Use of an internal reference for the quantitative HPLC-UV analysis of solid-phase reactions: A case study of 2-chlorotrityl chloride resin

Article abstract of DOI:10.1021/co4000024

Here we evaluated the use of internal reference compounds for the rapid assessment of reactions performed in solid-phase. An internal reference compound (commercially available) was bound to the resin, together with the substrate, and cleaved with the products after completion of the reaction. The peak area of the reference compound in the HPLC-UV chromatograms can be correlated directly with those of other compounds present in the reaction mixture, thereby allowing a quantitative interpretation of the chromatograms with respect to conversion and yield. The usefulness of this method was demonstrated by optimization of a protocol for the synthesis of proline-based tripeptides.

Full text of DOI:10.1021/co4000024

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Technology Note
Use of an Internal Reference for the Quantitative HPLC-UV Analysis of
Solid-Phase Reactions: A Case Study of 2-Chlorotrityl Chloride Resin
Jan Spengler, Anna-Iris Fernandez-Llamazares, and Fernando Albericio
ACS Comb. Sci., Just Accepted Manuscript • DOI: 10.1021/co4000024 • Publication Date (Web): 24 Mar 2013
Downloaded from http://pubs.acs.org on March 25, 2013
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Use of an Internal Reference for the Quantitative HPLC-UV Analysis of Solid-Phase Reactions:
A Case Study of 2-Chlorotrityl Chloride Resin
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Jan Spengler,†, ‡,* Anna-Iris Fernandez-Llamazares,† Fernando Albericio†,‡,&,¥*
†Institute for Research in Biomedicine, Barcelona Science Park, Baldiri Reixac 10, 08028-Barcelona,
Spain; ‡CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine,
Barcelona Science Park, Baldiri Reixac 10, 08028-Barcelona, Spain; &Department of Organic
Chemistry, University of Barcelona, Martí i Franqués 1-11, 08028-Barcelona, Spain; ¥School of
Chemistry and Physics, University of KwaZuluNatal, 4001 Durban, South Africa
jan.spengler@irbbarcelona.org; fernando.albericio@irbbarcelona.org
TOC Graphic
internal
reference
linker
linker
chemistry
cleavage
enhanced
HPLC-UV
analysis
internal
reference
+
chemistry
Abstract
Here we evaluated the use of internal reference compounds for the rapid assessment of reactions
performed in solid-phase. An internal reference compound (commercially available) was bound to the
resin, together with the substrate, and cleaved with the products after completion of the reaction. The
peak area of the reference compound in the HPLC-UV chromatograms can be correlated directly with
those of other compounds present in the reaction mixture, thereby allowing a quantitative
interpretation of the chromatograms with respect to conversion and yield. The usefulness of this
method was demonstrated by optimization of a protocol for the synthesis of proline-based tripeptides.
Keywords
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internal standard, reporter compound, analytical construct, Barlos-Resin, DKP-formation
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Solid-phase synthesis is widely used in several fields of chemistry, such as combinatorial sciences,
organic and peptide chemistry.1,2 However, the optimization of reactions performed on solid-phase
requires considerably more effort than when performed in solution. Essential information like the
conversion of starting material and product yield cannot normally be obtained directly. NMR, IR, and
mass spectroscopy methods applied to compounds on solid supports seldom provide information
about quantitative composition.3 The outcome of organic reactions on solid supports (usually resins of
organic polymers) is commonly monitored by HPLC-UV analysis of the products obtained from a
cleaved resin sample.
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The content of a compound is usually expressed as a percentage of its peak area from the total peak
area. However, UV-absorption coefficients can differ from compound to compound. In this case, the
relations of the area values of the different peaks do not directly reflect the molar concentrations of the
corresponding compounds.4 This makes it difficult to construe chromatograms from reaction mixtures
involving, for example, strongly absorbing protecting groups.
A second limitation of the conventional quantification is that the exact amount of resin the sample was
prepared from is not known. Consequently, the peak areas in a chromatogram from a test reaction
performed on a resin sample cannot be correlated with the peak areas in a chromatogram obtained
from another reaction run on another sample of the same resin.
