A new method testing the orthogonality of different protecting groups

Article abstract of DOI:10.1016/j.carres.2015.10.007

A new test was elaborated to identify a new set of orthogonal protecting groups. With the developed method eight different protecting groups were tested under various deprotection conditions and the complex reaction mixtures were analysed by HPLC. The dev

Full text of DOI:10.1016/j.carres.2015.10.007

Accepted Manuscript
Title: A new method testing the orthogonality of different protecting groups
Author: Károly Ágoston, Ágnes Ágoston, Colin R. Dorgan, Péter Fügedi
PII:
S0008-6215(15)00330-4
DOI:
Reference:
http://dx.doi.org/doi: 10.1016/j.carres.2015.10.007
CAR 7081
To appear in:
Carbohydrate Research
Received date: 24-6-2015
Revised date: 16-10-2015
Accepted date: 16-10-2015
Please cite this article as: Károly Ágoston, Ágnes Ágoston, Colin R. Dorgan, Péter Fügedi, A
new method testing the orthogonality of different protecting groups, Carbohydrate Research
(2015), http://dx.doi.org/doi: 10.1016/j.carres.2015.10.007.
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A new method testing the orthogonality of different protecting groups
Károly Ágoston[a,b]*, Ágnes Ágoston[c], Colin R. Dorgan[b] and Péter Fügedi[a]
[a]
Körútja 2, 1117-Budapest, Hungary
[b] K. Ágoston, C. R. Dorgan: Carbosynth LTD 8&9 Old Station Business Park, RG20
6NE-Compton, UK; E-mail: karoly.agoston@carbosynth.com
[c] Á. Ágoston: Lab-Comp LTD, Rákos Tér 23/A, 1142-Budapest, Hungary
K Ágoston, P Fügedi: Institute of Organic Chemistry, RCNS-HAS Magyar Tudósok
ARTICLE INFO
Received:
Highlights
 A method was developed to identify orthogonality of various protecting
groups.
 The method is based on the chemo-selectivity of protecting groups.
 The crude reaction mixtures were analysed by HPLC.
 With the method a new deprotection protocol for the propagyl group is
developed.
Graphical abstract
ABSTRACT
A new test was elaborated to identify a new set of orthogonal protecting groups. With
the developed method eight different protecting groups were tested under various deprotection
1
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conditions and the complex reaction mixtures were analysed by HPLC. The developed
method allows for quick identification of orthogonality using simple model structures.
Keywords
Protecting groups, Orthogonal protection, Method development
1.
Introduction
The synthesis of complex natural products generally requires the extensive use of
protecting groups. Although very elegant, there are few examples of natural product total-
synthesis which avoid the use of any protecting groups at all.1 These examples are exceptions
to the trends in synthetic chemistry. The number of protecting groups used during a synthesis
grows significantly with the increasing complexity of the targeted compound. The concept of
orthogonal sets of protecting groups has been established for more than 30 years2 for the
protection of amino groups in peptide synthesis. The principle has been generalized for
practically all kinds of protecting groups.3 The concept of orthogonal protection is particularly
useful for the synthesis of complex branched oligosaccharides. Already several orthogonal
sets have been reported and applied for the protection of the hydroxyl groups within
oligosaccharide synthesis.4 Most of these sets consist of 2-4 individual groups. We believe
there is still a need for new sets of orthogonal protecting groups especially with sets of more
than four members. To our knowledge there is only one example in the literature to use five
orthogonal protecting groups during a synthesis.5 The increasing number of newly developed
protecting groups makes it possible to identify similar orthogonal sets with more than four
orthogonal protecting groups. In order to develop new sets of orthogonal protecting groups
time consuming preparative work is necessary until a fully protected derivative is made. Our
intention was to shorten this procedure with a fast and simple test method.
2.
Results and discussion
The aim of this study was to develop a quick and simple method to test the
orthogonality of selected protecting groups. The basis of our method can be seen in Figure 1.
