Acm Group Research Articles

Sulfoxide‐Mediated Cys‐Trp‐Selective Bioconjugation that Enables Protein Labeling and Peptide Heterodimerization

AbstractA method was developed that enables the magnesium chloride (MgCl2)‐activated S‐acetamidomethyl cysteine sulfoxide (Cys(Acm)(O)) to induce the sp2(C−H) sulfenylation of the indole of Trp residues. The reaction operates under mild acidic conditions using acetic acid or an ionic liquid to give the Trp‐sulfenylated products. Other than Trp, all other proteinogenic amino acids are unreactive to the sulfenylation conditions. We demonstrated the successful application of this reaction to various peptides, including lysozyme. Furthermore, we achieved the Trp‐modification of a monoclonal antibody by a MgCl2‐mediated reaction in an acidic ionic liquid. The resulting antibody exhibited antibody performance comparable to the parent protein. The amide moiety in the Acm group contributes to the difference in chemical behaviors between S‐Acm and S‐p‐methoxybenzyl (MBzl)‐protected cysteine sulfoxides. This is because the S‐Acm sulfoxide is converted to S‐chlorocysteine responsible for Trp‐sulfenylation under less acidic conditions than those required for the reaction of S‐MBzl sulfoxide. Based on this rationale, we prepared a linker possessing S‐Acm and S‐MBzl oxide moieties and subjected the linker to heterodimerization of DNA‐binding MYC and MAX peptides containing a Trp handle. The one‐pot/stepwise Cys‐Trp conjugation between the linker and DNA‐binding peptides allowed the generation of a heterodimeric MYC/MAX DNA binder.

L-Methionine Selenoxide as an Oxidizing and Deprotection Reagent for the Synthesis of Multiple Disulfide Bonds in Peptides.

The regioselective synthesis of multiple disulfide bonds in peptides has been a significant challenge in synthetic peptide chemistry. In this work, two disulfide bonds in peptides were regioselectively synthesized via an approach of MetSeO oxidation and deprotection reaction (SeODR), in which the first disulfide bond was constructed through oxidation of dithiol by MetSeO in a neutral buffer, and the second disulfide bond was then directly constructed through the deprotection of two Acm groups or one Acm group and one Thz group by MetSeO in acidic media. Synthesis of two disulfide bonds by the SeODR approach was achieved through a one-pot manner. Moreover, the SeODR approach is compatible with the synthesis of peptides containing methionine residues. Both H+ and Br- drastically promoted the reaction rate of SeODR. The mechanistic picture for the SeODR approach was delineated, in which the formation of a stable Se-X-S bridge as the transition state plays a critical role. The SeODR approach was also utilized to construct the three disulfide bonds in linaclotide, conferring a reasonable yield.

P645: THE INFLUENCE OF ACTIVE CARDIAC MONITORING WITH REMOTE CONTROL ON THE SURVIVAL OF PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA, RECEIVING IBRUTINIB.