A theoretically simple solution to these problems might be the cleavage of the samples from resin
aliquots and the addition of a fixed amount of (an external) reference compound. However, there is no
straightforward way to obtain resin aliquots from reaction mixtures. In the course of a synthesis, the
resins are swollen in the solvents and drying until weight constancy causes a significant delay for the
evaluation of the outcome of a reaction. Furthermore, the molecular weight of the resin-bonded
compounds changes during synthesis, and consequently, so does the resin weight. An alternative
solution would be to run the reactions separately on a mini-scale on resin aliquots until an analysis is
required. Consequently, all the resin from a reactor has to be cleaved. Prior samples (for intermediate
analysis) cannot be taken. Generally, strategies that involve measuring the exact amount of resin
subjected to cleavage require much more material than for HPLC-analysis (1-5 mg). Moreover, they
are error-prone and imply time-consuming sample preparation for each analysis.
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Resins equipped with analytical constructs provide great improvements (Figure 1 A). Such a reporter
resin can be mixed in a small amount with the resin on which the synthesis is performed. The
analytical constructs have two orthogonal cleavage sites. By analytical cleavage, conversion of
starting material, yield and identity can be unambiguously determined as a result of the presence of
the analytical construct bound to the products, which combine a chromophore that absorbs at a
remote wavelength and a mass-sensitizer. For the synthesis, the product can be cleaved without
analytical construct. However, such useful tools are not readily available, and so far have been
developed for the preparation of carboxamides only.5,6 It should also be considered that this approach
can lead to erroneous results because the outcome of reactions on the reporter-resin with its extra-
linker should not be the same as on the resin used for the synthesis.
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Here we report a strikingly simple and straightforward method for the quantitative analysis of reactions
performed on solid-phase (Figure 1 B). A reference compound is bound to the resin, together with the
starting material, via the same functional group and is cleaved under the same conditions as the
products.7 Commercially available compounds can be used as reference. A suitable reference is
chemically inert to the reaction conditions applied. Its HPLC-retention time differs from those of the
other compounds and the reference substance can be detected at the same wavelength used for the
UV detection of products. This strategy is expected to be applicable to all types of resins and linkers,
and the synthetic steps will proceed on the same linker and resin as used for the synthesis. The area
of product peaks in HPLC chromatograms can be expressed as multiples of the reference peak area
and allows for comparison of chromatograms. Here we describe the use of an internal reference for
the straightforward optimization of a protocol for combinatorial synthesis.
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A:
linker 2 chemistry
chemistry
product
cleavage
in mixture
linker 1
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analytical
enhancer
linker 2 chemistry
analytical cleavage
analytical
enhancer
linker 2 chemistry
B:
internal
reference
linker
linker
chemistry
cleavage
internal
reference
+
chemistry
Figure 1
Figure 1. An analytical construct resin with two orthogonal cleavage sites is mixed with the resin used
for the synthesis (A). An internal reference can be introduced as "willful contamination", together with
the starting material, in the resin-loading step. This resin can be used to optimize the reaction
conditions (B).
Following a solid-phase peptide synthesis with an internal reference
As proof of principle, we synthesized the tripeptide Val-Arg-Phe in the presence of 1-pyreneacetic acid
(PAA) as reference compound. PAA is commercially available and can be easily identified because of
its characteristic PDA-absorption profile. Its peak retention time was separated from those of the
products obtained during the synthesis. As a simple carboxylic acid, PAA was expected to bind to the
resin as stably as amino acids and peptides and be cleaved under the same conditions. As solid
support, we used 2-chlorotrityl chloride (CTC) resin, a standard support for the synthesis of peptides,
pseudopeptides and non-peptide molecules.8,9 It was expected that the synthesis of Val-Arg-Phe on
this resin would work well under standard conditions.
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The solid-phase peptide synthesis (SPPS) started with the loading of CTC resin with Fmoc-Phe
containing 10 mol% PAA (Figure 2). A test cleavage from the resulting resin 1 gave a HPLC-
chromatogram with two peaks at 220 nm (a wavelength routinely used for product detection), namely
the small PAA peak and the peak of Fmoc-Phe, the latter with a 15-fold area with respect to the former
(Table 1, entry 1). We obtained the same result for a repeated test cleavage. The relation between the
peak areas remained constant as long as the main peak was not higher than one absorption unit (1
AU). Therefore, we ensured that the main peak did not exceed 1 AU for all the HPLC chromatograms.
