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The porphyrin handbook - Kadish K.M.

Kadish K.M. The porphyrin handbook - Academic press, 2000. - 368 p.
Download (direct link): kadishsmishgulilard2000.djvu
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Gel M
Gel
270
Sessler and Gale
Table 6. Elution Times for Various Cbz-Protected Amino Acids on Columns M
and
Elution times (min)
Amino acid Silica gel M b Silica gel c
Serine 4.4 (0.1) 5.0 (0.1)
Glutamine 4.6 (0.1) 5.0 (0.1)
Alanine 5.6 (0.1) 6.0 (0.1)
Phenylalanine 12.7 (0.1) 13.1 (0.1)
Tryptophan 15.6 (0.1) 21.1 (0.1)
Aspartate 24.7 (0.1) 25.4 (0.1)
Glutamate 32.5 ( 0.1) 30.6 (0.1)
Source: Data from Sessler. J. L.; Gale. P. A.; Genge, J. W. Chem. Eur. J.
1998. 4. 1095.
" All amino acids are N-carboxybenzyl protected.
i! Protected amino acids eluted using the following conditions: Mobile
phase. 70/30 30 mM acetate buffer. pH = 7.0/CH.iCN; Flow rate 0.3
ml/min.; Column temperature 25 C, Detection UV =- 254 nm.
' Protected amino acids eluted using the following conditions: Mobile
phase. 75/25 30 mM acetate buffer. pH = 7.0/CH^CN: Flow rate. 0.25
ml/min.: Column temperature 25 C. Detection UV - 254 nm.
AMP
Elution time (min.)
Figure 19. Separation of adenosine monophosphate, diphosphate and
triphosphate on a calixpyrrole-modified silica gel column, M. Flow rate
0.3 ml/min., mobile phase 200 mM sodium phosphate (isochratic), pH = 7.0,
column temperature 25 C, UV detection at 262 nm. (Reprinted with
permission from Chem. Eur. /. 1998, 4, 1095 Copyright (c) 1998 Wiley-VCH.)
phase liquid chromatography) in that the more highly charged nucleotide
(ATP) is retained longest without the use of ion-pairing agents.
Presumably, this convenient elution order reflects the fact that a higher
charge density is present in the di- and triphosphate anions and that
this, in turn, permits these substrates to interact more favorably with
the calixpyrrole subunits present on the column.
These results led to the more generalized proposal that the more
phosphate groups present on a given substrate, the longer the retention
time. This thinking then led to the prediction that calixpyrrole-derived
columns could be used to affect the separation of oligonucleotides based
on chain length. This prediction has in fact been borne out by
experiment.35 For instance, Figure 20 shows the separation of a mixture
of unprotected oligodeoxythymidylate fragments containing between 12 and
18 nucleotide subunits on
12
Elution lime (min.)
Figure 20. Separation of d^ 2_i8 on a calixpyrrole-modified silica gel
column, M. Flow rate 0.4 ml / min., mobile phase 50 / 50 CH3CN / 250 mM
sodium chloride: 50 mM sodium phosphate (v/v) (isochratic), pH = 7.0,
column temperature 25 C, UV detection at 265 nm. (Reprinted with
permission from Chem. Commun. 199B, 1. Copyright (c) 1998 The Royal Society
of Chemistry.)
Elution time (min.)
Figure 21. Separation of hexamers; TCTAGA, GCATGC, and CCCGGG on a
calixpyrrole-modified silica gel column, M. Flow rate 0.4 ml/min., mobile
phase 50/50 CH3CN / 50 mM sodium phosphate buffer (isochratic), pH = 7.0,
column temperature 25 C, UV detection at 265 nm. (Reprinted with
permission from Chem. Eur. j. 1998, 4, 1095. Copyright (c) 1998 Wiley-VCH.)
gel M. As expected, the species containing the higher numbers of
phosphate groups display the longer retention times. Interestingly, this
same column (M), as well as column B, worked to effect the HPLC-derived
separation of three oligonucleotide hexamers of equal charge and length
(Figure 21). In these latter instances, the separations reflect,
presumably, the fact that different hydrogen-bonding interactions are
possible for the various nucleobase-residue sequences.
A particularly simple class of conjugates, namely model compounds 16
and 17, were synthesized in order to ascertain the extent to which
binding interactions could be influencing the observed separation
phenomena.35 The
45/Calixpyrroles: Novel Anion and Neutral Substrate Receptors
271
relevant anion-coordination studies were carried out in dichloromethane-
c/? (for reasons of solubility) with the stability constants and binding
stoichiometries (which were found to be 1 : 1 in all cases) being derived
from 1H NMR titration analyses analogous to those discussed earlier.
These binding studies provide a basis for understanding the HPLC results
obtained with gels M and (Table 7). For instance, the fact that
phenylphosphate is readily separated from phenylsulfonate on both silica
gels M and can be rationalized by considering that dihydrogen phosphate
is far better bound by model compounds 16 and 17 than is hydrogensulfate.
Likewise, the fact that similar retention times are observed when either
gel M or gel is used could reflect that nearly identical Kas for this
substrate are displayed by both 16 and 17 in dichl oromethane-rf?
solution.
Since weak binding interactions can lead to good HPLC-based
separations, the ability of columns M and to affect the separation of
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