Anal. Chem. 1994,66, 4370-4374
Monitoring Formation of a Diamine Process Intermediate by Reversed-PhaseLiquid Chromatography Barbara S. Lord‘ and Rodger W. Stringham Process Research Facility, The DuPont Merck Pharmaceutical Company, Chambers Works (S l ) , Deepwater, New Jersey 08023-0999
A successful separation of solutes in a reaction mixture that includes a hydrophobic solvent and precursor, a relatively hydrophobic intermediate, and a product containiig two primary amine groups was performed on several different reversed-phase column packings. The problem was complicated by the presence of two labile triethylsilyl ether protecting groups which dominate hydrophobic retention. Resolution was found to require both ionic and hydrophobic interactions. Ionic interactions appear to be necessary to reorient the amine portion of the molecules toward the chromatographic surface, allowing selectivity. These interactions must be moderated to minimize peak broadening. The strength of ionic interactions was successfully modulated by varying pH and mobile-phase ionic strength for each of the three different chromatographic supports. I is an unstable intermediate in the synthesis of a new drug entity under development at DuPont Merck Pharmaceutical Co. A schematic representation of this molecule is given in Figure 1 showing two triethylsilyl (ES) protecting groups and two deprotected primary amines attached to a core. I is produced from a structurally similar precursor by the reductive removal of two carboxybenzyl (Z, ester protecting groups. It is critical for the next synthetic step that this reaction proceed to completion without loss of the TES protecting groups. Monitoring this reaction requires resolution of I (fw -500) not only from its diZ-di-TES precursor (fw -800), but also from the monoamine diTES intermediate, the deTESylated versions of I, and the reaction solvent, toluene. Initial attempts at monitoring this reaction using silica-based reversed-phase LC columns were unsuccessful due to extremely broad triangular peaks (Figure 2) obtained for both the diamine diester I and the monoZ intermediate. Removal of the Z groups yields primary amines which interact with residual silanols of the stationary phase. Use of a low-pH mobile phase to suppress ionization of the stationary phase was precluded by the acid lability of the TES protecting groups. Inclusion of an ion-pairing agent (octanesulfonic acid) was unsuccessful at ameliorating the problem of broad peaks and limited detector sensitivity by contributing to background absorbance. This challenging separation is further complicated by the possibility of blind portions of the molecule. Modem drug candidates are often peptide mimetics of sufficient size, rigidity, 4370 Analytical Chemistry, Vol. 66,No. 23,December 7, 7994
and complexity that portions of the molecule dominate chromatographic behavior. Regnier’ described such behavior for proteins where a small portion of the protein’s surface determines the speciticity and strength of interaction between the protein and the support. The size and location of this contact region vary depending upon the mode of separation employed. Peptide mimetics are small enough that their contact region would comprise a large portion of the molecule. Chromatographic interaction could then be visualized as a preferred binding orientation where one “side” of the molecule is in contact with the stationary phase and the other “side” is exposed only to the mobile phase. Differences in the mobile-phase “side” of the molecule would not be detected without alteration of this binding orientation. In a reversed-phase separation, I and its precursors would be expected to orient themselves to maximize contact between the TES groups and the hydrophobic stationary phase, destroying resolution of similarly protected impurities. Ionized groups would also favor this orientation by preferring exposure to the mobile phase. The broad peak shapes in Figure 2 indicate that the primary amines do interact with the stationary phase. That these amines interact with the stationary phase suggests that I will r e orient itself to achieve this interaction. The chromatographic contact region induced by ionic interactions would be different from that favored by hydrophobic interactions. If the strength of the ionic interaction could be controlled, it might be possible to induce a larger contact region combining both the ionic and hydrophobic contact regions. Control of the strength of ionic interactions could also allow chromatography of the primary amine compounds with acceptable peak shape. We hypothesized that mixed-mode chromatography offered the potential to modulate ionic interactions. These supports contain both hydrophobic alkyl ligands and titratable charged ligands. They have been used to enhance selectivity in the resolution of oligonucleotide^,^-^ peptide^,^ and proteins6 Mixed-mode supports have also been used to resolve mixtures of dissimilar analytes such as inorganic ions and carboxylic acids7 (1) Regnier, F. E. Science 1987,238,319. 117. (2) Bischoff, R; McLaughlin, L. W. J, Chromatogr, 1983,270, (3) Crowther, J.; Fazio, S.; Hartwick, R J. Chromatogr. 1983,282,619. (4) Bischoff, R: McLaughlin, L. W. J. Chromatogr. 1984,296,329. (5) Hirata, N.; Kasai, M.; Yanagihara, Y.; Noguchi, K j. Chromatogr. 1988, 434,71. (6) Kopaciewicz. W.; Rounds, M. A; Regnier, F. E. J Chromatogr. 1985,318,
157. (7) Saari-Nordhaus, R; Anderson, J. Anal. Chem. 1992,64,2283. 0003-2700/94/0366-4370$04.50/0 0 1994 American Chemical Society
Di-Zdi-TES
[I1
MOnO-Zdi--fES
(Di-TES monoamine)
(Di-TES diamine)
Figure I. 23M
ImO
L.