Background: Survival of chronic lymphocytic leukemia (CLL) patients receiving ibrutinib (Ib) is influenced by many factors, among which cardiovascular disease, both in history and during treatment with Ib, is of great importance. Aims: To assess the impact on 5-year overall survival (5-OS) of patients CLL treated with Ib two options for dynamic monitoring by a cardiologist: standard cardiac monitoring (SC) and active cardiac monitoring with remote control (ACM). Methods: We observed in dynamics for 5 years’ period 217 patients with CLL, constantly receiving therapy with Ib 420 mg/day. In the ACM group (n=89), in addition to the standard examination, every week or more often using messengers, we did active medical monitoring of the patient’s symptoms and well-being, assessment of blood pressure and heart rate, control of cardioprotective medicines taken, correction of the therapy, calling patients for examination and additional examination. The remaining 128 patients were examined in dynamics, but did not support remote control, constituting the SC group. The age of patients in the ACM group and in the SC group did not differ and was 66.0 (60.0–70.0) years and 66.0 (59.0–74.0) years, respectively (p = 0,39). There were slightly more men in the SC group (68.8%) than in the active cardiac monitoring group (53.9%), p = 0.026. The proportion of patients with CLL with the number of pretreatment lines from 0 to 2 (median -1) in the ACM group was 52.7%, in the SC group it was 58.1%; with the number of lines from 3 to 12 (median - 4) in the AKM group - 47.3%, in the SC group - 41.9% (p = 0.53). The AKM and SC groups did not differ in the results of the Geriatric 8 scale, Charlson index, and echocardiography parameters at visit 1. In the ACM group, there were more patients with cardiac problems: with arterial hypertension (AH) (p < 0.0001) and atrial fibrillation (p < 0.0001), receiving anticoagulants (p < 0.0001), a comparable number of patients with coronary artery disease. Results: In the ACM group, 70 out of 77 (90.9%) patients with CLL and AH achieved a stable level of target blood pressure values, in contrast to the SC group - 26 out of 66 (39.9%), p < 0.0001. Significantly more events requiring cardiac surgery (stenting of the coronary arteries, installation of a pacemaker) were detected in the ACM group – 12, versus 0 in the SC group (p = 0.0004). In the ACM group, despite a more pronounced cardiac comorbidity, 5-OS was significantly better, than in the SC group in both men (p < 0.0001) and women (p < 0.0001) with CLL and in patients older than 70 years old (p = 0.0004). 5-OS was also better in the ACM group than in the SC group in patients with CLL with a median number of previous lines of therapy equal to 1 (p<0.0001) and in patients with a median number of chemotherapy lines equal to 4 (p<0.0001), in patients with genetic abnormalities (p=0.004) and pretreated with fludarabine and/or anthracyclines (p < 0.0001). Summary/Conclusion: Early detection and correction of cardiovascular complications/events, achievement of stable target blood pressure values, constant monitoring of cardioprotective treatment in the ACM group explain the statistically highly significant differences in 5-OS in patients with CLL who are on constant Ib therapy. Conducting active cardiomonitoring with remote control makes it possible to achieve higher rates of total 5-OS in patients with CLL receiving Ib.

2-Hydroxy-4-methoxybenzyl as a Thiol-Protecting Group for Directed-Disulfide Bond Formation.

The chemical synthesis of disulfide-rich peptides such as toxins can be accomplished by using numerous orthogonal cysteine-protecting groups. Herein we report the use of the Hmboff/on protecting group for directed disulfide bond formation. Its combination with classical Trt, Acm, and Mob groups was studied for the synthesis of NMB-1 and phlotoxin-1 toxins highlighting new orthogonal strategies for directed disulfide bond formation.

Deprotection of S-Acetamidomethyl and 1,3-Thiazolidine-4-Carbonyl Protecting Groups from Cysteine Side Chains in Peptides by trans-[PtX2(CN)4]2–: One-Pot Regioselective Synthesis of Disulfide Bonds

In this study, we developed an efficient approach for disulfide bond formation in peptides utilizing the Pt(IV) complex trans-[PtBr2(CN)4]2- to mediate Acm and Thz deprotections. [PtBr2(CN)4]2- can oxidatively deprotect two Acm groups or deprotect one Thz group and one Acm group to directly form an intramolecular disulfide bond in peptides. Several disulfide-containing peptides with excellent yields were achieved via the deprotection method in an aqueous medium under aerobic conditions. Kinetic studies indicated that the dominant path of the reaction is of first-order in both [Pt(IV)] and [peptide]; moreover, the deprotection rate increased dramatically with the addition of NaBr. A mechanism including a bromide-bridge-mediated electron transfer process was proposed. Apamin, α-conotoxin SI, and the parallel homodimer of oxytocin, all containing two disulfide bonds, were synthesized regioselectively through a one-pot method by the combined use of the above deprotection approach with oxidants l-methionine selenoxide and [PtBr2(CN)4]2-. All of the reactions were completed within 30 min to afford good yields for these peptides.

2,2'-Dipyridyl diselenide: A chemoselective tool for cysteine deprotection and disulfide bond formation.

