Chiral NADH model systems functionalized with Zn(II)-cyclen as flavin binding site

Article abstract of DOI:10.1016/j.tet.2005.03.081

A series of chiral peptides has been prepared, bearing a 1,4-dihydronicotine amide and a zinc cyclen moiety. The metal complex reversibly binds flavins in aqueous solution, while the dihydronicotine amide serves as a NADH model transferring a hydride to the flavin within the assembly. The reaction rate of the redox reaction was monitored and determined by UV spectroscopy. The reaction rates of the substituted compounds were slower if compared to the non-substituted parent compound 1-H, but still show a 30-100 fold rate enhancement compared to the compound missing a flavin binding site. It was anticipated to probe the cryptic stereoselectivity of the hydride transfer from dihydropyridine to flavin. Spectroscopic data indicate that the introduction of deuterium labels upon reduction of the pyridinium salts to 1,4-dihydropyridine in D₂O proceeds diastereoselectively, but identical isotope effects on the rate of flavin reduction as with a non-chiral NADH model revealed that the hydride transfer within the assembly proceeds not stereoselective. A more rigid chiral NADH model compound must be prepared to achieve this goal.

Full text of DOI:10.1016/j.tet.2005.03.081

Tetrahedron 61 (2005) 5241–5251
Chiral NADH model systems functionalized with Zn(II)-cyclen as
flavin binding site
*
¨
Stefan C. Ritter, Martin Eiblmaier, Veronika Michlova and Burkhard Konig
¨
¨
¨
Institut fur Organische Chemie, Universitat Regensburg, Universitatsstr. 31, D-93040 Regensburg, Germany
Received 6 December 2004; revised 22 March 2005; accepted 23 March 2005
Available online 25 April 2005
Abstract—A series of chiral peptides has been prepared, bearing a 1,4-dihydronicotine amide and a zinc cyclen moiety. The metal complex
reversibly binds flavins in aqueous solution, while the dihydronicotine amide serves as a NADH model transferring a hydride to the flavin
within the assembly. The reaction rate of the redox reaction was monitored and determined by UV spectroscopy. The reaction rates of the
substituted compounds were slower if compared to the non-substituted parent compound 1-H, but still show a 30–100 fold rate enhancement
compared to the compound missing a flavin binding site. It was anticipated to probe the cryptic stereoselectivity of the hydride transfer from
dihydropyridine to flavin. Spectroscopic data indicate that the introduction of deuterium labels upon reduction of the pyridinium salts to
1,4-dihydropyridine in D2O proceeds diastereoselectively, but identical isotope effects on the rate of flavin reduction as with a non-chiral
NADH model revealed that the hydride transfer within the assembly proceeds not stereoselective. A more rigid chiral NADH model
compound must be prepared to achieve this goal.
q 2005 Elsevier Ltd. All rights reserved.
1. Introduction
(Scheme 1). All investigated dehydrogenases transfer
stereospecifically either the pro-R or the pro-S hydrogen
atom of NADH, with nearly equal distribution.4 Several
explanations for the observations have been proposed.
While historical models5 conclude the evolution of the
enzyme families from a common ancestor and random
selection of either stereospecificity, because it is a non-
adaptive property without selection pressure, this has been
criticized by Benner et al.4 If the specificity is without
biological or chemical function, it is a surprisingly
conserved property. All lactate dehydrogenases transfer
the pro-R hydrogen atom of NADH6 and, therefore, the
cryptic stereospecificity is older than the evolutionary
separation of life into bacteria, archaee and eucaryonts.
The substrate specificity, one of the most important
properties of an enzyme, shows a much faster drift.
Functional models try to explain the chemical phenomenon
with a biological function.7 The cryptic stereoselectivity is
now seen as an adaptive property under selection pressure.8
The strength as a reducing agent of NADH may be different
in its syn- and anti-conformation. Enzymes, that react with
easily reducible substrates may prefer one conformation,
while enzymes reacting with substrates that are difficult to
reduce use NADH in the other conformation.9 The high
complexity of enzymes makes it difficult to derive
conclusive experimental evidence for the phenomenon.
Therefore, we were interested to adapt our previously
reported chemical model to target stereochemical issues
of the hydride transfer from NADH to flavin under
Flavins and nicotine amides are the two most important
biological redox cofactors.1 Nicotine amide nucleotides2 are
the strongest reducing agents found in biology and can
transfer electrons to flavins in a thermodynamically allowed
process. The transfer of reduction equivalents is catalyzed
by enzymes, which bind both cofactors to allow very
efficient intramolecular electron transfer. We have recently
reported a chemical model system,3 which mimics this
process under physiological conditions.
Enzymatic reactions that involve nicotine amide cosub-
strates show stereospecificity in two aspects: the transfer of
a hydride to a prochiral substrate is usually highly
stereospecific and from the two hydrogen atoms available
in the dihydronicotine amide only the pro-R or pro-S
hydrogen atom is transferred. L-Lactate dehydrogenase, as
a prominent example, catalyzes the transfer of the pro-R
hydrogen atom of NADH to pyruvate, whereby only
L-lactate, and no D-lactate, is obtained. The arising NADC
contains the remaining pro-S hydrogen atom. The stereo-
specific transfer of the pro-S hydrogen atom from NADH
leads to the same NADC and the cryptic stereospecificity
of the process can only be observed by isotope labeling
Keywords: Flavin; NADH; Redox reaction; Zinc cyclen complex.
*
Corresponding author. Tel.: C49 941 9434576; fax: C49 941 9431717;
e-mail: burkhard.koenig@chemie.uni-regensburg.de
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.03.081

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S. C. Ritter et al. / Tetrahedron 61 (2005) 5241–5251
Scheme 1. Stereochemistry of L-lactate dehydrogenase and conformations of NADH in dehydrogenases.
physiological conditions. We report here the synthesis of
chiral NADH model compounds, the kinetics of the redox
reaction within NADH–flavin aggregates, the diastereo-
selective reduction of the NADC model compounds intro-
ducing deuterium atoms and observed deuterium isotope
effects in intra-assembly flavin reduction (Scheme 1).
In addition, a diastereoselective introduction of deuterium
in the reduction of the nicotinium amide may be possible.
The synthesis of compounds 1-Me, 1-Bn, 1-iPr and 1-
CH(OH)–CH3 uses 5 as a common precursor (Scheme 3).
The synthesis of 5 has been described previously.3 The
coupling of the Fmoc-protected amino acids 6 to 5
proceeded under standard conditions using HATU and
HOAt in high yield. The Fmoc protection group in 7 was
removed with TBAF in acetonitrile. These conditions
proved to be superior to standard piperidine, but aceto-
nitrile as solvent is essential. Benzylated nicotinic acid was
coupled to amines 8 using EDC and HOAt in DMF at room
temperature. Isolated product yields range from 75 to 95%
depending on the substituent R. The Boc-protecting groups
were removed with TFA and the free amine was generated
by eluation from a basic ion exchange resin. The stereo-
centers do not racemize under these conditions as confirmed
by control experiments.10 Finally, the zinc ion is introduced
by refluxing compounds 12 with zinc bisperchlorate in
ethanol. The choice of the solvent is important to obtain
quantitative complexation of the macrocyclic ligand. The
zinc complexes are stable salts. For kinetic measurements of
the flavin reduction, 13 is reduced to 1,4-dihydronicotin-
amide 1-R by treatment with sodium dithionite in aqueous
solution. Compounds 1-R must be handled under strict
exclusion of oxygen to avoid rapid reoxidation and
decomposition.