Compound A was selected as a starting material having one free hydroxyl function. (Protected
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monosaccharide derivative was selected for the study which is available in three steps from
raw materials.) R and R’ were persistent protecting groups and at least one of the groups is
aromatic providing good UV absorbance for detection by HPLC. The free hydroxyl function
of compound A was protected with different temporary protecting groups affording
derivatives such as B1 or B2. The applied temporary protecting groups were tested for their
orthogonal behaviour. Known literature methods to remove those temporary groups were
optimized on the clean Bi derivatives. A HPLC method was developed to separate all the Bi
derivatives and A starting material. Then all Bi derivatives were mixed in equal molar
concentrations resulting in a stock solution. The optimized conditions to remove temporary
protecting groups were applied to samples of the stock solution and after work up procedures
the crude reaction mixtures were analysed by HPLC methods.
Methyl 2,3,4-tri-O-benzyl-α-D-glucopyranoside (A)6 was selected as scaffold for
derivatization (Scheme 1.). Compound A is easily available from methyl α-D-glucopyranoside
in three steps with literature methods7 and protected with benzyl groups which are commonly
used as persistent protecting groups. Furthermore these groups provide high UV absorbance
for detection in HPLC analysis.
The temporary protecting groups selected for the test were chloroacetyl, levulinoyl, (o-
nitrophenyl)acetyl esters, Fmoc carbonate, t-butyldimethylsilyl, 1-naphtylmethyl, allyl and
propargyl ethers (Scheme 1). Some of these groups have been known to be orthogonal (e.g:
chloroacetyl, levulinoyl, Fmoc) to each-other for many years3 while other groups were proven
to be orthogonal recently.8 All of Bi derivatives were prepared based on literature procedures.
Known methods for the chemoselective cleavage of the protecting groups were optimized for
Bi derivatives (See the details in the experimental part of the supplementary info). All Bi
derivatives were mixed in a defined concentration resulted in a stock solution. A HPLC
method was developed to separate all derivatives including compound A. Calibration curves
were recorded to obtain more precise calculation. Unfortunately, there was no baseline
separation between 6-propargylated and 6-chloroacetylated derivative but this did not affect
the calculation based on the calibration curves. All deprotection conditions were performed on
the stock solution (See the details in the experimental part), and the HPLC results collated
into TABLE 1. The numbers in each field represent the percentage of the Bi present in the
mixture after performing the deprotection procedure. The numbers were rounded to the
3
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nearest 5% value for easier interpretation. (For the exact obtained numbers see Supporting
Info).
First the removal of the chloroacetate group was tested, and under the developed
9
conditions all B1 was transformed into compound A (Table 1, entry 1, for HPLC
chromatogram see Figure 2). As an undesired effect we observed the partial decomposition
(deprotection) of the silylated derivative (B5). Thiourea treatment alone on the pure B5 did not
result in any cleavage of the silyl protecting group, but the HCl generated in the reaction
between thiourea and the chloroacetylated derivative caused the partial cleavage of the silyl
ether.
This was completely suppressed by addition of pyridine into the reaction mixture
(Table 1, entry 2). This undesired, but expected side effect showed the effectiveness of our
test method as it is not only the added reagent which is able to cleave certain protecting
groups. Other reactive species can form during a reaction, which might interact with other
compounds causing side-reactions These circumstances would not be observable when
working with clean compounds separately therefore our method provides a more
comprehensive test of protecting group orthogonality.
The cleavage of the Lev ester group from compound B2 did not cause any degradation
of other protecting groups, and resulted in high recovery of the individual compounds. (Table
1, entry 3, for HPLC chromatogram see Figure 3). Under the conditions to cleave NPAc ester
8
group (B3  A) most of the protecting groups were not decomposed at all, but the two other
esters (chloroacetate and levulinate) present in the mixture were damaged to some extent.
(Table 1, entry 4) The decomposition of these ester groups was not significant, but already
visible on the HPLC chromatogram. Most probably the amine generated during the cleavage
was the reactive agent causing these decompositions. Cleavage of Fmoc carbonate (B4  A)
with diluted, hindered base (DBU) and the cleavage of silyl ether (B510  A) with diluted
4
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acid (camphorsulfonic acid) resulted in very clean reaction mixtures with no observed
decomposition of other protecting groups. (Table 1, entry 5 and 6, respectively).
