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Evidence for a Folded Conformation of Methotrexate in Solution.

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[ 121 The calculated isotopic shift for the PN vibration is about 41'0 greater
than the measured shift. We attribute this to an interaction between the
fundamental of the stretching vibration and the overtone of the unobserved deformation vibration (Fermi resonance). Since the deformation
vibration should absorb at 4 5 0 c m - ' , o n the basis of a comparison of
the corresponding frequencies of COS, COz and NzO (cf. [21]), an interaction of its overtone with the PN stretching vibration is conceivable.
Such an effect is frequently observed (see, e.g., Refs. [2, 211). For the PN
vibration this would lead to an experimentally determined isotopic shift
smaller than the calculated one. This is observed.
1 131 H. Siebert: Anwendungen der Schwrngungsspekrroskopie in der Anorganicrhm Chemre, Springer, Berlin 1966; E. B. Wilson, J. C. Decius, P. C.
Cross: Moleculur i'ibrarions. McGraw-Hill, New York 1955.
[I41 The "Karlsruhe version" of the Columbus program systems was used:
R Ahlrichs. H. J. Bohm, C. Ehrhardt, P. Scharf, H. Schiffer, H. Lischka,
M. Schindler, J Compur. Chem. 6 (1985) 200.
(151 R. Ahlrichs, P. Scharf, C. Ehrhardt, J . Chem. Phys. 82 (1985) 890; with a
larger basis set [P( 1 l,7,2,l) [6,4,2,1], N, 0 (9,5,2,1) [5,3,2,11 and q(d,d,O
0.7, 0.23. 0.5 (P); 1.78, 0.49, 1.085 (N); 2.47, 0.68, 1.5 (O)] the results
listed in Table 3 were obtained. From these results the following freTable 3 Results of CI calculations for NPO a n d PNO.
XYZ d ( X Y ) d ( Y Z )
[pm] [pm]
NPO 1498
PNO 152.3
1473
119.4
E(CPF)
[a.u ]
-470.565219
-470.573433
f(Xu)
f(YZ)
f(XY/YZ)
Imdyn k ' ][mdyn A-'I [mdyn A - ' 1
12.1
10.4
11.1
13.7
0.2
1.2
quencies were calculated for the XY and YZ vibrations: NPO 16041
1150 c m - '; P N O 1922/945 c m - ' . The frequencies ca. 10% higher than
the experimental values are consistent with expectation. They likewise
confirm the greater stability of the isomer PNO.
1161 Here, the electronic structure is elucidated by a population analysis on
the basis of occupation numbers: S E N (shared electron number) and
charge on the relevant atom (q). For further detailed literature see, e.g ,
ref. [3], for an introduction to this problem see R. Ahlrichs, C. Ehrhardt,
Chem. Unrerer Zeir 19 (1985) 12. The S E N is regarded as a reliable
measure of the strength of a covalent bond. To demonstrate this some
typical values are: strong a-bonds (CC, CH) 1.4; double bonds (CC) 2.2;
triple bonds (CC, N N ) ca. 3: lower SEN values are found for polar
( N a F 0.3) and weak bonds (F2 0.6).
1171 The experimentally determined structure is based o n the calculations for
NZO:d ( N N ) = 112.6, d ( N O ) = 118.6 pm: A. E. Douglas, C. K. Moiler, J.
Chem. Phy.c. 22 [ 1954) 275.
I181 a) The energy calculated for PN at the S C F level is -395.121777 a.u.;
d(PN)(calc.)= 146.2, d(PN)(exp.)= 149 p m ; b) the following PN distances are obtained for NPFz and NPC12 at the S C F level (singlet state,
CZt,symmetry): in NPFl 146.2, in NPCIZ 147.6 pm: H. Plitt, S. Schunk,
H. Schnockel, unpublished.
1191 See, e.g.. R. Ahlrichs, R. Becherer, M. Binnewies, H. Borrmann, M. Lakenbrink. S. Schunck, H. Schnockel, J. Am. Chem. SOC. 108 (1986)
7905.
1201 On critical assessment of the methods of calculation available today,
calculations which afforded comparable energies for P N O and NPO
would also support our experimental findings.
[21] Similar unusual bonding situations are present in SNO, which could be
prepared in a n Ar matrix. M. Hawkins, A. J. Downs, J . Phys. Chem. 88
( 1984) 3042.