The Fmoc-group was removed from resin 1, and a cleaved sample from the resulting resin 2 showed
two peaks, corresponding to Phe and PAA, with nearly equal area values (Table 1, entry 2). The
Fmoc-group exerts strong absorbance, and with regard to the reference signal, its impact can now be
roughly estimated as a 15-fold increase in the peak area. Throughout this manuscript, we will express
the term "x-fold multiple of the peak area respectively to the reference peak area" by "area ratio". Thus,
an area ratio of 15 implies that the peak area of the analyte is 15 times higher than that of the
reference compound.
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After coupling Fmoc-Arg(Pbf) to 2, the chromatogram of a sample from the resulting resin 3 shows
that the Fmoc-Arg(Pbf)-Phe area ratio was nearly 46 (Table 1, entry 3). This increase resulted from
absorption of the Fmoc- and the Pbf-group. The contribution of the former was expected to produce an
approx. 15-fold increase in absorption, so we assumed that the additional 30-fold increase was
caused by the Pbf-group. To test this notion, resin 3 was treated with 95% TFA. Under these
conditions, the Pbf-group was removed to give Fmoc-Arg-Phe with an area ratio of 14 (Table 1, entry
4). The Fmoc-group from 3 was removed to give resin 4, and the area ratio of Arg(Pbf)-Phe was found
to be 30 (Table 1, entry 5). Summing the contributions of the absorbance of the Pbf- and Fmoc-group
(30 and 14) gave 44. This value is in good agreement with the 46 found for the dipeptide Fmoc-
Arg(Pbf)-Phe (entry 3).
Treatment of resin 4 with 95% TFA yielded the dipeptide Arg-Phe, whose area ratio was 1.8 (Table 1,
entry 6). The increase with respect to Phe (entry 2) can be attributed to the additional peptide bond.
Coupling of Fmoc-Val to resin 4 rendered resin 5. A comparison of the area ratios of Fmoc-Val-
Arg(Pbf)-Phe, Fmoc-Val-Arg-Phe, Val-Arg(Pbf)-Phe and Val-Arg-Phe (entries 7, 8, 9 and 10,
respectively) with the those of the products from the previous coupling cycle (Fmoc-Arg(Pbf)-Phe,
Fmoc-Arg-Phe, Arg(Pbf)-Phe and Arg-Phe; entries 3, 4, 5 and 6, respectively) shows that these areas
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did not increase as expected, but remained constant or even decreased. This finding indicates the
loss of some product. In other experiments (vide infra), we found evidence that the activation of amino
acids with Oxyma / DIPCDI, as used throughout this synthesis, is not optimal for CTC resin.
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PAA
Fmoc-Phe-OH / PAA
/ DIEA to CTC-resin
CO2H
Table 1
entry 1
i)
i)
Fmoc-Phe-O-
-PAA
iii)
1
entry 2
H-Phe-O-
-PAA
iv)
2
i)
ii)
entry 3
entry 4
Fmoc-Arg(Pbf)-Phe-O-
-PAA
iii)
3
i)
entry 5
entry 6
H-Arg(Pbf)-Phe-O-
-PAA
iv)
4
ii)
i)
entry 7
entry 8
Fmoc-Val-Arg(Pbf)-Phe-O-
-PAA
iii)
5
ii)
i)
H-Val-Arg(Pbf)-Phe-O-
-PAA
ii)
entry 9
6
entry 10
H-Val-Arg-Phe-OH + PAA
Figure 2
Figure 2. Standard SPPS of the tripeptide Val-Arg-Phe in the presence of PAA as internal reference.
The results of intermediate product cleavages are summarized in Table 1. i) 2% TFA in DCM, 10 min;
ii) TFA / H2O (95:5), 90 min; iii) 20% piperidine in DMF, 10 min.; iv) 4 equiv. Fmoc-Xaa, 4 equiv.
Oxyma, 4 equiv. DIPCDI in DMF.
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Table 1. Summary of product compositions from intermediate cleavages during the synthesis of Val-
Arg-Phe (Figure 3). *see Figure 3; **Area ratio means the multiples of the PAA (reference) peak area;
for chromatograms see SI.