Cleavage of the naphtyl ether group under the oxidative conditions (B6  A)
developed for the clean product did not result in complete cleavage of the NAP ether group
when applied to the stock solution. (Table 1, entry 7) The amount of the reagent used for the
reaction and the reaction time had to be increased to achieve full conversion. (Table 1, entry
8). Due to the hydrolytic reaction conditions the silyl ether group was decomposed completely
even with the reduced amount of reagent.
Under the conditions to remove allyl ether protecting groups (B711  A) most of the
other protecting groups were untouched (Table 1, entry 9). One of the exceptions was the silyl
ether group which was completely cleaved due to the hydrolytic condition used to cleave the
rearranged enol-ether. The other compound which completely disappeared from the reaction
mixture was B8, the propargyl ether protected derivative. This observation could have been
expected since transition metal based catalyst might interfere with triple bonds. According the
accurate calculations on the amount of compound A not all B8 was transformed into A.
In the literature there are different conditions to remove propargyl ether,12 but most of
these are not compatible with ester protecting groups due to the strongly basic conditions. The
condition we selected was based on the formation of a pyramidal complex of the triple bond
with dicobalt-octacarbonyl.12h The formed complex can be hydrolyzed under acidic conditions
revealing the free hydroxyl function (B812a  A). Unfortunately, strongly acidic conditions
were needed to cleave the ether bond. This was modestly tolerated by other protecting groups
(Table 1, entry 10). There were two groups with relative high stability under this condition,
namely the Fmoc carbonate and the 1NAP ether protected derivatives, but we can conclude
that the conditions we used for the deprotection of the propargyl group is not highly
compatible with other protecting groups.
3.
Conclusion
A powerful method was developed for testing the stability of different protecting
groups under various reaction conditions. HPLC methods were applied to analyze the crude
5
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reaction mixtures. Eight different protecting groups were used for the study and all
deprotection conditions developed for the individual protecting groups were tested. New sets
of orthogonal protecting groups could be identified with the presented method. Based on the
results a new set of orthogonal protecting groups could be used such as: chloroacetyl,
levulinoyl, (o-nitrophenyl)acetyl esters, Fmoc carbonate, 1-naphtylmethyl and allyl ethers.
This model system is ideal to test the orthogonality of selected protecting groups before
starting any synthesis using an unknown combination of different protecting groups. The
method is based only on the chemo-selectivity of the various protecting groups. The main
limitation of this method is the use of a primary hydroxyl group for the test as primary
hydroxyls generally have higher reactivity than the more common secondary. We still assume
that the results generated on this system can be interpreted more generally by using different
reaction conditions (for example performing stability test using different concentration of
reagents and vary temperatures or reaction times). It is beyond the scope of this study to deal
with migration of acyl groups and further side reactions which are structure dependent
reactions, thus influenced by the orientation of the protected hydroxyl groups. The method, as
our referee suggested, could be potentially used to test more challenging set, such as selective
deprotection of different silyl ether groups. In addition to identifying a new set of orthogonal
protecting groups the test could provide new deprotection protocols for certain protecting
groups – as in our case turned out with the propagyl ether group. The investigation of
propagyl ether group deprotection with Wilkinson’s catalyst, as a new method, will be
published separately.
4.
Experimental
4.1. General. — Commercially available starting materials were used without further
purification. Solvents were dried according to standard procedures. Melting points
(uncorrected) were determined on a Griffin apparatus. Optical rotations were measured with a
Jasco-Optical activity AA-10R polarimeter. NMR spectra were recorded on a Varian Gemini
2000 (200 MHz for 1H and 50 MHz for 13C) and on a Varian Unity-Inova (300 MHz for 1H
and 75 MHz for 13C) spectrometer in CDCl3 as solvent. All chemical shifts are quoted in ppm
downfield from the characteristic signals (1H: 0.00 ppm (TMS), 13C: 77.00 ppm (CDCl3)).