Evidence for a Folded Conformation of
Methotrexate in Solution
By Peter Faupel and Volker Buss*
Methotrexate (MTX) is a powerful antimetabolite: by
complexing with dihydrofolate reductase (DHFR), it inhibits the reduction of dihydrofolate to tetrahydrofolate,
an essential step in the biosynthesis of nucleotide bases.
While the binding of this widely used chemotherapeutic
agent to DHFR of E. coli has been investigated in detail
using high resolution X-ray data,"' the crystal structure of
[*I Prof. Dr. V. Buss, DipLChem. P. Faupel
Fdchgebiet Theoretische Chemie der Universitdt
Lotharplatz I , D-4100 Duisburg (FRG)
Anyrw
Cheni I n r . Ed Engl. 2 7 (1988) No. 3
MTX itself was determined only recently:IZ1the molecule is
twisted through an angle of almost 90" at the CH, group
between the rings.
MTX
The question as to what the conformation in solution
might be has not yet been settled. We now present evidence, from low-temperature UV and C D spectroscopic
studies in nonpolar solvents, to show that under these
conditions the molecule adopts a folded conformation stabilized by intramolecular hydrogen-bonding.
The UV-spectrum of MTX in THF/CH2C12 (10: I ) exhibits a pronounced temperature-dependence (Fig. I , left):
on cooling to -130"C, the main absorption band at
290 nm is red-shifted by about 13 nm while the bands at
265 and 380nm disappear, making place for two new
bands at 253 and 350nm, respectively. These changes
strikingly resemble the spectral changes observed for
MTX in aqueous medium with decreasing pH-values, the
low-temperature spectrum in THF/CH2CIz corresponding
to the acidic form in water (Fig. 1, right).I3' There are several isosbestic points in the spectra, and we note a distinct
hypochromic effect in going from - 40 to - 60°C.
Fig. 1. Left: Temperature dependence of UV-spectra of MTX i n l H k /
CH2C12(10: I), c=7.7 x lo-' mol L - ' . Curves shown correspond to measurements at + 10, -40, -60, -80, - 110, and - 130°C, respectively. Arrows
indicate spectral changes with decreasing temperature; they are at 254, 265,
350, and 383 nm. Right: pH-dependence of UV-spectrum of MTX in water.
Curves shown correspond to pH-values of, respectively, I and 5.6
(c=S.Ox lo-' mol L - ' ) and > 10 (c=S.Ox
mol L-I). Arrows indicate
spectral changes with increasing acidity; they are at 244, 259, 335, and
373 nm.
The CD-spectra obtained under identical conditions reflect the spectral changes observed in the UV (Fig. 2, left).
At room temperature, there are three broad unstructured
bands of low intensities at 260, 290, and 350 nm. The lowtemperature spectra show three intense bands developing,
with alternating signs and maxima at 280, 307, and
345 nm.
Within the limits set by the solubility of MTX and experimental accuracy, these spectra are independent of concentration (Fig. 2, right). This suggests that the C D absorptions are not caused by intermolecular interactions such as
dimerization o r stacking processes, but result from a pre-
0 VCH Verlagsgesellrchaft mbH. 0-6940 Weinheim. 1988
0570-0833/88/0303-0423 $ 02.80/0
423
ferred molecular conformation evolving at low tempera
ture.
Fig. 2. CD-spectra of MTX in THF/CH2Cl2 (10: I), corrected for volunie
contraction. Left: Temperature dependence. Curves shown correspond to
measurements at + 10, -40, -60, -80, - 100,and - II5"C, c=X.Ox l o - '
mol L - '. Right: Concentration dependence: c = 10 x
(a), 8 x
(b),
and 4.5 x lo-' mol/L (c), T= - 120°C.
Both the UV and C D data can be interpreted by assuming a folded MTX conformation characterized by close
contact of the pteridine and benzene chromophores (Fig.
3). Evidence for electronic interaction between these two
groups has led to the proposal of a similarly folded conformation of folic acid in water at high d i l ~ t i o n . ' ~ ]
The folded MTX conformation is held together by a hydrogen-bond most probably involving the y-carboxyl
group of the glutamic acid and N1 of the pteridine moiety.
Protonation of this site has been suggested[51to account for
the observed spectral shifts of 2,4-diaminopteridine in acid
medium. In agreement with these observations, we calculatei6] for the protonated form a 30-nm blue-shift of the
380-nm absorption and a 20-nm red-shift of the 265-nm
band. This latter band presumably appears in the low-temperature spectra as a shoulder of the strong 300-nm absorption, which is essentially an excitation of the p-aminobenzoic acid chromophore. Also, according to STO-3G
calculation^^^^ N l is favored relative to the other ring nitrogens with respect to protonation, though steric constraints could easily overcome any differences in basicity.