$
3
Y$
1160
Y
a
5m IC In
u!
mc
2 -10D
Figure 2. Conditions: column, 250 mm ZORBAX RX C18; mobile phase, 20 min linear gradient, 80-100% methanol versus 10 mM sodium phosphate (pH 3.5).
that would otherwise require two diflerent supports. Ionic interactions in mixed-mode chromatography are modulated by titrating the fixed charges in the support or by altering the ionic strength of the mobile phase. Although this approach worked initially (Figure 3), difficulties with column reproducibility and availability prompted a search for a more rugged chromatographic approach. The 3M 218 column is a relatively new column packing material that allows the use of mobile phase pH's where the amines would not be charged, possibly allowing us to separate all components based solely on hydrophobicity, without the deterioration of peak shapes observed in Figure 2. There are indicationss-l1that use of a phosphate buffer mobile phase would (8) Schafer,W. A; Cam, P. W.; Funkenbusch, E. F.; Parson, K A]. Chromutogr 1991,587, 137.
give this column the desired cation exchange character. Although an adequate separation was achieved in this mode, an unexpectedly high ionic strength was needed in the mobile phase to moderate the ionic interaction. Use of this high ionic strength buffer also proved successful in moderating ionic interactions with silica reversed-phase columns. EXPERIMENTAL SECTION
Instrumentation. The liquid chromatograph used in this work was a Hewlett-Packard HP1090M diode array detector/liquid chromatograph. The temperature of the column oven was (9) Schafer, W. A; Cam,P. W. J. Chromatop. 1991,587,149. (10) Rigney, M. P.; Funkenbusch, E. F.; Cam, P. W. ]. Chromatogr. 1990,499, 291. (11) Rigney, M.P.; Weber, T.P.; Cam, P. W. J. Chromatop. 1989,484,273.
Analytical Chemistry, Vol. 66, No. 23, December 1, 1994
4371
L C R 22Ed4
550,180
of
L C A 22!,4
8381A02A.O
I
550.100
of
06119029.C
12001
r LI
/
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-
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Figure 3. Conditions: column, 150 mm mixed-mode RP18/Cation; mobile phase, 20 min linear gradient, 50-100% methanol versus 10 mM sodium phosphate (pH 3.5).
me [ m l , Figure 4. Conditions: column, 150 mm 3M 2-18; mobile phase, 25 min linear gradient, 50-100% methanol versus 25 mM sodium phosphate plus 100 mM ammonium chloride (pH 5.8).
Table 1. Effect of pH on Solute Retention for Mixed-Mode C18 Column
Table 2. Effect of pH on Solute Retentlon for 3M 2-18 Column
retention time (min)
retention time (min) compound
pH 2.1
pH 3.5
pH 5.3
compound
toluene diamine di-TES I monoamine di-TES di-Zdi-TESether
5.2 9.3 9.3 16.4
4.7 9.2 10.3 16.0
4.6 10.3a 12.0 15.9
toluene monoamine di-TES di-Zdi-TES diamine di-TES I
a
Broad peak.