There are many examples of bioactive, disulfide-rich peptides and proteins whose biological activity relies on proper disulfide connectivity. Regioselective disulfide bond formation is a strategy for the synthesis of these bioactive peptides, but many of these methods suffer from a lack of orthogonality between pairs of protected cysteine (Cys) residues, efficiency, and high yields. Here, we show the utilization of 2,2'-dipyridyl diselenide (PySeSePy) as a chemical tool for the removal of Cys-protecting groups and regioselective formation of disulfide bonds in peptides. We found that peptides containing either Cys(Mob) or Cys(Acm) groups treated with PySeSePy in trifluoroacetic acid (TFA) (with or without triisopropylsilane (TIS) were converted to Cys-S-SePy adducts at 37 °C and various incubation times. This novel Cys-S-SePy adduct is able to be chemoselectively reduced by five-fold excess ascorbate at pH 4.5, a condition that should spare already installed peptide disulfide bonds from reduction. This chemoselective reduction by ascorbate will undoubtedly find utility in numerous biotechnological applications. We applied our new chemistry to the iodine-free synthesis of the human intestinal hormone guanylin, which contains two disulfide bonds. While we originally envisioned using ascorbate to chemoselectively reduce one of the formed Cys-S-SePy adducts to catalyze disulfide bond formation, we found that when pairs of Cys(Acm) residues were treated with PySeSePy in TFA, the second disulfide bond formed spontaneously. Spontaneous formation of the second disulfide is most likely driven by the formation of the thermodynamically favored diselenide (PySeSePy) from the two Cys-S-SePy adducts. Thus, we have developed a one-pot method for concomitant deprotection and disulfide bond formation of Cys(Acm) pairs in the presence of an existing disulfide bond.

Pore Structure as a Response to the Freeze/Thaw Resistance of Mortars.

In this paper, the resistance to the freeze/thaw cycles for four groups of mortars (lime—LM, lime based—LBM, cement—CM, and aerated cement—ACM mortars) with different amount of mortar components within each group is quantified via a ratio of flexural/compressive strength after and before exposure to freeze/thaw cycles. Using a pore system obtained by three different methods (mercury intrusion porosimetry, X-ray micro-computed tomography analysis, and SEM (Scanning Electron Microscopy) analysis), an attempt was made to explain why some mortars achieved better resistance to freeze/thaw cycles than others. The mortars with lime as a binder in the composition (LM and LBM groups) did not survive the freezing and thawing regime, while no visible damage was recorded in samples of the CM and ACM group. It is concluded that the low initial value of the mechanical properties of the LM and LBM mortars, as well as the higher proportion of harmful pores (pores greater than 0.064 μm) compared to CM and ACM mortars are responsible for their poor durability. According the results of nanotomography, it is concluded that the most important factor influencing freeze/thaw resistance is pore connectivity—the higher the connectivity of the macropores, the higher the freeze/thaw resistance of the mortar. SEM analysis proved to be a very useful method for aerated cement mortars as it revealed the pore sizes that were not covered by mercury porosimetry and nanotomography.

Ekstrakranijalna karotidna ateroskleroza u genezi infarkta mozga u graničnoj oblasti supratentorijalne lokalizacije

Introduction: The emergence of brain infarction depends to a great extent on the status of collateral blood circulation and is associated with hemodynamic instability. This kind of brain infarction is usually found in border vascular zones. Infarction of borderline fields arises between vascular territories ACA and ACM or between ACM and ACP, deep paraventricular infarcts in internal border zones, wound or triangular cortical infarcts, as well as major infarctions involving the entire irrigated territory of ACM. Aim:To define the atherosclerotic stenosis of the inner carotid artery and its significance in brain infarction genes in the borderline of supratentorial localization. Material and methods: Inclusion criteria for inclusion in the study are the existence of an infarct lesion in a predetermined border cortical and subcortical area of supratentorial localization. Data on extracranial atherosclerotic carotid disease were obtained by ultrasonographic inspection of a commercially available apparatus, 7-9 and 9-14 MHz probes/catheter. As a criterion for assessing the degree of carotid stenosis, the formula of the apparatus software was used: 100 x (A - B) / wherein Ais the area of the cross-section of the artery at the site of the largest stenosis, and the B surface of the free lumen artery cross section on the largest stenosis site. Results: The study included 30 selected patients, 12 (40%) females, aged 47-79, (± 62.3 years) and 18 (60%) male, aged 43-79 (± 58.7 years). Cortical infarction of the frontal border region (ACA - ACM) was seen in 11 (36.6%), 15 (50%) ACM - ACP infarct, while subcortical infarction between the surface and deep branches a. Cerebri media ACM - ACM), seen in 4 (13.4%) patients. Carotid atherosclerosis in the form of occlusion or significant stenosis with unilateral localization was observed in 6 (26%) patients, while bilateral changes were present in 17 (74%) patients. In the ACA + ACM group (ACM + ACM), a statistically significant increase in bilateral carotid artery involvement was observed in the group of patients with cerebral infarction of the last ACM-ACP border region (p = 0.018). Conclusion: The two-sided significant steno-occlusive atherosclerotic disease of the inner carotid artery is a very common and statistically significant finding in patients with endocardial infarction of supratentorial localization. This finding corroborates the pathophysiological explanation of this entity and opens up adequate therapeutic perspectives in the prevention and treatment of this kind of stroke.