2. Results and discussion
2.1. Synthesis
Figure 1 shows the proposed conformation of the reversible
assembly of flavin and the NADH model for the redox
reaction. Model compounds with the depicted linker
between the zinc cyclen complex and dehydronicotine
amide gave the fastest hydride transfer reaction rates in
previous studies. Therefore, we chose compound 1 as a
starting point and introduced chirality into the linker.
Scheme 2 shows the structures of the chiral NADH model
compounds prepared and investigated in this study.
The syntheses of compounds 2 and 3 use threefold Boc-
protected cyclen 1411 as the starting material (Scheme 4).
Peptide coupling with Fmoc-protected Phe (6-Bn) or Val
(6-iPr) proceeds in nearly quantitative yields. Compound 15
was deprotected, again using TBAF in acetonitrile, and the
second amino acid was introduced yielding the isomeric
compounds 17-Bn-iPr and 17-iPr-Bn in good yield. The
nicotine amide was introduced after deprotection to yield
19. Removal of all Boc protecting groups and eluation from
a basic ion exchange resin12 set the stage for complexing the
macrocyclic ligand. Experiments using methanol as solvent
for the complexation reaction were unsuccessful. However,
Figure 1. Proposed conformation of the aggregate 1-flavin for the internal
redox reaction (RZribityl).
By introduction of chirality into the spacer between the
zinc–cyclen flavin binding site and the nicotine amide, the
methylene protons of the dehydronicotine amide become
diastereotopic and are, therefore, distinguishable by NMR.

S. C. Ritter et al. / Tetrahedron 61 (2005) 5241–5251
5243
Scheme 2. Structure of compound 1-H and of chiral model compounds 1–4 prepared and investigated in this study.
in acetonitrile solution complexes 22 are obtained in good
yields as stable salts. For the reduction to 2 and 3 only a
small excess of sodium carbonate is added to avoid
hydrolysis of the cyclen acyl bond, which has been observed
in other cases in basic aqueous solution. Acetonitrile must
be added to the reaction mixture to ensure sufficient
solubility of starting materials and products.
parent compound 1-H (see Table S-1; Supporting
information).
2.2. Kinetics of NADH–flavin redox reaction
The redox reaction of the NADH model compounds with
riboflavin in buffered aqueous solution was monitored
spectroscopically using the UV absorption at 450 nm. The
experimental set up and the methods to derive the reaction
rate constant were the same as described earlier.3 Table 1
summarizes the determined second order rate constants of
the redox reaction. The values of previously tested
compounds are given for comparison. With the exception
of compound 4 introduction of a substituent into the
dipeptide linker of 1-H, significantly reduces the reaction
rate. Most likely, substitution makes the conformation of
the peptide linker necessary for the arrangement of 1,4-
dihydronicotine amide and flavin for hydride transfer less
favourable. Due to the limited number of compounds
investigated, no predictive relation of molecular structure
and chirality, and the reaction rate could be derived.13
Dipeptide 4 is prepared from 16-iPr (Scheme 5). Reduction
of the amide bond with BH3–THF proceeds cleanly, but the
isolated yield of 23 was only 51%. Peptide coupling
with 6-Bn, deprotection and introduction of the nicotine
amide follows the previously described procedures. Com-
pound 26 was Boc deprotected and converted into the zinc
complex 28. Reduction using dithionite gave compound 4 in
good yield. A round bottom flask under argon atmosphere
was charged with the zinc-complex, sodium carbonate,
sodium dithionite, degassed water, and degassed aceto-
nitrile. Stirring of this mixture at room temperature for 3 h
under strictly exclusion of oxygen afforded a yellow
solution. The solvent was evaporated, degassed acetonitrile
was added, and the resulting suspension was filtered. The
filtrate was evaporated in vacuum to afford the dihydropyri-
dine as a yellow solid, which is highly sensitive to oxygen.
(See Supporting information for experimental details). The
absorption maxima of the substituted pyridinium salts and
1,4-dihydroniconine amides are similar to the values of the
2.3. Diastereoselective reduction of NADH model and
deuterium isotope effects in flavin reduction
The reduction step of the pyridinium salt to 1,4-dihydroni-
cotine amide in water was exemplarily investigated more

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S. C. Ritter et al. / Tetrahedron 61 (2005) 5241–5251
Scheme 3. Synthesis of compounds 1-Me, 1-Bn, 1-iPr and 1-CH(OH)–CH3 from 5. RZBn, iPr, Me and 1-R-1-hydroxyethyl.
closely for 1-CH(OH)–CH3. Upon reduction, the aromatic
resonances of the pyridinium ring between dZ7.8–9.4
almost disappear.14 Three new signals appear at dZ4.7, 5.9
and 7.1, which are assigned to the CH-proton resonances of
the dihydropyridine ring (see Supporting information for
spectra and assignment). The resonance of the methylene
group is in the 1 D proton spectrum in the same region as the
resonances of the other 20 methylene protons, but can be
identified from HSQC at dZ3.1–3.3 (see Supporting
information for spectrum). In the reduced form, some of
the compounds resonance signals broaden or show a double
set of signals. This process, which is reversed by oxidation
back to the pyridinium salt, indicates the formation of
conformers that slowly interconvert on the NMR time scale.
Intramolecular coordination of appended hydroxyl- or
carbonyl groups onto the Lewis-acidic zinc cyclen complex
has been observed in other cases. The reduction reaction in
D2O leads to a compound with nearly identical spectra. The
incorporation of deuterium is confirmed by an approxi-
mately half integral for the methylene resonance in the
proton spectrum and the multiplicity edited HSQC spec-
trum, which clearly indicates that only one proton is
attached to the methylene carbon (see Supporting infor-
mation for spectrum). To probe the stereochemistry of the
reduction reaction variable temperature spectra were
recorded to diminish signal broadening or doubling. At
393 K most resonances show coalescence giving a single
set of resonances for the compound, which may indicate
stereospecific deuteration yielding one diastereomere. A
similar diastereoselective non-enzymatic reduction has been
described for NADC.15 Traces of the pyridinium salt, the air
sensitivity, and thermal instability of the compound above
400 K unfortunately prevent a more rigorous structure
elucidation.
The reaction rate of deuterated 1-H, 1-CH(OH)–CH3 and 3
with flavin in aqueous buffer was measured and compared to
the rates of the corresponding non-deuterated compounds.
All determined isotope effects were identical within their
error limits: 1.29 for 1-H, 1.31 for 1-CH(OH)–CH3 and
1.27 for compound 3. This shows that either the deuterium
incorporation into the pyridinium ring was not diastereo-
selective, which we cannot finally exclude, or the hydride/
deuteride transfer within the assembly is not stereoselective.

S. C. Ritter et al. / Tetrahedron 61 (2005) 5241–5251
5245
Scheme 4. Synthesis of compounds 2 and 3. RZBn or iPr.
