Optimisation of conditions for O-benzyl and N-benzyloxycarbonyl protecting group removal using an automated flow hydrogenator

Article abstract of DOI:10.1002/adsc.200600558

A versatile, fully automated flow hydrogenator has been developed that is able to perform sequential flow optimisation experiments, flow library hydrogenation, or iterative scale-up hydrogenation. The behaviour of a palladium catalyst in effecting removal of O-benzyl and N-benzyloxycarbonyl protecting groups has been investigated. Significant observations relating to maintaining optimal throughput are reported. A small library of peptidic derivatives has been deprotected in high yield and purity.

Full text of DOI:10.1002/adsc.200600558

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DOI: 10.1002/adsc.200600558
Optimisation of Conditions for O-Benzyl and N-Benzyloxycar-
bonyl Protecting Group Removal using an Automated Flow
Hydrogenator
Kristian Rahbek Knudsen,a John Holden,b Steven V. Ley,a,* and Mark Ladlowc
a
University of Cambridge, Department of Chemistry, Lensfield Road, Cambridge, CB2 1EW, UK
Fax : (+44)-(0)1223-336-442; e-mail: svl1000@cam.ac.uk
Aitken Scientific Ltd, Oxford House, Oxford Road, Thame, OX9 2AH, UK
GlaxoSmithKline Cambridge Technology Centre, University of Cambridge, Cambridge, CB2 1EW, UK
b
c
Received: October 10, 2006
This paper is dedicated to Professor Masakatsu Shibasaki on the occasion of his 60th birthday.
Supporting information for this article is available on the WWW under http://asc.wiley-vch.de/home/.
Abstract: A versatile, fully automated flow hydro-
genator has been developed that is able to perform
sequential flow optimisation experiments, flow li-
brary hydrogenation, or iterative scale-up hydroge-
nation. The behaviour of a palladium catalyst in ef-
fecting removal of O-benzyl and N-benzyloxycar-
bonyl protecting groups has been investigated. Sig-
nificant observations relating to maintaining opti-
mal throughput are reported. A small library of
peptidic derivatives has been deprotected in high
yield and purity.
ther chromatographic purification. Continuous flow
hydrogenation[8] presents an attractive alternative ap-
proach that, following the introduction of the H-
CubeTM [9,10]
may be safely and conveniently per-
,
formed without the need for special facilities using a
portable device.
Here we report on the development and use of an
automated flow hydrogenation platform that facili-
tates the study and optimisation of catalytic flow pro-
cesses and present our findings in effecting the cata-
lytic hydrogenolysis of benzyl and Cbz protecting
groups with high conversion to afford high purity
products under flow-through conditions (Scheme 1).
A significant difference between the present platform
and a recently reported[11] combination of an H-
CubeTM with a robotic liquid handler resides in the
implementation of a simple single graphical user in-
terface (HydroMateTM) that controls all hardware de-
Keywords: automation; debenzylation; deprotec-
tion; flow hydrogenation; palladium; peptides
Microfluidic[1] and mesofluidic[2] flow processes are re- vices and enables an automated series of experiments
ceiving ever-increasing attention in an attempt to to be performed.[12]
identify and develop more efficient synthesis technol-
In order to develop an optimised procedure for O-
ogies[3] that assist in the rapid, early stage identifica- debenzylation that maximises throughput under con-
tion of potential small molecule modulators of emerg- tinuous flow hydrogenation conditions, we selected
ing therapeutic targets.[4] Key advantages associated O-benzyl protected N-Boc-tyrosine as a representa-
with flow processes in comparison with more tradi- tive substrate. A series of experiments was performed
tional batch techniques include the ability to inde- to probe the effects of flow rate, temperature and
pendently vary and precisely control reaction parame- concentration (Figure 1). The 10% Pd/Ccatalyst car-
ters such as stoichiometry, temperature, pressure and tridge was changed only for each new set of reaction
flow rate leading to high reproducibility and facilitat- conditions.
ed scale-up.[5] We are currently developing protocols
As expected the efficiency of the catalyst progres-
that invoke the use of supported reagents in reactor sively deteriorates with increasing throughput; the
cartridges[6] that may be combined to implement rate of deterioration being faster with increased con-
multi-step, flow-through compound synthesis.[7]
centration and flow rate. However, although increas-
Catalytic batch hydrogenation constitutes an impor- ing pressure (up to 60 bar; results not shown) had
tant synthetic method that is typically high-yielding little effect, increasing temperature was found to have
and affords clean products that often require no fur- a significant effect in maintaining catalyst activity.
Adv. Synth. Catal. 2007, 349, 535 – 538
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Kristian Rahbek Knudsen et al.
To study this phenomenon further, a series of ex-
periments at a concentration of 0.1M and flow rate of
1.0 mLminÀ1 was performed. For the first six runs the
temperature was maintained at 308Cand a rapid de-
terioration in catalytic activity was observed. Howev-
er, increasing the temperature to 608Clead to an im-
mediate recovery in catalytic activity giving high con-
version for the following eight runs (Figure 2). The
Scheme 1. Schematic representation of the automated flow
hydrogenation platform.
Figure 2. Sequential hydrogenation of 5-mL aliquots of a
0.1M solution of O-benzyl N-Boc tyrosine in a flow-through
catalytic hydrogenator at 308C(samples 1–6) and then at
608C(samples 6–14).
higher conversions observed under these conditions