RESULTS AND DISCUSSION
Alltech Mixed-Mode W-l8/Cation Column. An aqueous 10 mM sodium phosphate buffer at pH 3.5 was used with a linear 20 min 50-100% methanol gradient to produce the separation shown in Figure 3. Eluant pH affects the degree of ionization of the stationary-phase carboxylate groups and of the ionizable groups in the solute. With decreasing pH, the stationary phase should become less ionic while the di-TES diamine I and the monoamine intermediate become more ionic. Three different pHs 4372 Analytical Chemistty, Vol. 66, No. 23, December 1, 1994
pH 5.8
pH 8.0
5.2 19.7 20.6 25.2a
5.2 19.8 20.7 22.0
5.4 19.8 20.3 20.3
Broad, triangular peak. LC A 220.4
maintained at 40 "C. All data were recorded on a Hewlett-Packard HW9994A data station using Pascal Revision 3.25 software. Columns. (1) Alltech ( D e e ~ e l d ,IL) Mixed-Mode W18/ Cation column (150 mm x 4.6 mm, 5 pm). The column is packed with a multifunctional support that consists of a 100 A, 5 pm spherical silica substrate which is bonded with a single ligand containing both reversed-phase (C18) and cation (carboxylate) functionalities in a k e d 1:lratio. The material is not end capped. (2) Cohesive Technologies Inc. (Acton, MA) 3M Z-18 column (150 mm x 4.6 mm, 6 pm). The support material is zirconia (ZrOz) microspheres coated with polybutadiene. The Z18 packing is chemically stable in solvents ranging from pH 1to pH 14. (3) DuPont (Wilmington, DE) ZORl3AX Tu(. C8 column (150 mm x 4.6 mm, 5 pm) and ZORl3AX Eu(. C18 column (250 mm x 4.6 mm, 5 pm). Reagents. Eluants were prepared from HPLC grade methanol and ACS reagent grade sodium dhydrogen phosphate, disodium hydrogen phosphate, and ammonium chloride from J. T. Baker and Co. and HPLC grade water. The pH of the eluant was adjusted with phosphoric acid or aqueous sodium hydroxide solution as needed.
pH 3.0
550.100
o i
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B
A
-c 0
j Y 0
5
10 T i m e
'n
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29
Figure 5. Conditions: column, 150 mm ZORBAX RX C8; mobile phase, 25 min linear gradient, 50% to 100% methanol versus 25 mM sodium phosphate plus 100 mM ammonium chloride (pH 5.8).
were tested, and results are presented in Table 1. Between pH 3.5 and 2.1, retention of toluene increased, reflecting the loss of support ionization and concomitant increase in hydrophobicity. At pH 3.5, the mono- and diamine diTES ethers were resolved, while at pH 2.1, they were not. Resolution of this pair at pH 3.5 by increased retention of the monoamine indicates that the 2 group is interacting with the stationary phase. At pH 2.1, the Z group must be exposed only to the mobile phase. This is unexpected in view of the increased hydrophobicity of the stationary phase. Ionic interactions could induce this orientation by turning the amine into the stationary phase and the Z group outward, but ionic interactions should be minimal at pH 2.1. Possibly the increased strength of hydrophobic interaction restricts the orientation of both molecules such that both TES groups are firmly turned into the stationary phase leaving the Z and amine groups exposed to the mobile phase. At pH 5.3, the di-TES diamine peak was overly broad, reflecting excess ionic
Table 3. Effect of pH and Ionic Strength for ZORBAX RX C8
compound
pH 3.0 100 mM NH&l
pH 5.8 100 mM NH&l
toluene deTESylated diamine diamine di-TES I monoamine di-TES di-Zdi-TESether
7.8 14.5 21.2 21.6 25.3
7.9 15.7 21.6 23.9 25.2
retention time (mid pH 5.8 100 pH 5.8 50 mM NaCl mM NH&l 7.9 16.0 22.2 24.4 25.2
7.8 15.6 21.6 24.0 25.3
pH 5.8 25 mM NH&l
pH 5.8 0 mM NH&l
7.8 17.9 22.10 24.3 25.3
7.8 17.6 25.gb 24.6 25.3
Peak somewhat broadened and slightly split. * Broad, triangular peak.
