Coordination chemistry and biological activity of 5′-OH modified quinoline-B12 derivatives

Article abstract of DOI:10.1039/c1dt11161b

The consequences of structural modifications at the 5′-OH ribofuranotide moiety of quinoline modified B12 derivatives are discussed in regard of the coordination chemistry, the electrochemical properties and the biological behaviour of the compound.

Full text of DOI:10.1039/c1dt11161b

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Coordination chemistry and biological activity of 5¢-OH modified
quinoline–B12 derivatives†
Karel Zelenka,a Helmut Brandl,b Bernhard Spinglera and Felix Zelder*a
Received 20th June 2011, Accepted 27th July 2011
DOI: 10.1039/c1dt11161b
The consequences of structural modifications at the 5¢-OH
ribofuranotide moiety of quinoline modified B12 derivatives
are discussed in regard of the coordination chemistry, the
electrochemical properties and the biological behaviour of
the compound.
Vitamin B12 (B12) is essential for the metabolism of mam-
mals and most other forms of life, but only produced by few
microorganisms.1,2 Nature developed a complex, but efficient
transport of B12 into the cells.3–5 Thereabout, B12 runs through
multiple constitutional and electronical changes until it is con-
verted to the biologically active cofactors adenosylcobalamin
(Ado-Cbl) and methylcobalamin (Me-Cbl), respectively.4 These
cofactors are required in humans for the enzymes methionine
synthase and methylmalonylCoA mutase, respectively.6 In both
proteins, the cofactors are bound in the “base off-histidine on”
form. In contrast, some bacteria such as Lactobacillus delbrueckii
depend on AdoCbl dependent ribonucleotide reductase (RNR)
with the cofactor bound in the “base-on” constitution.7,8 Pio-
neering studies with modified B12 derivatives demonstrated that
structural changes of B12 may have biological consequences.9,10
In the last years, cell delivery of therapeutic and diagnos-
tic agents using B12-bioconjugates has regained considerable
attraction.5,11,12 Wuerges, Randaccio and co-workers suggested the
5¢-OH position of B12 was best-suited for further modifications
based on crystallographic studies of a transcobalamin (TC)-
(“base-on”) cobalamin complex which was further supported by
biological investigations.13,14
Scheme 1 B12 (1) and quinoline-B12 derivatives (2, 3) used in this study.
at the a-ribonucleotide moiety of B12 can destabilize the “base-
on” form.24,25 As part of our program to develop B12-derivatives
for medical applications, we decided to study the consequences of
structural modifications at the 5¢-OH moiety of B12 in respect to
the “base-on/base-off”equilibrium (i), the electronic properties at
the cobalt center (ii) and the biological activity of the derivative
(iii) (Scheme 2). Modifications of B12 with quinolines have been
chosen. Quinoline esters have been earlier successfully applied as
metal cleavable groups in amino acids or peptide nucleic acids.26,27
This behaviour was of interest for the biological studies.
Rational approaches towards the design of B12-based cell
delivery vehicles are also intensively followed by others.5,11,15–23
Little attention is paid in these biological studies on the
evaluation of the coordination chemistry of the modified B12-
derivative as well as the electronic properties at the cobalt center,
although alterations of these intrinsic properties might also effect
the biological behaviour. Kra¨utler et al. showed that modifications
aInstitute of Inorganic Chemistry, University of Zu¨rich, Winterthurerstr.
190, 8057, Zu¨rich, Switzerland. E-mail: zelder@aci.uzh.ch,http://www.
zelder.ch.vu; Fax: +41 44 63 56802; Tel: +41 44 63 54624
bInstitute of Evolutionary Biology and Environmental Studies, University of
Zu¨rich, Winterthurerstrasse 190, CH-8057, Zu¨rich, Switzerland
† Electronic supplementary information (ESI) available: Experimental
part and additional Tab. S1, S2 and Fig. S1–S9. CCDC reference number
820902. For ESI and crystallographic data in CIF or other electronic
format see DOI: 10.1039/c1dt11161b
Scheme 2 Evaluation of structural modification at the 5¢-OH moiety of
B12 (blue ball) on the “base-on/base-off” (left) and Co(III)/Co(II) (right)
equilibria as well as the biological activity of the compound in bacterial
growth tests with L. delbrueckii.
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Table 1 ‘Base-on/base-off’ (left), Co(III)/Co(II) (right) equilibria of 1
and 3 (Scheme 2)
Two related quinoline-B12 derivatives (2, 3) have been synthe-
sized attaching 8-hydroxy quinoline 2-carboxylate (HQCA) as
an ester or 8-amino quinoline (8-AQ) as a carbamate to B12
according to published procedures (Scheme S1, S2†).20,28,29,32 Cob-
cyano-O5R-[(8-hydroxyquinoline)-2-carboxyl] cobalamin (2) and
Cob-cyano-O5R-(8-aminoquinoline)-8-carbamoyl) cobalamin (3)
were isolated by preparative reverse phase C18 chromatography
and analysed with UV-vis, 1H-NMR and MS spectrometry.29
In the high-resolution mass spectra of 2 and 3, [M]2+ ions at
m/z 785.78821 (m/zcalc 785.78894) and m/z 785.29657 (m/zcalc
785.29663) were observed, consistent with molecular formulas
of C73H93CoN15Na2O16P and C73H94CoN16Na2O15P, respectively.
The UV-vis spectra of 1–3 were comparable and showed a
characteristic red shift compared to the absorption of aqua-cyano
cobinamide (Fig. S3†).29 This indicates that 2 and 3 are available
in their “base-on” forms.29 The chemical shifts of the protons at
C4R and C5R of the ribofuranosyl moiety in the 1H-NMR spectra