may be attributable to more effective solvolytic clean-
ing of the catalyst surface or an increase in catalytic
off-rate with increasing temperature. In general,
therefore, in order to maximise turnover it is advanta-
geous to perform flow-through catalytic hydrogena-
tion at elevated temperatures whenever possible.
Typically, under continuous flow hydrogenation
conditions the catalyst has no opportunity to regener-
ate in the absence of substrate. A series of automated
experiments was performed to contrast throughput
and conversion for both continuous and iterative loop
injection flow hydrogenation protocols.[13] In each of
the experiments shown in Figure 3, the total quantity
of substrate 1 processed was identical. However, by
Figure 1. Study of the effect of temperature, flow rate and
concentration on throughput for repeated catalyst use in an
automated flow-though hydrogenator.
536
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O-Benzyl and N-Benzyloxycarbonyl Protecting Group Removal
Figure 4. Optimisation of N-Cbz protecting group removal.
Table 1. Deprotection of eight N-Cbz protected compounds.
Figure 3. Comparison of a large scale reaction run as either
a continuous run fractionated by the liquid handler or a
series of single injections with recovery period for the cata-
lyst.
Substrate
Conversion [%][a] Yield [%]
allowing for a period of catalyst recovery in the ab-
sence of substrate, it is apparent that more concen-
trated solutions may be processed with full conver-
sion. Although, ultimately, maximum throughput was
obtained under continuous flow conditions, allowing
for a period of catalyst regeneration is beneficial in
preserving activity of the catalyst over an extended
period of time.
Cbz-Pro-OMe
Cbz-Piperazine
99
99
99
97
98
95
99[b]
80[b]
77
Cbz-Asp(OMe)-OMe
A
Cbz-(OBn)Tyr-OMe
Cbz-Ser-OMe
Boc-(N-Cbz)-Lys-O
Cbz-Thr-Tyr
83[c]
82
(Naph)
86
(O-t-Bu)-O-t-Bu 99
96
99
Cbz-Pro-tetrazole
99[d]
Finally, in order to establish general conditions that
would be suitable for hydrogenation of a small com-
pound library of N-Cbz derivatives, we selected e-[N-
Cbz] lysine tert-butyl ester 3 as a test substrate.
Again, optimal throughput was obtained at elevated
temperature, in this case 808Cat 0.05 M (Figure 4).
A small library of eight N-Cbz protected com-
[a]
Measured by 1H NMR and GC-MS; each reaction was
performed by automated processing of 3.5 mL of a
0.05M solution at 1.0 mLminÀ1 at 808C.
Isolated as hydrochloride salt.
Both protecting groups removed.
[b]
[c]
[d]
This reaction was also performed on 3-g scale; 30%
AcOH was added to the solvent, see ref.[14]
pounds, including the dipeptide Z-Thr-Tyr
A
t-Bu was selected and the compounds were subjected
to sequential automated deprotection under the opti- of elevated temperature and period regeneration
mised conditions (Table 1). In all cases the deprotect- were found to be beneficial in terms of maximising
ed derivatives were isolated in good yields and high throughput and extending the lifetime of the catalyst.
purities. In particular, both Cbz and benzyl protecting
groups were simultaneously removed from Z- and N-deprotection of a range of substrates including
(OBn)Tyr-OMe under these experimental conditions. amino acids, simple amines and dipeptides demon-
We have applied this methodology to effect the O-
In conclusion, we have shown that the optimisation strating that this strategy has utility in peptide synthe-
of flow-through hydrogenation processes may be fa- sis. This aspect is currently under further investigation
cilitated using an automated platform. A combination within our group.[15]
Adv. Synth. Catal. 2007, 349, 535 – 538
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Kristian Rahbek Knudsen et al.
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Experimental Section
Typical Experimental Procedure
A 40-mL sample vial containing 35 mL of a 0.1M N-Boc-
(OBn)-tyrosine solution in EtOH/EtOAc (1:1) was placed at
a designated sample position on the liquid handler robot. A
sequence of 7 reactions was programmed, in each case intro-
ducing a 5-mL aliquot into the flow hydrogenator using loop
injection . The system automatically stabilises at the desig-
nated temperature and flow rate (e.g., 608C, 1.0 mLminÀ1)
before commencing each experiment. For each reaction, the
product was collected as an individual fraction for the desig-
nated time before the robot progressed to the next experi-
ment. Conversion was measured by HPLC. HPLC: tR =
4.00 min (product), tR =4.52 min (starting material). All
other reactions were run in a similar manner except continu-
ous flow experiments where the system solvent was replaced
with a stock solution of substrate.
[3] a) I. R. Baxendale, S. V. Ley, G. K. Tranmer. J. J. Hay-
ward, ChemMedChem. 2006, submitted; b) A. Kirschn-
ing, W. Solodenko, K. Mennecke, Chem. Eur. J. 2006,
12, 5972.
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[5] a) N. G. Anderson, Org. Proc. Res. Dev. 2001, 5, 613;
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[6] S. V. Ley, I. R. Baxendale, R. N. Bream, P. S. Jackson,
A. G. Leach, D. A. Longbottom, M. Nesi, J. S. Scott,
R. I. Storer, S. J. Taylor, J. Chem. Soc., Perkin Trans. 1
2000, 3815.
Supporting Information
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[8] For other applications of flow hydrogenation see:
a) A. J. Sandee, D. G. I. Petra, J. N. H. Reek, P. C. J.
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More detailed description of the system configuration and
general experimental details.
Acknowledgements
K. R. Knudsen is grateful to the Carlsberg Foundation for fi-
nancial support and GlaxoSmithKline is thanked for gener-
ous financial support of this work.
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Adv. Synth. Catal. 2007, 349, 535 – 538