interaction. A pH of 3.5 was selected and used with fair success for monitoring this reaction. Unfortunately this separation could not be reproduced with several columns that were purchased later. Since a problem with column-to-column reproducibility was evident, other approaches to the analysis were investigated. 3M 2-18 Columns. This column is reputed to be an inert reversed-phase packing that produces symmetrical peaks for basic compounds. Previous work indicated that surface hydroxyl groups present on zirconia microspheres adsorb phosphate. The packing then exhibits cation exchange properties.8-11 Therefore, if a phosphate buffer is used with these columns, beneficial ionic interactions may be observed for the amines in the reaction mixture. These ionic interactions proved difficult to control. The diamine I was retained longer (Figure 4) than its more hydrophobic precursors, indicating the dominance of these interactions. Modulation of ionic interactions required addition of at least 80 mM ammonium chloride to the mobile phase. Below this level, the diamine peak broadened or did not elute. Effect of pH. One of the advantages of using zirconia supports is the ability to use alkaline pH’s where amines would not be expected to be charged. Mobile phases containing 100 mM ammonium chloride in 25 mM sodium phosphate buffers at three different pH’s were tested, using a linear 25 min 50-100% methanol gradient. Results are shown in Table 2. The impact of ionic interactions is shown by the longer retention of I relative to its more hydrophobic precursors. At pH 3.0, these interactions led to excessively broad peaks for I even in the presence of 100 mM ammonium chloride. As pH increased this interaction moderated, resulting in acceptable chromatography at pH 5.8 but loss of resolution between I and the di-Zdi-TES precursor at pH 8.0. This suggests that, in the absence of ionic interactions, only the TESylated portion of these molecules interacts with the hydrophobic stationary phase. Column-to-ColumnReproducibility. The second 3M 2-18 column on which these solutes were chromatographed produced a separation similar to Figure 4 after conditioning with 25 mM phosphate plus 100 mM ammonium chloride at pH 3.5. The mechanism by which this low-pH buffer conditions the 218 columns is unclear. It possibly facilitates the process of binding phosphate groups to the zirconia hydroxyl groups. Retention of the di-TES diamine increased after this treatment. Acceptable chromatography with this column was obtained at pH 5.8. A loss of resolution between toluene and de-TESylated I was observed for both columns at all pH values tested. This shortcoming led to the examination of another potential solution.
Zorbax RX CS and C18 Columns. The ability of ammonium chloride to moderate ionic interactions between the amines and the 3M 2-18 column led to testing the same approach on silicabased reversed-phase columns. A 15 cm ZORBAX RX C8 column was used with the linear 25 min 50-100% methanol gradient versus 25 mM phosphate plus 100 mM ammonium chloride buffer at pH 5.8. Under these conditions, reasonable peak shapes and good resolution of all components were observed (Figure 5). A pH of 3.0 was also tested as was the effect of substituting sodium chloride for ammonium chloride. Decreasing levels of ammonium chloride were examined. Data are presented in Table 3. The unchanging retention of toluene shows that the mobile phase does not alter stationary-phase hydrophobicity. Under the selected condition of 25 mM phosphate plus 100 mM ammonium chIoride buffer at pH 5.8, solutes elute in order of hydrophobicity as well-resolved, well-shaped peaks. At pH 3.0, the di-TES diamine and the di-TES monoamine are barely resolved, suggesting that the 2 protecting group is turned out to the mobile phase. At this pH there should be some residual silanol charge and a strong amine charge. The resulting ionic interaction is apparently strong enough to favor the undesired orientation (amine into the stationary phase, 2 group out) but not strong enough to distort peak shapes. No significant change was observed when sodium chloride was substituted for ammonium chloride. Buffer containing 50 mM ammonium chloride was also sufficient to produce the same separation. However, when only 25 mM ammonium chloride was added to the buffer, the di-TES diamine peak broadened noticeably and was slightly split. In the absence of added ammonium chloride, the Di-TES diamine peak shape deteriorated to a very broad, triangular peak. CONCLUSION
The successful separation of a reaction mixture containing a hydrophobic solvent and precursor, a relatively hydrophobic intermediate, and a product containing two primary amine groups was demonstrated on several different reversed-phase column packings. The key to this reversed-phase separation proved to be the ability to moderate ionic interaction via selection of mobilephase and stationary-phase conditions. Incorporation of moderate levels of ammonium chloride into a simple mobile phase allowed generation of acceptable peak shapes for amine-containing intermediates on a simple reversed-phase HPLC column. Elimination of all ionic interactions destroyed the required resolution as did overly strong ionic interactions. The requirement for moderated ionic interaction can be interpreted in terms of the Analytical Chemistry, Vol. 66, No. 23, December 1, 1994
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need to disturb preferred binding orientations, corroborating the premise that peptide mimetics exhibit chromatographic contact regions as described by Regnier (1) for proteins. Strong ionic interaction appeared to induce its own preferred binding orientation that disallowed resolution of a process intermediate. The implication of this goes beyond the prediction of chromatographic approaches to monitoring synthetic processes. Evaluation of
4374 Analytical Chemistry, Vol. 66,No. 23, December 1, 1994
peptide mimetic pharmaceuticals under a single set of chromatographic conditions may lead to a failure to detect significant impurities. Received for review April 25, 1994. Accepted September 2, 994*@ Abstract published in Advance ACS Abstracts, October 1, 1994.