The challenges of outreaching

According to Wikipedia, "outreaching is an activity of providing services to any populations who might not otherwise have access to those services" . Most people would have been the target of a fund raising campaign at some point, be it public radio during its annual fund raising or an alma mater fund raising event. While some of us may respond because it is a good cause or it aligns with our interests, out-reaching has always been very challenging. We recently met with some ACM groups that have been successful in getting members and outreaching to new people. This short paper summarizes what we learned from the conversations and discovered from our recent activities.

Cancer recurrence and mortality after pediatric heart transplantation for anthracycline cardiomyopathy: A report from the Pediatric Heart Transplant Study (PHTS) group.

We aimed to determine whether malignancy after pediatric HTx for ACM affects overall post-HTx survival. Patients <18y listed for HTx for ACM in the PHTS database between 1993 and 2014 were compared to those with DCM. A 2:1 matched DCM cohort was also compared. Wait-list and post-HTx survival, along with freedom from common HTx complications, were compared. Eighty subjects were listed due to ACM, whereas 1985 were listed for DCM. Although wait-list survival was higher in the ACM group, post-HTx survival was lower for the ACM cohort. Neither difference persisted in the matched cohort analysis. Primary cause of death in the ACM group was infection, which was higher than the DCM group. Malignancy rates were not different. All ACM malignancies were due to PTLD without primary cancer recurrence or SMN. Long-term graft survival after pediatric HTx for ACM is no different than for matched DCM peers, nor is there an increased risk of any malignancy. However, risk of infection and death from infection after HTx are higher in the ACM group. Further studies are needed to assess the effects of prior chemotherapy on susceptibility to infection in this group.

Effect of acupuncture combined with Chinese medicine on the expression of interstitial cells of Cajal, substance P and nerve nitric oxide synthase in diabetic mice with gastroparesis antrum

ObjectiveTo explore the effect of acupuncture combined with Chinese medicine on expression of interstitial cells of Cajal (ICC), substance P (SP) and nerve nitric oxide synthase (nNOS) in diabetic gastroparesis mice. MethodsEighty Kunming male mice were randomly divided into normal group (group A, 15 mice), diabetic gastroparesis model mice group (group B, 12 mice), acupuncture combined with Chinese medicine group (group C, 12 mice), Chinese medicine group (group D, 13 mice) and western medicine group (group E, 13 mice). The mice were intraperitoneally injected with steptozotocin and fed with high-fat diet-induced irregular except the normal group mice, that made mouse model of diabetic gastroparesis. And then, the ACM group were treated by acupuncture at the acupoints of Housanli (▪ ST 36) and Zhōngwăn (▪ CV 12) combined with Chinese medicine, the CM group were only treated by Chinese medicine; the WM group were treated by domperidone and vitamin B 6. Numbers and expression of ICC, SP and nNOS were detected by immunohistochemical methods. ResultsAfter treatment, compared with normal group, ICC and SP indexes of model group were significantly reduced. Compared with model group, all indexes in ACM and CM groups were improved significantly (P<0.05, P<0.01), but only the positive area and average optical density value in c-Kit and SP in WM group increased obviously (P<0.05). Compared with ACM group, no significant difference in c-Kit and SP of CM group were observed (P>0.05), but expression of nNOS were increased markedly (P<0.01). Besides, c-Kit and SP expressions of WM group were significantly induced was increased remarkably (P<0.01). ConclusionAcupuncture combined with Chinese medicine have much better therapeutic effects for diabetic gastroparesis mice, which may be due to repairing ICC damage, upregulating the expression of SP and lowering the expression of nNOS.