In any case is the investigated model system not suitable to
probe cryptic stereoselectivity of NADH reduction
reactions.
and nicotine amide. The redox reaction was followed by UV
spectroscopy and measurements revealed a decrease in
reaction rate in substituted compounds in comparison to the
non-substituted parent compound 1-H. We explain this by
substituent effects on the conformation of the linker, which
force the flavin binding site and 1,4-dehydropyridine in less
favorable relative orientation for the redox reaction with
flavin. Reduction of the pyridinium salts to 1,4-dihydro-
nicotine amides in D2O leads to deuterium incorporation.
For compound 1-CH(OH)–CH3 NMR measurements
indicate the formation of only one diastereomere, similar
to the non-enzymatic reduction of NADC. However,
identical deuterium isotope effects on the redox reaction
3. Conclusion
We have prepared a series of chiral NADH model
compounds to probe stereochemical effects on the rate of
the redox reaction between 1,4-dehydronicotine amide and
flavin within a reversible aggregate in buffered water. Chiral
a-amino acids were introduced into the linker tethering a
zinc cyclen complex, which serves as the flavin-binding site,

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S. C. Ritter et al. / Tetrahedron 61 (2005) 5241–5251
Scheme 5. Synthesis of compound 4.
with flavin show that the hydride transfer within the
assembly is not stereospecific. The pro-R and pro-S
hydrogen atoms of the 1,4-dehydropyridine are randomly
transferred to coordinated flavin, which reveals that the
conformational flexibility of the peptide linker between zinc
cyclen and 1,4-dehydropyridine does not sufficiently con-
fine the reactive conformation. A more rigid linker structure
within the NADH model restricting the rotation of the 1,4-
dehydropyridine is necessary to obtain a NADH model
compound, which will selectively transfer either its pro-R or
pro-S hydrogen atom to a bound substrate.
(EE/PE) afforded the fully protected compounds as colour-
less solids.
4.1.1. 10-{2-[2-(9H-Fluoren-9-yl-methoxycarbonyl-
amino)-2-benzyl-acetylamino]-ethyl}-1,4,7,10-tetraaza-
cyclododecan-1,4,7-tricarbonicacid-tri-tert-butylester
(7-Bn). The synthesis follows GP 1 using 5 (1.00 g,
1.94 mmol), 6-Bn (0.83 g, 2.13 mmol), HOAt (0.32 g,
2.35 mmol), HATU (0.89 g, 2.35 mmol) and collidine
(2.12 g, 2.3 ml, 17.5 mmol). CC with PE/EE (40:60) to
PE/EE (20:80) afforded 7-Bn in a yield of 1.66 g
(1.88 mmol, 97%); RfZ0.10 (EE/PEZ1:1); mp: 103–
1
105 8C. H NMR (600 MHz, CD2Cl2): dZ1.48 (s, 18H,
Boc-CH3), 1.49 (s, 9H, Boc-CH3), 2.56–2.65 (m, 6H, CH2),
3.09–3.11 (m, 2H, Phe-CH2), 3.24–3.51 (m, 14H, CH2),
4. Experimental
3
4.1. General procedure 1 (GP 1) for the synthesis of
compounds 7-Bn, 7-iPr, 7-Me, 7-CH(OH)–CH3, 15-Bn,
15-iPr, 17-Bn-iPr, 17-iPr-Bn, 24
4.23 (dd, JZ6.9 Hz, 1H, Fmoc-CH), 4.30–4.33 (m, 1H,
Fmoc-CH2), 4.44 (dd, 3JZ6.9 Hz, 2JZ10.5 Hz, 1H, Fmoc-
3
CH2), 4.47 (m, 1H, C*H), 5.66 (d, JZ7.0 Hz, 1H, NH),
6.67 (m, 1H, NH), 7.22–7.23 (m, 2H, arom. CH), 7.25–7.28
(m, 1H, arom. CH), 7.30–7.36 (m, 4H, arom. CH), 7.44 (dd,
3JZ7.5 Hz, 2H, arom. Fmoc-CH), 7.59–7.62 (m, 2H, arom.
A round bottom flask was charged with the amine
(1.0 equiv), Fmoc-protected amino acid (1.1 equiv), coup-
ling reagents HOAt and HATU (each 1.2 equiv) and
collidine (9.0 equiv). The compounds were dissolved in a
1:1-mixture of dry DMF and dry DCM. A minimum amount
of solvent was used. The yellow solution was stirred at
40 8C for 2 days and then diluted with 100 ml of DCM. The
reaction conversion was monitored by TLC. The mixture
was extracted with 50 ml of aqueous HCl (cZ1 mol/l), the
organic layer was dried over NaSO4 and concentrated under
reduced pressure. CC with ethyl acetate–petroleum ether
Fmoc-CH), 7.82 (d, 3JZ7.5 Hz, 2H, arom. Fmoc-CH); 13
C
NMR (150.1 MHz, CD2Cl2): dZ28.7 (C, Boc-CH3), 28.8
(C, Boc-CH3), 36.5 (K), 39.2 (K, Phe), 47.6 (C, Fmoc-
CH), 48.2 (K), 48.7 (K), 50.1 (K), 52.4 (K), 54.9 (K),
56.3 (C, C*H), 67.2 (K, Fmoc-CH2), 79.5 (Cquat, Boc),
79.8 (Cquat, Boc), 120.3 (C, 2 arom. Fmoc-C), 125.4 (C, 1
arom. Fmoc-C), 125.5 (C, 1 arom. Fmoc-C), 127.2 (C, 1
arom. C), 127.4, (C, 2 arom. C), 128.1 (C, 2 arom. Fmoc-
C), 128.8 (C, 2 arom. C), 129.9 (C, 2 arom. C), 137.2

S. C. Ritter et al. / Tetrahedron 61 (2005) 5241–5251
5247
Table 1. Reaction rate constants of the redox reaction of the respective NADH-model compound with 1 equiv of riboflavin tetraacetate in aqueous solution
(HEPES/KOH pH 7.4); cZ4.51$10K5 mol/l. UV detection at 447 nm. Rate constants are derived from a minimum of two independent measurements
Compound
k2 [l molK1 sK1
]
Relative rates
Ref. 3
22
1
Ref. 3
408G26
18
Ref. 3
671G37
29
1-H
3998G321
175
Ref. 3
646G67
28
1-Bn
643G132
706G141
725G65
28
31
32
1-iPr
1-CH(OH)CH3
3
783G151
806G155
2352G228
34
2
37
4
103

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S. C. Ritter et al. / Tetrahedron 61 (2005) 5241–5251
(arom. Cquat), 141.7 (arom. Cquat), 144.4 (arom. Cquat), 144.4
4.3. General procedure 3 (GP 3) for the synthesis of
compounds 10-Bn, 10-iPr, 10-Me, 10-CH(OH)–CH3,
19-Bn-iPr, 19-iPr-Bn, 26
(arom. Cquat), 155.8 (Cquat, urethane-C), 156.1 (Cquat
urethane-C), 156.3 (Cquat, urethane-C), 171.1 (Cquat
,
,
amide-C); UV–vis (CH3CN): l (log 3)Z205 nm (4.781),
265 nm (4.246), 289 nm (3.674), 300 nm (3.747); MS
(ESI, CH2Cl2/MeOH): m/zZ885.6 [MHC] (100%),
907.7 [MCNaC] (10%); MS-HR (FAB, CH2Cl2):
A round bottom flask was charged with the amine
(1.0 equiv), the nicotinic acid derivative (1.1 equiv),
coupling reagents HOAt and EDC (each 1.2 equiv) and N-
ethyldiiso-propylamine (1.2 equiv). The mixture was dis-
solved in the minimum amount of dry DMF. The yellow
solution was stirred at room temperature for 24 h and the
reaction conversion was monitored by TLC. The mixture
was evaporated and dried in vacuum. The resulting oil was
dissolved in 50 ml of DCM and extracted three times with
10 ml of aqueous HBr (cZ1 mol/l) to remove excess
coupling reagents and amine. The combined organic layers
were dried over NaSO4 and concentrated under reduced
pressure. CC (DCM/MeOH) afforded the fully Boc-
protected compounds as reddish solids.