are distinct from those of B12,30 but comparable to those of Cob-
cyano-O5R-acetyl cobalamin.28,29,31 The structural identity of 2
was further supported by crystal structure analysis.32 Compound
2 crystallized in the orthorhombic space group P212121 and the
quinoline unit was oriented underneath and parallel to the rim at
the C ring of the macrocycle of 2 (Fig. 1). This is a high quality
X-ray structure of a B12 derivative (the R1 value of the strong
Entry
Compound
pKbase-off
Epc* [V]a
1
2
1
3
0.1b
0.1
-1.126
-1.121
a Epc* = Epc - -E0¢ (see Supporting Information†); b Ref. 2
Co(III) center was not significantly affected by the modification at
the 5¢-OH moiety as indicated by the same pKbase-off value of 0.1
(Fig. S4†).2,29
The electrochemical reduction of the octahedral cobalt(III) to
the square-pyramidal cobalt(II) center of 1 and 3 was investigated
with cyclic voltammetry (CV) in water ([Tris-HCl] = 0.2 M, pH 8.0)
and indicated a comparable cathodic reduction potential (Epc*)
(Table 1; Fig. S5†).29
This behaviour is in agreement with recent studies of B12
derivatives having modifications at the f-side chain showing a
linear correlation between the pKbase-off value and the Co(III)/Co(II)
reduction potential.35
B12-dependant cell growth studies with L. delbrueckii showed
that bacterial cell growth was indeed strongly reduced with 2, 3 by
76 to 81% compared to 1 (Fig. 2).
˚
reflections up to 0.8 A is 4.92%). The Co–C bond length is 1.882(2)
˚
˚
A, the Co–N(benzimidazole) bond length is 2.052(3) A, the corrin
fold angle is 13.4(1)◦ and the base tilt angle is 11.8(2)◦. The overall
geometry of 2 resembles that of other cobalamines.33,34
Fig. 2 Growth of L. delbrueckii dependent on 1, 2, or 3 (c = 250 pg ml-1)
at 37 ◦C (n = 3).
Of note, the carbamate linker of 3 was not hydrolysed in the
assay medium as shown with HPLC (Fig. S1†).29 This excludes the
possibility that the observed activity of 3 derived from (hydrolysed)
B12.
Background hydrolysis of 2 to B12 and HQCA was also
negligible,29 but the addition of Zn(II) ions (25 eq) triggered
ester cleavage of 2 (130 mM) with a half-life time of 370 min
Fig. 1 X-Ray crystal structure analysis of 2. ORTEP representation with
C, N, O, P and Co colored in gray, blue, red, orange and green respectively.
Hydrogen atoms and most solvent molecules have been omitted for viewing
clarity. Hydrogen bonds between the HQCA moiety and a water molecule
are indicated.
following pseudo-first order kinetics (Scheme 3; Fig. S1†).29,36
A
The “base-on/base-off” and the Co(III)/Co(II)-redox equilibria
of 3 were investigated in more detail. Investigations with 2 were
disclaimed because of the instability of the ester functionality
under acidic conditions. Spectrophotometric titration with 3
showed that intramolecular coordination of the dmbz base to the
Scheme 3 Proposed mechanism for the zinc(II)-mediated quinoline-ester
hydrolysis.
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partly hydrolysed sample of 2 containing approx. one third of B12
(1) showed indeed an 30% increase in cell growth compared to
unhydrolyzed 2 as shown in Fig. S6.† This behaviour is in good
agreementwith thebiologicalactivityof the individualcompounds
1 and 2.
7 J. Stubbe, D. G. Nocera, C. S. Yee and M. C. Y. Chang, Chem. Rev.,
2003, 103, 2167–2201.
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Interestingly, when Zn2+ ions (1, 5, 10, 20 or 75 mg l-1) were
added as ZnSO4·7H2O at the beginning or 24 h after the start of
bacterial growth with 2 (250 pg ml-1), the biological activity of
the modified B12-derivative was not altered (Fig. S7†). This gives
evidence that 2 is not affected by zinc(II) mediated hydrolysis, most
likely due to protection by bacterial cellular uptake.
Competition experiments with B12 (250 pg ml-1) and increasing
amounts of 3 (0.1–10 000 eq) showed that at least an 100-fold
excess of 3 is necessary to obtain partial inhibition (Fig. S8†).
Co-crystal structures of B12 with the Cbl dependant RNRs of
Lactobacillus delbrueckii and Thermotoga maritima have been
reported.8,37 Superposition of the cofactor which is bound in the
active site of the latter structure with 2 suggests no steric strain
(Fig. S9†).29 We assume therefore that the reduced biological
activity of 2 is not caused by difficulties in Ado-Cbl dependant
RNR. Reasons may include a disturbed metabolism to the
organometallic cofactor (i) as well as lowered binding to other
participating proteins (ii).
In summary, modification at the 5¢-OH position of B12 with
quinoline did not alter intramolecular coordination and the
electronic properties at the metal centre. These properties are
desirable for the development of B12-bioconjugates as cell-delivery
agents. Bacterial uptake in L. delbrueckii was demonstrated with a
B12-quinoline derivative containing a metal-cleavable linker. The
strongly reduced biological activity in cell growth can therefore
be assigned to the structural modification at the 5¢-OH moiety.
This information is important for the developments of modified
B12-derivatives for biological and medical applications.
Support by R. Alberto and the Institute of Inorganic Chemistry
of the University of Zurich is acknowledged. The authors are
thankful to L. Bigler for recording of the HR-ESI-MS spectra
and to DSM Nutritional Products AG (Basel/Switzerland) for a
generous gift of vitamin B12.
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˚
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Notes and references
3
˚
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