In addition to any chirality originating from non-planar
conformations, MTX has a constitutional source of chirality in the asymmetric a-carbon atom of the glutamic acid
moiety which obviously can differentiate between other-
wise enantiomeric conformations. The reason why the absolute conformation depicted in Figure 3 should be preferred over the (distinguishable) inverted form is easily
seen: with the hydrogen atom of the asymmetric center
pointing towards the observer, the rr-carboxyl group points
down towards the C2-amino group, in perfect orientation
to make a second hydrogen bond.
The signs of the three prominent C D bands can be rationalized on the basis of the proposed structure (Fig. 3,
top right). According to our calculations, the direction of
the 300-nm band transition moment (1) lies inside the
acute angle formed by the transitions at 330nm (2) and
280 nm (3). This results in positive rotational strengths for
both the lower and higher energy absorptions from the interaction with the 300-nm band, while for the latter, negative rotational strengths are to be expected, in agreement
with the experimental CD spectrum.-Quantitative calculations are in progress to test the viability of the proposed
structure.
Received: October 14, 1987 [Z 2477 IE]
German version: Angew. Chem. 100 (1988) 422
[I] J. T. Bolin, D. J. Filman, D. A Matthews, R. C . Hamlin, J Kraut. J. Bfnl.
Chem. 257 (1982) 13650.
Hambley, H.-K. Chan, 1. Gonda, J. Am. Chem. Snc. 108 (1986)
2103: P. A. Sutton, V. Cody, G. D. Smith, ibid. 108 (1986) 4155.
131 The pH-dependence of the UV-spectra of MTX as well as of folic acid
have been documented before: M. Poe, J B i d . Chem. 248 (1973) 7025:
however, only the former shows the peculiar changes at low temperature
i n the UV.
[4] C.Thiery, Eur. J Biochem 37(1973) 100.
[5] G.Konrad, W. Pfleiderer, Chem. Ber. 103 (1970) 722.
[6] Reported values are based on C N D O / S calculations employing standard
parameters. Also, the transition moments in Fig. 3 are taken from these
calculations.
[7] J. E. Gready, J. Cnrnpuf Chem. 6 (1985) 377.
12)
T. W.
Preparation of 2,3,4-Tris(q5-cyclopentadienyl)-l,Sdiphenyl-l-phospha-2,3,4-tricobaltapentaborane(5) ;
Phenyl Group Migration from Phosphorus to Boron**
By Jiang Feilong, Thomas P. Fehfner,* and
Arnold L. Rheingold*
Only a few metal-rich metallaboranes ( M / B > I , direct
metal boron interactions) are knowd'l. We have shown
that the reaction of [CpCoLLl with BH3.THF is a source
of metal-rich cobaltaboranesrZ1. For low BH3.THF/
[CpCoLL'] ratios and L = L'= PPh3, PPh,(x= 1,2) fragments are incorporated into the cIustersi3l. We have now
demonstrated that C6H6 is a product of the preparative
reaction and is formed, presumably, by the reaction of
phenyl groups with h y d r i d e ~ [ ~ .Here
~ ' . we report the formation of a metal-rich phosphacobaltaborane 1 which
contains a BPh fragment in which the phenyl group on
boron is also derived from the PPh3 ligand of
[CpCo(PPh,),]. Hence, in addition to being eliminated as
C,H6, the Ph group on PPh3 can migrate from phosphorus
to boron[61.This behavior constitutes another manifesta1'1 Prof. T. P. Fehlner, J. Feilong
Fig. 3. Proposed structure of folded M T X conformation in solution. Open
circles represent carbon atoms, hatched circles nitrogen atoms and black circles oxygen atoms. Small figure, top right: transition moments of the two
chromophoric systems as obtained from CNDO/S calculations.
424
0 VCH Verfag.rqe.refl.crhafimhH. 0-6940 Weinheim. 1988
Department of Chemistry, University of Notre Dame
Notre Dame, IN 46556 (USA)
Dr. A. L. Rheingold
Department of Chemistry, University of Delaware
Newark. DE 19716 (USA)
[**I This work was supported by the National Science Foundation under
Grant C H E 8498251 and the Petroleum Research Fund, administered by
the American Chemical Society.
0570-0833/88/0303-0424
S
02 5 0 / 0
Angew. Chem. Ini. Ed. Engl. 27 (1988) No 3
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