Kieselgel 60 (E. Merck, Darmstadt, Germany) was used for column chromatography and DC-
Alufolien Kieselgel 60 F256 plates were used for TLC. MS spectra were recorded on an
Applied Biosystems 3200 QTRap spectrometer. HPLC chromatograms were recorded on
6
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Knauer Smartline system equipped with diode array detector. The detection was performed
both in 205 nm and 254 nm wavelength. YMC Pack ODS-AQ (150 × 4.6 mm) column with 3
µm particle size was used as stationary phase. A gradient was used from MeCN (65%) to
MeCN (80%) in water over 10 min, then to MeCN (98%) over 8 min. with a flow rate of 1.1
mL/min.
4.2. General deprotections:
 Stock solution was made as follows: 0.50 mmol of all Bi derivatives was measured
into a vial and were dissolved in CH2Cl2 resulting 100 mL solution. 10 µL of the stock
solution was used for HPLC control. 5 mL of the prepared stock solution was used for each
deprotections. The 5 mL sample was concentrated and dissolved on the solvent system used
for the deprotection procedure.
4.3. Entry 1:
5 mL of stock solution was concentrated and the residue was dissolved in the mixture of
MeOH (2.5 mL) and toluene (2.5 mL). Thiourea (4 mg) was added to the solution and stirred
for 3 h at 80 C. The volatiles were removed in vacuo and the residue was dissolved in
CH2Cl2 (20 mL), washed with water (25 mL), dried, filtered and concentrated. The residue
was dissolved in CH2Cl2 up to 5.00 mL volume and 20 µL was taken for HPLC analysis.
4.4. Entry 2:
5 mL of stock solution was concentrated and the residue was dissolved in the mixture of
MeOH (2.2 mL) toluene (2.2 mL) and pyridine (0.6 mL). Thiourea (4 mg) was added to the
solution and stirred for 3 h at 80 C. The volatiles were removed in vacuo and the residue was
dissolved in CH2Cl2 (20 mL), washed with 1N aq. HCl (25 mL), water (25 mL) and sat.
NaHCO3 solution (25 mL), dried, filtered and concentrated. The residue was dissolved in
CH2Cl2 up to 5.00 mL volume and 20 µL was taken for HPLC analysis.
4.5. Entry 3:
5 mL of stock solution was concentrated and the residue was dissolved in the mixture of
MeOH (0.5 mL) and CH2Cl2 (4.5 mL). Hydrazine acetate (20 mg) was added to the solution
and stirred for 1 h at rt. The volatiles were removed in vacuo and the residue was dissolved in
7
Page 7 of 13

CH2Cl2 (20 mL), washed with water (25 mL), dried, filtered and concentrated. The residue
was dissolved in CH2Cl2 up to 5.00 mL volume and 20 µL was taken for HPLC analysis.
4.6. Entry 4:
5 mL of stock solution was concentrated and the residue was dissolved in the mixture of
MeOH (5 mL) and THF (1 mL). Zn powder (80 mg) and NH4Cl (40 mg) were added to the
solution and stirred for 5 h at rt. The mixture was filtered and the filtrate was concentrated in
vacuo and the residue was dissolved in CH2Cl2 (20 mL), washed with water (25 mL), dried,
filtered and concentrated. The residue was dissolved in CH2Cl2 up to 5.00 mL volume and 20
µL was taken for HPLC analysis.
4.7. Entry 5:
DBU (50 µL) was added to the 5 mL stock solution and the mixture was stirred for 5 min at
rt. The mixture was diluted with CH2Cl2 (20 mL), washed with 1N aq. HCl (25 mL), water
(25 mL) and sat. NaHCO3 solution (25 mL), dried, filtered and concentrated. The residue was
dissolved in CH2Cl2 up to 5.00 mL volume and 20 µL was taken for HPLC analysis.
4.8. Entry 6:
5 mL of stock solution was concentrated and the residue was dissolved in the mixture of water
(1 mL) and THF (5 mL). Camphorsulfonic acid (20 mg) was added to the solution and stirred
for 8 h at 50 C. The mixture was diluted with CH2Cl2 (20 mL), washed with water (25 mL)
and sat. NaHCO3 solution (25 mL), dried, filtered and concentrated. The residue was
dissolved in CH2Cl2 up to 5.00 mL volume and 20 µL was taken for HPLC analysis.
