Phe-Arg)is a nonapepcidehaving
various physiological activities including regulation of blood
pressure. The conformations of this peptide in various solvents11-13and its complexation with polYphenols3have been
studied. Our NOE studies on die association of catechin (20
mmol dm-3) and bradykinin (14 mmol dm-3) showed significant cross peaks betWeenthe catechin A-ring protons and
the proline Cb as well as to the Ser C~ methyleneprotons. Also,
the catechin B-ring protons show correlations with the phenyl
protons of the phenylalanyl residues.A cross peak due to die
correlation of catechin A-ring protons and the phenyl protons
was also observed. The amino ~id residues in bradykinin
showing NOEs with catechin aromatic ring protons are
interestingly contained in the seq~nce Phe" Ser60Pro, and
Phes, where bradykinin interacts with micelles including
sodium dodecyl sulfate," and where the physiological ~tivity
of bradykinin is considered10be based.
Gly-Pro-Gly-Gly is a teb'apeptideknown as an inhibitor of
dipeptidyl peptidaseIV. This compoundhasalso beenreported
to inhibit the cotty of HIV into cells.'4 Aldtough the cis-isomer
(due to the asymmetryof proline nitrogen) is present.its content
is low so the mixture can be treated practically as the transisomer. The NOESYHG spectnlrn of the mixture of catechin
and Gly-Pro-Gly-Gly in water (SOmrnol dm-3 each) showed
correlations betWeen catechin A-ring protons and the CG
methylene protons of the C-terminal glycine residue. A cross
peak betweenthe catechin H-SB proton and methylene protons
.A
+
¥:~
~
( )
,~~.
OH
..('~-~i-H
B I OH
IH
"
~I
:
a-t
'""
H
H-
..-
~
..~-,(..
H
-;
H n
J
dr
~
~I
o/X: 0
OC
of the N-terminal glycine residue was also observed. Differentiation of die three sets of methylene protons of the glycine
residuesin die peptide was substantiatedby IH-IH aOOIH-IJC
long-range correlation specb'oscopy(COSY) measurements.
When measuredwith a mixing time of I s, NOEs indicating that
the A-ring preferentially associateswith the C-tenninal and the
B-ring with die N-tenninal glycine residues are observed
(Fig. I). The proline residue did not panicipate directly in the
complex. However, proline plays a role of die definition of the
relative spatial positions of the two glycine residues.
Catechin-(4Q- 8)-catechin(2), also showedNOE with GlyPro-Gly-Gly, but the combination of die association sites
betweendie polyphenol and the peptideare different from those
observedin die caseof catechin.~
measuredwith a mixing
time of 0.7 s, a cross peak with relatively strong intensity
attributable to NOE between H-8A. (die upper unit) and the
methylene protons of die N-tenninal glycine residue was
observed.E-ring protons, H-SEand H-2E of die dimer showed
cross peaks with die amide proton of the C-terminal glycine
residue(Fig. 2). Changesin I H chemical shifts were generally
unclear due in pan to die multiplicity of die peptide protons
(Table I). The use of NOE experiments is more sensitive for
defmition of complexation betweenoligomeric flavanoids and
polypeptides.
These results have shown that NOESYHG experimeie
reveal considerableselectivity in the hydrophobic interactions
between die aromatic protons of flavanoids and aliphatic and
phenyl protons of oligopeptidea. Analysis of the sites of
associationin thesecomplexesusing chemical shift differences
were comparatively insensitive. Strong selectivity for prolyl
residues is not seen; rather complexation is directed to
confOnnalionally accessiblehydrophobic regions so die molecular shapes of both die polYphenol and polypeptide are
important to selectivity.
This work was funded by USDA NRICGP Grant No.
94-03395.