[MHC]
(Calcd)Z885.5116,
[MHC]
(found)Z
885.5116G0.58 ppm; IR (KBr): n~ [cmK1]Z3064, 2970,
2932, 1683, 1534, 1462, 1250, 1161, 742; MF:
C49H68N6O9; MWZ885.12.
See electronic Supporting information for the synthesis and
characterisation of compounds 7-iPr, 7-Me, 7-CH(OH)–
CH3, 15-Bn, 15-iPr, 17-Bn-iPr, 17-iPr-Bn, 24.
4.2. General procedure 2 (GP 2) for the synthesis of
compounds 8-iPr, 8-Bn, 8-Me, 8-CH–(OH)CH3, 16-iPr,
18-Bn-iPr, 18-iPr-Bn, 25
4.3.1. 1-Benzyl-3-{2-phenyl-1-[2-(4,7,10-tris-tert-butoxy-
carbonyl-1,4,7,10-tertaaza-cyclododec-1-yl)-ethylcarba-
moyl]-ethylcarbamoyl}-pyridinium-bromide (10-Bn).
The synthesis follows GP 3 using 8-Bn (1.10 g,
1.66 mmol), 9 (0.54 g, 1.83 mmol), HOAt (0.27 g,
2.01 mmol), EDC (0.31 g, 0.36 ml, 2.01 mmol) and N-
ethyldiisopropylamine (0.26 g, 0.34 ml, 2.01 mmol). CC
with CH2Cl2/MeOH (98:2) to CH2Cl2/MeOH (87:13). After
concentrating the solution under reduced pressure and
drying in vacuum the obtained solid was diluted in as little
dry DCM as possible and stored at K18 8C over night. Any
separated silica gel was removed by filtration and the
obtained solution was dried in vacuum. This afforded 10-Bn
in a yield of 1.34 g (1.43 mmol, 86%); RfZ0.16 (CH2Cl2/
MeOHZ9:1); mp: 146–149 8C. 1H NMR (400 MHz,
CDCl3): dZ1.42 (s, 18H, Boc-CH3), 1.45 (s, 9H, Boc-
CH3), 2.67–2.74 (m, 6H, CH2), 3.26–3.48 (m, 16H, CH2),
4.87–4.92 (m, 1H, C*H), 6.05 (bs, 2H, Bn–CH2), 7.09–7.12
(m, 1H, arom. CH), 7.15–7.18 (m, 2H, arom. CH), 7.34–
7.36 (m, 2H, arom. CH), 7.41–7.45 (m, 3H, arom. CH),
7.60–7.62 (m, 2H, arom. CH), 7.67 (bs, 1H, NH), 7.98–8.02
(m, 1H, py-CH), 8.95 (d, 3JZ8.2 Hz, 1H, py-CH), 9.09 (d,
3JZ5.2 Hz, 1H, py-CH), 9.51 (m, 1H, NH), 10.22 (s, 1H,
py-CH); 13C NMR (100.6 MHz, CDCl3): dZ28.5 (C, Boc-
CH3, 6C), 28.7 (C, Boc-CH3, 3C), 35.4, (K, 1C), 38.2 (K,
Phe), 47.9 (K, 4C), 49.8 (K, 2C), 50.5 (K, 1C), 53.9 (K,
1C), 55.0 (K, 1C), 57.4 (C, C*H), 65.0 (K, Bn), 79.4
(Cquat, Boc, 1C), 79.6 (Cquat, Boc, 2C), 126.7 (C, 1 arom.
C), 127.9 (C, 1 py-C), 128.4 (C, 2 arom. C), 129.4 (C, 2
arom. C), 129.7 (C, 2 arom. C), 129.9 (C, 2 arom. C),
130.5 (C, 1 arom. C), 131.9 (arom. Cquat), 134.4 (arom.
Cquat), 137.3 (arom. Cquat), 144.7 (C, 1 py-C), 145.1 (C, 1
py-C), 145.3 (C,1 py-C), 155.5 (Cquat, urethane-C), 155.8
In a round bottom flask the Fmoc-protected product from
GP 1 is dissolved in a solution of tetrabutylammonium
fluoride–trihydrate (TBAF) in acetonitrile (cZ0.05 mol/l,
2.0 equiv of TBAF) and stirred at room temperature for
17 min. The reaction conversion was monitored by TLC.
Then 150 ml of DCM were added to stop the reaction.
The mixture was extracted twice with 75 ml of water. The
combined aqueous layers were extracted with 75 ml of
DCM, the combined organic layers were dried over
NaSO4 and concentrated under reduced pressure. CC with
EE/PE or methylene chloride–methanol (DCM/MeOH)
afforded the Fmoc-deprotected compounds as colourless
solids.
4.2.1. 10-[2-(2-Amino-3-methyl-butyrylamino)-ethyl]-
1,4,7,10-tetraazacyclododecan-1,4,7-tri-carbonicacid-
tri-tert-butylester (8-iPr). The synthesis follows GP 2
using 1.36 g (1.62 mmol) of 7-iPr and 1.03 g (3.24 mmol)
of TBAF. CC with EE/PE (70:30) to CH2Cl2/MeOH (95:5)
gave 8-iPr (0.87 g, 1.41 mmol, 87%); RfZ0.42 (DCM/
MeOHZ9:1); mp: 81–84 8C. 1H NMR (300 MHz, CDCl3):
dZ0.75 (d, 3JZ6.9 Hz, 3H, Val-CH3), 0.91 (d, 3JZ6.9 Hz,
3H, Val-CH3), 1.38 (s, 18H, Boc-CH3), 1.40 (s, 9H, Boc-
CH3), 1.63 (bs, 2H, NH2), 2.19 (dhept, 3JZ4.2, 6.9 Hz, 1H,
3
Val-CH), 2.60–2.65 (m, 6H, 3CH2), 3.12 (d, JZ4.2 Hz,
1H, C*H), 3.24–3.46 (m, 14H, 7CH2), 7.39 (m, 1H, NH);
13C NMR (75.5 MHz, CDCl3): dZ16.2 (C, Val-CH3),
19.67 (C, Val-CH3), 28.5 (C, Boc-CH3), 28.6 (C, Boc-
CH3), 30.8 (C, Val-CH), 35.2 (K, 1C), 48.0 (K, 4C), 49.8
(K, 2C), 51.9 (K, 1C), 54.2 (K, 1C), 55.3 (K, 1C), 60.2
(C, C*H), 79.3 (Cquat, Boc), 79.6 (Cquat, Boc), 155.4 (Cquat
urethane-C), 155.8 (Cquat, urethane-C), 156.1 (Cquat
,
,
(Cquat, urethane-C), 156.1 (Cquat, urethane-C), 160.7 (Cquat
,
urethane-C), 174.7 (Cquat, amide-C); MS (ESI, MeOH):
m/zZ615.6 [MHC] (100%), 1251.9 [2MCNaC] (0.7%),
1345.8 [2MCHCCCH3COOH] (1.0%); IR (KBr):
n~ [cmK1]Z3391, 2974, 2931, 1695; MF: C30H58N6O7;
MWZ614.83.