Effect of Antrodia cinnamomea on gene expression related to aortal endothelial injury in rats with hyperlipidemia

To investigate the effect of Antrodia cinnamomea on gene expression related to aortal endothelial injury of rats with hyperlipidemia. Fifty SD rats were randomly divided into five groups: the normal control group (NG), the model group (MG), the antrodia cinnamomea groups of low, middle and high doses (AC-LG, AC-MG, AC-HG, 250, 500, 1 000 mg x kg(-1)). The rats were fed with high-fat diets to establish the hyperlipidemia model. After the drug administration for 10 weeks, their serum lipid, SOD, MDA and ox-LDL, LOX-1, P38 MAPK and NF-kappaB mRNA and protein expression were respectively determined, and the aortal endothelial injury was observed under electron microscope. In the model group, the contents of TC, TG and LDL-C significant increased (P < 0.01), whereas the content of HDL-C significant decreased (P < 0.01). Compared with the model group, both the AC-M group and the AC-H group showed reduction in endothelial injury and significant decrease in the content of TC, TG and LDL-C (P < 0.05 or P < 0.01). The content of HDL-C increased, but with no significant difference. SOD activity in serum remarkably increased (P < 0.05 or P < 0.01), MDA and ox-LDL levels dramatically decreased (P < 0.05 or P < 0.01). A. cinnamomea can alleviate endothelial lipid injury by inhibiting the expressions of LOX-1, P38MAPK and NF-kappaB in aorta and better protect aortal endothelial cells from oxidative lipid injury.

Cytarabine plus mitoxantrone versus cytarabine plus clofarabine as second-line therapy in AML.

e18001 Background: There are no comparative studies that can provide guidance about the choice of second line therapy in AML patients (pts). Methods: We retrospectively evaluated 30 consecutive pts who underwent reinduction chemotherapy for AML with mitoxantrone and cytarabine (ACM) or cytarabine and clofarabine (ACC). Results: Baseline characteristics of the pts are described in the Table. Ten pts achieved remission after initial induction therapy in ACM group and 4 pts in ACC group (P=0.71). Median length of remission was 314 days (d) (81-874 d) in ACM group and 178.5 d (57-232 d) in ACC group (P=0.13).After reinduction in ACM and ACC groups 9 and 3 patients achieved remission respectively (P=0.4).One pt in each group underwent allogeneic stem cell transplant. After a median follow-up of 11.2 m (ACM) and 10.5m (ACC), 9 and 6 pts had died in ACM and ACC groups respectively. Causes of death were progression (ACM=2pts, ACC= 1pt), end organ failure (3 pts each) and unknown (ACM=4pts, ACC=2 pts). Median Overall survival (OS) was 18.4 months (m) in ACM and 13.1 m in ACC group (P=0.49). After reinduction therapy OS was 8.3 and 6.9 m in ACM and ACC groups (P=0.72). In our patient cohort we did not find any baseline characteristics that were associated with OS or achievement of remission after reinduction therapy. Conclusions: In our pt cohort both ACM and ACC regimens demonstrated the ability to induce remission as a possible bridging strategy to allogeniec stem cell transplant. Larger studies are needed to compare these two regimens as well as evaluate any baseline characteristics which correlate with OS or achievement of remission. [Table: see text]

Total synthesis of α-conotoxin MII using a soluble-tag-assisted method

Oxidative disulfide bond formations have been applied to soluble-tag-assisted method successfully to realize the total synthesis of α-conotoxin MII (4) that comprises 16 amino acid residues and possesses 2 disulfide bonds. Orthogonal peptide folding using DEAD and iodine oxidations in combination with Mmt and Acm groups for the side chain protection of cysteines could be carried out with no peptide aggregation.