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Entry*
Product
Area ratio**
Ph, CONH
Ph, CONH,
+ Fmoc
15
Ph, CONH,
+ Pbf
Ph, CONH
+ Fmoc, + Pbf
1
2
3
4
5
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7
8
9
10
Fmoc-Phe
Phe
1.0
1.8
2.2
Fmoc-Arg(Pbf)-Phe
Fmoc-Arg-Phe
Arg(Pbf)-Phe
Arg-Phe
46
43
14
13
30
30
Fmoc-Val-Arg(Pbf)-Phe
Fmoc-Val-Arg-Phe
Val-Arg(Pbf)-Phe
Val-Arg-Phe
The synthesis of the tripeptide H-Val-Arg-Phe-OH in the presence of a reference compound (PAA)
allowed us to obtain a series of HPLC-UV chromatograms, wherein the products peak areas were
quantified as multiples of the peak area of the reference compound. This approach allows for a
coherent interpretation of chromatograms during a synthetic sequence that involves strongly
absorbing temporary protecting groups.
Use of an internal reference for optimizing a solid-phase protocol for combinatorial synthesis
Here we demonstrated the use of a reference compound for the optimization of the synthesis of a
library of tripeptides type Yaa-Xaa-Pro. The target peptides have Pro at the C-terminus and a rather
sterically crowded amino acid (Yaa) at the N-terminus. We aimed to design a straightforward general
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protocol that works reliably with
position (Xaa).
L- and D-, as well with sterically hindered amino acids in the middle
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Figure 3 shows the envisaged synthesis of three model peptides Fmoc-Aib-Gly-Pro, Fmoc-Aib-
Pro, and Fmoc-Aib-Sar-Pro by stepwise SPPS in the presence of a reference compound. Aib
DLeu-
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represents the sterically crowded amino acid Yaa. The foreseeable critical step in the synthesis is the
coupling of Fmoc-Aib to the sequence Xaa-Pro. It is known that dipeptides with C-terminal Pro form
diketopiperazines (DKP) and that SPPS of these peptides is often hampered by this unwanted side-
reaction, which can cause dramatic reduction in yield.10 The DKP-prone sequences Gly-Pro and
DLeu-
Pro are representative examples for DKP-formation (cyclo-Gly-Pro; cyclo- Leu-Pro) under the basic
D
conditions of Fmoc-removal. Furthermore, an appropriate protocol should also give satisfactory results
for sterically hindered couplings, which are represented by the coupling of Fmoc-Aib to the sterically
hindered N-methylamino group on Sar-Pro-resin (Figure 3).
We show that an internal reference contributed to obtaining a reliable estimation of the extent of DKP-
formation and product yield under a range of coupling conditions, and thus indicates the best protocol
for library synthesis.
Fmoc-Pro-OH / BPA
/ DIEA to CTC-resin
CO2H
BPA
Fmoc-Pro-O-
-BPA
Ph
7
i)
Pro-O-
-BPA
8
Coupling to Pro: Table 2
Fmoc-Xaa-Pro-O-
ii)
-BPA
9a-c
Impact of Fmoc-removal
conditions on DKP
formation: Table 3
iii)
Xaa-Pro-O-
Coupling to Xaa: Table 3
Fmoc-Aib-Xaa-Pro-O-
-BPA
10a-c
iv)
-BPA
11a-c
Figure 3
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Figure 3. Stepwise SPPS strategy for the synthesis of compounds Fmoc-Aib-Gly-Pro, Fmoc-Aib-
Leu-Pro and Fmoc-Aib-Sar-Pro on CTC resin. Intermediate samples for HPLC-UV analysis were
D
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cleaved from resins 7, 9a-c and 11a-c with 2% TFA in DCM for 10 min (a: Xaa = Gly, b: Xaa = DLeu, c:
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Xaa = Sar). The results (Tables 2 and 3) indicate that the following protocols show the best
performance: i) 2 x 10 min 20% piperidine in DMF, wash with DCM; ii) 40 min 3 equiv. Fmoc-Xaa
activated with HBTU/DIEA, wash with DCM; iii) 5 min 20% piperidine in DMF, wash with DCM; iv) 40
min 3 equiv. Fmoc-Yaa activated with HATU/DIEA, wash with DCM.