amide-C), 171.0 (Cquat, amide-C); UV–vis (MeOH): l
(log 3)Z264 nm (3.755), 204 nm (4.577); MS (ESI, H2O/
MeOH/AcN): m/zZ429.9 [(MCCHC)2C] (20%), 858.6
[MC] (100%); IR (KBr): n~ [cmK1]Z3063, 2977, 2930,
2856, 1679, 1545, 1460, 1416, 1366, 1250, 1162, 749, 702;
[a]2D0 (MeOH)ZK2G18; MF: C47H68N7O8Br; MWZ
939.00.
See electronic Supporting information for the synthesis and
characterisation of compounds 8-Bn, 8-Me, 8-CH(OH)–
CH3, 16-Bn, 16-iPr, 18-Bn-iPr, 18-iPr-Bn, 25.
See electronic Supporting information for the synthesis and

S. C. Ritter et al. / Tetrahedron 61 (2005) 5241–5251
5249
characterisation of compounds 10-iPr, 10-Me, 10-CH(OH)–
CH3, 19-Bn-iPr, 19-iPr-Bn, 26.
4.5.1. 1-Benzyl-3-{1S-1-[2-(1,4,7,10-tetraazacyclododec-
1-yl)-ethylcarbamoyl]-ethylcarbamoyl}-pyridinium-
hydroxide (12-Me). The synthesis follows GP 5 using
245 mg (0.30 mmol) of 11-Me and 2.0 ml of the basic ion-
exchanger (1.80 mmol). This afforded 12-Me in a yield of
145 mg (0.29 mmol, 97%); mp: 85–87 8C. UV–vis
(CH3CN): lmax [nm] (log 3)Z322 (3.829); MS (ESI,
MeOH/CH3CNC0.1% TFA): m/z (%)Z196.4 (30)
[(KCCHC–Bn)2C], 216.9 (45), 241.5 (100) [(KCC
HC)2C], 392.1 (64) [(KCKBn)C], 482.3 (93) [KC],
596.4 (24) [KCCCF3CO2H)]; IR (KBr): nꢀ [cmK1]Z704,
735, 1179, 1212, 1279, 1348, 1413, 1456, 1540, 1665, 2830,
2934, 3424; [a]2D0 (CH3CN)ZK68G78; MF: C26H41N7O3;
MWZ499.66.
4.4. General procedure 4 (GP 4) for the synthesis of
compounds 11-Bn, 11-iPr, 20-Bn-iPr, 20-iPr-Bn, 27
In a round-bottomed flask the Boc-protected product from
GP 3 (1 equiv) was dissolved in DCM and treated with
trifluoroacetic acid (TFA) (42 equiv). The yellow solution
was stirred at room temperature for 24 h and was then
evaporated and dried in vacuum. This afforded the fully
deprotected compounds as yellow solids in sufficient purity
for use in subsequent steps.
4.4.1. 1-Benzyl-3-{2-phenyl-1-[2-(1,4,7,10-tertaaza-
cyclododec-1-yl)-ethylcarbamoyl]-ethyl-carbamoyl}-
pyridinium-trifluoroacetate-trihydro-trifluoroacetate
(11-Bn). The synthesis follows GP 4 using 1.28 g
(1.36 mmol) of 10-Bn dissolved in 60 ml CH2Cl2 and
6.53 g (4.4 ml, 57.30 mmol) of TFA. This gave 1.24 g
of 11-Bn (1.22 mmol, 90%); mp: 92–94 8C. 1H NMR
(400 MHz, CD3CN): dZ2.61–3.18 (m, 19H), 3.10 (dd, 2JZ
See electronic Supporting information for the synthesis
and characterisation of compounds 12-CH(OH)-CH3, 21-
Bn-iPr, 21-iPr-Bn.
4.6. General procedure 6 (GP 6) for the synthesis of
compounds 22-Bn-iPr, 22-iPr-Bn
3
In a round bottom flask Zn(ClO4)2$6H2O was dissolved in
acetonitrile and a suspension of the amine in acetonitrile
was slowly added. The resulting reddish solution was stirred
at room temperature for 16 h and then heated to reflux for
4 h. After cooling to room temperature the solvent was
evaporated to afford the crude product as orange oil. Ethanol
(2 ml) was added and the resulting pale red precipitate was
treated with ultrasound for 15 min. After filtration, the solid
was washed with 2 ml of ethanol and dissolved in 1.5 ml of
acetonitrile. Any resulting precipitate was removed by
centrifugation. Drying of the solution in vacuum afforded
the zinc-complex as a hygroscopic orange solid.
13.8 Hz, JZ11.9 Hz, 1H, Phe-CH2), 3.44–3.51 (m, 1H,
2
3
CH2), 3.56 (dd, JZ13.8 Hz, JZ3.4 Hz, 1H, Phe-CH2),
4.88 (ddd, 3JZ3.4, 8.6, 11.9 Hz, 1H, C*H), 5.86 (s, 2H, Bn–
CH2), 7.11–7.21 (m, 3H, arom. CH), 7.38–7.40 (m, 2H,
arom. CH), 7.45–7.49 (m, 3H, arom. CH), 7.54–7.57 (m,2H,
3
arom. CH), 8.01 (dd, JZ6.2, 8.1 Hz, 1H, py-CH), 8.23
3
3
(t, JZ6.0 Hz, 1H, NH), 8.82–8.83 (m, JZ6.2 Hz, 1H,
py-CH), 8.96 (dt, 3JZ8.1 Hz, 4JZ1.3 Hz, 1H, py-CH), 9.47
3
(d, JZ8.6 Hz, 1H, NH), 9.82 (m, 1H, py-CH); 13C NMR
(100.6 MHz, CD3CN): dZ37.6 (K, Phe), 39.1 (K, 1C,
CH2–NH), 43.1 (K, 4C, cyclen), 45.3 (K, 2C, cyclen), 50.2
(K, 1C, cyclen), 50.7 (K, 1C, cyclen), 55.7 (K, 1C, CH2–
N), 57.3 (C, C*H), 65.5 (K, Bn), 127.6 (C, 1 arom. C),
129.2 (C, 1 py-C), 129.3 (C, 2 arom. C), 130.4 (C, 2 arom.