Potential Risk of Artificial Cerebrospinal Fluid Solution Without Magnesium Ion for Cerebral Irrigation and Perfusion in Neurosurgical Practice

The effect of irrigation with artificial cerebrospinal fluid (CSF) containing various magnesium ion (Mg(2+)) concentrations on vasospastic arteries was investigated in the dog. Cerebral vasospasm was induced by the experimental subarachnoid hemorrhage model in 15 beagle dogs. Cisternal irrigation was performed for 1 hour via a microcatheter placed in the cisterna magna with commercially available artificial CSF (ARTCEREB®) with physiological concentration of Mg(2+) (2.2 mEq/l) (ACM group, n = 5), ARTCEREB solution without Mg(2+) (ACR group, n = 5), and ARTCEREB solution with higher Mg(2+) concentration (5 mEq/l) (ACMM group, n = 5). CSF electrolyte concentrations and the diameters of the basilar and vertebral arteries were measured. In the ACM group, no changes were detected in either CSF Mg(2+) concentration or arterial diameters. In the ACR group, the CSF Mg(2+) decreased significantly to 0.8 ± 0.07 mEq/l from the baseline value of 1.4 ± 0.03 mEq/l, and both basilar and vertebral artery diameters were significantly decreased to 0.61 ± 0.18 mm and 0.57 ± 0.23 mm from their baseline values of 0.74 ± 0.22 mm and 0.68 ± 0.17 mm, respectively. In the ACMM group, the CSF Mg(2+) significantly increased to 2.4 ± 0.15 mEq/l from the baseline value of 1.4 ± 0.05 mEq/l, and both basilar and vertebral artery diameters were significantly increased to 0.84 ± 0.19 mm and 0.90 ± 0.22 mm from their baseline values of 0.71 ± 0.21 mm and 0.69 ± 0.24 mm, respectively. Irrigation with artificial CSF solution without Mg(2+) causes vasoconstriction of the cerebral artery. Irrigation with artificial CSF with appropriate Mg(2+) concentration is essential, especially in patients with subarachnoid hemorrhage.

4-Methoxybenzyloxymethyl Group, a Racemization-Resistant Protecting Group for Cysteine in Fmoc Solid Phase Peptide Synthesis

The 4-methoxybenzyloxymethyl (MBom) group was introduced for sulfhydryl protection of Cys in combination with Fmoc chemistry. The MBom group proved to substantially suppress racemization of Cys during its incorporation mediated by phosphonium or uronium reagents. Furthermore, this group was found to significantly reduce racemization of the C-terminal Cys linked to a hydroxyl resin during repetitive base treatment, in comparison with the usually used trityl (Trt) and acetamidomethyl (Acm) groups.

On‐resin conversion of Cys(Acm)‐containing peptides to their corresponding Cys(Scm) congeners

The Acm protecting group for the thiol functionality of cysteine is removed under conditions (Hg(2+)) that are orthogonal to the acidic milieu used for global deprotection in Fmoc-based solid-phase peptide synthesis. This use of a toxic heavy metal for deprotection has limited the usefulness of Acm in peptide synthesis. The Acm group may be converted to the Scm derivative that can then be used as a reactive intermediate for unsymmetrical disulfide formation. It may also be removed by mild reductive conditions to generate unprotected cysteine. Conversion of Cys(Acm)-containing peptides to their corresponding Cys(Scm) derivatives in solution is often problematic because the sulfenyl chloride reagent used for this conversion may react with the sensitive amino acids tyrosine and tryptophan. In this protocol, we report a method for on-resin Acm to Scm conversion that allows the preparation of Cys(Scm)-containing peptides under conditions that do not modify other amino acids.