CTC resin was used as solid support because it prevents DKP formation.11 Biphenyl acetic acid (BPA)
was found to have a peak separable from the product peaks (PAA co-elutes with some products). 1 g
CTC resin with a maximal capacity of 1.6 mmol/g was loaded with 0.3 mmol BPA and 0.8 mmol Fmoc-
Pro. The area ratio between Fmoc-Pro and BPA and was 6.5 in a test cleavage from the test resin 7
(Table 2, entry 1).
After removal of the Fmoc-group, a Pro-peak from resin 8 was not observed by HPLC-UV analysis
and consequently its conversion to Fmoc-Xaa-Pro could not be followed directly. However, it is
reasonable to expect that the area ratio of the subsequent product Fmoc-Gly-Pro from resin 9a would
be similar to that of Fmoc-Pro from 7. Thus, in this case, the area ratio should be 6.5 or higher due to
the additional amide bond. Coupling of Fmoc-Gly activated with Oxyma / DIPCDI to resin 8 gave an
area ratio of 3.7 (Table 2, entry 2), which did not improve by a repeated coupling. A similar result was
obtained for HOAt / DIPCDI activation (area ratio 3.5, Table 2, entry 3). In contrast, by HBTU / DIEA
activation, the product was detected with an area ratio of 6.8 (Table 2, entry 4). Since the yield did not
increase by longer coupling time or by repeated coupling, we reasoned that the coupling under this
condition proceeded quantitatively and that the Oxyma / DIPCDI and HOAt / DIPCDI activation
conditions did not work well for coupling to 8. The first part of the synthesis was then accomplished by
coupling Fmoc-Gly, Fmoc-DLeu and Fmoc-Sar activated by HBTA / DIEA to give resins 9a-c, from
which Fmoc-Gly-Pro, Fmoc-
DLeu-Pro and Fmoc-Sar-Pro were obtained with area ratios higher than
the 6.5 (Table 2, entries 5, 6 and 7).
Table 2. Coupling conditions for the synthesis of resins 9a-c. *Area ratios corresponding to BPA.
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Entry
Protocol applied to 8
Area ratio* (product)
5
6
7
8
1
2
3
4
5
6
7
-
6.5 (Fmoc-Pro from 7)
3.7 (Fmoc-Gly-Pro)
3.5 (Fmoc-Gly-Pro)
6.8 (Fmoc-Gly-Pro)
6.8 (Fmoc-Gly-Pro)
Fmoc-Gly / Oxyma / DIPCDI, 40 min
Fmoc-Gly / HOAt / DIPCDI, 40 min
Fmoc-Gly / HBTU / DIEA, 40 min
Fmoc-Gly / HBTU / DIEA, 40 min
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Fmoc-
D
Leu / HBTU / DIEA, 40 min
7.2 (Fmoc-DLeu-Pro)
Fmoc-Sar / HBTU / DIEA, 40 min
6.9 (Fmoc-Sar-Pro)
The next step was the removal of the Fmoc-group from the resins 9a-c. DKP formation can proceed
rapidly under these basic conditions (20% piperidine in DMF). It was not possible to obtain area ratios
for the products Xaa-Pro from resins 10a-c, because of the peak shape of the corresponding
dipeptides. Fmoc-removal was subsequently followed by coupling of Fmoc-Aib activated by HBTU /
DIEA for 40 min over resins 10a-c. The products Fmoc-Aib-Gly-Pro (from 11a) and Fmoc-Aib-DLeu-
Pro (from 11b) were obtained with area ratios lower than the corresponding starting materials and
these ratios did not increase with coupling time (Table 3, entries 1 and 2 vs. Table 2, entries 5 and 6,
respectively). This finding indicates a yield decrease during Fmoc-removal. In contrast, the coupling of
Fmoc-Aib over resin 10c only reached an area ratio of 2.0 after 40 min , but increased after 2 h of
reaction time to 3.6 (Table 3, entries 3 and 4). This result indicates a slow coupling between the N-
methyl group of Sar and Fmoc-Aib.