C), 130.5 (C, 4 arom. C), 131.0 (C, 1 arom. C), 133.9
(arom. Cquat, 1C), 135.2 (arom. Cquat, 1C), 139.3 (arom.
Cquat, 1C), 145.8 (C, 1 py-C), 146.1 (C, 1 py-C), 146.9 (C,
1 py-C), 163.2 (Cquat, amide-C), 173.4 (Cquat, amide-C);
UV–vis (MeOH): l (log 3)Z264 nm (3.889), 205 nm
(4.626); MS (ESI, CH3CN): m/zZ391.9 [(MCCHCC
KClCCF3COOHCHCl)2C] (68%), 392.9 [(MCCHCC
KClCHBrC2HCl)2C] (75%), 419.4 (100%), 558.4 [MC]
(20%), 694.3 [MCCCF3COONa] (4%), 746.3 [MCC
KClCCF3COOH] (6%), 748.3 [MCCKClCHBrCHCl]
(8%); IR (KBr): n~ [cmK1]Z3432, 3070, 2970, 2856, 2362,
1676, 1545, 1497, 1458, 1200, 1135, 706; MF: [C32H47N7-
O2]4C(CF3COOK)4/C40H47N7O10F12; MWZ1013.83;
[a]2D0 (MeOH)ZK13G18.
4.6.1. 1-Benzyl-3-{1-[1-benzyl-2-oxo-2-(1,4,7,10-tetra-
aza-cyclododec-1-yl)-ethyl-carbamoyl]-2-methyl-pro-
pylcarbamoyl}-pyridinium-zinc-(II)-tri-perchlorate
(22-Bn-iPr). The synthesis follows GP 6 using 0.22 g
(0.60 mmol, 3 equiv) Zn(ClO4)2$6H2O dissolved in 5 ml
acetonitrile and a suspension of 21-Bn-iPr (0.13 g,
0.20 mmol) in 7 ml acetonitrile. This gave 22-Bn-iPr in a
yield of 0.17 g (0.18 mmol, 88%); mp: 230 8C (dis.).
1H NMR (400 MHz, CD3CN): dZ0.95 (d, 3JZ6.7 Hz, 3H,
3
Val-CH3), 0.98 (d, JZ6.7 Hz, 3H, Val-CH3), 2.05–2.13
3
(m, JZ6.7 Hz, 1H, CH), 2.31–2.36 (m, 1H, CH2), 2.48–
2.61 (m, 1H, CH2), 2.68–2.93 (m, 9H, CH2), 3.00–3.13 (m,
5H, CH2), 3.28–3.35 (m, 1H, CH2), 3.48–3.54 (m, 1H,
CH2), 3.67–3.71 (m, 1H, NH), 3.83–3.86 (m, 1H, NH),
3.95–3.97 (m, 1H, NH), 4.53–4.56 (m, 1H, Val-C*H), 4.71–
2
See electronic Supporting information for the synthesis
and characterisation of compounds 11-iPr, 20-Bn-iPr,
20-iPr-Bn, 27.
4.75 (m, 1H, Phe-C*H), 5.80 (d, JZ14.8 Hz, 1H, Bn–
2
CH2), 5.84 (d, JZ14.8 Hz, 1H, Bn–CH2), 7.22–7.31 (m,
6H, arom. CH), 7.44–7.52 (m, 4H, arom. CH), 7.82 (d, 3JZ
3
8.3 Hz, 1H, Val-NH), 8.06 (dd, JZ6.1, 8.0 Hz, 1H, py-
4.5. General procedure 5 (GP 5) for the synthesis of
CH), 8.05–8.08 (m, 1H, Phe-NH), 8.76 (d, 3JZ6.1 Hz, 1H,
compounds 12-Me, 12-CH(OH)CH3, 21-Bn-iPr, 21-iPr-Bn
py-CH), 8.93 (d, JZ8.0 Hz, 1H, py-CH), 9.66 (bs, 1H,
3
py-CH); 13C NMR (100.6 MHz, CD3CN): dZ18.5 (C, Val-
CH3), 19.3 (C, Val-CH3), 32.8 (C, Val-CH), 37.3 (K,
Phe), 42.8 (K, 1C), 45.2 (K, 1C), 45.5 (K, 1C), 46.1–46.4
(K, 3C), 46.7 (K, 1C), 48.9 (K, 1C), 55.1 (C, Phe-C*H),
59.4 (C, Val-C*H), 65.8 (K, 1C, Bn), 128.3 (C, 1 arom.
C), 129.2 (C, 1 py-C), 129.7 (C, 2 arom. C), 130.3 (C, 2
The TFA-salt (1 equiv) from GP 4 was dissolved in water
and passed over a strongly basic ion-exchanger column
(loading: 0.9 mmol/ml, 6 equiv). The obtained solution was
lyophilized to afford the corresponding amine as a pale
yellow solid.

5250
S. C. Ritter et al. / Tetrahedron 61 (2005) 5241–5251
arom. C), 130.4 (C, 1 arom. C), 130.5 (C, 1 arom. C),
130.8 (C, 1 arom. C), 133.8 (arom. Cquat), 135.6 (arom.
Cquat), 136.4 (arom. Cquat), 145.9 (C, py-C), 146.1 (C, py-C),
over a strongly basic ion-exchanger column (loading:
0.9 mmol/ml, 6 equiv). The obtained solution was lyophil-
ized to afford the corresponding amine as a pale yellow
solid. In a round bottom flask Zn(ClO4)2$6H2O was
dissolved in ethanol and a solution of the amine in 5 ml of
ethanol was slowly added. A white precipitate formed
immediately. The suspension was stirred at room tempera-
ture for 16 h and then heated to reflux for 2 h. The white
precipitate became an orange oil which separated from the
solution. The solution was removed and the oil was dried in
vacuum. Ethanol (1 ml) was added and the resulting pale red
precipitate was treated with ultrasound for 15 min. After
filtration, the solid was washed with 2 ml of ethanol and was
dissolved in 1.5 ml of acetonitrile. Any resulting precipitate
was removed by centrifugation. Evaporation of the solution
in vacuum afforded the zinc-complex as a hygroscopic
orange solid.
146.7 (C, py-C), 162.3 (Cquat, amide-C), 173.0 (Cquat
,
amide-C), 175.3 (Cquat, amide-C); UV–vis (CH3CN): l
(log 3)Z264 (3.813), 385 nm (2.609); MS (ESI,CH3CN):
m/zZ338.7 [(M3CKHC)2C] (90%), 356.6 [(M3CC
ClK)2C] (100%), 368.6 [(M3CCCH3COOK)2C] (35%),
388.6 [(M3CCClO4K)2C] (50%), 586.3 [(M3CKHC–
BnC)C] (20%), 712.4 [(M3CKHCCClK)C] (10%),
776.4 [(M3CKHCCClO4K)C] (6%), 876.4 [(M3CC
2ClOK4 )C] (4%); IR (KBr): n~ [cmK1]Z3386, 3078, 2966,
2928, 1642, 1539, 1497, 1455, 1367, 1096, 746, 703; [a]D20
(CH3CN)ZC5G18; MF: [C35H48N7O3Zn]3C(ClO4K)3/
C35H48N7O15Cl3Zn; MWZ978.54.