Footnotes
a-t
t ~
addressof T. H.: FKuiry of AIMm~-~
saa-s. Okayama
University,Tsusbima,Otaylma 700, JIp8L
i The solventcmlaiDl CD,oD (HzQ: CDzQD . 9: 1).
a-t
01
~
-
Ref«1ItC8
a-t
FiI- 11ntem1O1ecu18r
NOEssuuestina die pe~
of caleclliD-(40
8)<81ecbin
siresin the association
8Id Gly-PIo-Oly-Giy
T.bIt 1 0Iemica1 shifts of Gly-Pro-Gly-Oly pIUIXIS in die presaICe 8M!
abseIM:eof JX)1yJi-as (300 MHz; in HzO: PH.)med18IOl 9: I)
I H 0Iemical shifts (6)
Gly-Pn>Gly-Gly
--
.
8..59
7.91
4.47
-3.99
-3..
3.85
3.71
3.62
3.56
2.32
-2.04
2.00
Gly-Pft).
Gly-Oly + .-
GIy-Pro-
Gly-Olyz.
8..57
7.934.42
8.61
7.96t
4.4'
-3.98"
-3.97
3.861
3.~
3.S>
3-'3
2.29
-2.02
1.99
-3.951
-3.94
-
3.8"
3.773..57
3.49
2.26
-2.00
1.96
~
Assipment
0lY3 NH
Oly. NH
c.-H
~
OIYI CH2
OIY3 CH2
Oly. 012
OIy. ~
c.-H
~
c.-H
~
~
c.-H
~
~-H
~
4-H
--
The NOESYHG spcctrl showed conelations of die peptide protons wilh
diefoUowinIpoiyphad ~.
. H-6A(wea) ... H-8A(weal.' H-S..
J H-6A Ind H(8A.' H-s.. Ho2EMId H-6A (wea).fH-60
9 P. Ziealer. A Pr«tkDl GuiM 10 l-D GIld 2.D ~-"'s
Sy~.
Bruker 1DIInmeIIII. Bi1Ierk:a. M~'_as.
Chem. Comm"".. 1996
for ACAM
1991.
10 C. M. S.-:er. Y. Cai. R. M8tiD. T. H. LiIIey..s E. Hasl8D.J. CMm.
Soc..P,/'kill TrQ/lS.2. 1990.6.51.
11 L Denys.A. A. &-..By.G.
H. Ftlber8M1J.W. Ry8I.BiocMmimy.
1982.11.6531.
12 J. K. YOImIIIM! R. P. Hicks. Biopolymers.1994.34.611.
13 J. R. C-. X. Liu. J. No SleW. L Gera8M1G. KCMOYych.
Biopl._rs.
1994.34.869.
14 C. Ca1Jet*It.B. K... E. J~
8M1A. G. Hov8lelai8l. Sc;'lICr. 1993.
161. 204'.
MId H-8A (IUOIIII
Received,2nd Allgut J~;
. H-2..
2538
1 T. Okuda,T. YosbMia8M1T. HaI8KJ.Fonx-lv. Cum. Org. NallVst..
1995.66. I.
2 E. Haslam. Plant PoIypMlIOIs: Vegnable TaMilU Re.risited.
C~
University Press.C~.
1919.
3 E. Haslam.J. Nat. Prod.. 1996.59. 2OS.
4 N. J. Mmny. No P. Will~~
T. H. Liiky 11M!E. Haslam. Eur.
J. BiocMm.. 1994.11'.923.
5 H. N.Ir.ohim. T. Mura-i.
N. YIm
o.
H. SIbc8mi. S. Tanuma.
T. ~.
T. Yoshida8M! T. 0tIIda. AIIIi"irQ/ R,s.. 1992.11.91.
6 K. Miyamoto. M. Noawra. T. MuraYanIL T. FUnlkawa.T. Hatano.
T. Y~
R. KOIIImIlM! T. 0tIIda. Bioi. PAamI.BMlI.. 1993.16.
379.
7 E. C. Blfe-SmiIh. P.yf«umiltry. 1973.u. 903.
8 L. F. TIlstta. D. OK». W. R. Sa
..s W. L ~.
in Pill",
PoIypMlIol.s. S.y"'usu. PrOP'rti,s aM Sigllijicanc,. ed. R. W.
HcmiJIIWay..s P. E. Pats. PlenumPres&.New York. 1992.p. 335.
ClMI. 6/054821