The activation reagent HBTU was replaced by its more potent aza-analog HATU. The couplings over
resins 10a and 10b rendered the similar area ratios as when HBTU was used (Table 3, entries 1 vs. 5,
and entries 2 vs. 6, respectively). The coupling of Fmoc-Aib over 10c gave an area ratio of 7.4 for
Fmoc-Aib-Sar-Pro (Table 3, entry 7), which was higher than those obtained for the Gly (6.2) and DLeu-
analogs (6.0) (Table 3, entries 5 and 6). The area ratio did not increase with prolonged coupling. We
therefore reasoned that after 40 min the coupling was complete and that the differences between the
product area ratios corresponded to the susceptibility of the corresponding Xaa-Pro-sequence on
resins 10a-c to DKP formation under the Fmoc-removal conditions.
To test this, resins 9a-c were treated for 20 min with 20% piperidine in DMF, and Fmoc-Aib was
coupled for 40 min with HATU/DIEA activation. The highest impact of the prolonged basic treatment
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was observed for the DLeu-Pro sequence, which experienced a 50% reduction in yield (Table 3, entry
6 vs. entry 9). The sequence Gly-Pro was also obtained in significantly lower yield (Table 3, entry 5 vs.
entry 8). However, the same yield was achieved for the sequence Sar-Pro (Table 3, entry 7 vs. entry
10).
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Table 3. Coupling of Fmoc-Aib over resins 9a-c. *Prior Fmoc-removal by 20% piperidine in DMF for 5
min. **Prior Fmoc-removal by 20% piperidine in DMF for 20 min. ***Area ratios corresponding to BPA.
Entry
Conditions*,**
Area ratio*** (product, from resin)
1
2
3
4
5
6
7
8
9
10
*Fmoc-Aib / HBTU / DIEA, 40 min
*Fmoc-Aib / HBTU / DIEA, 40 min
*Fmoc-Aib / HBTU / DIEA, 40 min
*Fmoc-Aib / HBTU / DIEA, 2 h
*Fmoc-Aib / HATU / DIEA, 40 min
*Fmoc-Aib / HATU / DIEA, 40 min
*Fmoc-Aib / HATU / DIEA, 40 min
**Fmoc-Aib / HATU / DIEA, 40 min
**Fmoc-Aib / HATU / DIEA, 40 min
**Fmoc-Aib / HATU / DIEA, 40 min
6.4 (Fmoc-Aib-Gly-Pro, 11a)
5.9 (Fmoc-Aib-DLeu-Pro, 11b)
2.0 (Fmoc-Aib-Sar-Pro, 11c)
3.6 (Fmoc-Aib-Sar-Pro, 11c)
6.2 (Fmoc-Aib-Gly-Pro, 11a)
6.0 (Fmoc-Aib-DLeu-Pro, 11b)
7.4 (Fmoc-Aib-Sar-Pro, 11c)
5.3 (Fmoc-Aib-Gly-Pro, 11a)
3.1 (Fmoc-Aib-
DLeu-Pro, 11b)
7.4 (Fmoc-Aib-Sar-Pro, 11c)
Stability of carboxylic acids bound to CTC resin under a range of acylation conditions
Low yields of Fmoc-Gly-Pro were obtained when coupling Fmoc-Gly activated with Oxyma / DIPCDI
over resin 8 (Table 2, entry 2). These conditions also caused a yield decrease in the synthesis of Val-
Arg-Phe (Table 1). To check the stability of the reference compounds PAA and BPA, and peptides on
CTC resin toward different acylation conditions, CTC resin was loaded with an equimolar mixture of
BPA, PAA, Fmoc-Gly, Fmoc-Pro and Fmoc-Leu (0.1 mmol each for 500 mg CTC resin) to give resin
12 (Figure 4).
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Fmoc-Gly, Fmoc-Pro, Fmoc-Leu,
BPA and PAA / DIEA to CTC
5
6
7
8
9
Fmoc-Gly-O
BPA
12
PAA
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Fmoc-Pro-O
Fmoc-Leu-O
i) 20% piperidine in DMF
ii) acylation conditions
Fmoc-Gly-Gly-O
Fmoc-Gly-Pro-O
BPA
13
PAA
Fmoc-Gly-Leu-O
iii) 2% TFA in DCM
iv) + Z-Gly
HPLC-UV analysis
Figure 4
Figure 4. For conditions ii) see Table 4.