P‐Nitrobenzyl protection for cysteine and selenocysteine: A more stable alternative to the acetamidomethyl group

This study evaluated the acidic lability of the acetamidomethyl (Acm), trimethylacetamidomethyl (Tacm), and the p-nitrobenzyl (pNB) as protecting groups for cysteine and selenocysteine (Sec) during the tert-butyloxycarbonyl (Boc)-chemistry solid-phase peptide synthesis of oxytocin (OT). Two novel Sec building blocks (Nalpha-tert-butyloxycarbonyl-Se(acetamidomethyl)-L-selenocysteine (Boc-L-Sec(Acm)-OH) and Nalpha-tert-butyloxycarbonyl-S(4-nitrobenzyl)-L-selenocysteine (Boc-L-Sec(pNB)-OH)) were developed for this study. Six partially protected thio- and seleno-OT analogues were synthesized, purified, and exposed to neat trifluoroacetic acid (TFA) at temperatures of 25, 40, 50, and 60 degrees C for 1 h, and HF treatment at 0 degrees C for 1 h. Significant losses were observed for the Acm and Tacm group in TFA at temperatures greater than 25 degrees C and during HF treatment at 0 degrees C, whereas the pNB group remained intact. Removal of the pNB was achieved via reduction to the p-aminobenzyl group either with zinc in acetic acid in solution or via tin chloride in hydrochloric acid on solid support, followed by oxidative cleavage with iodine yielding the corresponding disulfide or diselenide bond. No major side reactions were observed. This study confirms the occasionally described Acm instability and underpins the development of the pNB group as an alternative for cysteine and Sec protection.

Toward the total chemical synthesis of the cancer protein NY‐ESO‐1

During the course of developing a synthetic route for the cancer protein NY-ESO-1 using native chemical ligation, a number of the required thioester polypeptide fragments were unable to be synthesized effectively using Boc solid phase peptide synthesis. Modification of the SPPS protocols to include an arginine tag at the C terminus linked via the thioester resulted in a better purity profile and enhanced solubility, facilitating purification by HPLC. During preparation of another reactive partner for ligation that contained an internal Cys(Acm) residue by Fmoc SPPS, extensive loss of the Acm group occurred during cleavage from the resin while substitution with Cys(tBu) resulted in no loss of protecting group. It was shown that native chemical ligation of N-terminal cysteine peptide 155-180 containing the Cys(tBu) residue with thioester 140-154 was slow, incomplete and led to extensive HPLC column fouling. Subsequent incorporation of a C-terminal arginine tag into the N-terminal NY-ESO-1 155-180 fragment joined by a base labile 4-hydroxymethylbenzoic acid (HMBA) linker facilitated rapid quantitative ligation. The HMBA linker was demonstrated to be stable to the conditions required for native chemical ligation, subsequent transformations and final purification. Importantly it was effectively removed at pH=10.

Clinical evaluation and induced corneal vascularization study by native and anionic collagen membranes in rabbits corneas

To evaluate the corneal vascularization (CV) and the clinical aspects induced by interlamellar graft with native (NCM) and anionic (ACM) collagen membranes in rabbits corneas. An interlamellar graft with a 0.25 x 0.25 cm NCM (group 1) or ACM (group 2) fragment was performed in the right eye (treated eye). In the left eye, an estromal tunnel was done (control eye). Sixteen rabbits were used, and they were subdivided into two experimental groups of eight animals each. The clinical evaluation was performed at the 1st, 3rd, 7th, 15th and 30th postoperative days. Corneal vascularization analysis was performed after 30 days by the Images Analizator System Leica Qwin-550. After 7 days, corneal vascularization was observed at about 2.25 +/- 0.71 mm (NCM) and at about 1.0 +/- 1.69 mm (ACM), respectively, from the limbus in direction to the central cornea. After 15 days, CV increased in both groups (5.25 +/- 1.03 mm - NCM; 2.0 +/- 2.39 mm - ACM) and then progressively decreased until day 30 (2.25 +/- 2.10 mm - NCM; 0.75 +/- 2.12 mm - ACM). The statistical analysis indicated that the averages of the distances from the limb vessels to the grafts observed after 7 and 15 days had not differed statistically (p=0.17), and after 15 and 30 postoperative days had a tendency to differ statistically (p=0.09). The control eyes did not present any changes. The interlamellar graft with native and anionic collagen membranes induced corneal vascularization when applied to rabbit corneas, but anionic collagen membrane induced a smaller corneal vascularization when compared to native collagen membrane. Although further studies are required, the results found in this study demonstrated the usefulness of interlamellar graft with native and anionic collagen membranes in keratoplasties. These membranes consists in one more graft option for the surgical treatment of corneal repair in rabbits and others animals, when other forms of medical and surgical treatment are not effective.