Peptides
`
`The Wave of the Future
`
`Proceedings of the Second International
`and the Seventeenth American
`Peptide Symposium
`
`Edited by
`
`Michal Lebl
`and
`Richard A. Houghten
`
`American Peptide Society
`
`MYLAN EXHIBIT - 1022
`Mylan Pharmaceuticals, Inc. v. Bausch Health Ireland, Ltd. - IPR2022-00722
`
`

`

`A C.I.P. Catalogue record for this book is available from the Library of Congress.
`
`ISBN 0-9715560-0-8 (American Peptide Society)
`ISBN 1-4020-0473-7 (Kluwer)
`
`Published by American Peptide Society,
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`
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`
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`Copyright ©2001 by American Peptide Society
`
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`

`

`Peptides: The Wave of the Future
`Michal Lebl and Richard A. Houghten (Editors)
`American Peptide Society, 2001
`
`• Systematic Investigation of the Aspartimide Problem
`
`M. Mergler, F. Dick, B. Sax, P. Weiler and T. Vorherr
`BACHEM AG, CH-4416 Bubendolf, Switzerland
`
`Introduction
`Aspartimide formation is one of the best-documented side reactions in peptide synthe-
`sis. Even bulky side-chain protecting groups such as OtBu do not prevent this unde-
`sired reaction. In Fmoc/tBu-based SPPS, the repetitive piperidine treatments needed
`for Fmoc removal lead to aspartimide formation and further by-products. Since the
`the worst-case scenario, the hexapeptide
`combination Asp-Gly represents
`Va1-Lys-Asp-Gly-Tyr-Ile (I) [1] was used in the present work to investigate parame-
`ters influencing aspartimide formation (Fmoc-cleavage conditions, nature of Asp side
`chain protection). Based on the idea of backbone-protection [2], Fmoc-Asp(OtBu)-
`(Hmb)-Gly-OH was also synthesised and included in this study.
`Results and Discussion
`For Asp side chain protection, the following groups were applied for synthesis of I as
`their Fmoc-derivatives: OtBu, 0-3-methylpent-3-y1 ester (OMpe) [3], 4-pyridyl-
`diphenyl-methyl ester (OPyBzh) [4], the bicyclic ortho-ester 4-methyl-2,6,7-trioxa-
`bicyclo-[2,2,2]-octane (OBO) [4] and, as already mentioned, the combination OtBu
`side chain protection plus Hmb-backbone protection. In the first step, several potential
`by-products, V KdGYI, VKd(GYI), VIUD(GYI), VKD(piperidide)GYI and
`VKD(GYI)-piperidide, resulting from the opening of the aspartimide cycle, were inde-
`pendently synthesized. Furthermore, RP-HPLC-optimization of these potential con-
`taminants was carried out to properly resolve and quantify the different side products.
`The OPyBzh-protecting group had ideal TFA lability. Unfortunately, high levels of
`aspartimide and piperidides were detected following synthesis of I. Another new de-
`rivative, the orthoester protected Asp derivative (OBO-protection), was designed to
`completely suppress nucleophilic attack at the I3-carboxy group. Surprisingly,
`a-piperidide was generated during synthesis and, in addition, large quantities of
`aspartimide were observed during the second stage of OBO removal, which consists
`of the saponification under basic conditions. Therefore, the disappointing results ob-
`tained on OPyBzh- and OBO-protection were not included in Table 1.
`
`Table 1. HPLC-analysis of crude products (Bakerbond C18 300 A, phosphate buffer pH 2.3,
`CH3CN as modifier).
`
`Protection
`
`Base'
`
`Product
`(96)
`
`D/L-Aspartimide
`(%)
`
`L-a-Piperidide
`(%)
`
`OtBu
`
`OMpe
`
`OtBuiHmb
`
`OtBu
`
`OMpe
`
`Pip.
`
`Pip.
`
`Pip.
`
`DBU
`
`DBU
`
`89.1
`
`93.9
`
`94.0
`
`52.1
`
`83.0
`
`3.0
`
`0.7
`
`<a3b
`
`21.8
`
`7.8
`
`1.5
`
`<0.3"
`<0.3"
`
`9.4
`
`1.9
`
`L-P-Piperidide
`(%)
`<cob
`
`<0.3b
`
`<0.3"
`
`0.6
`<cub
`
`<0.3"
`94.1
`<0.3"
`<0.3"
`DBU
`OtBualmb
`° Pip.: Piperidine/DMF 1 : 4, DBU: DBU/Piperidine/DMF 1 : 20: 79; b below detection
`limit.
`
`63
`
`

`

`Mergler et aL
`
`Two Fmoc-cleavage procedures, the standard protocol piperidine/DMF (1 : 4) and
`1% DBU in piperidine/DMF (1 : 4), and different protecting groups for the Asp resi-
`due in model peptide I (see Table 1) were employed to study aspartimide formation.
`Peptide I, synthesized according to the various strategies indicated above, was ob-
`tained after TFA assisted cleavage, and the crude products were subsequently ana-
`lysed by HPLC.
`In fact, Fmoc-removal in the presence of DBU worsened the effects already ob-
`served in the case of the standard piperidine treatment. However, no significant
`amounts of 13-peptide were detected. In the case of OtBu-protection, upon DBU treat-
`ment an as yet undefined component (5.8%) was observed in addition to the known
`compounds. The OMpe-protecting group showed a significant improvement with re-
`spect to aspartimide formation when compared to regular OtBu-protection. Most inter-
`estingly, if the Hmb-backbone protection approach was followed, neither under
`standard conditions nor in the presence of DBU, aspartimide or related side product
`was observed (detection limit 0.3%).
`
`Conclusions
`This systematic investigation clearly showed that in our test system, no detectable
`amounts of aspartimide were formed if limb-backbone protection was applied in addi-
`tion to standard OtBu-protection of the Asp side chain. However, the synthesis of all
`different limb protected amino acid derivatives followed by their incorporation into
`dipeptides would be quite labourious. Therefore, taking into account the markedly im-
`proved properties of Mpe-protection compared to the standard OtBu-group, this re-
`cently described variant should be considered for sequences prone to aspartimide
`formation.
`
`References
`1. Nicolas, E., Pedroso, E., Giralt, E. Tetrahedron Lett. 30, 497 (1989).
`2. Offer, J., Quibell, M., Johnson, T. J. Chem. Soc., Perkin Trans. 1 175 (1996).
`3. Karlstr0m, A., Linden, A. Tetrahedron Len. 37, 4243 (1996).
`4. Unpublished derivatives.
`
`64
`
`