See electronic Supporting information for the synthesis and
characterisation of compound 22-iPr-Bn.
4.8.1. 1-Benzyl-3-{2-phenyl-1-[2-(1,4,7,10-tetraaza-
cyclododec-1-yl)-ethylcarbamoyl]-ethyl-carbamoyl}-
pyridinium-zinc-(II)-tri-perchlorate (13-Bn). The syn-
thesis follows GP 8 using 101 mg (0.1 mmol) of 11-Bn and
74 mg (0.2 mmol, 2 equiv) Zn(ClO4)2$6H2O dissolved in
3 ml ethanol. This afforded 13-Bn in a yield of 92 mg
4.7. General procedure 7 (GP 7) for the synthesis of
compounds 1-Bn, 1-Me, 1-iPr, 1-CH(OH)–CH3, 2, 3, 4
A round bottom flask under argon atmosphere was charged
with the zinc-complex (obtained from GP 6, GP 8 or GP 9),
sodium carbonate, sodium dithionite, 4 ml of degassed
water and 2 ml of degassed acetonitrile. Stirring of this
mixture at room temperature for 3 h under strictly exclusion
of oxygen afforded a yellow solution. The solvent was
evaporated, 2 ml of degassed acetonitrile were added and
the resulting suspension was filtered and the filtrate
evaporate in vacuum to afford the dihydropyridine as a
yellow solid, which is highly sensitive to oxygen.
1
(0.1 mmol, 100%); mp: 142–145 8C. H NMR (300 MHz,
CD3CN): dZ2.70–3.17 (m, 19H, CH2), 3.32–3.55 (m, 6H,
CH2C3 NH), 4.80–4.87 (m, 1H, C*H), 5.80 (s, 2H, Bn–
CH2), 7.21–7.30 (m, 5H, arom. CH), 7.45–7.49 (m, 5H,
3
arom. CH), 7.99 (d, JZ6.6 Hz, 1H, Phe-NH), 8.10 (dd,
3JZ6.1, 8.0 Hz, 1H, py-CH), 8.21 (m, 1H, NH), 8.73–8.76
(m, 3JZ8.1 Hz, 1H, py-CH), 8.82–8.84 (m, 3JZ6.1 Hz, 1H,
py-CH), 9.09 (m, 1H, py-CH); 13C NMR (75.5 MHz,
CD3CN): dZ37.6 (K, 1C, Phe), 39.5 (K, 1C), 43.3 (K, 2C),
44.8 (K, 1C), 45.1 (K, 1C), 45.3 (K, 1C), 45.4 (K, 1C), 52.7
(K, 1C), 52.9 (K, 1C), 55.2 (K, 1C), 57.2 (C, C*H), 66.0
(K, 1C), 128.0 (C, 1 arom. C), 129.7 (C, 2 arom. C), 129.7
(C, 1 py-C), 130.3 (C, 2 arom. C), 130.6 (C, 2 arom. C),
130.6 (C, 2 arom. C), 131.1 (C, 1 arom. C), 133.5 (arom.
Cquat), 135.0 (arom. Cquat), 137.8 (arom. Cquat), 145.3
4.7.1. 1-Benzyl-1,4-dihydropyridin-3-carbonicacid-{[2-
phenyl-1-[2-(1,4,7,10-tetraaza-cyclododec-1-yl)-ethylcar-
bamoyl]-ethyl}-amide-zinc(II)-di-perchlorate (1-Bn). The
synthesisfollowsGP7using13-Bn(32 mg, 35 mmol), sodium
carbonate (15 mg, 139 mmol, 4 equiv) and sodium dithionite
(15 mg, 87 mmol, 2.5 equiv). This afforded 1-Bn in a yield of
1
(C, py-C), 145.4 (C, py-C), 147.4 (C, py-C), 163.0 (Cquat
,
26 mg (31 mmol, 90%). H NMR (300 MHz, CD3CN): dZ
2.50–3.67 (m, 27H, CH2), 4.30 (s, 2H, Bn–CH2), 4.60–4.67
amide-C), 177.1 (Cquat, amide-C); UV–vis (CH3CN): l
(log 3)Z265 nm (3.863); MS (pos. ESI, CH3CN): m/zZ
310.7 [(M3CKHC)2C] (100%), 530.2 [(M3CKHC–
BnC)C] (18%), 720.3 [(M3CKHCCClOK4 )C] (12%),
820.2 [(M3CC2ClO4K)C] (1%); MS (neg. ESI, CH3CN):
m/zZ918.1 [(M3CKHCC3ClOK4 )K] (100%), 1018.1
[(M3CC4ClO4K)K] (30%); IR (KBr): n~ [cmK1]Z3426,
3297, 3082, 2963, 2933, 1658, 1627, 1539, 1495, 1458,
1092, 748, 703; [a]2D0 (CH3CN)ZK24G28; MF: [C32H44-
N7O2Zn]3C(ClO4K)3/C32H44N7O14Cl3Zn; MWZ922.48.
(m, 1H, C*H), 4.74 (dt, 3JZ8.1, 3.3 Hz, 1H, CH), 5.86 (dd,
4
3
3JZ8.1 Hz, JZ1.3 Hz, 1H, CH), 6.08 (d, JZ7.0 Hz, 1H,
NH), 7.06 (d, 4JZ1.3 Hz, 1H, CH), 7.18–7.49 (m, 11H, arom.
CHCNH); 13C NMR (75.5 MHz, CD3CN): dZ22.9 (K),
36.6 (K, 1C), 38.5 (K, 1C), 43.3 (K, 2C), 44.9 (K, 2C), 45.4
(K, 2C), 52.8 (K, 2C), 55.2 (K, 1C), 56.8 (C, C*H),
57.6 (K, 1C), 103.6 (C), 127.8 (C, 1arom. C), 128.5 (C, 2
arom. C), 128.8 (C), 129.4 (C, 2 arom. C), 129.9 (C, 2
arom. C), 130.3 (C), 130.3 (C, 2 arom. C), 130.4 (C, 1
arom. C), 137.6 (Cquat), 139.5 (Cquat), 139.8 (Cquat), 169.2
(Cquat, amide-C), 175.2 (Cquat, amide-C); UV–vis (H2O):
lmax (log 3)Z361 nm (3.794); MF: [C32H45N7O2-
Zn]2C(ClOK4 )2/C32H45N7O10Cl2Zn; MWZ824.04.
See electronic Supporting information for the synthesis and
characterisation of compounds 13-iPr, 28.
4.9. General procedure 9 (GP 9) for the synthesis of
compounds 13-Me, 13-CH(OH)–CH3
See electronic Supporting information for the synthesis and
characterisation of compounds 1-Me, 1-iPr, 1-CH(OH)–
CH3, 2, 3, 4.
In a round bottom flask Zn(ClO4)2$6H2O was dissolved in
ethanol and a solution of the amine in 5 ml of ethanol was
added slowly. A white precipitate formed immediately. The
suspension was stirred at room temperature for 16 h and
then heated to reflux for 2 h. The white precipitate became
an orange oil which separated from the solution. The
4.8. General procedure 8 (GP 8) for the synthesis of
compounds 13-Bn, 13-iPr, 28
The TFA-salt (1 equiv) was dissolved in water and passed

S. C. Ritter et al. / Tetrahedron 61 (2005) 5241–5251
5251
solution was removed and the oil was dried in vacuum.