4.9. Entry 7:
5 mL of stock solution was concentrated and the residue was dissolved in the mixture of water
(0.5 mL) and MeCN (4.5 mL). CAN (42 mg) was added to the solution and stirred for 3 h at
r.t. The mixture was diluted with CH2Cl2 (20 mL), washed with water (25 mL) and sat.
NaHCO3 solution (25 mL), dried, filtered and concentrated. The residue was dissolved in
CH2Cl2 up to 5.00 mL volume and 20 µL was taken for HPLC analysis.
4.10. Entry 8:
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5 mL of stock solution was concentrated and the residue was dissolved in the mixture of water
(0.5 mL) and MeCN (4.5 mL). CAN (70 mg) was added to the solution and stirred for 8 h at
r.t. The mixture was diluted with CH2Cl2 (20 mL), washed with water (25 mL) and sat.
NaHCO3 solution (25 mL), dried, filtered and concentrated. The residue was dissolved in
CH2Cl2 up to 5.00 mL volume and 20 µL was taken for HPLC analysis.
4.11. Entry 9:
5 mL of stock solution was concentrated and the residue was suspended in EtOH (5 mL).
Rh(P(Ph)3)3Cl (20 mg) was added to the solution and stirred for 3 h at 80 C. The volatiles
were removed in vacuo and the residue was dissolved in the mixture of acetone (4.5 mL) and
1N aq. HCl (0.5 mL) and stirred for 1 h at 60 C. The volatiles were removed in vacuo and
the residue was dissolved in CH2Cl2 (20 mL), washed with water (25 mL) and sat. NaHCO3
solution (25 mL), dried, filtered and concentrated. The residue was dissolved in CH2Cl2 up to
5.00 mL volume and 20 µL was taken for HPLC analysis.
4.12. Entry 10:
Co2CO8 (30 mg) was added to the 5 mL of stock solution and stirred for 1 h at r.t.. The
volatiles were removed in vacuo and the residue was dissolved in the mixture of MeOH (4
mL), water (0.5 mL) and TFA (0.5 mL). The mixture was stirred for 48 h at 45 C, then TEA
(2 mL) was added and the mixture was filtrated through CELITE. The filtrate was diluted
with CH2Cl2 (50 mL) and washed with water (2 × 25 mL) and NaHCO3 solution (25 mL) the
organic phase was dried (MgSO4), filtrated and concentrated. The residue was dissolved in
CH2Cl2 up to 5.00 mL volume and 20 µL was taken for HPLC analysis.
Acknowledgements
Generous support from RCNS-HAS is gratefully acknowledged.
Supplementary data
Supplementary data associated with this article (Experimental procedures, NMR
spectra and HPLC chromatograms) can be found, in the online version, at…
References
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10
11
12
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11
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Scheme 1. The compounds used for method development.
Figure1. The principle of the test.
Figure 2. Overlapped HPLC chromatogram of Entry 1 and reference sample.
Figure 3. Overlapped HPLC chromatogram of Entry 3 and reference sample.
12
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Entry Conditions
B1
0%
Thiourea MeOH/tol./pyr. reflux 0%
B2
B3
B4
B5
B6
B7
B8
1
2
3
4
5
6
7
8
9
Thiourea MeOH/tol. reflux
35%
95% 95% 95%
95% 95% 95%
95% 95% 95% 95% 95% 95% 95%
0%
90% 90%
95% 95% 95%
H2NNH2×AcOH DCM/MeOH
Zn, NH4Cl MeOH/THF
1% DBU DCM
95%
95% 95% 95% 95% 95% 95%
0%
95% 95% 95% 95% 95%
0%
95% 95% 95% 95%
CSA THF/H2O
0%
0%
0%
0%
90% 90% 90% 90%
95% 95% 95% 95%
95% 95% 95% 95%
95% 95% 95% 95%
90% 90% 90%
CAN (3 eq.) MeCN/H2O, 3h
CAN (5 eq.) MeCN/H2O, 8h
i: Wilkinson’s cat., EtOH
ii: 1N HCl, acetone
50%
0%
95% 95%
95% 95%
0%
0%
95%
10
i: Co2CO8 DCM
50%
70%
20%
95%
0%
90%
50%
0%
ii: TFA MeOH/H2O
TABLE 1. Collected results of different deprotections.
13
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