Aliquots of washed and dried 12 (10 mg) were used to monitor the stability of the resin-bound
compounds towards a range of activation conditions for Fmoc-Gly coupling to give 13. After reaction
and cleavage of the corresponding products from the resin aliquot, a fixed volume of an external
reference (Z-Gly) solution was added. The area ratios of the products obtained from 13 are shown in
Table 4 and refer to the peak area of the external reference (Z-Gly). The area ratios corresponding to
the Fmoc-Gly-Xaa-peptides and internal references (BPA, PAA) after coupling of Fmoc-Gly activated
with HBTU / DIEA for 40 min were set to 100% (Table 4, entry 1). For all other conditions, the
compound peak areas were smaller with respect to the conditions of entry 1 (Table 4, entries 2-8, the
losses of compounds from resin with respect to entry 1 are given in parenthesis). The yields dropped
in all examples during "overnight"-couplings. The reduction in yield was dramatic (up to 60%) when
acidic coupling reagents (Oxyma, HOBt and HOAt) were used (Table 4, entries 3-8). The reference
compound PAA was found to be the most stable but in contrast, Fmoc-Gly-Pro on CTC showed the
lowest stability under the conditions tested. Because of the similar and even higher stability of the
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reference compounds PAA and BPA with respective to peptides, we were able to detect the
decreases in yield in the test reactions described above (Figures 2 and 3). With respect to SPPS on
CTC resin, on the basis of our findings we recommend that DIPCDI-mediated HOAt, HOBt, and
Oxyma-activation be carefully checked.12
10
11
12
13
14
15
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Table 4. Quantification of premature cleavage of compounds on CTC resin under a range of peptide
coupling conditions (HBTU / DIEA, 40 min and 15 h, entries 1 and 2; Oxyma / DIPCDI, 40 min and 15
h, entries 3 and 4; HOBt / DIPCDI, 40 min and 15 h, entries 5 and 6; HOAt / DIPCDI, 40 min and 15 h,
entries 7 and 8). *Area ratios with respect to the external standard Z-Gly.
Entry
Area ratio* (percent variation respectively to entry 1)
Fmoc-Gly-Gly
3.1 (+/-0%)
2.9 (-8%)
Fmoc-Gly-Pro
3.2 (+/-0%)
3.0 (-4%)
BPA
Fmoc-Gly-Leu PAA
1
2
3
4
5
6
7
8
1.3 (+/-0%)
1.3 (-4%)
1.4 (+1%)
1.3 (-7%)
1.4 (+1%)
1.2 (-9%)
1.3 (-1%)
0.7 (-50%)
3.3 (+/-0%)
3.2 (-3%)
3.1 (-4%)
2.8 (-14%)
3.0 (-7%)
2.6 (-20%)
2.7 (-18%)
1.7 (-48%)
1.9 (+/-0%)
1.9 (+1%)
1.9 (+/-0%)
1.9 (+/-0%)
2.0 (+7%)
1.8 (-3%)
3.1 (-1%)
2.6 (-19%)
2.3 (-26%)
1.9 (-41%)
1.7 (-47%)
1.9 (-39%)
1.2 (-63%)
2.7 (-13%)
3.0 (-4%)
2.5 (-21%)
2.9 (-8%)
1.9 (+4%)
1.1 (-43%)
1.8 (-43%)
Summary
The data presented in this study demonstrate the internal reference method to be a valuable tool for
optimizing conditions for solid-phase peptide synthesis and its possible applications in solid-phase
synthesis of other compounds. We wish to summarize how to use this method.
First, it must be noted that the internal reference does not improve the chromatographic properties of
resin-bound compounds. Its exactness depends on the integration of the peaks. With a suitable
internal reference, reaction control by quantitative HPLC-UV analysis proceeds in the same manner as
conventional HPLC-UV analysis (taking a resin sample from the reaction mixture, washing it, and
running an HPLC chromatogram directly from the cleaved products). The practical value of an internal
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reference is the rapid sample analysis, which allows comparison of chromatograms from resin
samples before and after reaction, or from different reaction conditions. Rather than providing
"absolute" values for single chromatograms, the validity of this approach is based on the comparison
of chromatograms.