Supplementary data
Ethanol (1 ml) was added and the resulting pale red
precipitate was treated with ultrasound for 15 min. After
filtration, the solid was washed with 2 ml of ethanol and was
dissolved in 1.5 ml of acetonitrile. Any resulting precipitate
was removed by centrifugation. Evaporation of the solution
in vacuum afforded the zinc-complex as a hygroscopic
orange solid.
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.tet.2005.03.
081
References and notes
4.9.1.
1-Benzyl-3-{1S-1-[2-(1,4,7,10-tetraazacyclo-
dodec1-yl)-ethylcarbamoyl]-ethylcarbamoyl}-pyridi-
nium-zink-(II)-tri-perchlorate (13-Me). The synthesis
follows GP 9 using 134 mg (0.36 mmol, 2 equiv)
Zn(ClO4)2$6H2O dissolved in 3 ml ethanol and 90 mg
(0.18 mmol) 12-Me. This afforded 13-Me in a yield of
135 mg (0.16 mmol, 91%), mp: 105–108 8C. 1H NMR
1. (a) How important they are, is illustrated best by the
consequences of non-sufficient supply in human nutrition.
Until the end of the 19th century the disease Pellagra (from
Italian ‘pelle agra’ for rough skin) was widespread. Since
1918 it was recognized that the disease resulted from
insufficient supply of riboflavine and nicotinic acid. From
1938 on nicotinic acid and amide was added to cereal products
in the USA, which drastically reduced the number of deaths
from Pellagra. (b) Offermanns, H.; Kleemann, A.; Tanner, H.;
Beschke, H.; Friedrich, H. In Kirk-Othmer Encyclopedia of
Chemical Technology, Vol. 24; Wiley: New York, 1984.
(c) Gopalan, C.; Rao, K. S. J. Vitam. Horm. 1975, 33, 505.
2. For a recent review on the mechanism of NADH to NADC
interconversion, see: Gebicki, J.; Marcinek, A.; Zielonka, J.
J. Acc. Chem. Res. 2004, 37, 379–386.
3
(400 MHz, CD3CN): dZ1.56 (d, JZ7.2 Hz, 3H, CH3),
2.66–3.11 (m, 18H, CH2), 3.35 (m, 1H, NH), 3.41–3.57 (m,
4H, 2CH2, 2 NH), 4.57 (dq, 3JZ1.5, 7.2 Hz, 1H, CH), 5.84
(s, 2H, CH2), 7.48–7.54 (m, 5H, CH), 8.10–8.11 (m, 1H,
NH), 8.16 (dd, 3JZ6.3, 6.6 Hz, 1H, CH), 8.38 (bs, 1H, NH),
8.85–8.87 (m, 1H, CH), 8.91–8.93 (m, 1H, CH), 9.27 (s, 1H,
CH); 13C NMR (100.6 MHz, CD3CN): dZ17.2 (C), 40.1
(K), 43.4 (K), 43.4 (K), 45.0 (2C, K), 45.3 (2C, K), 52.4
(C), 53.3 (2C, K), 56.0 (K), 66.0 (K), 129.7 (C), 130.5
(2C, C), 130.6 (2C, C), 131.1 (C), 133.6 (Cquat), 134.9
(Cquat), 145.6 (C), 145.8 (C), 147.5 (C), 163.4 (Cquat),
180.0 (Cquat); UV–vis (CH3CN): lmax [nm] (log 3)Z264
(3.713); MS (ESI, CH3CN): m/z (%)Z272.5 (100)
3. Reichenbach-Klinke, R.; Kruppa, M.; Koenig, B. J. Am. Chem.
Soc. 2002, 124, 12999–13007.
4. Benner, S. A.; Glasfeld, A.; Piccirilli, J. A. Top. Stereochem.
1989, 19, 127–207.
[(LCKHCCZn2C 2C], 454.1 (21), 644.3 (5) [(LCK
)
HCCZn2CCClOK4 )C], 746.3 (1) [(LCCZn2CC
2ClOK4 )C]; IR (KBr): nꢀ [cmK1]Z626, 706, 749, 1091,
1454, 1497, 1542, 1669, 2938, 3079, 3294, 3407; [a]D20
(CH3CN)ZC13G18; MF: (C26H40N7O2Zn)3C (ClO4K)3,
respectively C26H40N7O14Cl3Zn; MWZ846.39.
5. Glasfeld, A.; Leanz, F. G.; Benner, S. A. J. Biol. Chem. 1990,
265, 11692–11699.
6. You, K.-S. CRC Crit. Rev. Biochem. 1985, 17, 313.
7. Benner, S. A.; Nambiar, K. P.; Chambers, G. K. J. Am. Chem.
Soc. 1985, 107, 5513–5517.
8. It is assumed that equilibrium constants of an enzymatic
reaction approach their optimal values in evolution: Ellington,
A. D.; Benner, S. A. J. Theor. Biol. 1987, 127, 491.
9. Benner, S. A. Experientia 1982, 38, 633.
See electronic Supporting information for the synthesis and
characterisation of compound 13-CH(OH)-CH3.
10. Solutions of Boc-protected L-valine with equimolar amounts
of cyclen, basic ion-exchange resin or 10 equivalents of KOH
in water and methanol were prepared. Their optical rotation
was monitored over 24 h. While solutions with 10 equivalents
of KOH showed rapid racemization, the optical rotation does
not change of solutions with cyclen or basic ion-exchange
resin.
4.10. Electronic supporting information
The electronic Supporting information contains experi-
mental procedures and characterization of new compounds,
copies of proton and/or carbon NMR spectra of new
compounds, a table of the UV absorption maxima of
compounds 1–4, copies of multiplicity edited HSQC and
variable temperature spectra of deuterated 1CH(OH)–CH3.
11. Brandes, S.; Gros, C.; Denat, F.; Pullumbi, P.; Guilard, R. Bull.
Soc. Chim. Fr. 1996, 133, 65–73.
12. The possible risk of racemization under the basic conditions of
the ion-exchanger was excluded by a series of control
experiments (see Ref. 10). Under the experimental conditions,
no loss of stereochemical information could be detected.
13. Force field and semi-empirical calculations of minimum
energy conformations have been performed, but results from
gas phase cannot reliably predict conformations in aqueous
solution, which would be needed to predict and correlate
reaction rates.
Acknowledgements
The authors thank the Fond der Chemischen Industrie for
financial support and the Schering AG, Berlin, for a
donation of cyclen. V.M. thanks the International Quality
Network Medicinal Chemistry (IQN-MC) of the German
Academic Exchange Service (DAAD) and the graduate
college GRK 760 of the Deutsche Forschungsgemeinschaft
(DFG) for support.
14. A small amount of reoxidation is difficult to avoid due to the
high air sensitivity of the 1,4-dihydronicotine amide.
15. Pullman, M. E.; San Pietro, A. J. Biol. Chem. 1954, 206, 129.