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When improvement of a solid-phase synthesis is required, a test resin with internal reference can be
designed. There is no "general" internal reference available. However, here PAA and BPA, with high
retention times in RP-HPLC, were found to be useful in peptide synthesis on CTC resin. As
demonstrated, these compounds bound to the resin more stably or as stable as the peptides by the
same functional group. They gave sharp peaks on HPLC-UV which were well separated from product
peaks. However, there are no limitations for other compounds that fulfill these key requirements.
For quantification, the main peak should not exceed 1 AU in order to have a linear relation between
the peak areas of the reference and the analyte. As a result, the area ratio between products and the
reference should not be too large. The area ratios in this study were between 1 and 50. All analyses
should be performed on the same equipment. The peaks have to be clearly distinguished from the
baseline. Noisy baselines (caused by UV lamps with long running time) affect the accuracy.
We have demonstrated that such a quantitative analysis provides information rapidly and precisely on
the outcome of reactions that would otherwise be difficult to obtain. Once the appropriate protocol is
found, the synthesis can then be performed on a resin without a reference. Therefore we consider the
internal reference method a valuable addition to the toolbox for solid-phase synthesis.
Supporting Information Available Experimental procedures and HPLC-UV chromatograms. This
material is available free of charge via the internet at http:/www.pubs.acs.org.
Acknowledgment Peter Fransen (IRB Barcelona, for proposing the use of PAA), Brigitte Morales
(MIT, Internship 2011), and Isidro Casals (Barcelona Science Park). The work has been partially
financed by CICYT (CTQ2009-07758), the Generalitat de Catalunya (2009SGR 1024), the Institute for
Research in Biomedicine Barcelona (IRB Barcelona), and the Barcelona Science Park.
References
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1 Dörwald, F. Z. Organic Synthesis on Solid Phase, Wiley-VCH: Weinheim, 2002.
2 The Power of Functional Resins in Organic Synthesis, Tulla-Puche, J.; Albericio, F., Eds.; Wiley-VCH:
Weinheim, 2008.
3 Scicinski, J. J.; Congreve, M. S.; Kay, C.; Ley, S. V. Analytical Techniques for Small Molecule Solid
Phase Synthesis. Curr. Med. Chem. 2002, 9, 2103-2127.
4 Dong, M. W. Modern HPLC for Practicing Scientists, John Wiley & Sons, Inc.: Hoboken, NJ, 2006.
5 Congreve, M. S.; Ley, S. V.; Scicinski, J. J. Analytical Constructs for Analysis of Solid-Phase
Chemistry. Chemistry - Eur. J. 2002, 8, 1768-1776.
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6 Congreve, M. S.; Ladlow, M.; Marshall, P.; Parr, N.; Scicinski, J. J.; Sheppard, T.; Vickerstaffe, E.;
Carr, R. A. E. Reporter Resins for Solid-Phase Chemistry. Org. Lett. 2001, 3, 507-510.
7 We use the term "internal reference" in order to avoid confusion with the "internal standard" method
used predominantly for the analysis of complex biological samples. See ref. 4.
8 Barlos, K.; Gatos, D.; Kallitsis, J.; Papaphotiu, G.; Sotiriu, P.; Yao, W.; Schaefer, W. Preparation of
Protected Peptide Fragments Using Triphenylmethyl Resins. Tetrahedron Lett. 1989, 30, 3943-3946.
9 Hoekstra, W. J. The 2-Chlorotrityl Resin: A Worthy Addition to the Medicinal Chemist´s Toolbox. Curr.
Med. Chem. 2001, 8, 715-719.
10 Fischer, P. M. Diketopiperazines in Peptide and Combinatorial Chemistry. J. Peptide Sci. 2003, 9, 9-
35.
11 see ref. 9
12 Subirós-Funosas, R.; El-Faham, A.; Albericio, F. Use of Oxyma as pH Modulatory Agent in the
Prevention of Base-Driven Side Reactions and Its Effect on 2-Chlorotrityl Chloride Resin. Biopolymers
2012, 98, 89-97.
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